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HIV TESTING TECHNOLOGIES ELISA / WESTERN BLOT. There are numbers of tests They should be used in combination (strategies) Combinations must be consistent.

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Presentation on theme: "HIV TESTING TECHNOLOGIES ELISA / WESTERN BLOT. There are numbers of tests They should be used in combination (strategies) Combinations must be consistent."— Presentation transcript:

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 II Strategy 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. Strategy I Test all samples with one EIA Strategy II Strategy 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 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 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  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 Ab +Ag AbAg AbAg AbAg AbAg AbAg AbAg AbAg AbAg

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

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

11 The Use of Screening Assays Define samples as negative for a given analyte Enable high throughput 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 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 II Strategy 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 Diagnosis Surveillance Blood transfusion safety Objectives for HIV testing

16 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 Kinetics of Antibody Response to HIV

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19 DIRECT INDIRECT  PRE-TEST COUNSELING, INFORMED CONSENT, CONFIDENTIALITY. Different Test for HIV

20 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

21 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 Current HIV technologies

22 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 EIAs (Enzyme Immunosorbent Assays)

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24 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 competes against the unlabeled component in the patient sample for binding sites. Results Negative Reaction has color change Positive Reaction no color change

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26 IgG-Capture EIA Serum (1/100) Biotin-Labeled Ag Strepavidin- PeroxidaseSubstrate Goat-anti-Human-IgG Capture Antibody

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

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

29 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

30 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

31 Sources of Error for HIV EIA Tests Documented 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

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

33 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

34 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.

35 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.

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

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

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

39 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

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

41 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

42 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

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

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

45 HIV Western Blot Banding Pattern envgp160 gp120 gp 41 gagp55 p18 p24 polp65 p51 p31

46 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

47 * * *

48 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 Specificity is less than that of EIA A significant number of indeterminate blots are seen in low risk populations A significant number of indeterminate blots are seen in low risk populations

49 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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