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HIV TESTING TECHNOLOGIES ELISA / WESTERN BLOT

HIV TESTING TECHNOLOGIES ELISA / WESTERN BLOT. There are numbers of tests They should be used in combination (strategies) Combinations must be consistent. WHO Recommended Strategies. Strategy I Test all samples with one EIA

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HIV TESTING TECHNOLOGIES ELISA / WESTERN BLOT

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  1. HIV TESTING TECHNOLOGIES ELISA / WESTERN BLOT

  2. There are numbers of tests • They should be used in combination (strategies) • Combinations must be consistent

  3. WHO Recommended Strategies • Strategy I Test all samples with one EIA • Strategy IIStrategy I with all reactives retested in a more specific test with different principle and/or antigen. • Strategy III Strategy II with reactives tested in a third test differing from the first two tests.

  4. Testing Strategies AIM: To develop the logic used in establishing the use of HIV tests (testing strategies)

  5. Objectives of Testing Strategies • To achieve the correct diagnosis in the most efficient manner • To maintain consistency in testing • To develop baseline data for assessing changes • To deliver useful results

  6. Screening Assays • Are used to detect antibody-- specific or nonspecific • Are designed to handle large numbers of samples with rapid throughput • Must be high performance • Should include a full range of HIV antigens

  7. Ab + Ag AbAg

  8. AbAg Ab +Ag AbAg Ab +Ag Ab +Ag AbAg Ab +Ag AbAg AbAg Ab +Ag Ab +Ag AbAg Ab +Ag AbAg AbAg Ab +Ag

  9. SCREENING TEST, highly sensitive NEG REACTIVE SUPPLEMENTAL TEST, highly sensitive & higher specificity POS NEG NEG ADDITIONAL TESTS POS Serological Testing Strategy

  10. HIV Testing Strategy HIV1/2 SCREEN NEG SCREENING REACTIVE HIV-1 WB POS NEG NEG SUPPLEMENTAL ADDITIONAL TESTS POS POINT OF REPORTING

  11. The Use of Screening Assays • Define samples as negative for a given analyte • Enable high throughput

  12. Why Follow a Strategy?

  13. The Importance of Maintaining a Strategy • Consistency of laboratory records • Consistency of results • Clarity of results to doctors • Maintaining data base to assess performances • Avoiding common false reactivity • Avoiding technical errors • Reducing costs

  14. WHO Recommended Strategies • Strategy I Test all samples with one EIA • Strategy IIStrategy I with all reactives retested in a more specific test with different principle and/or antigen. • Strategy III Strategy II with reactives tested in a third test differing from the first two tests.

  15. Objectives for HIV testing • Diagnosis • Surveillance • Blood transfusion safety

  16. Kinetics of Antibody Response to HIV • KNOWLEDGE VIRAL STRUCTURE • STRUCTURAL PROTEIN OF HIV1 AND HIV 2 HIV 1 ENV – gp41, 120, 160 core – p55, 18, 24 pol – p31, 51, 65 • HIV 2 ENV – gp36, 140, core – p56, 26, 16 pol – 68, 53, 34 • Viral entry, Target cell (CD4) • Window period • IgM. IgG

  17. HIV STRUCTURE

  18. Different Test for HIV • DIRECT • INDIRECT • PRE-TEST COUNSELING, • INFORMED CONSENT, • CONFIDENTIALITY.

  19. Challenges of HIV Testing • Sensitivity - Early diagnostic ( window period) • Specificity- Cross reactivity • Easy to perform, low cost • Détection of HIV-1 & HIV-2 and discrimination between the two viruses • One test can not fulfill these requirements • Need to perform a combination of HIV tests for screening and confirmation

  20. Current HIV technologies Detection of antibodies • Screening tests • Enzyme immunosorbent assays (EIAs) • Simple/rapid immuno-diagnostics assays • Confirmatory or supplemental tests • Western blot (WB) • Alternatives to confirmatory tests • Repetitive EIA or rapid assays

  21. EIAs (Enzyme Immunosorbent Assays) • This term describes a variety of assays that are based on the binding of antibodies with their antigens and the detection of this reaction using a component conjugated with an active enzyme. • This enzyme acts on its substrate to produce a colour change.Test results are measured by measuring this colour. • Four immunologic principles • Indirect • Competition • Sandwich • Immuno-capture

  22. Indirect EIA

  23. Competitive EIA • A measured amount of known enzyme-labeled component (being measured) is added to the reaction at the same time patient sample is added. • The labeled component therefore competesagainst the unlabeled component in the patient sample for binding sites. • Results • Negative Reaction has color change • Positive Reaction no color change

  24. Sandwich EIA(Ab-Ag-Ab)

  25. IgG-Capture EIA Serum (1/100) Goat - anti - Human - IgG Capture Antibody Biotin - Labeled Ag Strepavidin - Substrate Peroxidase

