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LECTURE 3 (1)

This lecture covers the structure and function of antibodies, including the differences between monoclonal and polyclonal antibodies, and their interactions with antigens. It discusses various immunological assays, such as precipitation and agglutination tests, and their applications in diagnosing diseases and blood typing. Additionally, it emphasizes the importance of antigen-antibody reactions in laboratory investigations and the implications of blood group compatibility for transfusions and transplants.
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0% found this document useful (0 votes)
10 views28 pages

LECTURE 3 (1)

This lecture covers the structure and function of antibodies, including the differences between monoclonal and polyclonal antibodies, and their interactions with antigens. It discusses various immunological assays, such as precipitation and agglutination tests, and their applications in diagnosing diseases and blood typing. Additionally, it emphasizes the importance of antigen-antibody reactions in laboratory investigations and the implications of blood group compatibility for transfusions and transplants.
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LECTURE 3

ANTIGEN-ANTIBODY INTERACTIONS AND THEIR


APPLICATIONS IN LABORATORY INVESTIGATIONS

STRUCTURE OF ANTIBODY

Basic structure of Immunoglobulin – Y-shape

Four polypeptide chains – 2 light chain and 2 heavy chains which


are linked to form a Y-shape.

Constant region determines the Ab class.

Variable regions determine antigen binding .

Variable regions in both heavy and light chains consist of 3


Hypervariable regions (HV1, HV2, HV3) and 4 framework regions
(FR1 – 4).

HV regions are in direct contact with the antigens/epitopes and


therefore determine complementarity and antigen binding. Hence
HV regions are also called complementarity determining regions.

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Nature of Ag/Ab Reactions

• Lock and Key Concept

• Non-covalent Bonds

– Hydrogen bonds
– Electrostatic bonds
– Van der Waal forces
– Hydrophobic bonds

• Reversible

• Multiple Bonds

AFFINITY

•Strength of the reaction between a single antigenic determinant


and a single Ab combining site.

Affinity = attractive and repulsive forces.

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AVIDITY

•The overall strength of binding between an Ag with many


determinants and multivalent Abs.

SPECIFICITY AND SENSITIVITY

Specificity:
The ability of an individual antibody combining site to react with
only one antigenic determinant.
The ability of a population of antibody molecules to react with
only one antigen.

Sensitivity:
This term often applies to tests
It is the ability of a test to detect minimal levels of antibody of
antigen in a sample
Some tests are more sensitive than others i.e. some tests like
ELISA is more sensitive than precipitation. It can detect upto
nanograms of antibody in a sample, whereas a precipitation test
would detect 200ug/ml antibody in a sample and not below this.

CROSS REACTIVITY

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•The ability of an individual Ab combining site to react with more
than one antigenic determinant.

•The ability of a population of Ab molecules to react with more


than one Ag.

Cross Reactions

MONOCLONAL ANTIBODIES AND POLYCLONAL ANTIBODIES

Monoclonal antibodies:

Are homogenous mono-specific antibodies which bind to only to


one epitope on an antigen.

They are produced in a laboratory by a specific clone of B cells


through the hybridoma technique

Polyclonal antibodies:
Are poly-specific antibodies and consist of a mixture of antibodies
produced by multiple clones of B cells. Polyclonal antibodies are
capable of reacting with different epitopes and they vary in
affinity and specificity to different epitopes

Polyclonal antibodies are produced by repeatedly injecting


antigen in an animal – the serum will contain the polyclonal
antibodies.

HYBRIDOMA TECHNIQUE FOR PRODUCTION OF


MONOCLONAL ANTIBODIES

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AG-AB REACTIONS AND LATTICE FORMATION (MARRACK’S
HYPOTHESIS, 1934)

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Lattice formation is the process by which antigens and
complementary antibodies react to form (ag-ab) complex
networks eventually leading to insoluble precipitates.

In terms of the immune response, lattice formation is important


as it enhances phagocytosis, agglutination, opsonization
and elimination of the pathogen.

The optimum condition for extensive lattice formation occur at


the zone of equivalence – when there are almost equal
concentration of circulating antigen and antibody.

Excess antibody (prozone) or antigen (postzone) hinder


appropriate formation of the ag-ab lattice – thereby hindering
phagocytosis, agglutination, opsonization and elimination of the
pathogen.

ANTIGEN ANTIBODY CONCENTRATION AND LATTICE


FORMATION

ONLY POLYCLONAL ANTIBODIES ARE INVOLVED IN LATTICE


FORMATION

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IMMUNOLOGICAL ASSAYS

Development of various biological assays.


Based on presence of antigens or antibodies.

