Antigen antibody reactions and their application in diagnosis of infections

Antigen antibody reactions and their application in diagnosis of infections
By Uday
MSc. Clinical microbiology

1. Uday Antigen antibody reactions

2. The antigen-antibody reaction is a bimolecular association where the antigen and antibody combine with each other specifically and in an observable manner similar to an enzyme-substrate interaction, the only difference is, it does not lead to an irreversible alteration in either antibody or in antigen.

3. General properties of Ag-Ab reactions Specific Non-covalent bonding: Hydrogen bonds Electrostatic interactions Hydrophobic interactions • Van der Waals forces Strength: Affinity Avidity

4. Qualitative assays Quantitative assays

5. Stages: The interaction between an antigen and an antibody occurs in three stages: Primary Stage: The first stage involves the formation of the Ag-Ab complex. It is rapid and and reversible without any visible effects. Secondary Stage: It leads to observable phenomena such as agglutination or precipitation It is slow and irreversible with visible effects. Tertiary Stage: It involves the neutralization or destruction of the antigen.

6. Marrack’s hypothesis

7. Types of Ag-Ab reactions

8. Evaluation of the immune assays Sensitivity is defined as ability of a test to identify correctly all those who have the disease, i.e. true-positives Sensitivity = (True positive) / (True positives +False negatives) Specificity is defined as ability of a test to identify correctly all those who do not have disease, i.e. true negatives Specificity = (True negatives) / (True negative +False positives)

9. Types Precipitation reaction When a soluble antigen reacts with its antibody in the presence of optimal temperature, pH and electrolytes (NaCl) Flocculation reaction: When a drop of antigen is mixed with a drop of patient's serum, then the precipitate formed remains suspended as floccules. This test can be done on a slide or in a tube

10. Precipitation in gel (Immunodiffusion) Single diffusion in one dimension (Oudin procedure): When antigen solution is poured over a layer of gel containing antibody in a test tube, only the antigen diffuses in one direction towards antibody to form a band Double diffusions in one dimension (Oakley- Fulthorpe procedure): In the above test, if a column of plain agar is placed between the antigen layer and the layer of gel incorporated with antibody; then both antigen and antibody move towards each other in opposite directions and the precipitate band is formed at the line they meet in the plain agar (Fig. 12.2).

11. Single diffusion in one dimensions (Radial immunodiffusion): Double diffusions in one dimensions (Ouchterlony procedure): Examples of double diffusions in two dimensions include-Elek's test for detecting toxin of Corynebacterium diphtheriae and Eiken test to detect toxin of Escherichia Single diffusion in two direction (Radial Immunodiffusion) Double diffusions in two dimensions (Ouchterlony procedure)

12. Single diffusion in 2 dimensions Single and double diffusion in 1 dimension Double diffusion in 2 dimensions

13. Agglutination reaction When a particulate or insoluble antigen is mixed with its antibody in the presence of electrolytes at a suitable temperature and pH, the particles are clumped or agglutinated. Advantage: Agglutination is more sensitive than precipitation test and the clumps are better visualized and interpreted as compared to bands or floccules.

14. Slide agglutination (Eg.: Blood grouping, serotyping) Tube agglutination (Eg.: Widal test) Microscopic agglutination

15. Indirect or passive hemagglutination tests Coating the soluble antigen on the surface of acarrier molecule (e.g. RBC, latex or bentonite), so that the antibody binds to the coated antigen and agglutination takes place on the surface of the carrier molecule. Indirect Hemagglutination Test (IHA): It is a passive agglutination test where RBCs are used as carrier molecules Latex Agglutination Test (LAT) for Antibody Detection: Polystyrene latex particles (0.8-1 um in diameter) are used as carrier molecules which are capable of adsorbing several types of antigens Eg.: Anti-streptolysin O antibody (ASO) test, CRP, RA, etc.

16. Hemagglutination test It refers to the agglutination tests that uses RBCs as the source go Ag. Direct hemagglutination tests: Serum antibodies directly agglutinate with surface antigens of RBCs to produce a matt. Examples include: Eg.: Paul Bunnell test: It employs sheep RBCs as antigens to detect Epstein-Barr virus antibodies in serum Cold agglutination test: It uses human RBCs as antigens to detect Mycoplasma antibodies in serum. Test is performed in tubes Blood grouping (ABO and Rh grouping) Coombs test or antiglobulin test: to diagnose Rh incompatibility by detecting Rh antibody from mother's and baby's serum.

17. Viral Hemagglutination Test In strict sense, it is not an antigen-antibody reaction. The hemagglutinin antigens (HA) present on surface of some viruses (hemagglutinating viruses, e.g. influenza virus) can agglutinate with the receptors present on the surface of RBCs.

