Antigen – antibody reactions

complement fixation, immunoflourescence, ELISA

1. Antigen – Antibody reactions Part II Dr.M.Malathi Final year PG

2. Time to recollect !!! Antigen? Antibody? Antigen antibody reactions?

3. Complement Fixation Test (CFT) What is a complement ? Antigen antibody complex – bound by complement – mediates bacterial clearance, viral neutralisation , lysis of RBC

4. Im the source of complement

5. Procedure Heat inactivation of the antiserum – to inactivate the complementary activity of serum The source of the complement is guinea pig serum . Complement – heat labile Freshly draw the serum

6. Ingredients CFT has two steps and five reagents Antigen Antibody Complement Sheep erythrocytes Amboceptor ( Rabbit antibody to sheep red cells)

7. Amboceptor ( Rabbit antibody to sheep red cells)

8. Wassermann reaction Formerly used for the serodiagnosis of syphilis Procedure Lysis of erythrocytes – Negative CFT No lysis of erythrocytes – Positive CFT For every test, appropriate controls should be added.

9. Other complement dependent tests: Immune adherence- T.pallidum , V.cholerae Immobilisation test – Treponema pallidum Cytolytic or cytocidal test- V.cholerae

10. Conglutination Some donot fix the guinea pig serum complement Use indirect CFT or conglutination test Use sheep RBC with bovine serum Bovine serum – conglutinin – beta globulin – antibody to complement

11. NEUTRALISATION TESTS Virus neutralisation tests: Neutralisation of viruses by their antibodies and are demonstrated in cell cultures, eggs and animals Neutralisation of bacteriophages by the plaque inhibition test

12. Toxin neutralisation tests Bacterial exotoxins - induce neutralising antibodies(antitoxins)  protection against diseases such as diphtheria and tetanus. In vivo tests : 1. Toxigenicity test-detection of toxin of C.diphtheriae 2. Schick test In vitro tests: Anti streptolysin O (ASO) test Virus neutralisation test Nagler reaction-rapid detection of C.welchii

13. OPSONISATION TEST Opsonisation - process by which a particulate antigen becomes more susceptible to phagocytosis . Opsonin – is then identified as a complement. Bacteriotrophin – heat stable serum factor with similar activity  Specific antibody

14. Opsonic index  ratio of the phagocytic activity of the patients blood for a given bacterium, to the normal individual. Measured by incubating fresh citrated blood with the bacterial suspension at 37deg C for 15 minutes Phagocytic Index: Estimating the average number of phagocytosed bacteria per PMN from stained blood films

15. Immunofluorescence Fluorescence ? Immuno fluorescence ? Types – Direct and indirect The commonly used fluorescent dyes are: Fluorescin isothiocyanate (Blue green fluorescence) Lissamine rhodamine (orange red fluorescence)

16. Direct immunofluorescence test

17. Uses 1. It is commonly employed for detection of bacteria, viruses or other antigens in blood, CSF, urine, faeces , tissues and other specimens 2. It is a sensitive method to diagnose rabies by detection of the rabies virus antigens in brain smears Disadvantage Separate specific fluorescent labelled antibody has to prepared against each antigen to be tested

18. Indirect immunofluorescence test

19. RADIOIMMUNOASSAY (RIA) Berson and Yallow (1959) first described RIA Radioimmunoassay is widely-used because of its great sensitivity RIA detect antigens upto picogram (10 −12 g) quantities

20. RADIOIMMUNOASSAY(RIA)

21. Uses of RIA RIA have applications in biology and medicine- 1. Quantitation of hormones, drugs, tumour markers, 2. IgE and viral antigens

22. Disadvantages Radiation hazards: Uses radiolabelled reagent Radioactive waste disposal

23. ELISA Micro-plate reader 96-well micro-plate Positive result ENZYME LINKED IMMUNOSORBENT ASSAY

24. Types of ELISA Direct ELISA Indirect ELISA Competitive ELISA Sandwich ELISA Capture ELISA Cylinder or cassette ELISA

25. Types of ELISA

26. Immunochromatographic Tests A colored band appears at the second window Control also can be recorded

27. Chemiluminescence immunoassay (CLIA) Chemiluminescence - Chemical reaction emitting energy in the form of light Chemilumiscence compounds - Luminol or acridinium esters . Signal can be amplified, measured, and the concentration of the analyte calculated The method is fully automated Uses - where the volume of work is large - drug sensitivity testing of M.tuberculosis

28. CLIA

29. Immunoelectroblot / Westernblot Immunoblot or westernblot techniques combine the sensitivity of enzyme immunoassay with much greater specificity. Western blotting – Proteins Northern blotting – RNA Southern blotting - DNA

30. Western blotting ( Immunoblotting )

31. Western Blot for detection of HIV antibody HIV-1 Western Blot Lane1: Positive Control Lane 2: Negative Control Sample A: Negative Sample B: Indeterminate Sample C: Positive

32. Immunoelectronmicroscopic tests Immunoelectronmicroscopy : viral particles are mixed with specific antisera and observed under the electron microscope which are seen as clumps. Use: study of some viruses such as hepatitis A and viruses causing diarrhoea

33. Flow cytometry An instrument capable of analyzing single cells as they pass through an orifice at high velocity. Measures the properties of light scattering by the cells. Detection by the emission of light from flourescently labelled mAb bound to the surface of the cell

34. ELISA ? CLIA ? EIA ? RIA ? CFT ?

35. What are the types of ELISA?

36. Enumerate the antigen antibody reactions?

37. Enumerate the antigen antibody reactions? Precipitation Agglutination Complement fixation test Neutralisation test Opsonisation Radioimmunoassay (RIA) Enzyme immunoassay (EIA)

38. Chemiluminescence immunoassay (CLIA) Immunoelectroblot / western blot Immuno chromatographic test Immuno electron microscopic tests Immunoflourescence

39. THANK YOU