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Methods in
Molecular Biology 1161

Erica Spackman Editor

Animal
Influenza Virus
METHODS IN M O L E C U L A R B I O LO G Y

Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651
 Animal Influenza Virus

Second Edition

Edited by

Erica Spackman
Exotic and Emerging Avian Viral Diseases Unit, Southeast Poultry Research Laboratory,
US Department of Agriculture, Agricultural Research Service, Athens, GA, USA
Editor
Erica Spackman
Exotic and Emerging Avian Viral Diseases
Unit
Southeast Poultry Research Laboratory
US Department of Agriculture
Agricultural Research Service
Athens, GA, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic)

ISBN 978-1-4939-0757-1 ISBN 978-1-4939-0758-8 (eBook)
DOI 10.1007/978-1-4939-0758-8
Springer New York Heidelberg Dordrecht London

Library of Congress Control Number: 2014937456

© Springer Science+Business Media New York 2014

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
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Preface

Influenza A viruses are among the most important pathogens for humans, food animals,
and companion animals. Of the animal influenza viruses, avian, swine, and equine influenza
are likely to have the greatest economic impact internationally because of their value as food
animals or, with horses due to a large sport competition industry. Also, although the risk is
truly unknown, as domestic animals, poultry, swine, and horses have extended contact with
humans, which provides an interface for interspecies transmission, there is always the poten-
tial for these animal influenza viruses to become threats to public health.
Regardless of the possible implications for public health, influenza is highly significant
to poultry, swine, and equine health. Research and diagnostics with animal influenza viruses
are critical to animal health in its own right and it should be recognized that the needs and
goals of animal agriculture and veterinary medicine are not always the same as those of
public health. Even within these three examples of animal influenza viruses there are differ-
ences in the approach which may need to be taken, as the structure of the poultry, swine,
and equine industries are different and there are some biological differences of influenza
virus from each animal group as well. One of the aims of this book is to sort out those dif-
ferences and to provide host, strain, and lineage specific guidance and procedures.
The reader will also recognize that in some cases the same method is described for all
three of these animal viruses, for example real-time RT-PCR or hemagglutination inhibi-
tion (HI) assay. At first glance this may seem redundant; however there are often seemingly
minor, but crucial differences in the assay, such as sample processing for each species (e.g.,
how to treat for sera the HI assay) or the specificity of reagents (e.g., primer sequences for
RT-PCR; optimal laboratory host system for virus isolation). In contrast there are some
methods that will be unique to an animal influenza virus group and parameters will neces-
sarily vary. Certainly, assays may be adapted to individual study needs with proper optimiza-
tion or can simply be used as they are described. The aim of this book is to provide the
essential methods used in working with animal influenza viruses, and to compile more
advanced information that will guide the user in designing influenza studies.
Most importantly this book would not have been possible without the contributions of
the authors. The contributors are experts in their fields; therefore their input and knowl-
edge is invaluable with the details they provide from their extensive experience. I want to
gratefully acknowledge each of them for taking time from their busy schedules to contrib-
ute to this book. I would also like to thank the editorial team at Springer: John Walker,
MIMB series editor, and both Patrick Marton and David Casey for all their time and help
with completing this book.
Happy pipetting.

Athens, GA, USA Erica Spackman

v
Contents

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

PART I GENERAL
1 Hemagglutination Assay for Influenza Virus . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Mary Lea Killian
2 Hemagglutination-Inhibition Assay for Influenza Virus Subtype
Identification and the Detection and Quantitation of Serum
Antibodies to Influenza Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Janice C. Pedersen
3 Neuraminidase-Inhibition Assay for the Identification of
Influenza A Virus Neuraminidase Virus Subtype or Neuraminidase
Antibody Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Janice C. Pedersen
4 Reverse Genetics of Influenza Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Chang-Won Lee
5 Immunohistochemical Staining of Influenza Virus in Tissues. . . . . . . . . . . . . . 51
Mary J. Pantin-Jackwood

PART II AVIAN INFLUENZA

6 A Brief Introduction to Avian Influenza Virus. . . . . . . . . . . . . . . . . . . . . . . . . 61
Erica Spackman
7 Wild Bird Surveillance for Avian Influenza Virus. . . . . . . . . . . . . . . . . . . . . . . 69
Justin D. Brown, Rebecca Poulson, and David E. Stallknecht
8 Avian Influenza Virus Sample Types, Collection, and Handling . . . . . . . . . . . 83
Mary Lea Killian
9 Avian Influenza Virus RNA Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Erica Spackman and Scott A. Lee
10 Avian Influenza Virus Detection and Quantitation
by Real-Time RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Erica Spackman
11 Influenza Subtype Identification with Molecular Methods. . . . . . . . . . . . . . . . 119
Erica Spackman
12 Avian Influenza Virus Isolation, Propagation, and Titration
in Embryonated Chicken Eggs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Erica Spackman and Mary Lea Killian
13 Agar Gel Immunodiffusion Assay to Detect Antibodies
to Type A Influenza Virus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Terra A. Jenson

vii
viii Contents

14 Detection of Influenza A Antibodies in Avian Serum Samples by ELISA . . . . . 151

Len Chappell, Mary Lea Killian, and Erica Spackman
15 Conducting Influenza Virus Pathogenesis Studies in Avian Species . . . . . . . . . 169
Carol J. Cardona, David A. Halvorson, Justin D. Brown,
and Mary J. Pantin-Jackwood
16 Laboratory Methods for Assessing and Licensing Influenza
Vaccines for Poultry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
David E. Swayne
17 Detection of Cell-Mediated Immune Response
to Avian Influenza Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Darrell R. Kapczynski
18 Characterization of Cytokine Expression Induced by Avian
Influenza Virus Infection with Real-Time RT-PCR . . . . . . . . . . . . . . . . . . . . . 217
Darrell R. Kapczynski, Hai Jun Jiang, and Michael H. Kogut
19 Newcastle Disease Virus Detection and Differentiation
from Avian Influenza . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
Patti J. Miller and Mia Kim Torchetti

PART III SWINE INFLUENZA

20 A Brief Introduction to Influenza A Virus in Swine. . . . . . . . . . . . . . . . . . . . . 243
Amy L. Vincent, Kelly M. Lager, and Tavis K. Anderson
21 Sample Types, Collection, and Transport for Influenza A
Viruses of Swine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
Marie R. Culhane and Susan E. Detmer
22 Isolation of Swine Influenza Virus in Cell Cultures
and Embryonated Chicken Eggs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
Jianqiang Zhang and Phillip C. Gauger
23 RNA Extraction from Swine Samples and Detection of
Influenza A Virus in Swine by Real-Time RT-PCR . . . . . . . . . . . . . . . . . . . . . 277
Jianqiang Zhang and Karen M. Harmon
24 Hemagglutinin Inhibition Assay with Swine Sera . . . . . . . . . . . . . . . . . . . . . . 295
Pravina Kitikoon, Phillip C. Gauger, and Amy L. Vincent
25 Enzyme-Linked Immunosorbent Assay for Detection of Serum
or Mucosal Isotype-Specific IgG and IgA Whole-Virus Antibody
to Influenza A Virus in Swine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
Phillip C. Gauger, Crystal L. Loving, and Amy L. Vincent
26 Serum Virus Neutralization Assay for Detection and Quantitation
of Serum-Neutralizing Antibodies to Influenza A Virus in Swine . . . . . . . . . . 313
Phillip C. Gauger and Amy L. Vincent
27 Microneutralization Assay for Swine Influenza Virus in Swine Serum . . . . . . . 325
Pravina Kitikoon and Amy L. Vincent
 Contents ix

28 Analyzing Swine Sera for Functional Antibody Titers Against

Influenza A Neuraminidase Proteins Using an Enzyme-Linked
Lectin Assay (ELLA). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
Matthew R. Sandbulte and Maryna C. Eichelberger
29 Antibody Secreting Cell Assay for Influenza A Virus in Swine . . . . . . . . . . . . . 347
Pravina Kitikoon, Crystal L. Loving, and Amy L. Vincent
30 In Vivo Models for Pathotyping and Vaccine Efficacy
for Swine Influenza. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
Kelly M. Lager and Amy L. Vincent

PART IV EQUINE INFLUENZA

31 A Brief Introduction to Equine Influenza and Equine
Influenza Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
Thomas M. Chambers
32 Equine Influenza Diagnosis: Sample Collection and Transport . . . . . . . . . . . . 371
Thomas M. Chambers and Stephanie E. Reedy
33 RNA Extraction from Equine Samples for Equine Influenza Virus . . . . . . . . . 379
Udeni B.R. Balasuriya
34 Type A Influenza Virus Detection from Horses by Real-Time
RT-PCR and Insulated Isothermal RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . 393
Udeni B.R. Balasuriya
35 Equine Influenza Culture Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
Thomas M. Chambers and Stephanie E. Reedy
36 Equine Influenza Serological Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
Thomas M. Chambers and Stephanie E. Reedy

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423
Contributors

TAVIS K. ANDERSON • Department of Biology, Georgia Southern University, Statesboro,

GA, USA
UDENI B.R. BALASURIYA • Department of Veterinary Science, Maxwell H. Gluck Equine
Research Center, University of Kentucky, Lexington, KY, USA
JUSTIN D. BROWN • Department of Population Health, Southeastern Cooperative Wildlife
Disease Study, College of Veterinary Medicine, University of Georgia, Athens, GA, USA
CAROL J. CARDONA • College of Veterinary Medicine, University of Minnesota, Saint Paul,
MN, USA
THOMAS M. CHAMBERS • Department of Veterinary Science, Maxwell H. Gluck Equine
Research Center, University of Kentucky, Lexington, KY, USA
LEN CHAPPELL • Georgia Poultry Laboratory Network, Oak wood, GA, USA
MARIE R. CULHANE • Veterinary Diagnostic Laboratory, University of Minnesota, St. Paul,
MN, USA
SUSAN E. DETMER • Veterinary Pathology, Western College of Veterinary Medicine,
University of Saskatchewan, Saskatoon, SK, Canada
MARYNA C. EICHELBERGER • Division of Viral Products, Center for Biologics Evaluation
and Research, Food and Drug Administration, Bethesda, MD, USA
PHILLIP C. GAUGER • Veterinary Diagnostic Laboratory, Iowa State University, Ames, IA, USA
DAVID A. HALVORSON • Department of Veterinary Biomedical Sciences, College of
Veterinary Medicine, University of Minnesota, St. Paul, MN, USA
KAREN M. HARMON • Veterinary Diagnostic Laboratory, Iowa State University, Ames,
IA, USA
TERRA A. JENSON • Diagnostic Virology Laboratory, U.S. Department of Agriculture,
National Veterinary Services Laboratories, Animal and Plant Health Inspection Service,
Ames, IA, USA
HAI JUN JIANG • Exotic and Emerging Avian Viral Diseases Unit, Southeast Poultry
Research Laboratory, US Department of Agriculture, Agricultural Research Service,
Athens, GA, USA
DARRELL R. KAPCZYNSKI • Exotic and Emerging Avian Viral Diseases Unit, Southeast
Poultry Research Laboratory, US Department of Agriculture, Agricultural Research
Service, Athens, GA, USA
MARY LEA KILLIAN • Diagnostic Virology Laboratory, U.S. Department of Agriculture,
National Veterinary Services Laboratories, Animal and Plant Health Inspection Service,
Ames, IA, USA
PRAVINA KITIKOON • Virus and Prion Research Unit, U.S. Department of Agriculture,
National Animal Disease Center, Agricultural Research Service, Ames, IA, USA
MICHAEL H. KOGUT • Food and Feed Safety Research Unit, U.S. Department of
Agriculture, Southern Plains Agricultural Research Center, Agricultural Research
Service, College Station, TX, USA

xi
xii Contributors

KELLY M. LAGER • Virus and Prion Research Unit, U.S. Department of Agriculture,
National Animal Disease Center, Agricultural Research Service, Ames, IA, USA
CHANG-WON LEE • Food Animal Health Research Program, Ohio Agricultural Research
and Development Center, The Ohio State University, Wooster, OH, USA
SCOTT A. LEE • Exotic and Emerging Avian Viral Diseases Unit, Southeast Poultry
Research Laboratory, US Department of Agriculture, Agricultural Research Service,
Athens, GA, USA
CRYSTAL L. LOVING • Virus and Prion Research Unit, U.S. Department of Agriculture,
National Animal Disease Center, Agricultural Research Service, Ames, IA, USA
PATTI J. MILLER • Exotic and Emerging Avian Viral Diseases Unit, Southeast Poultry
Research Laboratory, US Department of Agriculture, Agricultural Research Service,
Athens, GA, USA
MARY J. PANTIN-JACKWOOD • Exotic and Emerging Avian Viral Diseases Unit,
Southeast Poultry Research Laboratory, US Department of Agriculture, Agricultural
Research Service, Athens, GA, USA
JANICE C. PEDERSEN • Diagnostic Virology Laboratory, U.S. Department of Agriculture,
National Veterinary Services Laboratories, Animal and Plant Health Inspection Service,
Ames, IA, USA
REBECCA POULSON • Department of Population Health, Southeastern Cooperative Wildlife
Disease Study, College of Veterinary Medicine, University of Georgia, Athens, GA, USA
STEPHANIE E. REEDY • Department of Veterinary Science, Maxwell H. Gluck Equine
Research Center, University of Kentucky, Lexington, KY, USA
MATTHEW R. SANDBULTE • Department of Veterinary Microbiology and Preventive
Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA, USA
ERICA SPACKMAN • Exotic and Emerging Avian Viral Diseases Unit, Southeast Poultry
Research Laboratory, US Department of Agriculture, Agricultural Research Service,
Athens, GA, USA
DAVID E. STALLKNECHT • Department of Population Health, Southeastern Cooperative
Wildlife Disease Study, College of Veterinary Medicine, University of Georgia, Athens,
GA, USA
DAVID E. SWAYNE • Exotic and Emerging Avian Viral Diseases Unit, Southeast Poultry
Research Laboratory, US Department of Agriculture, Agricultural Research Service,
Athens, GA, USA
MIA KIM TORCHETTI • Diagnostic Virology Laboratory, U.S. Department of Agriculture,
National Veterinary Services Laboratories, Animal and Plant Health Inspection Service,
Ames, IA, USA
AMY L. VINCENT • Virus and Prion Research Unit, U.S. Department of Agriculture,
National Animal Disease Center, Agricultural Research Service, Ames, IA, USA
JIANQIANG ZHANG • Veterinary Diagnostic Laboratory, Iowa State University, Ames, IA, USA
 Part I

General
 Chapter 1

Hemagglutination Assay for Influenza Virus

Mary Lea Killian

Abstract
The hemagglutination assay (HA) is a tool used to screen cell culture isolates or amnioallantoic fluid harvested
from embryonated chicken eggs for hemagglutinating agents, such as type A influenza. The HA assay is
not an identification assay, as other agents also have hemagglutinating properties. Live and inactivated
viruses are detected by the HA test. Amplification by virus isolation in embryonated chicken eggs or cell
culture is typically required before HA activity can be detected from a clinical sample. The test is, to some
extent, quantitative as 1 hemagglutinating unit (HAU) is equal to approximately 5–6 logs of virus. It is
inexpensive and relatively simple to conduct. Several factors (quality of chicken erythrocytes, laboratory
temperature, laboratory equipment, technical expertise of the user) may contribute to slight differences in
the interpretation of the test each time it is run. This chapter describes the methods validated and used by the
US National Veterinary Services Laboratories for screening and identification of hemagglutinating viruses.

