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ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGEN TS

ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGEN TS. ANTIGEN -ANTI BODY REA CTIONS WITH LABELED ANTI BODY. M. M - marker. A b. Fc. Fab. E NZYME - L INKED I MMUNO S ORBENT A SSAY (ELISA , EIA )- marker EN ZY M E. I MUNO F LUORESCEN CE (IF) - marker FLUORESCENT DYE.

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ANTIGEN-ANTIBODY REACTIONS WITH LABELED REAGEN TS

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  1. ANTIGEN-ANTIBODY REACTIONSWITHLABELED REAGENTS

  2. ANTIGEN-ANTIBODY REACTIONS WITH LABELED ANTIBODY M M - marker Ab Fc Fab ENZYME-LINKEDIMMUNOSORBENTASSAY (ELISA, EIA)-marker ENZYME IMUNOFLUORESCENCE (IF) - marker FLUORESCENT DYE RADIOIMMUNOASSAY (RIA) - marker RADIOACTIVE ISOTOPE

  3. ELISA Used for detection of antigen in liquide samples (e.g. serum) or antibodies (tipicallyin serum) Marker - enzyme, primary Ab (specificfor antigen) or secondary Ab (anti-Ig antibody) labeled Based on a change of colour ofthe substrateby an enzyme Signal detected by the spectrophotometer (absorbance measured) Bothqualitative andquantitaitve Performed in the plates for microtitration (96 well plate) enabling simultaneous analysis of large number of samples

  4. ELISA Ab detection sample serum Ag –coated plate

  5. ELISA Ab detection 12 6 Wash Incubation

  6. E E E E E E ELISA Ab detection labelled secondary Ab

  7. E E E E E E ELISA Ab detection 12 6 Incubation

  8. E E E E E E E E ELISA Ab detection Wash

  9. E E E E 12 ELISA Ab detection 6 Colorimetric detection

  10. Ag ELISA (Ag detection in liquid sample - SANDWICH ELISA) sample (serum) unlabelled Ag-specific Ab

  11. ELISA (Ag detection in liquid sample- SANDWICH ELISA) 12 6 Wash Incubation

  12. E E E E E ELISA (Ag detection in liquid sample- SANDWICH ELISA) 12 labelled Ag-specific Ab 6 Wash Incubation

  13. E E ELISA (Ag detection in liquid sample- SANDWICH ELISA) 12 adding supstrate 6 Colorimetric detection

  14. ELISA (plate with positive samples)

  15. Immunofluorescence (IF) Used for detectionof antigen in solid samples (e.g. on/in cells/tissues) or antibodies (tipically in serum) Marker - fluorochrome, primary Ab (specific for antigen) or secundary Ab (anti-Ig antibody) labeled Based on emission of visible light (colour) after exposure to a light of a defined wave length (UV light) (fluorescein –light green, rodamin – red ...) Signal detected using fluorescent microscope or flow cytometer Qualitative, semi-quantitative or quantitative

  16. Detection of Agon/in cells/tissues DIRECT (DIF) (primary Ab labeled) IMMUNOFLUORESCENCE INDIRECT (IIF) (secondary Ab labeled)

  17. F F F F DIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample) Ag-specific labeled - Ab Fixed sample

  18. F F F F DIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample) 12 6 Washing Incubation

  19. F F DIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample) Excitation Fluorescence

  20. DIF Chlamydia trachomatis

  21. Detection of Agon/in cells/tissues DIRECT (DIF) (primary Ab labeled) IMMUNOFLUORESCENCE Detection of Agon/in cells/tissues INDIRECT (IIF) (secondary Ab labeled)

  22. INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample) Ag-specific unlabeled Ab Fixed sample

  23. INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample) 6 Washing Incubation

  24. F F F F F F F F INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample) Secondary labeled Ab Incubation

  25. F F F F F F F F INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample) 12 6 Incubation

  26. F F F F F F F F INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample) Washing

  27. F F F F INDIRECT IMMUNOFLUORESCENCE (Ag detection in solid sample) Excitation Fluorescence

  28. Detection of Agon/in cells/tissues DIRECT (DIF) (primary Ab labeled) IMMUNOFLUORESCENCE Detection of Agon/in cells/tissues INDIRECT (IIF) (secondary Ab labeled) Detection of Abs in serum

  29. INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum) Tested serum Fixed antigen

  30. INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum) 6 Washing Incubation

  31. F F F F F F F F INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum) Ig-specific labelled Ab Incubation

  32. F F F F F F F F INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum) 12 6 Incubation

  33. F F F F F F F F INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum) Ispiranje

  34. F F F F INDIRECT IMMUNOFLUORESCENCE (Ab detection in patient‘s serum) Excitation Fluorescence

  35. IIF ANCA (Anti-neutrophil cytoplasmic antibody) Neutrophils (pANCA) Perinuclear staining pattern Neutrophils (cANCA) Cytoplasmic staining pattern

  36. IIF (Anti-glomerular basement membrane antibody) Glomerulonephritis Goodpasture’s syndrome

  37. Ag detection DIRECT (DIF) (primary Ab fluorescently labeled) specificity IMUNOFLUORESCENCIJA Ag detection INDIRECT (IIF) (primary Ab fluorescently labeled) sensitivity Ab detection

  38. Radio-imunotest (RIA) Nobelova nagrada za fiziologiju ili medicinu 1977 Rosalyn Yalow "for the development of radioimmunoassays of peptide hormones" Very sensitive quantitative test Used for measuring low quantities of Ag in a solution (e.g.blood hormone levels) Marker is radioactive isotope (e.g.125I) Bound radioactivity is measured by a counter Difficulties: radiation exposure during work and waste diposal

  39. Rapid pregnancy test(demonstration)

  40. RAPID PREGNANCY TEST (principle) detection of beta HCG in urine of a pregnant women three antibodies used: 1. anti-HCG antibody – labeled with colloidal gold (free) 2. anti-HCG antibody – unlabeled (fixed on a membrane) 3. anti-Ig antibody – unlabeled (fixed on a membrane)

  41. RAPID PREGNANCY TEST (results) + sample (urine) positive – sample (urine) negative

  42. Z Z Z Z Z Z Z RAPID PREGNANCY TEST (positive result) sample (urine) positive sample (urine) unlabeled anti-HCG Ab anti-Ig Ab + HCG diffusion diffusion labelled anti-HCG Ab Z Z Z Z

  43. Z Z Z Z Z RAPID PREGNANCY TEST (negative result) negative sample (urine) sample (urine) unlabeled anti-HCG Ab anti-Ig At – diffusion diffusion labeled anti-HCG Ab Z Z Z Z

  44. 1. ELISA is used for detection of a. enzyme 2. Antibody in ELISA is labeled by b. colorimeter 3. ELISA is done c. antigen or antibody in liqid samples 4. Substrate degraded by the enzyme in ELISA is detected by d. indirect imunofluorescenceor ELISA is used 5. In ELISA, the amount of antigen or antibody is measured by e. degraded substrate colour intensity 6. In indirect immunofluorescence, by fluorescent dye is labelled f. radioactive isotope 7. For detection ofChlamydiain cervical swab is used g. 96 well - plate 8. For detection of auto-antibodies in serum h. RIA 9. RIA as a marker utilises i. direct imunofluorescence 10. For blood hormone level determination is used j. anti-imunoglobuline antibody c a g b e j i d f h

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