Immunodiagnostic assays are useful in:


Diagnosing diseases.
Monitoring level of humoral immune response.
Identification of molecules of bio/med interest.
 The assays differ in speed and sensitivity.
 Some e.g. agglutination and precipitation assays depend on
lattice formation. Others e.g. ELISA does not.

SENSITIVITY OF VARIOUS IMMUNOASSAYS

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A:PRECIPITATION TESTS

Precipitation occurs when a soluble antigen reacts with the


complemetary antibody in the presence of electrolytes, under
appropriate conditions of temperature and pH, to form a an
insoluble antigen-antibody complex in the form of a
precipitate.
Precipitation tests are dependent on lattice formation and
follow the principles of Marrack’s hypothesis on lattice formation.
Optimal precipitation occurs at the zone of equivalence
Antigen antibody concentration and lattice formation.
Precipitation tests are semi-quantitative tests which detect the
present of antigens and are used widely in diagnosis of
pathogens.
Precipitation tests may be carried out in fluids or on agar.

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1. The ring precipitin test

The test is performed in a capillary tube to test for antigen in


test sample.
The serum or antibody preparation is put in the tube and then
test sample is layered over the antibody.
If the sample is positive, the antigen will bind complementary
antibodies leading to lattice formation and precipitation.
A visible ring of precipitate will form in the capillary tube at
the zone of equivalence.
This procedure is applied in the Ascoli’s thermoprecipitin test
to diagnose anthrax.

2.The slide test

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This test can detect either antigen or antibodies
This is the test applied for in diagnosis of syphilis i.e. the VDRL
test.
The VDRL tests involves detecting antibodies against syphilis
bacteria,Treponema pallidum
A drop of the patient’s blood or serum is placed on a glass slide.
A drop of the VDRL reagent, which contain T.pallidum cardiolipin
antigen is added. The drops are mixed.
Formation of precipitate indicates presence of anti-T.pallidum
antibodies and hence a positive diagnosis for syphilis.

• The VDRL test becomes positive


1-2 weeks after appearance of
(primary lesion) chancre.
• The test becomes weakly
reactive (50-75%) in the late
phase of primary syphilis due to
excess antigen and low antibody
levels
• In the secondary syphilis the
test becomes highly reactive
(100%). Reactivity decreases
thereafter due to excess antibody
and reduced levels of antigen
(75%).
• Treatment in the early stages of
infection maycompletely suppress
production of antibodies and

IMMUNODIFFUSION TESTS

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In this method, the antigen-antibody precipitation occurs in a
gel
Usually 1% soft agar is used.
The precipitation is observed as a line or arc (precipitin line) in
the gel.
The advantage of immunodiffusion tests over previously
discussed methods of fluid precipitation is that because the
results are on a solid phase, they can be preserved for future
reference or for further quantitative or other testing procedures.

1. Single immunodiffusion (Oudin procedure)

This procedure is useful for detecting the presence of single and


multiple antigens in a sample.
The antibody preparation or serum is mixed with agar in a tube.
The test sample or antigen(s) is layered over the antibody-agar
preparation in the tube.
Complementary antibodies and antigen diffuse toward each
other in agar and react forming a visible line of precipitation.
For each different antigen, a separate precipitation line is formed
– It is possible to analyze different antigens in a single sample.

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2.Radial immunodiffusion (mancini procedure)

This is a quantitative procedure for measuring antigen


concentration.
An agar solution is mixed with known dilution of antibodies or
serum and spread onto a glass plate and allowed to set.
Wells are formed on the agar and antigen preparation/test
sample is added to each well. The antigen is allowed to diffuse
into agar containing the antibodies.
A Precipitation or precipitin ring forms around the well as the
antigen diffuses and reacts with complementary antibody forming
precipitate.
The area is proportional to the antigen concentration. To
determine the antigen concentration in sample, the area of the
precipitin ring is compared with a standard curve.

3.Double immunodiffusion (Ouchterlony procedure)

This method determines relationship between antigens.


Agar is poured on the glass slide and three identical wells are
made using a template.
The central well is filled with antibody preparation of serum
The adjacent wells are filled with antigens whereby one of the
wells contains a known antigen, which acts as a positive control
and the other well contains the test antigen.
If the two antigens are identical or related, they diffuse each
other forming an arc.

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Pattern of precipitation arc indicates whether antigens share
epitopes or have cross-reacting components i.e:
i) A single precipitin line indicates that the antigens have identical
epitopes.
ii) Two crossing precipitin lines indicate that the antigens are non-
identical.
iii) If the precipitin line is a curved spur, there is partial identity in
the epitopes.

