Excellnet serological tests in identification of infectious agents

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  • Antigen = p24 One of first proteins responded to
  • If only 3 bands, do PCR
  • Excellnet serological tests in identification of infectious agents

    1. 1. Serological tests involved in identification of infectious agents Prof M.I.N. Matee
    2. 6. Precipitation Tests Lattice Formation
    3. 7. PRECIPITATION
    4. 9. Precipitation reactions in fluids (p. 142) Precipitation reaction can be seen
    5. 10. Radial Immunodiffusion (Mancini) <ul><li>Interpretation </li></ul><ul><ul><li>Diameter of ring is proportional to the concentration </li></ul></ul><ul><li>Quantitative </li></ul><ul><ul><li>Ig levels </li></ul></ul><ul><li>Method </li></ul><ul><ul><li>Ab in gel </li></ul></ul><ul><ul><li>Ag in a well </li></ul></ul>Ag Concentration Diameter 2 Ag Ag Ag Ag Ab in gel
    6. 11. I mmunoelectrophoresis <ul><li>Method </li></ul><ul><ul><li>Ags are separated by electrophoresis </li></ul></ul><ul><li>Interpretation </li></ul><ul><ul><li>Precipitin arc represent individual antigens </li></ul></ul><ul><ul><li>Ab is placed in trough cut in the agar </li></ul></ul>Ag - + Ag Ab Ag Ab
    7. 12. Countercurrent electrophoresis <ul><li>Method </li></ul><ul><ul><li>Ag and Ab migrate toward each other by electrophoresis </li></ul></ul><ul><ul><li>Used only when Ag and Ab have opposite charges </li></ul></ul><ul><li>Qualitative </li></ul><ul><ul><li>Rapid </li></ul></ul>Ag Ab - +
    8. 13. I mmunoelectrophoresis <ul><li>Method </li></ul><ul><li>Interpretation </li></ul><ul><li>Qualitative </li></ul><ul><ul><li>Relative concentration </li></ul></ul>
    9. 14. Agglutination Tests Lattice Formation
    10. 15. Agglutination/Hemagglutination <ul><li>Definition - tests that have as their endpoint the agglutination of a particulate antigen </li></ul><ul><ul><li>Agglutinin/hemagglutinin </li></ul></ul>Y Y Y +  <ul><li>Qualitative agglutination test </li></ul><ul><ul><li>Ag or Ab </li></ul></ul>
    11. 16. Agglutination/Hemagglutination <ul><li>Quantitative agglutination test </li></ul><ul><ul><li>Titer </li></ul></ul><ul><ul><li>Prozone </li></ul></ul>1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 1/1024 Pos. Neg. Titer 64 8 512 <2 32 128 32 4 Patient 1 2 3 4 5 6 7 8
    12. 17. Agglutination/Hemagglutination <ul><li>Definition </li></ul><ul><li>Qualitative test </li></ul><ul><li>Quantitative test </li></ul><ul><li>Applications </li></ul><ul><ul><li>Blood typing </li></ul></ul><ul><ul><li>Bacterial infections </li></ul></ul><ul><ul><ul><li>Fourfold rise in titer </li></ul></ul></ul><ul><li>Practical considerations </li></ul><ul><ul><li>Easy </li></ul></ul><ul><ul><li>Semi-quantitative </li></ul></ul>1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512
    13. 18. Passive Agglutination/Hemagglutination <ul><li>Definition - agglutination test done with a soluble antigen coated onto a particle </li></ul><ul><li>Applications </li></ul><ul><ul><li>Measurement of antibodies to soluble antigens </li></ul></ul>Y Y Y + 
    14. 19. Hemagglutination Used to identify blood group antigens or antibodies to them Variations: Chemically couple haptens to RBC Use other particles - bacteria, antigen-coated latex beads
    15. 20. Agglutination Inhibition Assay for HCG
    16. 21. Coombs (Antiglobulin)Tests <ul><li>Incomplete Ab </li></ul><ul><li>Direct Coombs Test </li></ul><ul><ul><li>Detects antibodies on erythrocytes </li></ul></ul>+  Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Patient’s RBCs Coombs Reagent (Antiglobulin)
    17. 22. Coombs (Antiglobulin)Tests <ul><li>Indirect Coombs Test </li></ul><ul><ul><li>Detects anti-erythrocyte antibodies in serum </li></ul></ul>Y Y Y Y Y Patient’s Serum Target RBCs +  Step 1 +  Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Coombs Reagent (Antiglobulin) Step 2
    18. 23. Coombs (Antiglobulin)Tests <ul><li>Applications </li></ul><ul><ul><li>Detection of anti-Rh Ab </li></ul></ul><ul><ul><li>Autoimmune hemolytic anemia </li></ul></ul>
    19. 24. Agglutination/Hemagglutination Inhibition <ul><li>Definition - test based on the inhibition of agglutination due to competition with a soluble Ag </li></ul>Y Y Y +  Prior to Test Y +  Y Y + Test Patient’s sample
    20. 25. Agglutination/Hemagglutination Inhibition <ul><li>Applications </li></ul><ul><ul><li>Measurement of soluble Ag </li></ul></ul><ul><li>Practical considerations </li></ul><ul><ul><li>Same as agglutination test </li></ul></ul><ul><li>Definition </li></ul>
    21. 27. Immunofluorescence Antibodies can be labeled with fluorescent dye Can localize binding sites on cells Dyes: Fluorescein, rhodamine, phycoerythrin can be conjugated to Fc region of Ab (so antigen binding is unaffected) Absorb at one wavelength and emit at another