Key words Hemagglutination, Avian influenza virus, Type A influenza, Influenza detection, Virus titer

1 Introduction

The hemagglutinin protein on the surface of influenza virus parti-

cles is capable of binding to N-acetylneuraminic acid-containing
proteins on avian and mammalian erythrocytes [1, 2]. When com-
bined, if the influenza virus is present in a high enough concentra-
tion, there is an agglutination reaction and the erythrocytes link
together to form a diffuse lattice (see Fig. 1). The hemagglutina-
tion assay (HA) is a classic diagnostic test used to screen cell cul-
ture supernatant or amnionic–allantoic fluid (AAF) harvested from
embryonated chicken eggs.
The HA is not an identification assay. Other types of viruses
(e.g., paramyxoviruses, adenovirus-127) and certain bacteria also
have hemagglutinating properties [3–5]. The HA should be
followed by a hemagglutination-inhibition assay (see Chapter 2) to
determine the type and/or subtype of virus. The HA does not nec-
essarily indicate the presence of a viable virus [1]. It is also capable

Erica Spackman (ed.), Animal Influenza Virus, Methods in Molecular Biology, vol. 1161,
DOI 10.1007/978-1-4939-0758-8_1, © Springer Science+Business Media New York 2014

3
4 Mary Lea Killian

Fig. 1 Illustration of the hemagglutination process. The virus binds red bloods
cells (RBCs) and forms a matrix by linking RBCs together which prevents the
RBCs from settling in the diluent (often PBS)

of detecting viral particles that have been degraded or inactivated

and are no longer infectious.
Generally, there is enough virus present following a single pas-
sage in culture for detection of influenza virus by HA [4, 5].
Additional passages increase the chance for cross-contamination in
the laboratory. On average, a 105–106 50 % egg infectious dose
(EID50)/ml is required for detection by HA [5]. At the time of
collection, AAF that contains blood should be discarded as there
may be some adsorption of the virus by the erythrocytes, decreas-
ing the overall titer of the virus in the fluid or inhibiting the ability
to detect the virus by HA assay.
The following test procedure is used at the US National
Veterinary Services Laboratories (NVSL). The World Animal
Health Organization (OIE) Manual of Standards for Diagnostic
Tests and Vaccines outlines a slightly different procedure, however
the results are equivalent. The OIE procedure utilizes different
volumes of reagents and a different concentration of chicken eryth-
rocytes, and the steps are performed in a slightly different order.
For this reason, steps or reagents from the NVSL procedure and
the OIE procedure should not be combined in the same test.

2 Materials

1. Sterile bottle with lid.

2. Anticoagulant, Alsever’s solution or acid citrate dextrose.
(a) Alsever’s solution: Weigh out reagents into a conical flask:
0.55 g of citric acid, 0.8 g of sodium citrate, 2.05 g of D-
glucose, and 0.42 g of sodium chloride. Dissolve in distilled
water to a final volume of 100 ml. Dispense into sterile 10 ml
 HA Assay for AIV 5

bottles, do not tighten lids and sterilize by autoclaving at

116 °C for 10 min. Use slow exhaust. Allow to cool, then
tighten the lid and label the bottle. Store at 4 °C.
(b) Acid citrate dextrose (ACD): Weigh out reagents into a
conical flask: 4 g of citric acid, 11.3 g of sodium citrate,
and 11 g of D-glucose. Dissolve in 300 ml of distilled water
then add distilled water to a final volume of 500 ml.
Dispense into 100 ml bottles and do not tighten the lids.
Sterilize by autoclaving at 116 °C for 10 min. Use slow
exhaust. Allow to cool, then tighten the lid and label the
bottle. Store at 4 °C.
3. 50 ml Conical tubes.
4. U-bottom or V-bottom 96-well plate with lid.
5. Single and multichannel pipettes and pipette tips to deliver
50 μl volumes.
6. Liquid reagent reservoirs.
7. 0.1 M phosphate-buffered saline (PBS), pH 7.2.
(a) To prepare PBS, combine the following ingredients: 8.5 g
of sodium chloride, 1.33 g of sodium phosphate dibasic,
and 0.22 g of sodium phosphate monobasic. Dissolve in
distilled water to a final volume of 1 l. Mix thoroughly and
adjust pH to 7.2 ± 0.1.
8. Chicken erythrocytes, 0.5 % in PBS (see Chapter 2).
9. Positive control influenza antigen.
10. Mylar microtiter plate sealers (Thermo) or equivalent.
11. Test material: AAF harvested from eggs inoculated with AIV
or an AIV suspect sample. Sufficient quantity of AAF should be
harvested to provide enough material to conduct the HA assay
and subsequent characterization assays. Cell culture superna-
tant may also be used.

3 Methods

3.1 Collection and 1. Rooster red blood cells (RBCs) collected from specific
Preparation of Rooster pathogen-free (SPF) chickens are preferred for use in the HA
Red Blood Cells test. Red blood cells collected from hens may contain hor-
mones that interfere with hemagglutination. Chicken eryth-
rocytes are typically used because the settling time is quicker
and the settling patterns are typically clearer than with cells
from other species [2]. Certain AI viruses may not hemag-
glutinate chicken erythrocytes before adaptation in embryo-
nated chicken eggs; these viruses may be more sensitive to
hemagglutination with turkey erythrocytes or guinea pig
erythrocytes.
6 Mary Lea Killian

2. Fresh erythrocytes should be prepared regularly. Red blood

cells will begin to hemolyze after 5–7 days, causing inaccura-
cies in the HA test [6]. The erythrocyte suspension should be
mixed gently before use to ensure a uniform distribution of
cells in each of the test wells.
3. Prepare a sterile bottle with a lid containing an anticoagulant
(either ACD or Alsever’s solution). Use one volume ACD to
three volumes blood or one volume Alsever’s solution to one
volume blood.
4. Before collecting blood, draw a small amount of anticoagulant
into the syringe and expel again. This will coat the syringe
which will keep the blood from coagulating in the syringe
before addition to the anticoagulant in the bottle.
5. Collect 3–5 ml blood from the wing vein or by cardiac punc-
ture from an SPF rooster in accordance with the appropriate
animal care and use procedures. Animals should not be vacci-
nated for Newcastle disease or other pathogens as this may
interfere with some serological hemagglutination-inhibition
tests. Add the blood to the anticoagulant. Rotate the bottle
gently to mix thoroughly. Always treat the red blood cells gen-
tly to avoid hemolysis.
6. Add the suspension to a 50 ml conical tube and add a sufficient
quantity of PBS to total 50 ml. Rotate gently to mix.
7. Centrifuge at approximately 800 × g for 10 min to pellet RBCs.
8. Aspirate supernatant and surface layer of white cells (buffy coat)
from the tube without disturbing the pellet of erythrocytes.
9. Wash the erythrocytes a total of three times in PBS by repeat-
ing steps 6–8.
10. Add 1 ml packed red blood cells to 199 ml PBS for a final RBC
concentration of 0.5 %.

3.2 Hemag- 1. Orient a microtiter plate (U-bottom or V-bottom) so that

glutination Assay samples will be diluted either 8 wells or 12 wells across as
needed. Number the rows on each plate so that the contents of
each row are uniquely identified.
2. Add 50 μl PBS to every well on the plate.
3. Add 50 μl AAF (see Note 1) or cell culture fluid to be tested
(“test material”) to the first well in each row (or column) to be
tested. Note that this will result in a 1:2 dilution of test mate-
rial. Positive control antigen and cell control (no antigen,
where PBS is added instead of antigen) wells with RBCs must
be included each time the test is performed. If multiple plates
are being tested at the same time, one positive control antigen
and one RBC only control well should be included on a mini-
mum of every fifth plate.
 HA Assay for AIV 7

4. Dilute the test material: Mix the contents of the first well by
pipetting up and down slowly (avoid generating bubbles).
Transfer 50 μl from the first well to the second well in the row or
column as appropriate for the plat orientation. Continue to make
twofold dilutions of the virus suspension across the entire row or
column. Discard the excess 50 μl after the last row or column.
All wells should have a final volume of 50 μl after this step.
5. Starting at the end of the plate with the highest dilution add
50 μl 0.5 % erythrocyte suspension to every well. Tap the plate
gently to mix.
6. Apply adhesive plate sealer to each plate. Once the sealer is
applied, the plates may be removed from the biological safety
cabinet after decontaminating the surface.
7. Allow 20–30 min for the RBC to settle (see Note 2).

3.3 Interpretation 1. The HA plate should be read when the erythrocytes in the cell
of Results control wells have settled to form a solid “button” in the bot-
tom of the well (hemagglutination negative) (see Note 3) (see
Chapter 24, Fig. 1). When the plate is tilted at approximately
45°, the RBCs will stream in a “tear-drop” fashion [2, 7]. Test
fluids that are HA negative will also form solid buttons in all
wells of the corresponding row. These buttons should teardrop
at the same rate as the cell control. Because 105–106 EID50/ml
is required for hemagglutination to occur, an additional pas-
sage of negative material in embryonated chicken eggs may be
optionally performed to confirm that isolations are not missed
because of low levels of virus in the sample.
2. Samples showing complete hemagglutination in one or more
test wells should be considered positive for a hemagglutinating
agent (see Fig. 2). Hemagglutination positive samples may be
further characterized by testing in the hemagglutination-
inhibition assay (see Chapter 2) using monospecific antibodies or
may be confirmed as influenza with another assay (e.g., rRT-PCR).
3. Incomplete hemagglutination may be observed as buttons that
do not teardrop, have fuzzy margins, or form a donut-shaped
ring in the bottom of the well (incomplete hemagglutination
may not be observed if using V-bottom plates). Incomplete
hemagglutination usually indicates an unbalanced proportion
of erythrocytes and virus particles allowing partial settling of
the erythrocytes. The incomplete reaction may be recorded
but should be interpreted as negative.
4. The endpoint of the virus titration is the highest dilution
causing complete hemagglutination (initial dilution is 1:2).
The endpoint dilution is considered 1 HA unit (HAU),
and the number of HAUs/50 μl is the reciprocal of the highest
dilution. Example: For 6 wells of complete hemagglutination
with an endpoint dilution of 1:64, there are 64 HAU/50 μl.
8 Mary Lea Killian

Fig. 2 Illustration of microtiter plate with positive and negative hemagglutination

results. Sample ID is designated on the left side of the plate (+C indicates positive
control antigen and CC indicates RBC control), the dilution factor is designated
across the top. Results for each sample: (1) positive hemagglutination 128 HAU;
(2) positive hemagglutination 32 HAU; (3) positive hemagglutination 64 HAU with
incomplete hemagglutination in 128 HAU well; (4) negative hemagglutination; (5)
negative hemagglutination; (6) positive hemagglutination 16 HAU with incomplete
hemagglutination in 32 HAU well; (7) negative hemagglutination; (8) positive hem-
agglutination 32 HAU; (9) positive hemagglutination 128 HAU with prozone effect
in 2, 4, and 8 HAU wells; (10) negative hemagglutination

4 Notes

1. All steps with infectious or potentially infectious material must

be performed in a Class II biological safety cabinet. Aerosol-
resistant tips and aseptic technique should be used any time an
aliquot is taken from the original sample tube containing virus.
Material carried on the surface of a pipette is a common source
of laboratory contamination.
2. Assay plates should not be left too long before reading results
[1]. The neuraminidase protein present on influenza viruses
acts to break virus-cell bonds, and may eventually begin to
break apart the lattice formed by the virus and erythrocytes.
Some virus strains have very high neuraminidase activity and
may not allow proper hemagglutination. When these viruses
are encountered, the assay should be performed at 4 °C to
 HA Assay for AIV 9

decrease neuraminidase activity, and the incubation period

should be increased to 45–60 min before reading the plate.
3. Troubleshooting.
(a) If the erythrocytes do not settle in cell control wells after
20–30 min possible causes are as follows: (1) The erythro-
cytes are not equilibrated to room temperature; (2) the
erythrocyte suspension is poor quality or hemolyzed; (3)
the PBS is poor quality or incorrect pH; (4) contamina-
tion of wells with viral antigen has occurred.
(b) If the positive control is negative possible causes are as fol-
lows: (1) The positive control antigen was not added to
control well; or (2) the erythrocytes have been allowed to
settle for longer than 30 min.
(c) When a sample is negative for hemagglutination in the first
wells (where the highest virus concentration should be)
and positive in the last wells (lower concentration wells) it
is called the prozone effect. The prozone phenomenon is a
high dose effect where reactions may be weak or negative
in the first wells and stronger in the higher dilutions. This
is caused when the virus in the high concentration wells is
proportionately higher than the available receptors on the
erythrocytes therefore cannot crosslink into a lattice. They
may appear as either complete buttons or a partial button
with fuzzy margins in the bottom of the well. Reactions
may be interpreted as false negative if the dilution series is
not carried out far enough (see Fig. 2). A dilution of 28 is
normally sufficient to avoid a false-negative reading due to
a very high virus concentration.

