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Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
Serology - Prac. Microbiology
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Serology - Prac. Microbiology

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  • 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.
  • 2. ANTIGENANTIBODY INTERACTIONS
  • 3. Antigen-Antibody reactions in vitro = Serology Diagnosis of diseases Identification of Ag /Ab Quantitation of Ag / Ab
  • 4. METHODS OF DETECTION OF ANTIGEN-ANTIBODY REACTIONS Reactions Accompanied by Visible Phenomena Reactions Detected by Labeled Reagents
  • 5. Reactions Accompanied by Visible Phenomena Resulting Ag-Ab complexes seen directly • By naked eye • By microscope
  • 6. A- AGGLUTINATION Ag: cellular or particulate antigen = “agglutinogen” antibody = “agglutinin”
  • 7. AGGLUTINATION
  • 8. Agglutination Slide Agglutination. 1 Tube Agglutination. 2
  • 9. 1. Slide agglutination (Qualitative Test) Unknown Ag  Add known antiserum (Ab) 
  • 10. 2. Tube agglutination (Quantitative Test) Detection of Ab against a certain pathogen = indirect evidence for diagnosis of disease = serological diagnosis 
  • 11. Tube Agglutination 1/10 1/20 1/40 1/80 1/160 1/320 1/640
  • 12. B- PASSIVE AGGLUTINATION Soluble antigen + antibody → precipitation Not very sensitive!! How can we convert: precipitation agglutination ?? more sensitive !!!
  • 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. Soluble known antigen coated on latex particle + Unknown antibody (IgM)
  • 15. (Soluble) known antibody coated on latex particle + Unknown soluble antigen
  • 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. C- COOMBS (ANTIGLOBULIN) TEST Non-agglutinating antibodies Examples:   Anti-Rh antibodies Abs in autoimmune haemolytic anaemia
  • 18. Direct Coombs Test Done for cases of: - Newborn with erythroblastosis foetalis - Patients with haemolytic anaemia (RBCs have attached Abs already)
  • 19. Indirect Coombs Test Done to detect non-agglutinating antibodies in serum of Rh-negative mother sensitized with Rh antigen
  • 20. D- HAEMAGGLUTINATION INHIBITION Some viruses agglutinate RBCs in vitro. Antibodies (if present) prevent haemagglutination = Haemagglutination inhibition
  • 21. E- PRECIPITATION • Ag: soluble Applications: Agar gel diffusion a. Double diffusion b. Single radial immunodiffusion
  • 22. Agar gel diffusion a. Double Immunodiffusion Semi-solid medium Precipitation Line Ag well Ab well
  • 23. Elek’s Test (double diffusion)
  • 24. Elek’s Test
  • 25. b. Single radial immunodiffusion • Method: - Ab in media - Ag in well Antibody Ag Precipitation Ring
  • 26. b-Single radial immunodiffusion
  • 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. • 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. F- FLOCCULATION Antigen: Small insoluble particulate Examples: • Venereal Disease Research Laboratory (VDRL) test • Rapid plasma regain (RPR) test
  • 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. 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. 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. 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. 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. ANTIGEN-ANTIBODY REACTIONS Reactions Accompanied by Visible Phenomena Reactions Detected by Labeled Reagents
  • 36. A. Immunofluorescence B. ELISA C. RIA
  • 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. i) Direct Immunofluorescence • Detects unknown Ag e.g.: rabies virus in brain of dead animal • Tissue (Ag?) + labeled known Ab → binding → fluorescence
  • 39. Positive test
  • 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. 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. • Colour change = +ve test. • Colour intensity (by spectrophotometer) is proportional to the amount of Ag or Ab (quantitative). • Many variations in test procedure.
  • 43. Direct Method (Double Antibody Technique) For detection and quantitation of Ag
  • 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. ELISA
  • 46. ELISA plates
  • 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. MCQs
  • 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. 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. 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. 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. 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. 6. Direct ELISA requires: a. Known Ab b. Known Ag c. Complement d. Patient’s Ab e. Sheep RBCs

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