ANTIGEN & ANTIBODY REACTION (Ag-Ab reaction)
Antigen Antibody Reactions
Basis of serologic reactions:
of antigens & antibodies are highly specific
specificity helps in identifying one by using the other
results of many immunologic tests are expressed as a titer.
Titer: is the highest dilution of the specimen, that gives a positive reaction in the test.
NATURE OF ANTIGEN-ANTIBODY REACTIONS A. Lock and Key Concept The combining site of an antibody is located in the Fab portion of the molecule and is constructed from the hypervariable regions of the heavy and light chains. B. Non-covalent Bonds The bonds that hold the antigen to the antibody combining site are all non-covalent in nature. These include – Hydrogen bonds Electrostatic bonds Van der Waals forces Hydrophobic bonds
Multiple bonding between the antigen and the antibody ensures that the antigen will be bound tightly to the antibody.
C. Reversibility Since antigen-antibody reactions occur via non-covalent bonds, they are by their nature reversible.
Uses of serologic tests: 1. Diagnosis of infectious diseases When organisms cannot be cultured e.g. syphilis & hepatitis A,B, & C
When the organism is too dangerous to culture e.g. Rickettsial disease When culture techniques are not readily available e.g. HIV, EBV When the organism takes too long to grow e.g. Mycoplasma
2. Diagnosis of autoimmune disease: Antibodies against various normal body components are used e.g. antibody against DNA in systemic lupus erythematosus antibody against human IgG (rhematoid factor) in rheumatoid arthritis
3. Determination of blood type and HLA type Known antibodies are used to determine ABO and Rh blood types.
Known antibodies are used to determine class I class II HLA proteins prior to transplantation. 4. It is helpful in epidemiological studies.
General features of Ag-Ab reactions
Reaction is specific.
Entire molecules react
Only surface antigen participate in the Ag –Ab reaction Reaction is firm and reversible Ag & Ab can combine in varying proportions
Affinity: Is the intensity of attraction between antigen and antibody molecules Avidity: Is the binding strength after the formation of antigen- antibody complexes. Sensitivity: Ability of the test to detect even minute quantity of antigen or antibody. Specificity: Ability of the test to detect reactions between homologous antigen and antibody
Types of Antigen-Antibody reactions: Precipitation reaction:
Def: When a soluble Ag combines with its Ab in optimal proportions, in the presences of electrolytes, at a suitable temperature & pH then the Ag-Ab complexes form an insoluble precipitate.
Instead of sedimenting, the precipitation remains suspended as floccules the reaction is known as Flocculation.
Precipitation results when a large lattice is formed consisting of alternating Ag & Ab molecules – this is seen when both of them are in equivalent proportion- Zone of equivalence
If the number of Ab molecules are more when compared to Ag, then few valencies of antibodies are satisfied, resulting in little or no precipitation – Prozone phenomenon Similarly when Ag is more in number – Postzone phenomenon is seen.
Application of precipitation test: Precipitation tests are highly sensitive for detecting antigens. Precipitation test can be done in solution or in semisolid medium (agar). Precipitation tests in solution: Ring test- C- reactive protein (CRP) test Slide test- VDRL test (floccules are formed) for syphilis Tube test- Khan test previously done for syphilis
Precipitation in agar: This is done as single or double diffusion. It can also be done in the presence of an electric field. 1. Single Diffusion: Radial immunodiffusion: ►Antibody incorporated in agar
filled with different concentration of Ags
moves radially and where it meets Ab at optimal concentration a precipitation ring is formed.
Uses: To measure IgM, IgG, Complement components in patient’s serum
Double Diffusion: Double Immunodiffusion (Ouchterlony method): ► Both antigen and antibody are placed in adjacent wells made on agar-coated plates.
Both the antigen and antibody diffuse towards each other establishing a concentration gradient. At the zone of equivalence, a visible line of precipitation forms. This method indicates whether antigens are identical, related but not identical or not related.
Fuse : When antibodies in X and Y recognize the same antigen, the two precipitin lines will fuse completely.
When antibodies in X and Y partially recognize the same antigen, the two precipitin lines will form a spur. Cross : When antibodies in X and Y recognize two different antigens, the two precipitin lines will cross.
Immunoelectrophoresis: ►Immunoelectrophoresis is the method used for the separation of antigen on a gel with the help of electricity.
combines - separation by electrophoresis and identification by double immunodiffusion.
of an antigen mixture is done to separate components by an electric charge. are then cut into the agar gel parallel to the direction of the electric field, and antiserum is added to the trough.
agar gel is then incubated in a humid chamber during which time antigen and antibody diffuse towards each other.
formation of specific bands identifies individual antigen components.
is widely used in clinical laboratories to detect the presence or absence of proteins in serum.