References
1. Grimes S (2002) A basic laboratory manual for poultry, 11th edn. Iowa State University Press,
the small-scale production and testing of I-2 Ames, IA, pp 135–155
newcastle disease vaccine. FAO & APHCA. 5. Swayne DE, Senne DA, Beard CW (1998)
http://www.fao.org/docrep/005/AC802E/ Avian influenza. In: Dufour-Zavala L (ed) A
ac802e00.htm. Accessed 2 June 2013 laboratory manual for the isolation and identifi-
2. Webster RG, Cox N, Stöhr K (2002) WHO cation of avian pathogens, 4th edn. American
manual on animal influenza diagnosis and sur- Association of Avian Pathologists, Kennett
veillance. http://www.who.int/csr/resources/ Square, PA, pp 128–134
publications/influenza/whocdscsrncs20025 6. Barrett T, Inglis SC (1985) Growth, purifica-
rev.pdf. Accessed 11 Jul 2005 tion and titration of influenza viruses. In: Mahy
3. Flint SJ, Enquist LW, Racaniello VR, Skalka BWJ (ed) Virology: a practical approach. IRL,
AM (2004) Virus cultivation, detection, and Washington, DC, pp 119–150
genetics. In: Flint SJ (ed) Principles of virology: 7. World Animal Health Organization (OIE)
molecular biology, pathogenesis, and control of (2012) Avian influenza. In: Manual of stan-
animal viruses, 2nd edn. ASM dards for diagnostic tests and vaccines. http://
4. Swayne DE, Halvorson DA (2003) Influenza. www.oie.int/fileadmin/Home/eng/Health_
In: Saif YM, Barnes HJ, Gilsson JR, Fadly AM, standards/tahm/2.03.04_AI.pdf. Accessed 1
McDougald LR, Swayne DE (eds) Diseases of June 2013
 Chapter 2

Hemagglutination-Inhibition Assay for Influenza

Virus Subtype Identification and the Detection
and Quantitation of Serum Antibodies to Influenza Virus
Janice C. Pedersen

Abstract
Hemagglutination-inhibition (HI) assay is a classical laboratory procedure for the classification or subtyping
of hemagglutinating viruses. For influenza virus, HI assay is used to identify the hemagglutinin (HA)
subtype of an unknown isolate or the HA subtype specificity of antibodies to influenza virus. Since the HI
assay is quantitative it is frequently applied to evaluate the antigenic relationships between different influ-
enza virus isolates of the same subtype. The basis of the HI test is inhibition of hemagglutination with
subtype-specific antibodies. The HI assay is a relatively inexpensive procedure utilizing standard laboratory
equipment, is less technical than molecular tests, and is easily completed within several hours. However
when working with uncharacterized viruses or antibody subtypes the library of reference reagents required
for identifying antigenically distinct influenza viruses and or antibody specificities from multiple lineages of
a single hemagglutinin subtype requires extensive laboratory support for the production and optimization
of reagents.

Key words Hemagglutination, Hemagglutination-inhibition, Avian influenza, Type A influenza,

Influenza subtype identification

1 Introduction

Influenza viruses agglutinate erythrocytes through the interaction

of the virus surface glycoprotein, the hemagglutinin (HA), with
receptors on the surface of the erythrocyte. If viral particles are in
sufficient quantity, the interaction of the HA protein with erythro-
cytes will form a complete network of linked erythrocytes prevent-
ing erythrocytes from settling out or precipitating as a small pellet
in the bottom of a tube or microtiter plate. Agglutination of eryth-
rocytes is the basis of the hemagglutination assay (see Chapter 1),
and inhibition of the agglutination reaction by HA subtype-specific
antisera is the basis of the hemagglutination-inhibition (HI) assay
[1–3] (see Fig. 1).

Erica Spackman (ed.), Animal Influenza Virus, Methods in Molecular Biology, vol. 1161,
DOI 10.1007/978-1-4939-0758-8_2, © Springer Science+Business Media New York 2014

11
12 Janice C. Pedersen

Fig. 1 Hemagglutination-inhibition assay. (1) Virus and antibody are mixed and incubated; (2) if the antibody is
an antigenic match to the virus, it will bind the virus; (3) red blood cells are then added to the assay; (4) since
the antibody is binding the virus, the virus can not bind the red blood cells and hemagglutination is blocked

A panel of serum prepared against the 16 distinct HA subtypes

is used in the HI assay to confirm the HA subtype identity of influ-
enza viruses and may be used to evaluate antigenic relatedness [4].
Typing is facilitated by using antisera raised against influenza iso-
lates with neuraminidase (NA) subtypes which are heterologous
with the test virus. The use of heterologous NA test virus elimi-
nates false-positive reactions due to steric inhibition caused by the
interaction of homologous neuraminidase antigen and antibodies
[5]. For example a false positive could be produced by testing an
H5N2 isolate with H7N2 sera, but should not cross-react with
H7N3 sera.
Reference laboratories maintain a library of antigens and anti-
sera of each HA subtype for isolate identification and testing anti-
genic relatedness. Production of antibodies to only the HA protein
with DNA vaccines has been reported [6] and offers an alternative
method for the production of sera without NA interference.
Importantly, with diagnostic and surveillance specimens an
influenza virus with a novel HA subtype would not be detected or
would produce a false-negative result in tests using antisera to the
known HA subtypes [7]. Therefore, it is essential to confirm that a
hemagglutinating agent that is negative by HI assay is not influenza
by another test such as commercial antigen immunoassay, rRT-
PCR, sequencing, or agar gel immunodiffusion assay.
In addition to virus subtype identification, the HI assay may be
used to detect and quantitate HA subtype-specific antibodies in
serum, plasma, or egg yolk following infection or vaccination. The
presence of AI virus-specific antibodies may be detected as early as
7 days after infection by HI assay [4]. Antibody positive serum
will inhibit the HA activity of an antigen of the same HA subtype,
 HI Assay 13

and negative serum will have no effect. Inhibition is evidenced by

the formation of a “button” of red blood cells in the bottom of the
tube or microtiter plate which will flow and form a “tear-drop”
when the plate or tube is tilted (see Chapter 24, Fig. 1).
The titer of serum is calculated as the reciprocal of the highest
dilution that produces complete HI [1]. The sera of many mam-
malian species contain nonspecific inhibitors that may interfere
with the specificity of the HI test (see Chapters 24 and 36).
However, sera from the majority of avian species do not contain
nonspecific inhibitors and do not need to be treated prior to test-
ing [1]. Sera which are or which may be problematic should be
pre-treated to reduce or destroy such activity, although it should
be recognized that some treatments may lower specific antibody
levels. The two most commonly used treatments for these inhibi-
tors have been receptor-destroying enzyme (RDE) (see Note 1) or
treatment with potassium periodate [1].
In addition to nonspecific inhibition of hemagglutination, sera
from some birds such as turkeys, chickens, and geese may cause
nonspecific agglutination (auto-agglutination) of chicken erythro-
cytes used in the HI test. Auto-agglutination will mask low levels
of HI activity. Auto-agglutination activity can be removed by pre-
treatment of the serum with erythrocytes of the same species as the
serum being tested [1] or with erythrocytes from the same species
that are used in the test.
The HI titer of either virus or antibodies is somewhat depen-
dent upon the antigenic relatedness of the isolate that is the source
of the antigen and the specific serum being used. This is because
inhibition relies on the relationship of antibodies to a virus antigen
which can be highly variable, even within a subtype [7]. Therefore
the level of antibody in a serum specimen may be underestimated
when using a distantly related virus of the same subtype as the anti-
gen source. Because of this, interpretation of HI assay results can
be challenging. The use of reagents of the same subtype from a
closely related lineage will give the most accurate quantitative
results; acceptable qualitative results can usually be obtained by
using reagents of the same HA subtype.
Additional complications may arise when one animal (particu-
larly in wild bird species) has antibodies to multiple subtypes or
may even have concomitant active infections with different sub-
types of AI virus. Finally, steric inhibition from antibodies to the
neuraminidase protein can affect the interaction of the HA protein
and the antibodies directed to it. Molecular techniques such as
real-time RT-PCR (rRT-PCR) [8] and nucleotide sequence analysis
of the hemagglutinin gene are being used by some reference labo-
ratories as an alternative to HI assay for subtype identification for
isolates (see Chapter 11); however this does not give information
on the antigenic relationship of different viruses which can be used
for vaccine selection.
14 Janice C. Pedersen

2 Materials

2.1 Collection 1. Alsever’s solution.

of Erythrocytes 2. Avian influenza antibody and avian influenza infection-free
chickens (see Note 2).
3. Syringes and needles appropriate for the size of animal and
amount of blood to be collected.

2.2 Preparation of 1. Nine to 11-day-old embryonated specific pathogen-free

Reference Antigens chicken eggs.
2. 1 cc Syringe with 26 G needle.
3. Egg punch.
4. 70 % Ethanol or iodine/70 % ethanol solution (1:1 ratio) to
disinfect egg shell.
5. Glue.
6. Antigen source: the appropriate influenza virus strain.
7. Incubator (37 °C with passive humidity).
8. Beta-propiolactone (BPL).
9. Cryo-vials.
10. Centrifuge tubes and centrifuge.
11. Litmus paper for the pH 4–8 range.
12. Sodium Bicarbonate.
13. Pipets, standard and aerosol resistant pipette tips.
14. Phosphate-buffered saline (PBS), 0.1 M, pH 7.2.
15. Egg candler.
16. Class II Biological safety cabinet (BSC).
17. “U”-bottom 96-well microtiter plates, plate covers, and plate
sealing tape.
18. 0.5 % Suspension of chicken erythrocytes (see Subheading 3.1).
19. HA Subtype-specific reference antigen (see Subheading 3.2)
and antisera (see Subheading 3.3).
20. 0.4 % BSA-PBS-SA.
21. Multiple and single channel pipettes calibrated to dispense
0.025–0.05 ml.
22. 0.05 M sodium citrate buffer solution: 1.92 g citric acid in
200 ml distilled water. Adjust pH to 4.5 with NaOH and filter
prior to use.

2.3 Preparation of 1. Packed chicken erythrocytes.

Reference Antisera 2. Centrifuge.
 HI Assay 15

3. Phosphate-buffered saline (PBS), 0.1 M, pH 7.2 or 0.4 %

bovine serum albumin-phosphate buffered saline-sodium azide
(0.4 % BSA-PBS-SA). Add 4 ml 4 % BSA and 1.0 ml of sodium
azide 10 % solution to 96 ml PBS (see Note 3).
4. 100 units/ml RDE prepared according to manufacturer’s
instructions (optional).

2.4 HI Assay for the 1. “U”-bottom 96-well microtiter plates, plate covers, and plate
Identification of sealing tape.
Influenza Virus Isolate 2. 0.5 % Suspension of chicken erythrocytes (see Subheading 3.1).
HA Subtypes
3. Standardized HA subtype-specific reference antisera (see
Subheading 3.3).
4. Multiple and single channel pipettes calibrated to dispense
0.025–0.05 ml.
5. Aerosol resistant and standard pipette tips.
6. 100 units/ml RDE prepared according to manufacturer’s
instructions (optional).
7. PBS.
8. BSC.

2.5 HI Assay 1. “U”-bottom 96-well microtiter plates, plate covers, and plate
for the Identification sealing tape.
HA Subtype Antibody 2. 0.5 % Suspension of chicken red blood cells (see Subheading 3.1).
Specificity and
3. Standardized HA subtype-specific reference antigen (see
Antigenic Comparison
Subheading 3.2).
4. Multiple and single channel pipettes calibrated to dispense
0.025–0.05 ml.
5. Aerosol resistant and standard pipette tips.
6. 100 units/ml RDE prepared according to manufacturer’s
instructions (optional).
7. PBS.
8. BSC.

3 Methods

3.1 Preparation 1. Collect whole blood in an equal volume of Alsever’s solution

of 0.5 and 10 % for preservation of erythrocytes (see Note 2).
Erythrocyte 2. Wash the erythrocytes to remove buffy coat and Alsever’s
Suspension solution [3, 4] by adding 20 ml of blood to a 50 ml centrifuge
tube and filling the tube with PBS. Gently invert the tube
several times to wash the erythrocytes. Centrifuge at 800 × g
for 10 min. Aspirate the PBS and buffy coat from the tube.
16 Janice C. Pedersen

Refill the tube with fresh PBS, mix by inversion, and repeat the
wash and centrifugation cycle two additional times for a total
of three washes. Washed erythrocytes can be stored at 4 °C for
up to 1 week. The suspension should be discarded if the eryth-
rocytes show evidence of hemolysis.
3. Prepare a 0.5 % suspension of erythrocytes with PBS for use in
the HI assay and a 10 % suspension for treatment of serum
(see Note 4).

