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ANTIGEN-ANTIBODY REACTIONS Im m unoaglutina tion Im m unoprecipita tion

ANTIGEN-ANTIBODY REACTIONS Im m unoaglutina tion Im m unoprecipita tion. Ag-A b rea ctions ( general features ). Reaction between antigen and specific antibodies detectable in vitro.

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ANTIGEN-ANTIBODY REACTIONS Im m unoaglutina tion Im m unoprecipita tion

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  1. ANTIGEN-ANTIBODY REACTIONS Immunoaglutination Immunoprecipitation

  2. Ag-Ab reactions (general features) Reaction between antigen and specific antibodies detectable in vitro The presence of antigen (using known antibody) or antibodies (using known antigen) in the sample (e.g. serum, cerebrospinal fluidcell culture…) is tested Ag - infectious agentor its part (e.g. HbsAg of hepatitis B virus) - other (e.g. cytokines,hormons, tumor-markers, Ig...) Ab - total Ab in the sample (e.g. total IgG in serum) - specific Abfor certain Ag (e.g. IgM for hepatitis A virus) Traditionally called serologic reactions

  3. Ag-Ab reactions (general features) Can be: Result: 1.Qualitative POSITIVE NEGATIVE 2.Quantitative Ag - CONCENTRATION(e.g. 2 mg/ml ili IU) Total Ab - CONCENTRATION(e.g. IgG 13,5 mg/ml) Specifc Ab - TITAR (npr. 1/64 ili 64) TITERis a measure of quantity of specific antibodies. It represents the highest dilution which gives the positive result. - positive Controls Test is invalid without controls - negative

  4. Primary and secondary humoral immune response Following pathogenencounter First pathogen encounter I more IgG little IgM I I IgG IgM IgA i IgE I IgM, IgG Serum Ab concentration Time (weeks)

  5. IMMUNOAGLUTINATION

  6. IMMUNOAGLUTINATIONis a reaction betweenparticulate (insoluble) antigen and specific antibodies Antigen is tipicallyon the surface of the cell (e.g. bacterial) or it is a solubile moleculboundfor inert insolubleparticle (carrier) Result of reaction isaglutinate, visible structurecreated by aggregation, i.e. aglutination of particles It is tipically done on a slide (e.g. microscopic) Maximalamount of aglutinateis observed when the concentrations of antigen and antibody are approximately equal

  7. EXCESS OF ANTIBODIES EXCESS OF ANTIGEN ZONE OF EQUIVALENCE Ag-AB COMPLEXES: AGLUTINATION: RESULT: SMALL ABSENT UNRELIABLE (FALSE NEGATIVE) PROZONE LARGE PRESENT RELIABLE (POSITIVE) SMALL ABSENT UNRELIABLE (FALSE NEGATIVE)

  8. Detection of Ab Application (Example) Identification of bacteria (enterobacteria) Detection of Ag DIRECT BAB test (Brucella sp.) IMMUNOAGLUTINATION INDIRECT (passive)

  9. Direct aglutination (identification of bacteria)

  10. Detection of Ab Application (Example) Identification of bacteria (enterobacteria) Detection of Ag DIRECT BAB test (Brucella sp.) IMMUNOAGLUTINATION Detection of Ab (Ag bound to a carrier) INDIRECT (passive) Detection of Ag (Ab bound to a carrier)

  11. Indirect aglutination Ab detection (Ag bound to a carrier particle) Ag detection (Ab bound to a carrier particle)

  12. Detection of Ab Application (Example) Identification of bacteria (enterobacteria) Detection of Ag DIRECT BAB test (Brucella sp.) IMMUNOAGLUTINATION Latex RF test (reumatoid factor) Detection of Ab (Ag bound to a carrier) INDIRECT (passive) Detection of Ag (Ab bound to a carrier) HBsAg test (hepatitis B virus)

  13. IMMUNOPRECIPITATION

  14. IMMUNOPRECIPITATIONis a reaction betweensoluble antigen and specific antibody Antigen is some product of a microbe (e.g. toxin) or plasma protein (immunoglobulins, complement componentsetc.) As a result of Ag-Ab reaction soluble Ag is precipitated by an antibody Maximalamount ofpreciptateis observed when the concentrations of antigen and antibody are approximately equal

  15. Immunoprecipitation can be performed in: in fluid fast diffusion (precipitate forms after minutes) in semisolid medium (gel) slow diffusion (precipitate forms after days) electrophoresis (precipitate forms after hours)

  16. Example RING test (qualitative) Antrax detection (history) IN FLUID Nephelometry (quantitative) IMMUNOPRECIPITATION IN SEMISOLID MEDIUM (GEL)

  17. Interfacial (interphase) RING test (principle)

  18. Example RING test (qualitative) Antrax detection (history) IN FLUID determination of plasma protein concentration (e.g. Ig) Nephelometry (quantitative) IMMUNOPRECIPITATION IN SEMISOLID MEDIUM (GEL)

  19. Nephelomety (principle)

  20. Example RING test (qualitative) Antrax detection (history) IN FLUID determination of plasma protein concentration (e.g. Ig) Nephelometry (quantitative) IMMUNOPRECIPITATION Double immunodiffusion (qualitative) Diphteric toxin detection (history) IN SEMISOLID MEDIUM (GEL) RID test (quantitative)

  21. Double immunodiffusion – Diphteric toxin detection (principle)

  22. Example RING test (qualitative) Antrax detection (history) IN FLUID determination of plasma protein concentration (e.g. Ig) Nephelometry (quantitative) IMMUNOPRECIPITATION Double immunodiffusion (qualitative) Diphteric toxin detection (history) IN SEMISOLID MEDIUM (GEL) determination of plasma protein concentration (e.g. Ig) RID test (quantitative)

  23. Radial immunodifusion – RID test (principle) Abin gel 2 Ag in wells: 3 1 RID IgG 4 5

  24. Quantification (principle) Standard (calibration) curve D2 (mm2) 3 2 1 4 5 Ag – concentration (g/l)

  25. Example RING test (qualitative) Antrax detection (history) IN FLUID determination of plasma protein concentration (e.g. Ig) Nephelometry (quantitative) IMMUNOPRECIPITATION Double immunodiffusion (qualitative) Diphteric toxin detection (history) IN SEMISOLID MEDIUM (GEL) determination of plasma protein concentration (e.g. Ig) RID test (quantitative)

  26. 1. Aglutination is a. nephelometry and radial immunodiffusion 2. Prozone is b. direct immunoaglutionation 3. For final identification ofEntrobacteriaceaefamilyis used c. false negative result in presense of excessive Ab concentration 4. Latex agglutination is d. liquid or gel 5. Solubilan antigen cannot be measured by e. qualitative technique 6. Immunoprecipitation can take place in f. in liquid 7. Ring test is an example of immunoprecipitation in g. direct immunoaglutination Nephelometry is h. quantitave technique 8. 9. Double immunodifusion is an example of i. indirect immunoaglutination 10. C3 complement component in blood can be measured by j. imunoprecipitation in gel 1._____ e 2._____ c 3._____ b 4._____ i 5._____ g 6.____ d 7.____ f 8.____ h 9.____ j 10.____ a

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