Rocket Immunoelectrophoresis: The electrophoresis of an antigen is done in an agarose gel in which the antibody has been incorporated.
height of the rocket is proportional to the conc. of the antigen.
technique permits the quantitation of antigen levels as low as 0.2 mcg/ml. limitation of rocket electrophoresis is the need for the antigen to be negatively charged for electrophoretic movement within the agar.
Counterimmunoelectrophoresis (Countercurrent – immunoelectrophoresis, CIE or CIEP): ►It is one directional double electroimmunodiffusion.
test is based on movement of antigen and antibody in opposite direction.
on glass slide layered with agarose & wells are cut on the surface.
well is filled with antigen & other with antibody.
move to cathode, Ab move to anode
The antigen & antibody move toward each other resulting in a precipitation line at a point between them. It is more sensitive than standard immunodiffusion technique. Use: for detection of bacterial fungal polysaccharide from CSF, Hepatitis B Ag or Ab in the serum.
Uses of precipitation reaction:
Identification of bacteria e.g. detection of group specific polysaccharide substance Identification of antigenic component of bacteria in infected animal tissue e.g. Bacillus anthracis (Ascoli test) Standardization of toxin & antitoxin Demonstration of antibody in serum e.g. Kahn test- syphilis
Agglutination reaction: Def: When particulate antigens combine with their specific antibodies at optimal proportions, in the presence of electrolytes, at a suitable temperature & pH, the antigen – antibody complexes are clumped or agglutinated. Agglutination tests are highly sensitive for detection of antibodies. Tests can be performed on1. Slides 2. Tubes
Types of agglutination reactions :
1. 2. 3. 4.
Slide agglutination test Tube agglutination test The Antiglobulin (Coombs) test Heterophile agglutination test
Weil-Felix Reaction Paul- Bunnel test
Passive Agglutination test Latex Agglutination test Haemagglutination test Coagglutination test
Slide agglutination test: Widely used to detect unknown antigen. Uniform suspension of antigen is made in a drop of saline on glass slide and to it a drop of antiserum is added. Clumping occurs within seconds when agglutination is positive Uses: 1. It is a routine procedure to identify the bacterial strains isolated from the clinical specimens. 2. It is also used for blood grouping
Tube Agglutination test:
is the standard quantitative method for determination of antibodies.
is diluted serially by doubling dilution in test tubes.
equal volume of particulate antigen is added to all tubes
highest dilution of serum at which agglutination occurs is the antibody titer.
Uses: 1. Enteric fever- Widal test 2. Typhus fever – Weil- Felix reaction 3. Infectious mononucleosis – Paul-Bunnell test
Antiglobulin (Coombs) test: It is used for the detection of incomplete anti-Rh antibodies There are two types of Coombs test: i) Direct Coombs Test ii) Indirect Coombs test Sera containing incomplete anti-Rh antibodies are mixed with corresponding Rh- positive erythrocytes. The incomplete antiglobulin coats the erythrocytes but agglutination does not occur. When such coated erythrocytes are treated with antiglobulin or Coombs serum (rabbit antiserum against human gamma globulin) the cells are agglutinated.
Coombs test can be used to detect antibody on the surface of an infant's Rh+RBC or ► To detect Rh antibody in the mother's serum against the infant's Rh+cells. ► The direct Coombs test is used to detect the antibody already on the infant's RBCs. ► The anti-human γ-globulin would be added directly to the cells and they would agglutinate. ► The indirect Coombs test would be using mother's serum to detect antibody against the RhD+cells. ► The reaction would involve known RhD+cells+mother's serum+anti-human γ-globulin.
For detection of anti-Rh antibodies
For demonstration of any type of incomplete antibody e.g. brucellosis
Heterophile Agglutination test: Heterophile antibodies have a property to react with microorganisms or cells of unrelated species due to common antigenic sharing. 1. Weil- Felix reaction: Some Proteus (OX19, OX2, OXK) strains are agglutinated by sera rickettsial infections due to antigenic sharing. 2. Paul- Bunnel test: Sheep erthryocytes are agglutinated by sera of infectious mononucleosis
Passive Agglutination By attaching a soluble antigen to the surface of carrier particles, it is possible to convert the precipitation test into agglutination test, which is more convenient and more sensitive for the detection of antibodies. When instead of antigen, antibody is absorbed on the carrier particles for estimation of antigens- reversed passive agglutination.