3.2 Preparation 1. All work with live virus should be performed in a biological
of Reference Antigen safety cabinet. Prepare dilutions of the virus isolate to be used
as the antigen source in tris-buffered tryptose broth or PBS.
The exact virus dilution will vary depending on the virus strain,
virus titer, and virus adaptation to growth in chicken embryos.
A 1:1,000 dilution is a recommended starting dilution.
2. Inoculate six embryonated chicken eggs with 0.1 ml diluted
virus by the chorio-allantoic sac route with a 1 cc syringe with
a 26 G needle (see Chapter 12). Seal the egg with glue.
3. Incubate the eggs for 4 days at 37 °C with passive humidity
and candle daily for mortality. Discard eggs that die within the
first 24 h post inoculation (PI). Store eggs which die between
2 and 4 days PI at 4 ºC.
4. Harvest the amnio-allantoic fluid (AAF) from dead and live
embryos within 48 h of egg death and check for virus replica-
tion by the HA assay (see Chapter 1).
5. Once virus replication and sufficient virus titer has been con-
firmed inactivate the virus material (the antigen) with beta-
propiolactone (BPL): add BPL to a final concentration of
0.1 %. Incubate for a minimum of 4 h at room temperature
(RT) while mixing on a rocker. Adjust the pH of the antigen
to 7.0 with sodium bicarbonate. Test for inactivation by inocu-
lating six embryos with 0.3 ml of the pH corrected, BPL-
treated AAF preparation as described in step 2. Incubate and
harvest as described in steps 3 and 4. Test for virus growth
with HA assay. A negative HA assay indicates that the virus has
been inactivated.
6. Clarify the antigen by centrifugation at 1,500 × g for 20 min
prior to bottling and storage.
7. Reference antigens and test virus including the positive control
must be standardized to a concentration of 8 HAU/50 μl
(equivalent to 4 HAU/25 μl). The initial concentration of
undiluted reference antigen is determined by the HA assay (see
Chapter 1) using 50 μl of the undiluted specimen. The num-
ber of HAUs present is equal to the endpoint of the HA titra-
tion, which is the highest dilution of the antigen/virus causing
 HI Assay 17

complete hemagglutination. For example if the 1:128 dilution

is the highest dilution to cause complete agglutination in the
HA assay, the HA titer is 128 and there are 128 HAU/50 μl
(if the HA assay is performed in 50 μl). The virus dilution con-
taining 8 HAU is determined by dividing the total HAU by 8.
For this example that would be 128÷8 = 16, therefore for
8 HAU/50 μl one would make a 1:16 dilution. If the virus
isolate does not contain 8 HAU/50 μl (±1 HAU), an addi-
tional passage of the virus in embryos should be performed to
increase the virus titer.
8. Once the dilution factor has been determined the reference
antigens may be diluted with PBS or BSA-PBS-SA. BSA-PBS-SA
will stabilize the virus and extend storage life at 4 °C up to
2–4 weeks.
9. To ensure that the virus has been diluted to the correct con-
centration a back titration is performed by using the diluted
virus in an HA assay where the HA titer should be 8 HAU/50 μl.

3.3 Preparation 1. In order to remove natural serum agglutinins from reference

of Reference Antisera sera produced in SPF chickens the serum needs to be treated
with chicken erythrocytes. Add 0.1 ml packed erythrocytes per
1 ml serum.
2. Incubate for 30 min at RT with occasional mixing to keep the
erythrocytes suspended.
3. Centrifuge the serum with packed erythrocytes at 800 × g for
10 min. Decant the serum and discard the erythrocyte pellet.
The serum can be stored for a week at 4 °C or −20 °C long term.
4. Using 8 HAU/50 μl (or 4 HAU/25 μl) of homologous anti-
gen determine the HI titer of the serum by the HI assay as
described (see Subheading 3.5) and standardize the serum to
an HI titer of 1:32–1:64 by dilution in PBS or 0.4 % BSA-
PBS-SA (see Note 5).

3.4 HI Assay 1. Each unknown isolate should be tested against antiserum to

for the Identification each of the 16 known subtypes of AI virus.
of HA Subtype 2. The test virus must be standardized to a concentration of
of a Virus Isolate 8 HAU/50 μl (equivalent to 4 HAU/25 μl) prior to testing
with the HI assay to identify the HA subtype. The procedure
is the same as that used to standardize the reference antigen
(see Subheading 3.2).
3. Working in a BSC dispense 25 μl of standardized test virus for
each unknown isolate into 3 wells (in triplicate) in a “U” bottom
microtiter plate. Also, dispense 25 μl of the corresponding HA
subtype positive-control antigen at 8 HAU/50 μl concentration
18 Janice C. Pedersen

into the positive control wells. The positive control sera should
be homologous to the antigen.
4. Add 25 μl of the appropriate standardized antiserum to the
first well of an HA subtype series.
5. Serially dilute (25 μl carry back and discard the excess 25 μl from
the final row) the antiserum in the antigen wells beginning with
the first well. In this format the serum is diluted in standardized
antigen (see Note 6). Each subtype series should be diluted as
soon as possible after the addition of the antiserum for that series.
After this step there should be 25 μl in each well.
6. Cover plate and incubate for 30 min at RT.
7. Add 50 μl 0.5 % erythrocyte suspension to each well and gently
shake/agitate the plate to mix. Mix the erythrocyte solution
periodically during this step to ensure that the erythrocytes are
evenly suspended during the dispensing process.
8. Seal the plate with microtiter plate sealing tape (plates can be
removed from the BSC after being sealed) and incubate at RT
until a distinct button has formed in the positive control wells.
This usually takes 20–30 min. The assay plates should be first
observed after about 20 min of incubation and checked fre-
quently after that for evidence of hemagglutination. Because
some isolates may begin to elute (detach from erythrocytes) in
as little as 30 min, the time window where the assay results may
be evaluated may be short for some isolates (see Note 7).

3.4.1 Analysis 1. Wells with complete hemagglutination are recorded as positive

of Test Results for HA, wells with a distinct button formation are recorded as
negative for HA or positive for HI; wells with partial button
formation (fuzzy margins or donut-like appearance) are
recorded as incomplete hemagglutination. When interpreta-
tion between complete and incomplete inhibition is difficult,
tilt the microtiter plate at a 45° angle for 20–30 s and look for
a “tear drop” of the erythrocytes to form. The tear-drop will
only form in the wells with complete inhibition; wells with
partial inhibition will not form a “tear drop” (see Note 7).
Inhibition at the initial 1:2 dilution of the virus is considered
positive.
2. The test should be repeated if (see Note 8):
(a) The back titration of the antigen does not result in
8 HAUs/50 μl (±1 HAU).
(b) The samples has the same NA subtype as the antigen used
in the HI assay.
(c) The positive control sera do not inhibit hemagglutination
to the expected titer (±1 dilution) and/or the RBC only
control does not button.
 HI Assay 19

3.5 HI Assay 1. This test can be used to identify the HA specificity of AI virus
for the Identification antibodies in a serum sample or can be used quantitatively to
HA Subtype Antibody evaluate the antigenic relatedness of AI virus isolates. As men-
Specificity and tioned previously, when possible, perform the HI assay, with
Antigenic Comparison antigens and antiserum which do not have the same NA speci-
ficity to avoid problems with steric inhibition due to the inter-
action of homologous neuraminidase antibodies and antigen
[3, 5]. Serum is the preferred sample for the HI test although
plasma, test samples extracted from egg yolk (see Note 9) or
dried blood on filter paper strips (see Note 10) can also be used
in the HI test [9] (see Chapter 14, Subheading 3.1). Under
certain conditions plasma samples coagulate, rendering the
sample unusable.
2. Nonspecific inhibitors of hemagglutination and nonspecific
agglutinins must be removed from sera, plasma, and yolk sam-
ples before the HI assay is performed. Nonspecific agglutinins
prevent buttoning of the erythrocytes and lead to false-negative
results. The presence of natural agglutinins is monitored in the
serum control. To remove nonspecific agglutinins prepare one
part serum, two parts PBS, and one part of a suspension of
10 % washed erythrocytes (see Subheading 3.1). Mix thor-
oughly on a shaker and incubate at RT for 30 min mixing every
10 min to keep the erythrocytes suspended. Centrifuge the
plate or test tube at 500 × g for 10 min to pellet the erythro-
cytes. Decant the serum and discard the erythrocyte pellet.
Sufficient serum should be treated to test each serum against
the all subtype-specific antigens selected for the test (e.g., at
least 16 if the sample(s) will be tested for all subtypes).
3. Dispense 25 μl of standardized HA antigen into 3 wells (in
triplicate) of a “U” bottom microtiter plate which have been
designated for their respective HA subtype.
4. Add 25 μl of serum which has been treated for nonspecific
agglutinins to the first well designated for each sample. Because
of the treatment in step 2 this will give an initial serum dilution
of 1:8. Prepare a serum control (25 μl test serum and 25 μl PBS,
i.e., no antigen) for each serum sample to further ensure that the
specimen does not contain nonspecific agglutinins.
5. Serially dilute the serum (25 μl carry-back) beginning with the
first well (1:8 serum dilution), through the third well (1:32
serum dilution) with a multi-channel pipet. Discard the excess
25 μl after the final dilution. After this step there should be a
total of 25 μl in each well. Similar to the test for virus subtype
identification (see Subheading 3.4), serum is diluted in antigen
(see Note 6). A minimum of three dilutions of each serum can
be tested against standardized antigens for subtype specificity
testing. When evaluating antigenic relatedness, further dilutions
20 Janice C. Pedersen

may be necessary; the end point will need to be determined

empirically (see Note 11).
6. Include a positive control (standardized reference antigen and
antisera which are homologous) for each HA subtype in the
test and a cell or erythrocyte control (50 μl PBS and 50 μl
0.5 % erythrocytes) in each test. The reference antisera are
treated in the same manner as the unknown sera. The control
sera are serially diluted in the standardized reference antigen as
described in step 5.
7. Cover plate and incubate for 30 min at RT.
8. Add 50 μl 0.5 % erythrocyte suspension to each well and gently
shake/agitate the plate to mix. Mix the erythrocyte solution
periodically during this step to ensure that the erythrocytes are
evenly suspended during the dispensing process.
9. Seal the plate with microtiter plate sealing tape and incubate it at
RT until a distinct button has formed in the positive control
wells. This usually takes 20–30 min. The assay plates should be
first observed after about 20 min of incubation and checked fre-
quently after that for evidence of hemagglutination (see Note 7).

3.5.1 Interpretation 1. Wells with complete hemagglutination are recorded as positive

of Test Results for hemagglutination; wells with a distinct button forma-
tion are recorded as negative for hemagglutination or posi-
tive for HI; wells with partial button formation (fuzzy margins
or donut-like appearance) are recorded as incomplete hemag-
glutination. When interpretation between complete and
incomplete inhibition is difficult, tilt the microtiter plate at a
45° angle for 20–30 s and look for a “tear drop” of the eryth-
rocytes to form. The tear-drop will only form in the wells
with complete inhibition (HI positive); wells with partial
inhibition will not form a “tear drop.” The erythrocytes in
the cell control should button in the same period of time it
takes the positive control to form a distinct button (20–30 min)
(see Note 7).
2. Samples are considered positive (i.e., indicate prior infection
with AI virus of the respective inhibiting HA subtype) if inhibi-
tion of hemagglutination is observed at the 1:8 dilution or
higher. When evaluating the antigenic relatedness of virus iso-
lates the endpoint of the HI titer with homologous serum is
compared with that of the heterologous serum.
3. Endpoints are reported as the highest serum dilution causing
complete inhibition of hemagglutination.
4. The test should be repeated if (see Note 8):
(a) The back titration of the antigen does not result in
8 HAUs/50 μl (±1 HAU).
 HI Assay 21

(b) The samples have the same NA subtype as the antigen

used in the HI assay.
(c) The positive control sera do not inhibit hemagglutination
to the expected titer (±1 dilution) and/or the RBC only
control does not button.