18. COMPLEMENT FIXATION TEST Complement fixation test (CFT) detects the antibodies in patient's serum that are capable of fixing with complements. It was once very popular, now is almost obsolete. Applications Wasserman test was the most popular CFT, used for the diagnosis of syphilis In addition, CFT was also widely used for detection of complement fixing antibodies in Rickettsia, Chlamydia, Brucella, Mycoplasma infections and some viral infections, such as arboviruses, rabies, etc. Complements are also used for various serological tests other than CFT, such as: Treponema pallidum immobilization test Sabin-feldman dye test for Toxoplasma Vibriocidal antibody test.

19. Principle of complement fixation test

20. Neutralization tests Viral neutralization test: • Detects the presence of neutralizing antibody in patient’s serum • Serum is mixed with a live viral suspension and poured onto a cell line, specific serum antibody neutralizes the surface antigen, making the virus unable to infect a cell line Plaque inhibition test: This is done for bacteriophages Toxin–antitoxin neutralization test: Schick test: It is a diphtheria toxin–antitoxin neutralization test Nagler’s reaction: It is used for detection of α-toxin of Clostridium perfringens ASO test: Antistreptolysin O antibody was detected before by neutralization method. Now replaced by latex agglutination Hemagglutination inhibition (HAI) test: Antibodies in patient’s sera can agglutinate with the hemagglutinin antigens present on the surfaces of some viruses. Eg.: influenza

21. ELISA ELISA is so named because of its two components: Enzyme is used to label one of the components of immunoassay (i.e. antigen or antibody) Immunosorbent: Here, an absorbing material is used (e.g. polystyrene, polyvinyl) that specifically absorbs the antigen or antibody present in serum A substrate-chromogen system is added at the final step of ELISA Eg. Horseradish Peroxidase ——- Hydrogen peroxide—— Tetramethyl benzidine (TMB) Urease ——- Urea ——-Bromocresol ß-Galactosidase———ONPG——ONPG

22. Types of ELISA   • Direct ELISA • Used for detection of antigen in test serum • Primary antibody (targeted against the serum antigen) is labeled with the enzyme • • Indirect ELISA • Used for detection of antibody or antigen in serum • Differs from the direct ELISA in that the secondary antibody is labeled with enzyme instead of primary antibody

23. Advantages of ELISA • Large number of samples can be tested together using the 96 well microtiter plate. • Economical, takes 2–3 hours for performing the assay •  ELISA has a high sensitivity Disadvantages of ELISA • less preferred  In small laboratories having less sample load • Takes more time (2–3 hours) compared to rapid tests which take 10–20 minutes • Needs expensive equipment such as ELISA washer and reader.

24. • Can be used both for antigen and antibody detection Ø Antigen detection: • Hepatitis B [hepatitis B surface antigen (HBsAg) and pre-core antigen (HBeAg)], • NS1 antigen for dengue Ø Antibody detection : hepatitis B, hepatitis C, HIV, D engue, EBV, HSV, toxoplasmosis, leishmaniasis

25. Enzyme linked fluorescent assay An modification of ELISA, differs from ELISA in two ways Automated system all steps are performed by the instrument itself Ag-Ab enzyme complex is detected by fluorometric method Advantages: an automated system easy to perform less contamination chance more sensitive and specific Disadvantages: Expensive Can run only 12–24 number of tests at a time

26. Applications: Infectious diseases: Markers of hepatitis viruses and HIV (Ag and Ab) Ab to TORCH infection Measles, mumps, varicella H. pylori Antigen to C. difficile Rotavirus Other uses: Biomarkers(e.g.procalcitonin) Hormones (e.g. thyroid) Tumor markers Cardiac markers Screening for allergy

27. Immunofluorescence assay A t echnique similar to ELISA, but differs by some important features: Fluorescent dye is used instead of enzyme for labeling of antibody Detects cell surface antigens It is also used to detect antibodies bound to cell surface antigens, unlike ELISA which detects free antigen or antibody

28. Chemiluminescence assay Chemiluminescence refers to the emission of light (luminescence), as a result of a chemical reaction Principle of CLIA is similar to that of ELISA - Chromogenic substance is replaced by chemiluminescent compounds (e.g. luminol and acridinium ester) that generate light during a chemical reaction (luxogenic) —light (photons) can be detected by a photomultiplier, also called as luminometer

29. • Detection of antigens or antibodies against various infections such as hepatitis viruses • HIV • TORCH infections • Biomarkers such as procalcitonin

30. RDT Simple to perform ,rapid (takes 10–20 minutes) Require minimal training do not need any sophisticated instruments. Also called Point ­of­ care (POC) tests Two principles of rapid tests are available—lateral flow assay and flow through assay Both the formats are available for the diagnosis of various diseases such as malaria, hepatitis B, hepatitis C, HIV, leptospirosis, Helicobacter pylori , syphilis