HIV

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

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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.

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Testing Strategies

• AIM To develop the logic used in establishing
  the use of HIV tests (testing strategies)

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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

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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

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Ab Ag AbAg
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AbAg
Ab Ag
Ab Ag
AbAg
Ab Ag
AbAg
Ab Ag
AbAg
AbAg
Ab Ag
Ab Ag
AbAg
Ab Ag
AbAg
AbAg
Ab Ag
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Serological Testing Strategy
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HIV Testing Strategy
HIV1/2 SCREEN
NEG
SCREENING
REACTIVE
HIV-1 WB
POS
NEG
NEG
SUPPLEMENTAL
ADDITIONAL TESTS
POS
POINT OF REPORTING
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The Use of Screening Assays

• Define samples as negative for a given analyte
• Enable high throughput

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Why Follow a Strategy?
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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

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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.

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Objectives for HIV testing

• Diagnosis
• Surveillance
• Blood transfusion safety

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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

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HIV STRUCTURE
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Different Test for HIV

• DIRECT
• INDIRECT
• PRE-TEST COUNSELING,
• INFORMED CONSENT,
• CONFIDENTIALITY.

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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

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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

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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

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Indirect EIA
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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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Sandwich EIA(Ab-Ag-Ab)
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IgG-Capture EIA
Serum (1/100)
Goat
-
anti
-
Human
-
IgG
Capture Antibody
Biotin
-
Labeled Ag
Strepavidin
-
Peroxidase
Substrate
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Reason for EIA

• This test supplied as kit
• Easy to Perform
• Use to screen large number of sample
• Sensitive
• Specific
• Cost effective

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Reason for EIA

• This test supplied as kit
• Easy to Perform
• Use to screen large number of sample
• Sensitive
• Specific
• Cost effective

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Components of Commercially Available EIA Kits

• SolidPhase 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

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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

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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

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Equipment 1- Pipettes

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

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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

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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.

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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.

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Source of False Positive Results

• MULTIPLE PREGNANCY
• MULTIPLE TRANSFUSION
• AUTO IMMUNE DISORDER
• CHRONIC HEPATITIS,
• CHRONIC ALCOHOLIC
• HBV VACCINATION
• ANTIBODY TO POLYSTERENE

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Cross contamination

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

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Sample Quality

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

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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

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

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• 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

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Sample requirement

• Serum sample
• Maximum 8 days
• Stored 2o C 8oC or frozen at 25oC
• Lipemic sample must be centrifuged well
• Avoid heating

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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
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HIV Western Blot Banding Pattern
env gp160 gp120 gp 41 gag p55 p18 p24 pol p6
5 p51 p31
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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

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Western blot Banding



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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

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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