4 Notes

1. Receptor-destroying enzyme (RDE) Treatment. The sera of

many species contain nonspecific inhibitors that lead to
false-positive results. As a rule, sera from the majority of avian
species do not contain nonspecific inhibitors and do not need
to be treated prior to testing. However, problematic sera
should be treated to reduce or destroy nonspecific inhibitors.
RDE treatment may lower specific antibody levels.
(a) Add 400 μl RDE (100 units/ml) to 100 μl of serum and
incubate in a 37 °C water bath overnight.
(b) Add 300 μl of sodium citrate and incubate in a water bath
at 56 °C for 30 min.
(c) Add 200 μl of PBS (this will result in a final dilution of the
serum of 1:10).
(d) Adsorb any nonspecific agglutinins by adding 50 μl washed
undiluted erythrocytes or 10 % suspension of erythrocytes
and incubate at room temperature for 30 min with peri-
odic mixing.
(e) To remove the erythrocytes centrifuge the treated serum
at 800 × g for 10 min.
(f) Collect treated serum supernatant and proceed with the
HI assay as described in (see Subheadings 3.4 or 3.5).
2. Erythrocytes should be collected from specific pathogen-free
(SPF) chickens and mixed with an equal volume of Alsever’s
solution for preservation. Either rooster or hen erythrocytes
can be used in the test; however hormones present in hen’s
blood can reduce reproducibility of the test. If SPF chickens
are not available blood may be collected from birds that are
regularly monitored and shown to be free from antibodies to
avian influenza virus.
3. Diluting serum with BSA-PBS-SA, instead of plain PBS, will
prevent the growth of bacteria and extend the storage life of
the reagent at 4 °C.
4. The HI assay format described here was standardized using
50 μl of 0.5 % erythrocytes collected from SPF chickens (the
final concentration of erythrocytes: 50 μl of 0.5 % versus 25 μl
of 1 % is the same as 25 μl of test volume). The HI procedure
22 Janice C. Pedersen

has been described by a number of diagnostic manuals [1–3, 5,

10–14] and the volume and concentration of the erythrocyte
suspension may vary. For example, the World Organisation for
Animal Health (OIE) recommends using 25 μl of a 1 % eryth-
rocyte solution [13]. The procedure presented here has been
used routinely by the USDA, National Veterinary Services
Laboratories, as well as other diagnostic laboratories and pro-
vides equivalent results; it has been shown to be very effective
in subtyping avian influenza viruses.
5. To conserve time and resources and to identify the hemagglu-
tinin subtype, each isolate can be tested against just three
dilutions (1:8, 1:16, and 1:32) of antiserum for each of the
subtypes. However, when evaluating isolate antigenicity or
relatedness, specimens should be diluted further to establish an
endpoint.
6. With some procedures the sera is serially diluted in 25 μl PBS
in the plate, then 25 μl of standardized antigen is added to
each well. The results are considered equivalent.
7. In addition to the HA protein, influenza viruses have neur-
aminidase enzyme or the NA protein on the virion surface
which can remove sialic acid from the ends of oligosaccharide
chains. In virus replication, since the HA protein binds to sialic
acid on the cell surface during virus attachment, the function
of the NA serves to break the bonds between virus and cells
allowing newly replicated virus to disperse. By this same mech-
anism a fully agglutinated sheet of erythrocytes will eventually
collapse in vitro. The rate of neuraminidase activity varies by
virus strain and some virus strains have a very high neuramini-
dase activity and may not allow proper agglutination. To avoid
this or to compensate for a virus with high neuraminidase
activity the assay may be performed at 4 °C instead of room
temperature [10]. Also, viruses with highly active neuramini-
dase will not form a stable sheet of agglutinated erythrocytes
and should not be selected as seed stock for the production of
reference antigen.
8. Troubleshooting:
(a) If the erythrocytes in the cell control do not settle into a
well-defined button: (1) the PBS pH or salt concentration
may be incorrect, (2) excessive evaporation of PBS from
plates during the test has occurred as PBS exposed to air
or blowing air from a BSC exhaust fan can cause the eryth-
rocytes to form a button more slowly (greater than
20–30 min), (3) the erythrocytes are too old and have
started to deteriorate, or (4) the concentration of erythro-
cytes is incorrect.
 HI Assay 23

(b) Incomplete or negative inhibition test results, for example

if the unknown virus has been shown to be positive by a
type A influenza test (antigen detection, rRT-PCR, or
AGID) or the serum has tested positive for type A influ-
enza antibody by the ELISA or AGID test, but the HI test
did not identify the HA subtype, one of several situations
may be occurring: (1) the HI reference reagents were not
adequately antigenically related to the unknown sera or
isolate, therefore repeat the HI test with different refer-
ence reagents of the same HA subtypes, (2) the antibody
titer of the serum was too low for detection with the HI
test, (3) the HA titer of the reference antigen was too high
(i.e., >8 HAU/50 μl ± 1 HAU), therefore the test serum
has an insufficient concentration of antibody to inhibit the
elevated hemagglutination activity of the antigen, the titer
of the positive control sera is lower than expected, or (4)
the test serum contains natural agglutinins which have
agglutinated the erythrocytes, thus preventing the detec-
tion of inhibition or the erythrocytes in the serum control
do not button.
(c) Elevated virus titers in control reagents may mean an
incorrect concentration of erythrocytes. Lower concentra-
tions of erythrocytes in the assay will give a falsely high
virus titer, it is important that the erythrocyte suspension
should be thoroughly suspended before use.
(d) False-positive results can be due to: (1) nonspecific inhibi-
tors present in the sera tested in the HI antibody test, (2)
nonspecific inhibition due to NA steric inhibition in HI
antigen test, or (3) the reference antigen is not standard-
ized correctly to 8 HAU/50 μl and/or the reference anti-
serum is not standardized to 1:32–1:64 HI titer.
(e) False-negative results may occur due to: (1) the presence of
nonspecific agglutinins in the test serum in HI antibody
test, (2) the reference reagents are antigenically distinct
from unknown specimen(s), or (3) the reference antigen is
not properly standardized to 8 HAU/50 μl and/or refer-
ence antiserum is not standardized to a 1:32–1:64 HI titer.
9. Procedure for the extraction of antibodies from egg yolk by
separating the antibody containing aqueous phase from the
lipid phase. The resulting sample can be used in the HI assay
to evaluate the immune status of a flock.
(a) Carefully break the test egg and pour the contents into a
shallow bowl without breaking the yolk. Insert a 1 cc
syringe without a needle into the yolk sac and withdraw
1.0 ml yolk material (see Chapter 14, Fig. 1).
(b) Expel the yolk into a tube with 1.0 ml PBS.
24 Janice C. Pedersen

(c) Vortex the yolk–PBS mixture at maximum speed for

10–15 s.
(d) Incubate the yolk–PBS mixture at RT for 1 h. Then repeat
the mixing step (step 9c).
(e) Centrifuge the yolk–PBS mixture at 1,500 × g for 30 min
at 4 °C.
(f) Collect the supernatant (aqueous phase) and discard the
lipid portion. Note that this procedure results in a 1:2
dilution of the yolk. The supernatant may be used in the
HI assay.
10. Procedure for the filter paper method of blood collection:
avian blood samples can be collected and preserved on filter
paper. Blood collected in this manner can be used in place of
serum in the HI assay. The technique is especially useful in field
situations where aseptic collection and adequate preservation
of serum and/or plasma may be difficult (see Chapter 14,
Subheading 3.1.3 for the procedure with Nobuto strips).
(a) Cut filter paper into 1.2 × 10 cm strips.
(b) Collect the blood sample by puncturing the brachial wing
vein or cut a toe nail of the bird to be sampled in accor-
dance with the appropriate animal care and use
procedures.
(c) Briefly allow blood to pool on the skin at the puncture
site. The blood should not be allowed to clot before soak-
ing the filter paper strip.
(d) Saturate the distal 1.2–2.5 cm of a filter paper strip with
blood by soaking the flat side (not the edge) in the blood
that has pooled at the puncture site. Collect sufficient
blood so that both surfaces of the strip are saturated.
(e) Allow the strips to air dry.
(f) Place the dry strips in a plastic bag and seal.
(g) Store dried strips at 4 °C until processed.
(h) To elute the antibodies punch three 5 cm discs from a
saturated portion of the filter paper strip and transfer the
discs with forceps to microtiter plate well.
(i) Dispense 200 μl of PBS into the well containing the dried
blood discs.
(j) Place the microtiter plate on a shaker or orbital mixer for
1 h at a high speed, but take care not to displace liquid
from wells. Cover plates to prevent evaporation.
(k) Incubate the plate at 4 °C overnight to allow the maxi-
mum elution of blood. Collect the supernatant. The eluted
sample is equivalent to a 1:10 dilution of serum.
 HI Assay 25

11. When numerous viruses or serum samples are being compared

in order to evaluate antigenic variation or relatedness, the test
sera may be standardized to a specific homologous HI titer, for
example each serum sample in a test will have a titer of 256
against the virus it was raised against. This helps to control for
variation in antibody levels among different serum samples.

References
1. Beard CW (1980) Hemagglutination- Suarez DL (2002) Development of a real-time
inhibition. In: Hitcher SB, Domermuth CH, reverse transcriptase PCR assay for type A
Purchase HG, Williams JE (eds) Isolation and influenza virus and the avian H5 and H7 hem-
identification of avian pathogens. American agglutinin subtypes. J Clin Microbiol 40:
Association of Avian Pathologists, Kennett 3256–3260
Square, PA, pp 331–336 9. Brugh M, Beard CW (1980) Collection and
2. Centers for Disease Control and Prevention processing of blood samples dried on paper for
(1982) Concepts and procedures for labora- microassay of Newcastle disease virus and avian
tory based influenza surveillance. CDC, U.S. influenza virus antibodies. Am J Vet Res
Department of Health and Human Services, 41:1495–1498
Washington, DC 10. Barrett T, Inglis SC (1985) Growth, purifica-
3. Palmer DF, Coleman MT, Dowdle WD, Schild tion and titration of influenza viruses. In:
GO (1975) Advanced laboratory techniques Mahy BWJ, Rickwood D, Hames BD (eds)
for influenza diagnosis. In: Immunology series Virology, a practical approach. IRL, Oxford,
no. 6. U.S. Department of Health, Education pp 119–150
and Welfare, Public Health Service, CDC, 11. Hanson RP, Appleton GS, Chute HL,
Atlanta, GA Grumbles LC, Hitchner SB, Peacock GV,
4. Swayne DE, Halvorson DA (2003) Influenza. Pomeroy BS, Rosenwald AS (1971)
In: Saif YM, Barnes HJ, Gilsson JR, Fadly AM, Identification and differentiation of avian
McDougald LR, Swayne DE (eds) Diseases of viruses. In: Methods for examining poultry
poultry, 11th edn. Iowa State University Press, biologics and for identifying and quantifying
Ames, IA, pp 135–155 avian pathogens. National Academy of
5. Kendal AP (1982) New techniques in anti- Sciences, p 261–290
genic analysis with influenza viruses. In: Beare 12. Mahy BWJ, Kangro H (1996) Hemagglutination-
AS (ed) Basic and applied influenza research. inhibition. In: Virology methods manual,
CRC, Boca Raton, FL, pp 51–78 Academic, London, p 114–116
6. Lee CW, Senne DA, Suarez DL (2006) 13. World Animal Health Organization (OIE)
Development and application of reference (2012) AvianInfluenza. In: Manual of standards
antisera against 15 hemagglutinin subtypes of for diagnostic tests and vaccines. http://www.
influenza by DNA vaccination of chickens. oie.int/fileadmin/Home/eng/Health_
Clin Vaccine Immunol 13:395–402 standards/tahm/2.03.04_AI.pdf. Accessed 1
7. Easterday BC, Hinshaw VS, Halvorson DA June 2013
(1977) Influenza. In: Calnek BW, Barnes HJ, 14. Swayne DE, Senne DA, Beard CW (1998)
Beard CW, McDougald LR, Saif YM (eds) Avian influenza. In: Dufour-Zavala L (ed) A
Diseases of poultry, 10th edn. Iowa State laboratory manual for the isolation and identi-
University Press, Ames, IA, pp 583–605 fication of avian pathogens, 4th edn. American
8. Spackman E, Senne DA, Myers TJ, Bulaga LL, Association of Avian Pathologists, Kennett
Garber LP, Perdue ML, Lohman K, Daum LT, Square, PA, pp 128–134
 Chapter 3

Neuraminidase-Inhibition Assay for the Identification

of Influenza A Virus Neuraminidase Virus Subtype
or Neuraminidase Antibody Specificity
Janice C. Pedersen

Abstract
The neuraminidase-inhibition (NI) assay is a laboratory procedure for the identification of the neuraminidase
(NA) glycoprotein subtype in influenza viruses or the NA subtype specificity of antibodies to influenza virus.
A serological procedure for subtyping the NA glycoprotein is critical for the identification and classification
of avian influenza (AI) viruses. The macroprocedure was first described in 1961 by Aminoff and was later
modified to a microtiter plate procedure (micro-NI) by Van Deusen et al. (Avian Dis 27:745–750, 1983).
The micro-NI procedure reduces the quantity of reagents required, permits the antigenic classification of
many isolates simultaneously, and eliminates spectrophotometric interpretation of results. Although, the
macro-NI has been shown to be more sensitive than the micro-NI, the micro-NI test is very suitable for
testing sera for the presence of NA antibodies and has proven to be a practical and rapid method for virus
classification. This chapter provides an overview of the USDA validated micro-NI procedure for the iden-
tification of subtype-specific NA in AIV and antibodies.