Carrier particles used are RBCs, latex particles, bentonite. 1. Latex agglutination test: polysterene latex paricles are widely employed to absorb several types of antigens. Uses: For detection of Hepatitis B antigen, ASO –Anti streptolysin O CRP- C Reactive Protein. Used for bacterial typing (N. meningitidis)
Many viruses clump red blood cells from one species or another- Active hemagglutination
Erythrocytes can be sensitized with antigens for detection of antibodies- Passive hemagglutination. e.g. TPHA- syphilis
Hemagglutination inhibition: Clumping of RBC can be inhibited by antibody, specifically directed against the virus to measure the titer of antibody
human viruses (e.g. Arbo, influenza, measles) are able to agglutinate red cells because they possess hemagglutinins on their outer surfaces.
helps in detection of antibodies against these viruses
Abs against these viruses in the patients serum will block hemagglutinins & therefore inhibit hemagglutination
Coagglutination : It is based on the presence of protein A on the surface of some strains of Staph. aureus (especially Cowan I strain). Specific IgG is coated on these Cowan I strains of
Staph. aureus .
Fc portion binds to the protein A, Fab terminal is free.
When corresponding antigen is mixed with these coated cells, Fab terminal binds to antigen resulting in agglutination.
Uses: To detect bacterial antigens in blood, urine & CSF.
N. gonorrhoeae, Strept. pyogenes, & H. influenzae
antigens can be detected by this method.
Uses of Agglutination Reaction:
Identification of bacteria e.g. serotyping of Salmonella & Shigella with known antisera Serological diagnosis of infection e.g. Widal test for typhoid
Complement fixation test: Principle: Ag-Ab complex fixes the complement. It does not have any visible effect. It is therefore necessary to use indicator system consisting of RBC coated with antisheep red cell antibody (amboceptor). Complement lyses antibody coated red cells.
The test is performed in two stepsFirst step: To activated patient’s serum add the antigen and complement. This mixture is incubated for 1 hour at 37°C.
Second step: After incubation the indicator system is added and again incubated for 1 hour at 37°C and observed .
Complement + test serum (contains antibody) + complement
+ Haemolytic system
Result – No haemolysis Positive test
Complement + test serum (contains NO antibody) + complement + Haemolytic system
Complement NOT fixed
Result –haemolysis Negative test
If the test is positive – hemolysis is not seen.
Uses: 1. Wasserman test – syphilis 2. Other complement dependent serological tests are – i. Immobilisation test- syphilis ii. Immune adherence. 3. Rickettsial disease – Typhus fever 4. Parasitic disease – Kalaazar Hydatid cyst Amoebiasis 5. Viral disease- Lymphogranuloma venerum
Neutralization test: to detect presence of Ab by their ability to neutralize the biological effects of microbes or their products. Neutralization of toxins Infectivity of viruses or enzymes. They can be used in cell cultures.
Radioimmunoassay (RIA): This test can detect antigens upto picogram(10-12 g) quantity It is based on competition for a fixed amounts of specific antibody between a known radiolabeled antigen and unknown unlabeled (test) antigen.
The complexes formed between antigen and antibody can be separated and the amount of radioactivity measured. It is highly sensitive method Uses: To assay hormones or drugs in serum. Detection of HBsAg & anti-HAV IgM.
The radioallergosorbent test (RAST) is specialized RIA used to measure the amount of IgE antibody which reacts with known allergen.
Enzyme linked Immunosorbent Assay (ELISA): This method is used for quantitation of either antigens or antibodies in patients specimens.
Here the enzyme is linked covalently to a known antigen or antibody.
Reacting the enzyme linked material with the patient’s serum & then assaying for enzyme activity by adding the substrate of the enzyme to give a color end point.
It is as sensitive as RIA. The enzymes used - Horse radish peroxidase Alkaline phosphatase
Specific substrate: O-phenyl-diaminase dihydrochloride - peroxidase. P-nitrophenylphosphate - alkaline phosphatase Types: Direct ELISA Sandwich ELISA
Detection of HIV antibodies in serum Detection of Mycobacterial antibodies in tuberculosis Detection of Rotavirus in faeces Detection of Hepatitis B markers in serum Detection of enterotoxin of Eschericihia coli in faeces
Immunofluorescence (Fluorescent antibody): Tests which detect either Ag or Ab using fluorescent dye labeled Ab or Ag. Principle:
Fluorescence is the property of certain dyes which absorb light in the UV region (200-400nm) & emit characteristic wave length of light (500-600nm) in the visible region.
