Serology - Prac. Microbiology


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Serology - Prac. Microbiology

  1. 1. Diagnosis of infectious diseases INDIRECT METHOD DIRECT METHOD (Serology) Specimen Detection of -IgM -rising titre of IgG Microscopy Culture technique Identification e.g. -Microscopy -Bioch. reactions - Serology - DNA probes. Antibiotic sensitivity Non-culture technique - Molecular methods - Serology.
  3. 3. Antigen-Antibody reactions in vitro = Serology Diagnosis of diseases Identification of Ag /Ab Quantitation of Ag / Ab
  4. 4. METHODS OF DETECTION OF ANTIGEN-ANTIBODY REACTIONS Reactions Accompanied by Visible Phenomena Reactions Detected by Labeled Reagents
  5. 5. Reactions Accompanied by Visible Phenomena Resulting Ag-Ab complexes seen directly • By naked eye • By microscope
  6. 6. A- AGGLUTINATION Ag: cellular or particulate antigen = “agglutinogen” antibody = “agglutinin”
  8. 8. Agglutination Slide Agglutination. 1 Tube Agglutination. 2
  9. 9. 1. Slide agglutination (Qualitative Test) Unknown Ag  Add known antiserum (Ab) 
  10. 10. 2. Tube agglutination (Quantitative Test) Detection of Ab against a certain pathogen = indirect evidence for diagnosis of disease = serological diagnosis 
  11. 11. Tube Agglutination 1/10 1/20 1/40 1/80 1/160 1/320 1/640
  12. 12. B- PASSIVE AGGLUTINATION Soluble antigen + antibody → precipitation Not very sensitive!! How can we convert: precipitation agglutination ?? more sensitive !!!
  13. 13. Both Ag & Ab are soluble One known! The other not known! + ↔ Soluble known reactant is coated onto inert particles e.g. Latex particles Known reactant becomes particulate (instead of soluble) Reaction becomes Agglutination (instead of precipitation)
  14. 14. Soluble known antigen coated on latex particle + Unknown antibody (IgM)
  15. 15. (Soluble) known antibody coated on latex particle + Unknown soluble antigen
  16. 16. Examples:  Rheumatoid arthritis: (Patient produces Abs against IgG) Latex-IgG (known Ag) + serum from patient (Ab?)  C-Reactive protein: (inflammatory conditions) Latex-anti-CRP (known Ab) + serum (CRP?)  Pregnancy Test (Test for HCG) Latex-anti-HCG (known Ab) + urine (HCG?)
  17. 17. C- COOMBS (ANTIGLOBULIN) TEST Non-agglutinating antibodies Examples:   Anti-Rh antibodies Abs in autoimmune haemolytic anaemia
  18. 18. Direct Coombs Test Done for cases of: - Newborn with erythroblastosis foetalis - Patients with haemolytic anaemia (RBCs have attached Abs already)
  19. 19. Indirect Coombs Test Done to detect non-agglutinating antibodies in serum of Rh-negative mother sensitized with Rh antigen
  20. 20. D- HAEMAGGLUTINATION INHIBITION Some viruses agglutinate RBCs in vitro. Antibodies (if present) prevent haemagglutination = Haemagglutination inhibition
  21. 21. E- PRECIPITATION • Ag: soluble Applications: Agar gel diffusion a. Double diffusion b. Single radial immunodiffusion
  22. 22. Agar gel diffusion a. Double Immunodiffusion Semi-solid medium Precipitation Line Ag well Ab well
  23. 23. Elek’s Test (double diffusion)
  24. 24. Elek’s Test
  25. 25. b. Single radial immunodiffusion • Method: - Ab in media - Ag in well Antibody Ag Precipitation Ring
  26. 26. b-Single radial immunodiffusion
  27. 27. b. Single radial immunodiffusion Interpretation: Diameter of ring is proportional to concentration of Ag in sample. 1 2 3 Diameter2 Note: Standards (1-4)of known concentrations are included in the test to create a curve. Ab in gel Ag Concentration 4
  28. 28. • Used to quantitate various immunoglobulin classes • Immunogloblin acts as an Ag. Standard 3 Standard 2 Pt. 2 Pt. 3 • In well: patient’s serum (Ag?) • In gel: anti-IgG, anti-IgM, ……etc Pt. 1 Standard 1
  29. 29. F- FLOCCULATION Antigen: Small insoluble particulate Examples: • Venereal Disease Research Laboratory (VDRL) test • Rapid plasma regain (RPR) test
  30. 30. VDRL: slide flocculation test for diagnosis of syphilis Cardiolipin used as antigen instead of Treponema antigen Antibody detected is a heterophil Ab called Reagin Negative Positive Microscopical aggregates
  31. 31. RPR rapid plasma reagin   Small carbon particles added → easier visibility (by naked eye)                -ve control +ve control +ve Test -ve control +ve control -ve Test
  32. 32. H- Complement fixation: • Test depends on the fact that: Ag - Ab → complement fixation • Sensitive • Used to: Detect and quantitate antibody Detect and quantitate antigen (less commonly) • Many applications in diagnosis of diseases caused by bacteria, viruses, fungi, etc. • Famous Test: Wassermann Test for serologic diagnosis of syphilis
  33. 33. I- Viral Neutralization: Certain viruses cause cytopathogenic (CPE) effect on certain cell cultures Uninfected cells Viral-infected cells, showing  cytopathic effect (CPE).    Fusion of infected cells→  multinucleated giant cells CPE are inhibited by virus-neutralizing Abs.