Key words Neuraminidase, Neuraminidase-inhibition, Avian influenza, Type A influenza, Influenza

subtype identification

1 Introduction

Influenza virus coats contain two subtype-specific glycoproteins on

their surface: the hemagglutinin (HA) and the neuraminidase (NA).
The HA mediates cell-surface sialic acid receptor binding to initiate
virus infection, while the NA contains a head domain which is enzy-
matically active [1–4]. The NA is important both for its biological
activity in removing N-acetyl neuraminic acid (NANA) or sialic acid
from sugar chains of glycoproteins and as a major antigenic deter-
minant [2–4]. After virus replication, NA removes sialic acid from
the virus and cellular glycoproteins by catalytic cleavage of the
NANA from adjacent D-galactose or D-galactosamine sugar chains
facilitating virus release and the spread of infection to new cells.
It is expected that all natural isolates of influenza A viruses have

Erica Spackman (ed.), Animal Influenza Virus, Methods in Molecular Biology, vol. 1161,
DOI 10.1007/978-1-4939-0758-8_3, © Springer Science+Business Media New York 2014

27
28 Janice C. Pedersen

Fig. 1 Basic premise of the neuraminidase-inhibition test; the top reaction is negative, the bottom reaction is
positive. (1) Test antibody and virus are mixed and incubated. (2) Fetuin, a substrate for neuraminidase is
added, if the antibody is an antigenic match to the virus (bottom) it will bind the influenza neuraminidase pro-
tein, blocking the neuraminidase from breaking down the fetuin into free NANA. (3) Periodate is added. If the
antibody does not match the virus (top), the neuraminidase will have produced free NANA from the fetuin.
Periodate reacts with the free NANA to create beta-formylpyruvic acid. (4) Na arsenate is added. If beta-
formylpyruvic acid is present (top) because the antibody and virus do not match a pink color will form. If the
antibody does match the virus (bottom) there is no beta-formylpyruvic acid, therefore no color change

this enzyme activity. Nine antigenically distinct neuraminidase

subtypes (N1–N9) have been identified [5, 6]. Despite the anti-
genic diversity of the NA glycoprotein, all influenza neuraminidases
bind the carboxylate of NANA [4].
The neuraminidase-inhibition (NI) assay has been used by
numerous reference laboratories for identification of the NA sub-
type for avian viruses and sera. However, it may have limited value
for turkey sera where vaccination of commercial turkey breeders
against swine lineage viruses, including H3N2, is practiced. Data
from the US National Veterinary Services Laboratories (NVSL)
suggests that the use of normal (i.e., antibody-free) turkey sera
produces a false-positive result with swine lineage H3N2 viruses.
The swine lineage of N2 may be detected in turkey sera with
reagents produced against avian viruses of the N2 subtype with
valid positive and negative chicken sera controls.
The inhibition of viral enzymatic activity by specific antibodies
forms the basis of the NI assay (see Fig. 1). The micro-NI assay is
performed in a microtiter plate and is evaluated based on a colori-
metric change where the substrate produces a dark pink color when
the NA is active. Specifically, the enzymatic release of NANA is
 NI Assay 29

detected when the β-formol pyruvic acid, the aldehyde to NANA

is produced by periodate (test reagent) oxidation. The pink chro-
mophore forms when the β-formol pyruvic acid reacts with thio-
barbituric acid (TBA), another NI test reagent [1, 2, 4, 7]. When
the NA catalytic activity is inhibited, such as with neutralizing anti-
body, the color change does not occur and a light pink color is
observed [4]. The micro-NI procedure is conducted in opaque
white 96-well microtiter plates which allows the color to be more
easily visualized and eliminates the need for interpretation of results
by spectrophotometry [4]. Although both macro- and micro-NI
procedures have been described, the micro-NI procedure is more
practical as it permits the testing of several viruses or sera simulta-
neously and greatly reduces the volume of reagents and time
required to conduct the test [2, 4]. The micro-NI procedure can
be conducted spectrophotometrically using the same general pro-
cedure and reagents described below in the non-spectrophotometric
procedure. Fetuin controls (containing all test reagents except
virus) are included for determining background signal, and a virus
control is included for non-inhibited signal. Warrenoff N-butanol
reagent is added after incubation with TBA to extract the color
into the organic (butanol) phase. The optical density of the upper
phase is read at 549 nm.
The NI assay can either be performed directly on virus isolates
(allantoic fluid or cell culture fluid) with subtype-specific reference
antisera for the determination of NA subtype, or may be used with
serum or plasma to identify antibody NA subtype specificity with
serum or plasma by using subtype-specific reference antigens.
Antigenic diversity and relatedness must be considered in the selection
of reference antigens and antisera as the NI procedure is directed to an
antigenically variant epitope. Using reference sera or antigen from
viruses with the same HA subtype as the test materials will improve
specificity by limiting variability to the NA subtype [4].

2 Materials

1. White polystyrene 96-well microtiter plates.

2. 12.5 mg/ml fetuin from fetal calf serum: dissolve 250 mg
fetuin in 10 ml sterile distilled water. Add 10 ml sterile 0.4 M
phosphate-buffered saline (PBS—0.4 M), pH 5.9. Store fetuin
at −20 °C.
3. Periodate reagent (sodium (meta) periodate): dissolve 4.28 g
sodium m-periodate in 38 ml distilled water. Carefully add
62 ml concentrated phosphoric acid and mix. Store at room
temperature (RT) in a dark glass (amber or foil-covered) bottle
in a cool, dark place.
30 Janice C. Pedersen

4. 50 % Arsenite reagent (sodium meta-arsenite): dissolve 50 g

sodium meta-arsenite in 100 ml distilled water. Slowly add
1.5 ml concentrated sulfuric acid (caution: add acid slowly)
and mix. Store at RT.
5. 0.6 % Thiobarbituric acid (2-Thiobarbituric acid): dissolve 0.6 g
thiobarbituric acid in 100 ml distilled water by heating in a boil-
ing water bath and stirring frequently or on a hot plate with a
magnetic stirring mechanism. Store at room temperature.
6. 0.01 M PBS, pH 7.2.
7. 0.4 M PBS, pH 5.9.
8. Bovine serum albumin (BSA), fraction V.
9. BSA-PBS-SA (4 % BSA in PBS (0.01 M, pH 7.2) containing
0.1 % sodium azide): dispense 10 ml 4 % BSA and 89 ml PBS
into a flask. Add 1.0 ml 10 % sodium azide to BSA-PBS.
10. 10 % Sodium azide solution: add 10 g sodium azide to 100 ml
distilled water.
11. Sulfuric acid.
12. 85 % Phosphoric acid.
13. Microtiter plate sealing tape.
14. Antiserum to each of the nine neuraminidase subtypes and a
known negative serum.
15. Inactivated viral antigen for each of the nine neuraminidase
subtypes and a negative antigen (see Chapter 2).
16. Calibrated multiple and single channel pipettes (0.025–
0.10 ml).
17. Aerosol resistant and standard pipette tips.

3 Methods

3.1 Preparation 1. Reference antigens for each of the nine neuraminidase subtypes
and Optimization of are propagated in embryonated chicken eggs (see Chapter 2).
Reference Reagents Either live or inactivated virus can be used as a reference reagent;
however use of inactivated virus or antigen allows the antibody
test to be conducted outside of a biosafety cabinet. Live virus
can be inactivated with beta-propiolactone (BPL) at a final
concentration of 0.1 % (see Chapter 2, Subheading 3.2, step 5).
A safety test for virus viability must be conducted in chicken
embryos to confirm that the virus has been inactivated.

3.1.1 Antigen Titration 1. Dispense 25 μl PBS into rows 2–6 of a white opaque 96-well
“U”-bottom microtiter plate.
2. Add 50 μl antigen into the first well of row 1.
 NI Assay 31

3. Make serial twofold dilutions with 25 μl of antigen, starting

with the first well (undiluted) through well 6 (well 6 will have
a 1:32 dilution).
4. Add 25 μl 0.01 M, pH 7.2 PBS to all wells.
5. Mix on a microtiter plate shaker for 10–15 s.
6. Add 25 μl fetuin to each well.
7. Mix on a microtiter plate shaker for 10–15 s. Cover and incu-
bate at 37 °C for 3 h. Incubation times may vary depending on
the enzymatic activity of the neuraminidase. For some isolates,
the incubation time may need to be increased by 1–2 h.
8. Add 25 μl periodate reagent to each well.
9. Mix on a microtiter plate shaker for 10–15 s. Cover and incu-
bate at room temperature for 20 min (±2 min).
10. Add 25 μl 50 % sodium arsenite reagent to each well. The addi-
tion of sodium arsenite reagent will cause a dark-brown pre-
cipitate to form.
11. Mix on a microtiter plate shaker until the dark-brown color
fades (this may take several minutes).
12. Add 100 μl 0.6 % thiobarbituric acid reagent to all wells. Cover
the plate with plate sealing tape. Puncture the tape (small pin-
hole) over each well to allow for expansion.
13. Float the sealed plate in a 56 °C water bath for 30 min.
14. Read results by carefully tilting the plate to view color through
the sealing tape.
15. The optimum antigen dilution is the highest dilution which
has a “medium” pink color or the dilution prior to a reduction
in pink color. All reference antigens should produce a uniform
“medium” pink color. The inhibition controls should produce
a uniform “light” pink color which can be easily differentiated
from the reference antigen control “medium” pink (see Fig. 2).
Reference antigen 1 should be diluted 1:4 and reference anti-
gen 2 should be diluted 1:8. Unknown isolates may be stan-
dardized before testing by titration if needed.

3.1.2 Optimization of 1. The optimum dilution for each reference antiserum is deter-
Reference Antisera mined by titration of the serum against a homologous antigen.
The optimal dilution is the highest dilution that causes
inhibition when compared to a positive control standard. The
optimal serum dilution usually ranges from 1:4 to 1:10. The
negative serum dilution should be the same as the lowest
dilution used for the reference antisera, for example if the
dilutions used for the reference antisera are 1:4, 1:8, and 1:10,
the negative control serum should be used at a dilution of 1:4.
32 Janice C. Pedersen

Fig. 2 Antigen titration for the neuraminidase-inhibition test. The optimum anti-
gen dilution is the highest dilution which has a “medium” color or the dilution
prior to a reduction in pink color. The “medium” color should be easily differenti-
ated from the lighter color observed in control wells with no inhibition and should
be uniform for all reference antigens. Reference antigen 1 should be diluted 1:4
and reference antigen 2 should be diluted 1:8

Reference antisera and antigens should be diluted with 0.4 %

BSA-PBS-SA.
2. Heat inactivate the sera at 56 °C for 30 min prior to testing.
Diluted sera and antigens may be stored for several weeks at
4 °C or frozen at −20 °C for long-term storage.

3.2 Neuraminidase- 1. Preparation of specimen virus isolates: Start with a 1:12 dilu-
Inhibition Assay tion of amniotic-allantoic fluid (AAF) containing the unidenti-
fied influenza A virus in 0.4 % BSA-PBS (e.g., 50 μl AAF and
3.2.1 Subtype
550 μl PBS). This dilution will work with most isolates; lower
Determination of Virus
or higher dilutions may be needed for some isolates and will
Isolates
need to be determined empirically. A standardized concentra-
tion of the unknown isolate can be determined by titration as
described in Subheading 3.1.1.
2. Add 25 μl of previously standardized neuraminidase antiserum
into corresponding vertical columns labeled N1–N9 for each
isolate and in the positive control row (see Fig. 3).
3. Dispense 25 μl negative control serum in the negative control
column and in the first 10 wells of the negative control row.
4. Add 25 μl of diluted AAF, for each unknown, into the first 10
wells of the corresponding horizontal row.
5. Dispense 25 μl of each positive control AAF into the corre-
sponding positive and negative control wells.
6. Mix on a microtiter plate shaker for 10–15 s and incubate at
room temperature for 1 h (±15 min). Cover plate during
incubation to prevent evaporation.
7. Add 25 μl fetuin to each well.
8. Mix on a microtiter plate shaker for 10–15 s. Cover and incubate
at 37 °C for 3 h. Incubation time may vary depending on the
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business flourished to such an extent that it swallowed
up the old one, and our draper at length became a
banker proper, and no more a shopkeeper.
“Such was the origin of the Smiths. First confined
to the town of Nottingham, afterwards extended to
Hull and Lincoln, the business of the firm required a
London correspondent entirely in their interest, and
such they found in the late Mr. Payne.”
And thus was founded the well-known firm of Smith, Payne, and
Smith, whose business has recently been amalgamated with that of
the Union Bank of London.
Many are the tales told of the wit and shrewdness of the early
country bankers, and the following anecdote, related in Mr. Maberley
Phillips’s interesting work on Banks, Bankers, and Banking in
Northumberland, Durham, and North Yorkshire, is on a par with the
well-known tale of the private Irish banker, who became so very
unpopular, that to show the contempt in which he was held, the
inhabitants of his district gathered together all his notes which they
could lay hands on, and made a bonfire of them in front of his
house; much to the banker’s amusement and gain.
Mr. Phillips’s story is of Jonathan Backhouse, a Quaker, who,
though originally a linen and worsted manufacturer in Darlington,
founded “Backhouse’s Bank” in 1774, in partnership with his father.
This institution only went out of existence in 1896, when it was
amalgamated with Messrs. Barclay and Company, Limited.
 “Before the time of railways, near the beginning of
the century, the commercial traveller of that day made
his visits to the towns of the county of Durham either
by mail coach or other conveyance, and sojourned for
some days in each town, where he was an important
person, especially at the head hotel or hostelry of the
place. It so happened that one of these gentlemen,
after having dined freely at the ‘King’s Head,’ Barnard
Castle, was boasting to a company present in the
commercial room of his own importance and wealth,
and exhibiting in proof a sheaf of bank-notes taken on
his journey.
“Jonathan Backhouse, attired in the usual dress of
the Society of Friends, unknown to the rest of the
company, was in the room quietly reading his
newspaper, when he was attacked by the wealthy
commercial, and by a series of sarcastic remarks held
up to ridicule as a man out of harmony with the spirit
of the time and place. Following up this raillery the
commercial, displaying his handful of notes, offered to
bet the Quaker £5, or any sum, that he could not
produce as much money as he was exhibiting. Mr.
Backhouse, after a great deal of banter, said he did not
bet, but to show his indifference to money offered to
put a £5 note in the fire if the commercial would do
the same. Suiting the action to the word, Mr.
Backhouse took out a £5 note and put it into the fire.
The commercial, not wishing to be behind, did the
same. Mr. Backhouse offered to repeat the process,
but the commercial, considerably cowed, declined;
when Mr. Backhouse quietly thanked him for having
burned one of his (Mr. Backhouse’s) bank-notes for
which he had received £5, while the note he (Mr.
Backhouse) had burned was on his own bank, and only
cost him the paper.”
 CHAPTER IV
THE BANK CHARTER ACT OF 1844,
AND ITS SUSPENSIONS