  26. Reason for EIA • This test supplied as kit • Easy to Perform • Use to screen large number of sample • Sensitive • Specific • Cost effective

  27. Reason for EIA • This test supplied as kit • Easy to Perform • Use to screen large number of sample • Sensitive • Specific • Cost effective

  28. Components of Commercially Available EIA Kits • Solid–Phase Support • Antigens bound to polystyrene microtiter plates (passive absorption) • Blocking is necessary to reduce nonspecific binding (test accuracy) • 96 microwell format • Antigens • The use of cloned antigens has reduced non- specific binding • Antibodies • Monoclonals of high titer, affinity and avidity

  29. Components of Commercially Available EIA Kits • Conjugates • Ab conjugated with enzyme (without effecting binding site) • Enzyme • HRP (horseradish peroxidase) • Substrate (chromogenic) • Colorless chromogen reacts with enzyme ( color) • Stop Solution • Typically an acid, stabilizes the color for a limited time

  30. Sources of Error for HIV EIA TestsDocumented Sources of False Negative Results Operator Error • FAIL TO ADD SERUM OR REAGENT TO THE CORRECT WELL • REAGENT DILUTED IN WRONG DILUENT IN WRONG DILUTION

  31. Equipment : 1- Pipettes • Single and Multi channel Pipettes should be calibrated on a monthly basis. • This can be done using a balance. • Inaccurate pipetting

  32. Equipment : 2- Microplate Washers • Daily • Prime the washer with wash solution before running sample plates • Set the washer to wash the recommended number of times (with correct volume) • Check for accurate dispensing and complete aspiration in each plate well, if not clean the washer head • Listen for changes in the sound the washer makes, this can indicate a vacuum leak • At the end of the day prime the washer with DI water

  33. Equipment : Micro-plate Washers • Weekly • If a washer is not used during the week rinse it out with DI water to reduce microbial growth. • Monthly • Run a 10% solution of ethanol through the washer to disinfect. This can also be done if the washer exhibits signs of contamination (high background). • Thoroughly rinse the washer after alcohol is used.

  34. Equipment : Micro-plate Reader • Daily • Each time a reader is turned on it runs a self test, it will then report any errors. • Weekly • Run a control plate weekly. Variations in positive or negative specimens could be a sign of a bad diode or a spill on a diode.

  35. Source of False Positive Results • MULTIPLE PREGNANCY • MULTIPLE TRANSFUSION • AUTO IMMUNE DISORDER • CHRONIC HEPATITIS, • CHRONIC ALCOHOLIC • HBV VACCINATION • ANTIBODY TO POLYSTERENE

  36. Cross contamination Can be caused by: • Reusing pipette tips (contaminated with + plasma) • Splashes from one well to another • During removal of plate covers

  37. Sample Quality • Properly collected (no haemolysis) • Transport conditions • Storage conditions • Number of freeze/thaw cycles • Age of sample

  38. Validation and Interpretation of Results • Product inserts provide guidelines • Positive and Negative controls must fall within a certain range. • Controls are used to calculate a cut-off. • Samples below cut-off are negative, those above are positive

  39. Western Blot (Immunoblotting) Solid-phase EIA with immobilized viral antigens to detect antibodies to specific HIV proteins.

  40. Principle • AIDS is caused by at least 2 etiological agents HIV-1 & HIV-2 • Inactivated and denatured protein of HIV-1 are fractioned by polyacrylamide gel electrophoresis • Protein bands are transferred into nitrocellulose strips • HIV-1 sample diluted with buffer are then incubated with the strip

  41. Conjugate peroxidase labeled anti human IgG is added • It will bind to the antibodies already bound to the strip • Chromogen is then added forming color reaction • Reaction is then stopped by aspiration and reaction

  42. Sample requirement: • Serum sample • Maximum 8 days • Stored 2o C – 8oC or frozen at – 25oC • Lipemic sample must be centrifuged well • Avoid heating

  43. Creating Western Blot Strips Proteins are transferred (blotted) onto the surface of a membrane HIV lysate proteins are separated by size using gel electrophoresis Strips are incubated with patient serum and antihuman IgG conjugated with an enzyme (and chromagen) The membrane is cut into strips

  44. HIV Western Blot Banding Pattern envgp160 gp120 gp 41 gag p55 p18 p24 pol p65 p51 p31

  45. Interpretation of Results(General Consensus) Negative: No bands present Positive: 2 ENV band present (WHO Guidelines) Indeterminate Any bands present but do not meet criteria for positive

  46. Western blot Banding * * *

  47. When should WB be used? • Western Blot assay should not be used as a screening test. • WB should be viewed as a supplemental test which can be used to confirm positive results obtained from EIA. • HOWEVER: • Specificity is less than that of EIA • A significant number of indeterminate blots are seen in low risk populations

  48. Advantages • Specific interaction of antibody and antigen can be directly visualized. Disadvantages • Technically demanding • Expensive • Subject to interpretation • Presence or absence of bands • Intensity of those bands

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