Fluorescent dyes e.g. Fluorescein isothiocyanate- blue green Lissamine rhodamine – orange red
The dyes can be covalently attached to antibody molecules and made visible by UV light in the fluorescence microscope
Such labeled antibody can be used to identify antigens On the surface of bacteria e.g. Streptococci & Treponemes. In cells in histologic sections & in other specimens. Types: Direct Indirect
Direct IF: Ag is detected in clinical specimen with known flurochrome labeled Ab.
Blood film or smear from clinical specimen is fixed on a slide & overlaid with labeled Ab. If Ag is present, labeled Ab will bind them. Washing the smear will remove unbound labelled Ab. The smear is observed under fluorescent microscope.
Uses : Detection of various viral Ag in clinical sample. e.g. Rabies Ag in corneal impression smear. Detection of bacterial Ag. e.g. T. pallidum in serous exudates Chlamydial Ag in urethral exudates. Adv. Rapid diagnosis Acute disease diagnosis. Dis adv: Separate specific fluorescent labelled antibody has to be prepared against each antigen to be tested.
Indirect IF: This is a double layer technique. Principle: The Ab against various infectious agent present in clinical samples are first made to react with known Ag. The Ag- Ab complexes are detected by using a fluorescent dye labeled antihuman globulin Indirectly demonstrating the presence of Ab in the patient’s serum. Uses: To detect treponemal Abs in syphilis.
1. 2. 3. 4.
Rapid serological diagnosis of number of bacteria Detection of antitoxoplasma antibody Detection of leptospira in human & animal muscle. Detection of viruses in cells.
Ab detects individual proteins (Ag) in complex mixture. It is used to determine whether a positive result in a screening immunologic test is true positive or a false positive. e.g. Patients who are positive in the screening ELISA for HIV infection should have a Western blot test performed.
procedure consists of separating viral antigens by SDS (sodium dodecyl sulfate)- polyacrylamide gel electrophoresis and blotting them onto nitrocellulosepaper.
serum with immunoglobulins is added. If specific HlV antibodies are present, they will bind. bound IgG antibodies are detected by adding enzyme-coupled anti-human Ig. substrate stains the paper when converted to a product.
are detected to numerous antigens, but the significant antigens are gp120, gp41, and p24. positive test results, when the patient has antibodies to of the above listed antigens.
no response is observed, then the result is negative.
any activity to any antigen is observed that is not consistent with a positive result, the result is reported to be indeterminate.
Fluorescent – Activated Cell Sorting Flow Cytometric Analysis
procedure allows for rapid analysis of cell types present in a blood sample.
using specific, fluorescent-labeled antibody to different receptors on cells. is possible to rapidly identify exact cell contents and types by passing through laser light beam.
The number of cells that fluoresce is counted by use of a machine called fluorescence-activated cell sorter (FACS) e.g. It is used in HIV – infected patients to determine the number of CD4+ T cells.
1. Antibody titer refers to the:
A. Absolute amount of specific antibody. B. Affinity of specific antibody. C. Avidity of specific antibody. D. Concentration of specific antibody.
E. Highest dilution of antibody still able to give a positive result in a test system.
2. The affinity of an antibody can be determined by measuring: A. Its concentration.
B. The valency of antigen binding. C. The amount of antibody bound at various antigen concentrations.
D. Its ability to neutralize bacterial toxins.
E. The sedimentation coefficient of the antibody.
3. Latex particles are often used in:
A. Agglutination tests. B. Affinity chromatography. C. Affinity measurements. D. Adjuvants.
E. Neutralization assays.
4. Characterization of antigens by electrophoresis and immunofixation relies on the reaction of antigen and antibody in (or on):
A Agar. B Streptavidin. C Gold-plated sensor chip.
D Latex particles.
E Plastic microtiter plates.
5. Western blots are primarily used to detect:
A. Protein. B. Carbohydrate. C. Lipid. D. RNA.
6. The Radioallergosorbent test (RAST) measures:
A. Antigen concentration. B. IgE antibodies. C. IgM antibodies. D. Agglutination.
E. IgG antibodies.
7. Direct Coomb's test was performed on a baby in its 7th month (30 weeks). The mother has had trouble with two early pregnancies and she has had no RhoGAM. The physician is concerned about a possible erythroblastosis case. What ingredients would be involved in a procedure to prove a positive Direct Coomb's test? A. Rh+ RBCs + mother's serum + Coombs' reagent B. Rh+ RBCs from the baby + Coombs' reagent C. Mother's serum + Rh- RBCs + Coombs' reagent
D. Mother's serum + RhoGAM + Coombs' reagent
E. RhoGAM + Rh+ RBCs from the baby
References : Levinson – 7th edition- chapter 64