A
fter the renewal of the Charter in 1833, the directors of the Bank
of England laid down as a principle on which their future
operations were to be guided, that one-third of their liabilities
should be kept in cash and bullion, and the remaining two-thirds in
securities. If this principle had been acted on, the Bank would have
been saved from many of the troubles which shortly assailed it; but
though the intentions of the directors were good, circumstances
were too strong for them, and the actual proportions of cash and
securities to liabilities respectively, often differed materially from the
standard laid down. This was notably the case during the periods of
financial pressure which were experienced in the years 1836 and
1837.
In the year 1839 matters assumed a very serious aspect. In the
early part of this year the amount of cash held by the Bank was
about one-third of the amount of securities, but during the year the
amount invested in securities increased at the expense of the
amount held in cash; and by September we find that securities stood
at nearly £29,000,000, while the cash was reduced to a tenth of that
figure, and stood at £2,936,000 only. In order to avert a calamity
which appeared to be impending, the Bank arranged loans in Paris
and Hamburg to the extent of between three and four millions.
This manifest exhibition of weakness on the part of the Bank led
to the appointment of a committee of the House of Commons to
inquire into the matter. The committee condemned the principles on
which the Bank was working, but were powerless to effect any
alteration, owing to the Charter of the Bank not expiring till 1844.
On the expiry of the Charter, however, Sir Robert Peel brought
forward his famous Act for remodelling the Bank, and regulating the
issues of the country banks throughout England and Wales.
The Act was passed on the 19th July, 1844, and continues
without alteration to the present day. The main provisions enacted
thereby, briefly stated, are as follows:—
I. The Issue Department and the ordinary
Banking Department of the Bank of England were
to be entirely separated as from the 31st August,
1844.
II. On such separation taking place, securities to
the value of £14,000,000 (including the debt due to
the Bank from the Government) were to be
transferred to the Issue Department, together with
so much gold coin and bullion that the total so
transferred should equal the total amount of notes
then outstanding. Thereafter (with the exception
noted below) the Issue Department must not issue
any notes in excess of a total of £14,000,000
except in exchange for gold coin or bullion.
III. The Issue Department might not at any
time hold more silver than one-fourth part of the
gold held. As a matter of fact, the Issue
Department holds no silver.
IV. Notes might be demanded from the Issue
Department by any person in exchange for gold at
the rate of £3 17s. 9d. per standard ounce.
V. If any banker having the power of issue on
the 6th May, 1844, should relinquish such issue,
the Issue Department may be authorised to
increase its issue of notes against securities to the
 extent of two-thirds of the issue so relinquished;
but all the profits on such increased issue against
securities were to belong to the Government.
VI. The Bank must issue a weekly statement of
the position of both its Issue and Banking
Departments, in a prescribed form.
VII. Bankers having the right to issue their own
notes on the 6th May, 1844, might continue such
issue under certain conditions, and to an agreed
amount; but no provision was made compelling
such bankers to keep any reserve either in cash or
securities against their issues. If any issue lapsed,
from any cause, it could not be resuscitated; and
no institutions could acquire the right of issue in
the future.
VIII. Banks consisting of more than six
partners, though within the sixty-five-mile radius of
London, might draw, accept, or endorse bills of
exchange not being payable to bearer on demand.
The first return issued by the Bank in accordance with the
regulations of the new Act was that of the 7th September, 1844, and
was as follows:—

ACCOUNT OF THE LIABILITIES AND ASSETS OF

THE BANK OF ENGLAND
For the Week ending 7th September, 1844

Dr. ISSUE DEPARTMENT Cr.

£ £
28,351,295 Government 11,015,100
Notes issued
debt
Other securities 2,984,900
 Gold coin and 12,657,208
bullion
Silver bullion 1,694,087
£28,351,295 £28,351,295

Dr. BANKING DEPARTMENT Cr.

£ £
14,553,000 Government 14,554,834
Proprietors’ capital
securities
Rest 3,564,729 Other securities 7,835,616
Public deposits 3,630,809 Notes 8,175,025
8,644,348 Gold and silver 857,765
Other deposits
coin
Seven-day and 1,030,354
other bills
£31,423,240 £31,423,240

For comparison we append the Return of the 2nd September,

1903.

Dr. ISSUE DEPARTMENT Cr.

£ £
51,831,835 Government 11,015,100
Notes issued
debt
Other securities 7,434,900
Gold coin and 33,381,835
bullion
Silver bullion Nil
£51,831,835 £51,831,835

Dr. BANKING DEPARTMENT Cr.

 £ £
14,553,000 Government 18,260,841
Proprietors’ capital
securities
Rest 3,740,209 Other securities 24,969,260
Public deposits 7,393,580 Notes 22,322,875
41,872,061 Gold and silver 2,119,339
Other deposits
coin
Seven-day and 113,465
other bills
£67,672,315 £67,672,315

The provisions of the 1844 Act, above noted, are the principal
ones which affected banking in general, and the Bank of England in
particular—they were the food for much debate and discussion
before they became law, and it may be added that several of the
provisions then enacted have been the food for much debate and
discussion ever since. Taken as a whole the Act has worked well, and
has succeeded, in combination with greater knowledge and
foresight, in maintaining our banking system in a sound condition.
The regulations as to the country bank-note issues were framed
with the idea of ultimately eliminating entirely such issues; but
though the amount of private notes in circulation has decreased, and
also the number of bankers who have the power of issue—by lapses,
bankruptcy, and amalgamations—the time when there are no
country bank-notes has not arrived, notwithstanding that the
framers of the Act confidently anticipated that such a result would
be achieved long before now.
The main point of contention between the supporters and
opponents of the Act lies in its want of elasticity in time of need.
Under no circumstances can the Bank increase its issue of notes
against securities beyond the prescribed limit, without a breach of
the law; but on three occasions in the past the law has been broken,
though with the consent of the Government, and subsequent
confirmation of Parliament.
Under the laws regulating the Imperial Bank of Germany such
procedure would not have been necessary. The German Bank Law
has been framed largely on the same lines as our own, but it gives
the Reichsbank power to increase the amount of notes issued
against securities on a payment to the Government of a fine of 5 per
cent. per annum on the excess issue. This fine is sufficient in
ordinary times to act as a complete check on overissue, but in times
of trouble it acts as an efficient safety-valve by relieving the minds of
business people from the fear that “there will not be enough to go
round.” If it is known that money can always be had at a price, the
probability of a crisis developing into a panic is almost entirely
obviated.
We will now briefly review the three occasions on which the Bank
Act was suspended, and the effect of such suspensions.
The first of these occasions was during the panic in the year
1847—known as the “railway panic.” Shortly previous to this year a
great accumulation of capital had led to a demand for new
investments, which were duly provided for the public by those
concerned with such matters. Added to this, interest rates had ruled
low for some time, and this conduced to a period of speculative
activity. Too much capital was put into fixed investments—chiefly
railways—and in one session of Parliament sanction was asked for
various railway schemes involving a total capital of £340,000,000.
Wild gambling in railway stocks ensued, credit was inflated above all
reason, and then the turn came. This was primarily due to a bad
harvest and potato crop causing a heavy importation of corn, and
consequent export of gold.
During the panic which ensued, the reserve of the Bank of
England fell to £1,600,000, but when the panic was at its height, the
Act, passed only three years before, was suspended. The Bank was
authorised to increase its accommodation to the public by
exceeding, to an indefinite extent, the limit fixed for the issue of
notes not secured against gold. The effect of this suspension of the
Act was immediate and complete. The fear that “there was not
enough to go round” passed from men’s minds. As a matter of fact,
the issue on this occasion did not exceed the normal limit, the mere
knowledge that the Bank was empowered to exceed this limit
proving sufficient to allay the panic.
The second suspension of the Bank Act was due to the crisis of
1857, a crisis that was brought about by reckless overtrading, and
came upon the public very suddenly and with practically no warning.
The reserve of the Bank of England had been allowed to fall
dangerously low during the course of the year, and large financial
operators had carried on vast transactions with hardly any capital—
only credit—relying for assistance on the Bank. Bad news came to
hand from America in September, detailing how there had been a
serious financial collapse in that country; failures had occurred,
shaking commercial credit to its core, and about one hundred and
fifty banks had stopped payment.
A heavy drain of gold from here to America commenced, and by
the middle of October credit was curtailed, and distrust was rife in
England. Within a very short space of time many of our banks and
financial houses were crippled and failed. In November heavy
demands were made for gold for Scotland and Ireland, and on the
11th November the failure of Sanderson and Company—a great
London discount house—was announced, with liabilities of upwards
of five millions. Utter rottenness appeared to pervade the
commercial world, and general bankruptcy seemed imminent, when
for the second time the Bank Act was suspended. This took place on
the 12th November, and at once had the effect of quieting the public
mind. On this occasion the Bank had to make use to a large extent
of its temporary authority to issue notes above the normal limit
without holding gold against them. The severity of this crisis can be
seen by the fact that in November the Bank reserve fell to under
£600,000, while the bankers’ balances at the Bank of England alone
stood at about five and a half millions, and the Bank Rate was as
high as 10 per cent. This was also the rate charged by the Bank of
France at the time, showing that the panic had spread, and was not
solely confined to ourselves and America.
The third suspension of the Bank Act took place in 1866. Many
elements of disturbance to the Money Market had been in force
during two or three preceding years. The Civil War in America had
resulted in gold being sent to this country; but the stoppage of the
supply of cotton from America, owing to the war, disorganised one of
our staple national industries, and supplies of cotton had to be
obtained from elsewhere at high prices, and paid for in cash. Hence
a drain of gold set in on a large scale. In addition, a large
speculation had been built up on credit in the stocks and shares of
the many new limited liability companies which were formed at that
time.
General uneasiness began to prevail towards the end of 1865; in
January, 1866, the Bank raised its discount rate to 8 per cent., and a
crisis began to develop rapidly.
Speculators tried to sell their securities and found no market for
them, several large railway contractors failed, and many of the newly
formed limited liability companies succumbed and were wound up.
The failure of the Joint Stock Discount Company, followed shortly by
that of Barned’s Bank of Liverpool, brought matters to a head; the
distrust became universal and culminated in panic. On the 9th May
the Bank Rate was raised to 9 per cent. On the 10th May the failure
of Overend, Gurney, and Company—for upwards of ten millions—was
announced, and the Bank Rate went to 10 per cent. This failure was
not made known till after business hours, so it was not till Friday, the
11th May, 1866—known as “Black Friday”—that the crisis reached its
height.
The stoppage of this large house affected the whole world, and
general failure seemed imminent, when, in the afternoon of the day
on which the failure became known, it was announced that the Bank
Act was again suspended, and calm began to take the place of
mania. But though the panic was allayed, many failures shortly took
place, which delayed the quick restoration of a sense of security.
Among these failures may be mentioned the Bank of London, the
Consolidated Bank, and Agra and Masterman’s Bank. All these three
institutions were perfectly solvent as a matter of fact, but they found
themselves in the dangerous position of having no available assets.
The two last-named banks subsequently resumed business.
From the above brief records of the financial tragedies of the
past, we see that on each occasion reckless speculation and
overtrading had been allowed to reach a dangerous height before
any steps were taken to check them, and on each occasion the
check came too late. But we also see the marvellously quick effect
which the suspension of the Act had on the situation. Although a
period of nearly half a century has elapsed since the time of the last
suspension, the position remains the same, and it is only owing to
greater knowledge and greater caution that such catastrophes have
been averted.
In contemplating any future catastrophe of the kind which may
come upon us, it is generally assumed that the Act would be again
suspended; but delays are dangerous. By the time the situation had
developed to such an extent that the Government might deem it
expedient to give the Bank the necessary powers, a panic such as
has never before been known might overtake us; whereas if the
suspension of the Act were to a certain extent automatic, and
responsible people knew for certain that money could always be had
at a price, the probability of such a termination of any crisis would
be very remote.
 CHAPTER V
THE DEVELOPMENT OF LONDON AS THE
FINANCIAL CENTRE OF THE WORLD