IMMUNOELECTROPHORESIS

This is a qualitative technique combines electrophoresis with


immunodiffusion .
It is used to identify antigen composition in a sample or to identify serum
proteins.
The technique is undertaken in two steps:

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Separation of the proteins in the test sample by molecular weight and
charge (electrophoresis).
Precipitation of the separated proteins with antibodies through
immunodiffusion
This technique is commonly applied to investigate the purity of a protein
sample.
It also useful in identifying the antibody composition of a serum sample,
whereby the serum antibodies are separated by eletrophoresis and
subsequently probed with anti-IgG, anti-IgM, anti-IgA, anti-IgD and anti-IgE
antibodies.

IMMUNOELECTROPHORESIS TECHNIQUE

B: AGGLUTINATION TESTS

 Antibody-Antigen interactions result into clamping.

 Depend on lattice formation.

 Excess antibody inhibits agglutination (prozone effect) due to


lack of cross-linking and lattice formation

In humans, haemagglutination reactions are common, whereby


the agglutination reactions involve red blood cells.

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 Haemagglutination tests have many applications including:
Blood group typing, blood transfusion matching, diagnosis of
bacterial and viral infections, diagnosis of congenital
abnormalities e.g. erythroblastosis foetalis.

I. HAEMAGGLUTINATION AND BLOOD GROUPING

There are over 200 antigens present in human blood hence


blood grouping can be a complex affair.
So far, the International Society for blood transfusion has
identified > 30 different blood grouping systems.
However, the ABO and Rhesus blood grouping systems are
the most commonly used systems for transfusion and
transplant purposes because of the consistency and
predictability of presence or absence of the corresponding
antigens and antibodies.
Blood grouping must always be performed on donors and
recipients prior to blood transfusion, organ transplant, on
mothers and neonates if erythroblastosis foetalis due to blood
incompatibility is suspected and on father and child for paternity
testing.

ABO BLOOD GROUPING

This system is based on the presence or absence of the ABO


antigens on the surface of the red blood cells.

The ABO genes are encoded on human chromosome 9 and the


genes are inherited from both parents.

Genes A and B are co-dominant i.e. if an individual inherits


either of the genes from a parent, it will always be expressed
either as A, B or AB.

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Gene O is recessive i.e. for an individual to be blood group O,
he must express the OO allele i.e. must inherit O gene from both
parents.

The AB and O antigens are glycolipids


(glycosyltransferases) found on the surface of RBCs. ABO
antigens are structurally related.

INHERITANCE OF ABO GENES

NB: Alleles A and B are co-dominant whereas allele O is recessive.

ABO ANTIGENS AND ANTIBODIES

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Q) Why do blood group A individuals have anti-B antibodies and not anti-A
antibodies?

ABO BLOOD GROUPING PROCEDURE

The process of blood group typing involves a 2-step procedure:


Detection of ABO antigens i.e. forward typing.
Detection of ABO antibodies i.e. reverse typing/backtyping or
serum confirmation.
In forward typing:
Two drops of the individual’s blood or RBCs are taken into
three wells or slides. Anti-A and Anti-B monoclonal antibodies
are added to the blood. Positive agglutination confirms presence
of corresponding antigen and hence blood group.

In reverse typing:
Two drops of the individual’s serum are taken into three wells
or slides. Antigens A and B are added as per manufacturer’s
indications to the serum. Positive agglutination confirms presence
of corresponding antibody and hence confirmation of blood group.
ABO BLOOD GROUPING INTERPRETATION

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THE RHESUS BLOOD GROUPING SYSTEM

Rhesus blood grouping is based on the presence or absence of


the Rhesus factor – individuals are either Rh+ve or Rh-ve.
The Rhesus factor is present on the RBC surface at high
percentage in the human population i.e. 85% in Caucasians and
94% in blacks.
The rhesus factor is encoded in the Rhesus D gene locus where
two alleles are involved i.e. DD and dd alleles.
The alleles are inherited from both parents and the combination
determines status i.e DD and Dd will be Rh+, whereas dd will be
Rh-.
The Rhesus grouping is combined with the ABO blood grouping
system and individuals are classified A+, A- or B+, B- or AB+, AB-
or O+, O-
Rhesus factor must be taken into account for transfusion
purposes.

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THE COMBINED ABO-RHESUS BLOOD GROUPING SYSTEM

BLOOD GROUPING, BLOOD TRANSFUSION AND TISSUE


TRANSPLANTATION

It is therefore important to perform ABO typing on both donor


and recipient to confirm compatibility and ensure success of
blood transfusion and tissue transplantation.

Blood group incompatibility may lead to:


(a) Adverse transfusion reactions (acute, delayed, allergic).
(b) Hemolytic disease of the newborn also referred to as
erythroblastosis foetalis.
Blood incompatibility disorders are covered in details in lecture
4: “Hematological disorders.”

