15.antigen - Antibody Reactions Part 2

ANTIGEN ANTIBODY REACTIONS Part -II
By Dr. Dinesh Jain

CLASSIFICATION
• Precipitation
• Agglutination
• Coagglutination
• Complement fixation test (CFT)
• Neutralization Test
• Opsonisation
• Radioimmunoassay(RIA)
• Enzyme Immunoassay(EIA)
Enzyme linked immunosorbent assay(ELISA)
• Chemiluminescence Immunoassay (CLIA)
• Immunochromatographic Tests
• Immunofluorescence
AGGLUTINATION
Agglutination
Particulate antigen + Antibody + Electrolytes - Suitable

temperature
Agglutination optimal - Ag + Ab - Equivalence
APPLICATIONS
Slide Agglutination
• Uniform suspension of particulate antigen and appropriate
antiserum
• Positive result - Clumping of particles and clearing of the
drop
• Typing of bacterial isolates
• Blood groups and typing
TUBE AGGLUTINATION
Standard quantitative method

• Particulate antigen and equal volume of serial dilution of
antiserum
• Agglutination titre
• Diagnosis - Typhoid, brucellosis, typhus fever
TUBE AGGLUTINATION
Widal Test: ‘H’ - flagellar antigen - large, loose, fluffy clumps

‘O’ - somatic antigen - tight compact deposits
Tube agglutination – Brucella - prozone
HETEROPHILE AGGLUTINATION
Weil–Felix - Typhus
Typhus, rickettsiae and Proteus
Streptococcus MG agglutination - Primary atypical pneumonia
Paul-Bunnel test – Infectious mononucleosis
AGGLUTINATION USING RBC
Paul-Bunnel Test: Infectious mononucleosis adsorbed by ox

RBC not by guinea pig
Cold Agglutination Test: Mycoplasma pneumoniae agglutinate
human O group at 4°C reversible at 3°C
ANTIGLOBULIN (COOMBS) TEST
• Coombs, Mourant and Race (1945) detection of anti-Rh antibodies

that do not agglutinate Rh-positive erythrocytes in saline
• Serum - Incomplete anti-Rh antibodies mixed with Rh-positive red
cells
• Antibody globulin coats the surface of erythrocytes (not agglutinated)
• Erythrocytes coated with antibody washed free of unattached protein
treated with rabbit antiserum against human gamma globulin
(Coomb’s serum), cells agglutinate
COOMBS TEST
Direct or Indirect
Direct: Sensitisation of erythrocytes in vivo
Example: Hemolytic disease of newborn
Indirect: Sensitisation of erythrocytes in vitro
PASSIVE AGGLUTINATION TEST
Attach antigen to surface of carrier particles

Carrier particles - Red cells, latex, bentonite
Passive hemagglutination - Rose-Waaler - Rheumatoid
arthritis - RA factor
Antigen-sheep erythrocytes sensitised with subagglutinating
dose of rabbit antisheep erythrocyte antibody (amboceptor)
PASSIVE AGGLUTINATION
• Latex agglutination test – polysterene latex

• Example: ASO, CRP, RA factor
• Co-agglutination – Protein A bearing Staphylococci coated
with any IgG antiserum will be agglutinate if mixed with
corresponding antiserum
COMPLEMENT FIXATION TEST
• The ability of antigen antibody complexes to fix complement is made use of
in CFT.
• Very sensitive test-detects as little as 0.04mg of antibody nitrogen and 0.1mg
of antigen.
• Involves two steps and five reagents-
-Antigen
-Antibody
-Complement (source-guinea pig serum)
-Sheep erythrocytes
-Amboceptor( rabbit antibody to sheep red cells)
• Each reagent has to be separately standardized.
• Classical example is Wasserman reaction( formerly routine method for
syphilis)
Steps
• Interpretation:
Negative CFT
Lysis of erythrocytes complement not fixed i.e. serum did
not have antibody
Positive CFT
No lysis complement fixed i.e. serum contained
antibody
NEUTRALIZATION TEST
• Definition : The ability of specific antibodies to reduce the
effect of toxin or infectivity of viruses.
• Most microbes use molecules in their envelope or cell
walls to bind and gain entry into the host cells.
• Antibodies may attach to the microbial epitopes and toxins
preventing microbes and toxins from binding to the host
cell.
• Neutralization test are broadly of two types-
1.Virus neutralization test
2.Toxin neutralization test
Virus Neutralization Test
• Definition: Neutralization of viruses by their specific antibodies is called
as virus neutralization test.
• When virus specific antibodies are injected into these systems replication
and growth of viruses is inhibited forming the basis of this test
• Examples-virus hemagglutination inhibition test used for diagnosis of
influenza, mumps and measles
• Neutralization may be quantified on
- chorioallantoic membrane of chick embryo
- By enumeration of plaque on monolayer tissue culture
Toxin Neutralization Test
• Definition: Biological action of toxin is neutralized on reacting with
specific neutralizing antibodies called a anti toxin
• Toxicity of endotoxin is not neutralized by endotoxins
• Toxin tests can be done in vivo or in vitro
IN VIVO: injecting toxin antitoxin mixture and estimating least
amount of antitoxin that prevent disease. E.g. Schick test for
diphtheria
IN VITRO: inhibition of demonstrable toxin effect. E.g.