B
efore proceeding to examine the Money Market and banking
system of more modern days, it will be well to glance at the
causes which contributed to the predominance of London among
international financial centres, and made it the clearing house of the
world. At the present time this predominance is being assailed from
several quarters—notably Paris, Berlin, and New York—but there is
no doubt that London still holds a good lead, and with knowledge,
activity, and perseverance there appears to be no reason why it
should not continue to be in the forefront in the future.
In the Wealth of Nations Adam Smith points out that the increase
and riches of commercial and manufacturing towns contribute to the
improvement and cultivation of the countries to which they belong,
and that commerce and manufacture introduce order and good
government, and with them the liberty and security of individuals.
In our own history we can trace the truth of these remarks. We
have already drawn attention to the flourishing state of trade which
developed during the reign of Elizabeth. New industries were
started, villages grew into towns, and new markets for our wares
were opened up in various parts of the then known world. In
consequence of the wars on the Continent, many wealthy merchants
came or were exiled to our shores, and settling here, helped to swell
the growing business of London. We were fortunate in the fact that
this “greater liberty and security of individuals,” enabling our
forefathers to devote their attention and wealth to the further
development and expansion of the commerce of the country, came
about with us long before a similar state of affairs began to have
effect with our continental neighbours, and this gave us a good start
commercially. While our neighbours were still in the throes of
continual disputes and unrest, we were laying the foundations of a
world-wide system of trade. We were in a position to supply the
Continent with their various needs as they arose, to our own
immediate benefit and the further advancement of our commerce.
In connection with the more rapid development of commerce
with us than with continental nations must be remembered the
energy and activity of the race forming the population of our country
—an energy and activity not only in starting and conducting new
industries, but in spreading abroad to new lands and forming new
colonies, thus enlarging the sphere of our operations and the
number of our markets.
Our geographical position has assisted materially in our
commercial development. The disturbances arising from the fear and
risk of invasion have been less pronounced than with continental
nations, and our extended coast-line, embracing many natural
shelters, has engendered our vast shipping industry, without which
we could never have attained to our present position. We have also
had the advantage of another natural circumstance in the proximity
of our coal and iron fields to each other. This gave us for a long
period an unparalleled command of certain trades dependent on this
fact. Thus we see that, owing to good government, national
character, and natural conditions, our trade was built up on a firm
foundation, and was spread abroad until its ramifications extended
into every country of the world.
Turning now from the trade point of view to the monetary
position, we find that as our trade grew, so did our means and
capacity of dealing with that trade. Although in early days we were
rather backward in our banking system, yet that system developed
quicker with us than with other countries. The eighteenth century
embraced many periods of financial distress, but the Government
never defaulted in any of its engagements, and our leading
merchants became renowned as men of honour and integrity. During
the early part of the nineteenth century our position improved by
leaps and bounds, our gold standard of currency was firmly
established, our merchants’ names became known and honoured
throughout the world, and direct financial operations between
London and all foreign parts became matters of daily occurrence.
These monetary transactions were very essential in the financial
growth of London, and were a large factor in our capital becoming
the centre of finance. Owing to the vast extent of our trade, both as
to its amount and diffusion, we were in a position to conduct
financial transactions the world over. From this it followed that when
commerce began to expand and grow among continental nations, it
was found that the easiest way of settling the financial business
arising therefrom was through the intermediary of London. England
bought from and sold to all nations, but in the first half of the
nineteenth century reciprocal transactions between foreign centres
of trade were few and far between. For instance, suppose a New
York merchant shipped produce to Hamburg, he would draw on
some appointed London house for payment; the merchant in
Hamburg who had bought the goods would then have to find the
means of providing this London house with the necessary funds to
meet the draft from New York. This he would do by buying a draft in
Hamburg from some merchant there who had shipped goods to
London and was wanting payment. This draft he would remit to the
London house on which the American exporter had drawn, and the
London house would collect the money represented by the Hamburg
draft, thus settling both transactions. This is only a rough example,
but it is typical of what came to be a regular custom.
Various countries may trade together, but unless they have direct
mutual financial transactions, they cannot settle their indebtedness
without remitting precious metal of one kind or another, or
employing the agency of another country which has direct financial
dealings with both. In the time of which we are speaking London
was the only centre which was in a position to carry out such
transactions. In the latter half of the past century, and especially
towards the close of the century, our position suffered through direct
financial operations between various countries becoming established,
thus eliminating the need of a middleman; but it may still be said of
London that it has direct financial and trading connection with every
country of the world, which at present cannot be said of either Paris,
Berlin, or New York. The very fact of the establishment in London of
branches of the leading banks and finance houses of France,
Germany, and the United States, emphasises the position which
London has attained and still holds in the financial world.
 CHAPTER VI
FACTORS OF THE MONEY MARKET

H
aving now surveyed the history and development of our financial
system up to a point when “system” can really be said to have
started, and also having glanced at the causes which have
placed London in the forefront of all financial centres, we will
consider the formation, as a whole, of what is called the “Money
Market”; and then more carefully examine certain of the more
important factors which help to form that market.
There is no definite “market” for money in the sense of a “place
of purchase and sale,” like a cattle market or a corn market; when
we speak of the “Money Market” we refer to the body or aggregation
of large dealers in money—bankers, bill-brokers, etc.—who either
have money to lend or who require to borrow money, and by whom
the rate to be charged for the use of money is largely settled, as a
result of their mutual transactions.
This body of money-dealers is not clearly defined into two classes
—lenders and borrowers—as an ordinary market is divided into two
classes—buyers and sellers; but with money-dealers all are
practically both buyers and sellers; that is, all are ready to sell the
use of money at a certain price, and to buy the use of money at
another price.
The Bank of England in bygone days was the predominant factor
in the Money Market; but now, in ordinary times, it has somewhat
fallen from its high estate in that respect. It is only at certain times
that its funds find their way into the Money Market to any large
extent. But the Bank still has the power, when occasion arises, to
make its influence predominate, as it constitutes the final reserve, in
case of need, of our banking system. The Bank likewise has the
power to make its influence felt when the directors deem it advisable
to obtain control of the Money Market, for the purpose of
maintaining the monetary position on a basis of safety. As we shall
see when dealing with the subject of the foreign exchanges, if, in
order to check an outflow of gold, the directors of the Bank wish to
raise the value of money in London—that is, to raise the rate at
which money can be borrowed or lent—they raise the official rate of
the Bank of England. If the outside market lags behind, or does not
keep in line with the movement, they force it to do so by themselves
borrowing large sums from the market, thus reducing the available
supply of money in the hands of the market, and consequently
enhancing the value of money.
The chief factor in the formation of the Money Market is the body
of the joint-stock and private banks of London, and through them of
the bankers of the kingdom. Practically all the working capital of the
country and the floating money of private individuals, together with
moneys awaiting permanent investment, are now in the hands of
our bankers.
Of this vast accumulation of capital held by bankers—amounting
in the United Kingdom to some £800,000,000—a certain part is
retained in actual cash, besides a balance which is kept with the
Bank of England or a London agent, some is invested in securities,
and the balance is used in lending to those that require the use of
further capital for their business or private needs. Of this balance so
lent, a large percentage is advanced to individual customers by way
of loan, overdraft, or in the discounting of bills; and the remainder is
used in the Money Market proper, or what has been aptly called “The
Short Loan Fund.” The rate of interest which private individuals have
to pay for advances from time to time is largely based on the
prevailing official rate of the Bank of England as regards loans, and
on the “market rate” as regards the discounting of first-class bills.
 For the greater part of the money in a banker’s hands no interest
whatever is paid, that is, for practically the whole of the current
account balances. For the remainder, the money on deposit, only a
small interest is paid; but a banker must always keep before him the
fact that nearly all his liabilities are repayable in cash on demand.
Thus he must always keep himself prepared for eventualities, and
his first line of defence consists of cash and balance with the Bank of
England or London agent, and he reckons his advances to the Money
Market as his next most quickly convertible and available asset.
For the money advanced to the Money Market bankers are
content to receive a low rate of interest, provided that the advances
are absolutely safe, and can quickly be called in when necessary.
These conditions can be obtained by lending money at “call” (that is,
repayable on demand) or at a few days’ notice to the bill-brokers,
who deposit as security for such loans, first-class bills, or certain of
the highest class of securities, such as Consols, etc.
The bill-brokers and discount houses of London form the second
most important factor in the Money Market. These firms and
institutions practically act as middlemen or intermediaries. Many of
them possess large capital themselves with which to conduct their
business, but the bulk of the funds which they employ consists of
borrowed money. This money is borrowed from various sources; the
greater part from the banks, some from the India Council, and some
from our merchant princes and finance houses, who of themselves
really constitute another factor in the Money Market. Besides these
sources for borrowing money, the bill-brokers further increase their
working funds by receiving money on deposit from the public. With
the funds so collected they buy bills, usually only those of a first-
class character, and these they either hold until maturity or
rediscount with the banks, and occasionally with the Bank of
England.
The British Government is at certain times a factor in the Money
Market, that is, when on account of any extraordinary outlay, or
when expenditure is temporarily exceeding revenue, it issues
Treasury Bills and Exchequer Bills. If these bills are bought by the
Money Market, it follows that the amount of money in the hands of
the Market is, at least for the time, decreased by the amount of
money paid for the bills (which goes into the Bank of England and
helps to increase “Public Deposits”), and consequently the rates for
money in the open market are inclined to rise or “harden.” When
these bills are repaid the contrary effect is produced, market
supplies are increased and rates are inclined to droop.
Another factor which has to be taken into account in this matter
is the India Government. The India Government has, from time to
time, large funds lying here which are not required for immediate
use, nor are they available to lend for long periods; these funds
practically constitute a floating balance. Use is made of this money
by lending it out to the market through a well-known house, much in
the same manner that banks lend their floating balances. The money
is usually lent in sums of not less than £50,000, for periods from a
fortnight to a month; and it is generally stipulated that the securities
deposited against the advances shall consist of either Consols, or
Indian Securities of certain kinds, such as rupee paper and the
guaranteed debentures of a few of the first-class Indian railways.
The India Government generally manages to obtain a very fair return
for the money so lent.
The Stock Exchange is another element which requires
consideration, although it is a rather one-sided element, inasmuch
that it is nearly always a borrower. In busy times on the Stock
Exchange enormous sums are borrowed from the banks for the
purpose of speculation of one kind or another. Stocks are bought by
various persons who have not the money to pay for them, in the
anticipation that they will increase in value; and these persons
arrange with their brokers to “take up” the stock for them—that is,
that the brokers shall find the money to pay for these purchases—
and this ultimately results in a banker advancing the money. During
periods when the rates of interest are low also, large amounts of
stock bearing a higher interest are then “taken up,” for the purpose
of securing the difference in the amount of the interest paid for the
loan and the interest received from the stock, and the money for
these purchases is largely borrowed from banks. These Stock
Exchange loans are made from “account to account”—that is, from
one settling day on the Stock Exchange to the next—and as there
are two settling days every month, the loans are nominally granted
for about a fortnight each. The interest charged is fixed at the
beginning of each account for that account, and varies according to
the prevailing conditions at each renewal.
Lastly, we have a somewhat new factor entering into the Money
Market, but one which is increasing in importance, and that is the
establishment in London of branches of many powerful and rich
continental banks, who make use of the London Money Market for
employing their surplus funds when they can do so to advantage.
They lend money to the bill-brokers much as do ordinary London
banks; and at times, when the conditions are favourable, they invest
their funds in English bills, occasionally absorbing considerable
amounts of Treasury and Exchequer bills. These purchases of English
bills increase the money in the market for the time being, and the
competition of these foreign bankers tends to depress the rate
charged for discount. It must be remembered, however, that this
course of business gives the banks in question power to draw gold
from us heavily on the maturity of the bills, or at any time when they
see fit, by selling their bills before maturity.
From a consideration of these various factors, we see how largely
they are interwoven together and dependent on each other. The
banks gather in deposits from all quarters and lend to the bill-
brokers, while leaving a large amount with the Bank of England; and
the bill-brokers borrow from the banks and buy up bills from all
quarters; but if the banks “call” their money from the bill-brokers the
latter are driven into the arms of the Bank of England, to reborrow
the money which the banks have called from them. This is also the
case if the India Government call in their loans, and the money to
repay the same cannot be borrowed from the banks. Similarly with
the Stock Exchange, if much money is absorbed in this quarter, the
banks will reduce their accommodation to the bill-brokers, who may
by this action again have to rely on the Bank of England; and again,
lastly, if the foreign banks commence to draw money from us, the
strain comes as usual on the Bank of England.
All these factors work round the Bank of England as a centre,
and the need of strength on the part of that institution becomes at
once apparent when it is seen what mighty interests are dependent
on it, that the financial credit of the country rests ultimately upon its
stability, and that its policy and actions involve consequences of weal
or woe to the community at large.
 CHAPTER VII
THE BANK RETURN

W
e will now examine the weekly “Return” issued by the Bank of
England in accordance with the requirements of the Act of
1844, and consider the significance of the various items
appearing therein. The Return is made up to the close of business
on every Wednesday, and is published on Thursday. Though the
figures given always attract attention, and are regularly made the
subject of analysis and criticism in the financial articles of the Press,
yet the interest taken in the Return varies considerably from time to
time. During periods of financial quiescence this interest is
somewhat of an academical character; but in times of doubt and
distrust a very real and practical interest is exhibited, not only by the
group of bankers, bill-brokers, and merchants constituting the
“Money Market,” but by that section of the general public who have
financial transactions at stake, and who possess the requisite
knowledge to understand the true import of the figures of the
Return.
For the purpose of our analysis we will take the Return of the
2nd September, 1903.

ISSUE DEPARTMENT
£ £
51,831,835 Government 11,015,100
Notes issued
debt
Other securities 7,434,900
Gold coin and 33,381,835
bullion
 £51,831,835 £51,831,835

BANKING DEPARTMENT
£ £
14,553,000 Government 18,260,841
Proprietors’ capital
securities
Rest 3,740,209 Other securities 24,969,260
Public deposits 7,393,580 Notes 22,322,875
41,872,061 Gold and silver 2,119,339
Other deposits
coin
Seven-day and 113,465
other bills
£67,672,315 £67,672,315

ISSUE DEPARTMENT
In a previous chapter we saw that under the Act of 1844 the
Issue Department was to be separated from the Banking
Department, and that it was at liberty to issue £14,000,000 of notes
against securities, of which the Government Debt, amounting to
£11,015,100, was to form a part. Any issue of notes above this
amount of £14,000,000 was to be secured by an equal amount of
coin or bullion, with the proviso, however, that the issue of notes
against securities might be increased from time to time to the extent
of two-thirds of the amount of any lapsed country issue.
In the return before us we see the result of nearly sixty years of
the working of the Act. The notes issued stand at £51,831,835, and
are secured by the Government Debt of £11,015,100 (as at the
passing of the Act) and other securities amounting to £7,434,900
(against £2,984,900 in 1844), the balance being made up of gold
coin and bullion, no silver being now held. We thus see that
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