    22. 28. Immunofluorescence <ul><li>Direct </li></ul><ul><ul><li>Ab to tissue Ag is labeled with fluorochrome </li></ul></ul>Ag Y Fluorochrome Labeled Ab Tissue Section
    23. 29. Immunofluorescence <ul><li>Indirect </li></ul><ul><ul><li>Ab to tissue Ag is unlabeled </li></ul></ul><ul><ul><li>Fluorochrome-labeled anti-Ig is used to detect binding of the first Ab. </li></ul></ul><ul><li>Qualitative to Semi-Quantitative </li></ul>Ag Y Y Fluorochrome Labeled Anti-Ig Tissue Section Unlabeled Ab
    24. 30. ELISA- enzyme-linked immunosorbent assay Based on RIA Nearly as sensitive Cheaper and safer Many detection systems have been developed Many variations of the assay have been developed
    25. 31. All ELISAs use an antibody conjugated with an enzyme that turns a colorless substrate into a colored product Direct- detects antigens using a single labeled antibody against that antigen Relatively few applications and permutations
    26. 32. ELI SA Enzyme- L inked I mmunosorbent A ssay (ELISA) © 1998 Gold Standard Multimedia Inc. 1. Antigen HIV Protein 2. Test antibody Human serum 3. Developing Ab Enzyme-Goat anti-Human IgG 4. Substrate Colorless > Blue
    27. 33. E nzyme- L inked I mmunosorbent A ssay (ELISA) HIV Ag Human Serum AP-Goat Anti- Human IgG Colorless > Yellow AP = alkaline phosphatase
    28. 36. Western Blot for HIV Antibodies + - Patient sera
    29. 37. Complement Fixation <ul><ul><li>Ag mixed with test serum to be assayed for Ab </li></ul></ul><ul><ul><li>Standard amount of complement is added </li></ul></ul><ul><ul><li>Erythrocytes coated with Abs is added </li></ul></ul><ul><ul><li>Amount of erythrocyte lysis is determined </li></ul></ul>Ag Ag <ul><li>Methodology </li></ul>Y Y Patient’s serum Y Y Y Y Y Y Y Y Ag No Ag
    30. 38. Tests for Cell Associated Antigens Lattice formation not required
    31. 39. Immunofluorescence <ul><li>Direct </li></ul><ul><ul><li>Ab to tissue Ag is labeled with fluorochrome </li></ul></ul>Ag Y Fluorochrome Labeled Ab Tissue Section
    32. 40. Immunofluorescence <ul><li>Indirect </li></ul><ul><ul><li>Ab to tissue Ag is unlabeled </li></ul></ul><ul><ul><li>Fluorochrome-labeled anti-Ig is used to detect binding of the first Ab. </li></ul></ul><ul><li>Qualitative to Semi-Quantitative </li></ul>Ag Y Y Fluorochrome Labeled Anti-Ig Tissue Section Unlabeled Ab
    33. 41. Radioimmuoassays (RIA) Enzyme-Linked Immunosorbent Assays (ELISA) Lattice formation not required
    34. 42. Competitive RIA/ELISA for Ag <ul><li>Method </li></ul><ul><ul><li>Determine amount of Ab needed to bind to a known amount of labeled Ag </li></ul></ul><ul><ul><li>Use predetermined amounts of labeled Ag and Ab and add a sample containing unlabeled Ag as a competitor </li></ul></ul>Y Y +  Prior to Test Labeled Ag Y Y +  Test + Patient’s sample Labeled Ag +
    35. 43. Competitive RIA/ELISA for Ag <ul><li>Method cont. </li></ul><ul><ul><li>Determine amount of labeled Ag bound to Ab </li></ul></ul><ul><ul><ul><li> NH 4 SO 4 </li></ul></ul></ul><ul><ul><ul><li> anti-Ig </li></ul></ul></ul><ul><ul><ul><li>Immobilize the Ab </li></ul></ul></ul><ul><li>Quantitative </li></ul><ul><ul><li>Most sensitive test </li></ul></ul><ul><ul><li>Concentration determined from a standard curve using known amounts of unlabeled Ag </li></ul></ul>Y Y +  Test + Patient’s sample Labeled Ag + Solid Phase Solid Phase
    36. 44. Solid Phase Non-Competitive RIA/ELISA <ul><li>Ag detection </li></ul><ul><ul><li>Immobilize Ab </li></ul></ul><ul><ul><li>Incubate with sample </li></ul></ul><ul><ul><li>Add labeled antibody </li></ul></ul><ul><ul><li>Amount of labeled Ab bound is proportional to the amount of Ag in the sample </li></ul></ul><ul><li>Quantitative </li></ul>Solid Phase Y Ag Immobilized Y Ag in Patient’s sample Labeled Ab
    37. 45. Tissue Immunofluorescence/Immunohistochemistry
    38. 47. NEUTRALIZATION
    39. 50. Immunofluorescence <ul><li>Flow Cytometry </li></ul><ul><ul><li>Cells in suspension are labeld with fluorescent tag </li></ul></ul><ul><ul><ul><li>Direct or Indirect Fluorescence </li></ul></ul></ul><ul><ul><li>Cells analyzed on a flow cytometer </li></ul></ul>Flow Tip Laser FL Detector Light Scatter Detector
    40. 51. CD4 CD8
    41. 52. Immunofluorescence <ul><li>Flow Cytometry cont. </li></ul><ul><ul><li>Data displayed </li></ul></ul>Green Fluorescence Intensity Number of Cells Unstained cells FITC-labeled cells One Parameter Histogram Red Fluorescence Intensity Green Fluorescence Intensity Two Parameter Histogram

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