  34. 34. I. Viral Neutralization (cont.): First step: Serum (virus-neutralizing Ab?) + known Virus Second step: Cell culture inoculated with mixture: No Ab (in serum) + Virus → no neutralization → CPE Ab ( in serum) + Virus → neutralization → No CPE
  35. 35. ANTIGEN-ANTIBODY REACTIONS Reactions Accompanied by Visible Phenomena Reactions Detected by Labeled Reagents
  36. 36. A. Immunofluorescence B. ELISA C. RIA
  37. 37. A. Immunofluorescent Techniques: Principle: Fluorescent substances (e.g. Fluorescein isothiocyanate) attached to known Ab → seen by fluorescence microscope using UV light → fluorescence = positive reaction
  38. 38. i) Direct Immunofluorescence • Detects unknown Ag e.g.: rabies virus in brain of dead animal • Tissue (Ag?) + labeled known Ab → binding → fluorescence
  39. 39. Positive test
  40. 40. ii) Indirect Immunofluorescence • Detects Ab in serum → indirect diagnosis of disease e.g. Syphilis • Known antigen (Treponema pallidum) bound to slide + serum (Ab against Treponema?) • Labeled antihuman globulin added → fluorescence
  41. 41. B.Enzyme-Linked Immunosorbent Assay (ELISA) • • • • • Very sensitive Very specific Used to detect Ag or Ab Label used: Enzyme Example: Horseraddish peroxidase or alkaline phosphatase • Enzyme can be conjugated to Ag or Ab • Ag-enzyme or Ab-enzyme is called conjugate • Enzyme acts on colorless substrate → colored product
  42. 42. • Colour change = +ve test. • Colour intensity (by spectrophotometer) is proportional to the amount of Ag or Ab (quantitative). • Many variations in test procedure.
  43. 43. Direct Method (Double Antibody Technique) For detection and quantitation of Ag
  44. 44. Indirect Method For detection and quantitation of Ab in serum. Unknown Ab in test serum Known Ag Enzyme labeled anti-human Ig Substrate is added
  45. 45. ELISA
  46. 46. ELISA plates
  47. 47. C. Radioimmunoassay (RIA) • Same idea as ELlSA but different label: Radioactive isotope (instead of enzyme) • Measurement of Degree of radioactivity (instead of degree of colour change) • As sensitive as ELISA • Disadvantage: Hazards of radioactivity • Applications: – Measurement of biological substances (Ags) (e.g. drugs, hormones, tumour markers) – Measurement of antibodies
  48. 48. MCQs
  49. 49. 1. If you mix bacteria with specific Abs, it would result in: a) Cell lysis b) Agglutination c) Haemagglutination d) Precipitation e) Fluorescence
  50. 50. 2. In passive agglutination reaction, soluble antigens or antibodies coat: a) Latex particles b) White blood cells c) Platelets d) Carbon particles e) None of the above
  51. 51. 3. Regarding direct Coomb's test: a) Maternal RBCs are used b) Group O Rh +ve RBCs are used c) Anti-human globulins are added directly to foetal RBCs d) Mother's serum is used e) Foetal serum is used
  52. 52. 4. In Elek’s test: a) The antigen is mixed with the agar gel. b)Lines of precipitate will form in positive cases. c) The antibody is incorporated in the agar. d) A strip of filter paper is soaked with the antigen. e) Agglutination can be observed.
  53. 53. 5. Single radial immunodiffusion test: a) Is an example of an agglutination reaction b) Utilizes agar gel mixed with the antigen c) Depends on complement fixation during Ag-Ab reaction d) Is used to detect non-agglutinating antibodies e) Is a precipitation reaction
  54. 54. 6. Direct ELISA requires: a. Known Ab b. Known Ag c. Complement d. Patient’s Ab e. Sheep RBCs