LECTURE 3 (1)
LECTURE 3 (1)
STRUCTURE OF ANTIBODY
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Nature of Ag/Ab Reactions
• Non-covalent Bonds
– Hydrogen bonds
– Electrostatic bonds
– Van der Waal forces
– Hydrophobic bonds
• Reversible
• Multiple Bonds
AFFINITY
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AVIDITY
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.
Cross Reactions
Monoclonal antibodies:
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
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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.
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IMMUNOLOGICAL ASSAYS
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A:PRECIPITATION TESTS
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1. The ring precipitin 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.
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.
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2.Radial immunodiffusion (mancini procedure)
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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
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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
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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.
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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.
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Q) Why do blood group A individuals have anti-B antibodies and not anti-A
antibodies?
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
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THE COMBINED ABO-RHESUS BLOOD GROUPING SYSTEM
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ABO-RH SYSTEM AND BLOOD TRANSFUSION
COMPATIBILITY
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II A) QUANTITATIVE BACTERIAL AGGLUTINATION TEST
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Concept of agglutination inhibition test
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ENZYME-LINKED IMMUNOSORBENT ASSAYS
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Application of ELISA in HIV testing
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)
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
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FACS – APPLICATION IN CLINICAL RESEARCH
END OF LECTURE 3
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