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ABO-RH SYSTEM AND BLOOD TRANSFUSION
COMPATIBILITY

II. BACTERIAL HAEMAGGLUTINATION

Detects presence of antibody to bacterial infection.


 Patient’s serum serially diluted in tubes and mixed with
bacteria.
 Last tube showing visible agglutination reflects the serum
antibody titre of the patient.
Application:
i.diagnosis of bacterial infections.
ii.Typing of bacteria.

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II A) QUANTITATIVE BACTERIAL AGGLUTINATION TEST

IIB) AGGLUTINATION INHIBITION

Definition – A variation of the agglutination test. It is based on


the inhibition of agglutination due to competition with a soluble
Ag

Basis early home pregnancy kits

Also used to determine exposure to illegal drugs e.g. cocaine

Also used in clinical laboratories to determine exposure

to viruses causing agglutination of RBC e.g. Rubella virus


In agglutination inhibition, no agglutination indicates a positive
result, because the agglutination reaction is inhibited by the test
sample

Agglutination indicates a negative result because it means the


test sample does not contain the appropriate competitive antigen.

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Concept of agglutination inhibition test

Agglutination inhibition enables detection of minimal amounts of Antigen –


more sensitive in comparison to direct agglutination reaction. This is why it is
used in early pregnancy detection.

APPLICATION OF AGGLUTINATION INHIBITION IN


PREGNANCY TESTING

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ENZYME-LINKED IMMUNOSORBENT ASSAYS

Enzyme-Linked Immunosorbent Assays (ELISA)


 Is dependent on antigen-antibody reaction but not on lattice
formation
 Can be used to detect both antigen and antibody in a test
sample. The detection system depends on reaction of an enzyme-
susbtrate reaction, whereby the enzyme is conjugated to a
secondary antibody.
 Different variations of ELISA allowing detection of either
antibody or antigen:
i) Indirect ELISA
ii) Sandwich ELISA
iii) Competitive ELISA
iv) Chemiluminescence
v) ELISPOT assay.

ELISA testing procedures

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Application of ELISA in HIV testing

LIMITATIONS OF ANTIBODY TESTING IN REFERENCE TO HIV


DIAGNOSIS

False positives:
Where ELISA test gives a positive result when the individual is
HIV-negative.
False positives occur in 1% - 0.1% of tests.
May be due memory B cells from a previous exposure to HIV
which the immune system encountered and cleared.
Less frequently due to cross-reacting antibodies.

False negatives:
Where ELISA test gives a negative result when the individual is
HIV-positive.
May occur in early infection when antibody response is not
established or when antibody titre is lower than the detection
threshold of the test. This is known as the window period.
May also occur if an individual is incapable of raising an antibody
response against virus. This is rare.

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NB:In HIV testing confirmatory tests should be
undertaken for both positive and negative results.
RADIOIMMUOASSAY (RIA)

• Principle involves competitive binding of a radio- labeled antigen and


unlabelled antigen to high affinity antibody.
• Excess of labeled antigen mixed with antibody + excess of unlabelled
antigen.
• Decrease in amount of labeled antigen is measured to determine amount
of antigen in test sample.

RADIOIMMUOASSAY (RIA)

Method
Determine amount of Ab needed to bind to a known amount of
labeled Antigen (Ag).
Use predetermined amounts of labeled Ag and Antibody (Ab)
and add a sample containing unlabeled Ag as a competitor.

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IMMUNOFLUORESCENCE

Direct immunoflourescence:
• Ab to tissue Ag is labeled with fluorochrome and immune
complex detected by colored emmision. A secondary antibody
that binds to the primary antibody (as in ELISA) may also be
conjugated.

Direct Indirect

Indirect immunoflourescence:
Ab to tissue Ag is unlabeled.
Fluorochrome-labeled anti-Ig is used to detect binding of the first
Ab.

Applications of Immunofluorescence
Flow cytometry
Also called flourescence

Activated Cell Sorting (FACS).


– Cells in suspension are labelled with fluorescent tag.
– Cells are sorted and analyzed on a flow cytometer.
–Cells are separated by charge, size and flourescent tags.

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FACS – APPLICATION IN CLINICAL RESEARCH

 Occupies key position in immunology and cell biology


 Important clinical tool e.g. for detection and classification of
leukemia
 Type and quantity of WBC in patients blood samples
 Determine number of cells (e.g. T cells) in total WBC population.
This is important in diagnosis infection and also blood cancers e.g.
leukaemia and lymphomas
 Analyzing cell distribution
 Analyzing Cell sizes
 Determination of cell surface markers e.g. CD14 on
Macrophages Further reading on FACS: See accompanying
article “overview of FACS_Herzenberg et al, 1976”

END OF LECTURE 3

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