Antistreptolysin O test for streptococcus pyogenes , Naegler’s
reaction for alpha toxin of Clostridium perfringens
OPSONISATION
• Name ‘Opsonin’ given by Wright (1903) to a heat labile substance present
in fresh normal sera, which facilitated phagocytosis
• Heat stable serum factor with similar activity called ‘bacteriotropin’
• Term Opsonin now used to refer both these factors.
• Opsonic index is used to study the progress of resistance during course of
disease
• Defined as the ratio of the phagocytic activity of the patient’s blood for a
given bacterium, to the phagocytic activity of blood from normal
individual.
• Measured by incubating fresh citrated blood with the bacterial suspension
at 37ºC for 15min and estimating average no. of phagocytosed bacteria per
polymorphonuclear leucocyte from stained blood films.
RADIOIMMUNOASSAY (RIA)
• First ligand assay method developed by Bernson and Rosalyn Yalow in 1959 to
detect and quantify human insulin using antihuman insulin antibodies
• In 1977, Yalow was awarded Nobel prize for the discovery
• Extremely sensitive method
• Can be used for quantization of any substance that can be labelled with
radioactive isotopes
• Principle: It is a competitive binding assay in which fixed amount of antibody and
radiolabelled antigen react in the presence of unlabeled antigen. The labelled
and unlabeled antigen competes for the limited binding site on the antibody. This
competition is determined by the level of unlabeled( test) antigen present in the
reacting system. After the reaction, antigen is separated into free and bound
fraction and their radioactive count measured. The concentration of test antigen
can be calculated from ratio of bound and total antigen labels using standard
dose response curve.
Steps in RIA
• Bound and free fractions subjected to a partition steps. These include:
A) Salting out of protein( using ammonium or sodium sulphate)
B) Protein denaturation/precipitation by solvent(methanol/ethanol/acetone)
C) Precipitation by polyethylene glycol (PEG)
• Measurement
Measurement method is radioactive counting, which depends on the type of
radiation emitted by label. A liquid scintillation counter is used for alpha and
beta emitter and solid crystal gamma counter for gamma emitters.
• Concentration of total Ag can be calculated from ratio of bound and total
labeled Ag using standard dose response curve.
Uses of RIA
• Quantization of hormones, drugs, tumour markers, IgE and

viral antigens
• Radioimmunoprecipitation –to measure antibodies against
HBsAg, coxiella burnetti
• Widely used in clinical chemistry, clinical toxicology and clinical
endocrinology
Contd…
• Advantages:
A) Precision and high sensitivity
B) Ease of isotope conjugation
C) Unlike enzymes, isotopes labeled are not likely to disrupt
Ag activity
• Disadvantages:
A) I 125 labeled reagent in common use have limited shelf life
B) Potential hazard
C) Expensive
IMMUNOCHROMATOGRAPHIC TESTS
• It is a one step ,qualitative immunochromatographic technique has wide
application in serodiagnosis due to its simplicity, economy and reliability.
• For example, routinely used for HBsAg detection.
• The test system is a small cassette containing a membrane impregnated
with anti-HBsAg antibody colloidal gold dye conjugate.
• The membrane is exposed at 3 windows on the cassette
– Test serum dropped into first window
– As the serum travels upstream by capillary action, a coloured band is produced at
2nd window(test site) if the serum contains HBsAg, due to formation of an HBsAg
antibody conjugate complex. Indicates positive reaction
– A coloured band should appear in 3rd window, which forms an inbuilt control, in
the absence of which the test is invalid.
• Test is nearly as sensitive and specific as EIA tests.
IMMUNOFLUORESCENCE
• Fluorescence is the property of absorbing light rays of one particular
wavelength and emitting rays with a different wavelength.
• Fluorescent dyes show up brightly under ultraviolet light as they convert
ultraviolet to visible light.
• This technique is generally used to locate presence of the specific Ag on
the cell membrane(receptors).
• Can also be used to detect Ags present inside the cells.
• Commonly used fluorescent dyes are fluorescein isothiocyanate which
emits yellow green light at 517nm, rhodamine B which emits deep red light
at 546nm, and phycoerythrin, an accessory light absorbing protein pigment
of photosynthetic algae which emits red light at the wavelength of 578nm.
• All these 3 dyes mentioned above stain the protein
Contd…
• Dyes for DNA are DAPI(emitting blue light at 456nm), and

propedium iodide which emits orange red light at 615nm.
• Fluorescence tests can be:

– Direct Immunofluorescence test
– Indirect Immunofluorescence test
– Immunohistochemical technique
– Flow cytometry
Direct Immunofluorescence test
• Can be used for identification of bacteria, viruses or other antigens

using the specific antiserum labelled with a fluorescent dye . For
example, routinely used as a sensitive method of diagnosing rabies.
• Disadvantage:
Separate fluorescent conjugates have to be prepared against each antigen
to be tested
Indirect Immunofluorescence test
• Here a single antiglobulin fluorescent conjugate can be

employed for detecting human antibodies to any antigen.
• An example is the fluorescent treponemal antibody test for

diagnosis of syphilis
Thank you

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ANTIGEN ANTIBODY REACTIONS Part -II

By Dr. Dinesh Jain

Particulate antigen + Antibody + Electrolytes - Suitable

Standard quantitative method

Widal Test: ‘H’ - flagellar antigen - large, loose, fluffy clumps

Paul-Bunnel Test: Infectious mononucleosis adsorbed by ox

• Coombs, Mourant and Race (1945) detection of anti-Rh antibodies

Attach antigen to surface of carrier particles

• Latex agglutination test – polysterene latex

IN VITRO: inhibition of demonstrable toxin effect. E.g.

• Quantization of hormones, drugs, tumour markers, IgE and

• Dyes for DNA are DAPI(emitting blue light at 456nm), and

• Fluorescence tests can be:

• Can be used for identification of bacteria, viruses or other antigens

• Here a single antiglobulin fluorescent conjugate can be

• An example is the fluorescent treponemal antibody test for

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