1. IMMUNOTECHNIQUES D.HAMSA MPhil BIOCHEMISTRY

2. CONTENTS Primary interaction Secondary interaction Radio immuno assay ELISA Western blotting

3. ANTIGEN ANTIBODY INTERACTIONS Antigen – antibody (ag-ab) Similar to enzyme substrate interaction - resembles the lock and key mechanism No irreversible reaction Non covalent type of interaction Detect the presence of either antigen or antibody Identifying molecules of biological or medical sensitivity Basis of Humoral or antibody mediated response

4. TYPES Primary interaction Secondary interaction

5. PRIMARY INTERACTION 1.Immune complex 2.Specificity of Ag-Ab reaction 3.Binding sites of antigen and antibody 4.Binding forces of antigen and antibody 5.Avidity 6.Affinity 7.Bonus effect 8.Cross-reaction

6. IMMUNE COMPLEX Antigen and antibody brought together and bind to form a complex

7. Specificity of Ag-Ab reaction Compared to lock and key concept

8. Binding sites of antigen and antibody Antigen combines with antibody – epitope or antigenic determinant Antibody combines with antigen – paratope or antigen binding sites

9. Binding forces of antigen and antibody Factors:  Closeness between antigen and antibody  Intermolecular forces  Affinity of antibody Forces: 1.Electrostatic forces 2.Hydrogen bonding 3.Hydrophobic bonding 4.Vander walls bonding

10. Avidity Capacity of an antiserum containing various antibodies to combine with whole antigen that stimulated the production of antibodies nAb+mAg Abn Agn n Ab = number of antibodies m Ag = Antigenic determinants

12. Affinity The strength of the reaction between a single antigenic determinant site of antigen and a single combining site on the antibody Higher affinity more stable

13. Bonus effect Giving extra strength to the antigen - antibody complex by the binding of two antibodies to two antigen molecules. Highly possible – antigens are multivalent and there are many types of antibodies

14. Cross reaction Antiserum raised against a given antigen by sometimes re act with another closely related antigen. Presence of one or more identical antigen

15. SECONDARY INTERACTION Precipitation Agglutination Radio immuno assay ELIZA Western blotting

16. Precipitation Precipitin curve 1.Zone of antibody excess 2.Zone of equivalence 3.Zone of antigen excess

17. Types Precipitation in solution 1.Ring test 2.Slide test Precipitation in gels 1.Immunodiffusion 2.Immunoelectrophoresis

18. PRECIPITATION IN GELS Precipitation occurs in solution Antigen and antibody are allowed to meet each other in a gel like agar or agarose – precipitation line 1.Single diffusion in one dimension 2.Single diffusion in two dimensions 3.Double diffusion in one dimensions 4.Double diffusion in two dimensions

19. SINGLE DIFFUSION IN ONE DIMENSION(OUDIN TECHNIQUE) In 1952, oudin PROCEDURE Molten agar with antibody antigen overlaid on agar gel precipitation ring (equivalence)

20. Applications Find unknown antigen Find approximate concentration Antigenic components present in the antigen solution against polyclonal antiserum – precipitation lines indicates the number of antigenic components present in the antigen solution

21. SINGLE DIFFUSION IN TWO DIMENSIONS (MANCINI TECHNIQUE) In 1965 – mancini and his co-workers Also called radial immunodiffusion (RIA) or single radial immunodiffusion (SRID) PROCEDURE: Here antigen allowed to diffuse in the gel that has incorporated by antibody Antigen can diffuse in two dimensions

22. APPLICATIONS Hospitals and laboratories to measure the concentration of IgG,IgM and IgA (agammaglobulin aemia)

23. DOUBLE DIFFUSION IN ONE DIMENSION(OAKLEY – FULTHORPE) Similar to oudin tube method PROCEDURE: Antigen and antibody diffuse into agar Meet each other at zone of equivalence Application same as oudin technique

24. DOUBLE DIFFUSION IN TWO DIMENSIONS(ouchterlony) Widely used immunological technique PROCEDURE: Small wells are punched in the gel Antigen and antibody allowed to diffuse towards each other Zone of equivalence

25. APPLICATIONS Identity of two given antigens against the polyclonal antiserum Find titre of the antiserum

26. IMMUNOELECTROPHORESIS In 1953, Graber and Williams – diagnosis of multiple myeloma Here gel normally takes place overnight to observe the result Fast movement of antigen and antibody by the influence of current TYPES: 1.Counter immunoelectrophoresis(CIEP) 2.Rocket electrophoresis 3.Two – dimensional immunoelectrophoresis

27. COUNTER IMMUNOELECTROPHORESIS Otherwise crossover immunoelectrophoresis PROCEDURE Ag in one well and ab in other well(pH> 8.0) Move towards each other by influence of current Ag (- charge) move towards anode Ab (-charge) move towards cathode slow mobility) –electroendosmosis Ag and ab meet and form precipitin line

28. ROCKET ELECTROPHORESIS (laurell technique) Single dimensional immunoelectrophoresis PROCEDURE: Antiserum with agar in large plate Wells filled with standard solutions of antigen or unknown solution Electric current across agar drives the antigen precipitates with ab - equivalent zone Height of rocket = conc of ag, unknown referred by standard

30. TWO – DIMENSIONAL IMMUNOELECTROPHORESIS Combination of electrophoretic separation of ag mixture in first dimension and running a rocket electrophoresis in second dimension second dimension First dimension

31. AGGLUTINATION REACTIONS Interaction between antibody and a particulate ag results in visible clumping Red blood cells are agglutinated – Heamagglutination Bacterial cells are agglutinated – Bacterial agglutination

32. AGGLUTINATION TEST Examination of clump formation when particulate antigen and its antibodies are combined Wide application in clinical field To test blood groups Infections diseases

33. APPLICATIONS ABO blood grouping RH blood grouping Widal test for typhoid Well felix test for typhus Coombs test for identification of anti-Rh ab Brucella agglutination test for leptospirosis Cold agglutination test for pneumonia,malaria,trypanosomiasis Haemagglutination inhibition test for diagnosis of viral and parasitic diseases

35. RADIO IMMUNO ASSAY(RIA) Sensitive - detecting the antigen or antibody, which is radioactively labeled Quantitation of hormones such as thyroxin and insulin Principle – competitive binding of radio labeled antigen and unlabeled antigen to high affinity

36. PROCEDURE Ag in saline is incubated on tube and small quantities become absorbed on the surface Free ag if washed away Test ag is added, which binds to ag Unbound ag are washed away Ab is detected by a radiolabellad ligand Radioactivity of the plate is counted on a gamma counter

37. RADIO IMMUNOSORBANT TEST (RIST) The ab content of the patient serum can be assessed by ability of ab to bind antiglobin, which has been immobilized on a solid surface by adsorption Solid surface – polycarbonate tube or nitrocellulose paper discs Usually done for detection of IgE ab in allergic patients

38. RADIO ALLERGOSORBANT (RAST) Measure the amount if IgE to specific allergen in patient serum Specific allergen is adsorbed on to solid surface and treated with patients serum The mixture is incubated and excess of serum is washed with saline Specific allergen is immobilized, bound IgE is detected by added radio labeled anti – IgE as RIST

39. ENZYME LINKED IMMUNOSORBENT ASSAY(ELIZA OR EIA) Similar to RIA but depends on an enzyme rather than radioactive label An enzyme conjugated to an ab reacts with a colorless substrate to generate a colored reaction product Enzyme – alkaline phosphatase,horse radish peroxidase and P- nitrophenyl phosphatase Less cost, number of variations of ELIZA based on the detection and quantitation of either ag or ab

41. Types of ELISA Indirect ELISA sandwich ELISA Competitive ELISA

42. Ab can be detected or quantitated –indirect ELIZA Ag can be detected or quantitated – sandwich ELIZA Variation of quantifying ag – competitive ELIZA

43. WESTERN BLOTTING Identification of specific protein in a complex mixture Here protein mixture is electrophoretically separated n PAGE in the presence of SDS Protein bands are transferred to nitrocellulose membrane by electrophoresis Individualprotein bands are identified by flooding the nitrocellulose membrane with radiolabelled polyclonal or monoclonalab specific for protein interest

44. Cont… Ag – ab complex are visualized by autoradiography If labeled specific antibody is not available, ag – ab complexes can be detected by adding a secondary antiisotype ab that is either radio labeled or enzyme labeled. It can also identify a specific ad in a mixture