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

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.
 Testing Strategies

AIM: To develop the logic used in

establishing the use of HIV tests
(testing strategies)
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
 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
Ab + Ag AbAg
Ab +Ag AbAg
Ab +Ag AbAg
Ab +Ag AbAg
Ab +Ag AbAg
Ab +Ag AbAg
Ab +Ag AbAg
Ab +Ag AbAg
Ab +Ag AbAg
Serological Testing Strategy

SCREENING TEST, highly sensitive

NEG
REACTIVE
SUPPLEMENTAL TEST,
POS highly sensitive & higher NEG
specificity

ADDITIONAL NEG
POS TESTS
 HIV Testing Strategy
G
IN
HIV1/2
N
NEG
EE

SCREEN
R
SC

REACTIVE
POS HIV-1 NEG
WB
L
TA
EN

NEG
ADDITIONAL
EM

POS
L

TESTS
PP
SU

POINT OF REPORTING
The Use of Screening Assays
Define samples as negative for a
given analyte
Enable high throughput
Why Follow a Strategy?
 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
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.
Objectives for HIV testing

Diagnosis

Surveillance

Blood transfusion safety

 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
 Different Test for HIV

DIRECT

INDIRECT

PRE-TEST COUNSELING,
INFORMED CONSENT,
CONFIDENTIALITY.
 Challenges of HIV Testing
Sensitivity - Early diagnostic ( window period)

Specificity- Cross reactivity

Easy to perform, low cost

Dtection 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
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
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
 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
 IgG-Capture EIA

Serum (1/100)

Goat-anti-Human-IgG
Capture Antibody Biotin-Labeled Ag

Strepavidin-
Substrate Peroxidase
 Reason for EIA
This test supplied as kit

Easy to Perform

Use to screen large number of sample

Sensitive

Specific

Cost effective
 Reason for EIA
This test supplied as kit

Easy to Perform

Use to screen large number of sample

Sensitive

Specific

Cost effective
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
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
 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

Equipment :
1- Pipettes

Single and Multi channel Pipettes should be

calibrated on a monthly basis.

This can be done using a balance.

Inaccurate pipetting
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

 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.
 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.
Source of False Positive Results
MULTIPLE PREGNANCY

MULTIPLE TRANSFUSION

AUTO IMMUNE DISORDER

CHRONIC HEPATITIS,

CHRONIC ALCOHOLIC

HBV VACCINATION

ANTIBODY TO POLYSTERENE
 Cross contamination
Can be caused by:

Reusing pipette tips (contaminated with + plasma)

Splashes from one well to another

During removal of plate covers

 Sample Quality
Properly collected (no haemolysis)

Transport conditions

Storage conditions

Number of freeze/thaw cycles

Age of sample
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
 Western Blot (Immunoblotting)

Solid-phase EIA with immobilized viral antigens

to detect antibodies to specific HIV proteins.
 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
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
Sample requirement:
Serum sample
Maximum 8 days
Stored 2o C 8oC or frozen at 25oC
Lipemic sample must be centrifuged well
Avoid heating
 Creating Western Blot Strips

HIV lysate Proteins are

proteins are transferred
separated by (blotted) onto
size using gel the surface of a
electrophoresis membrane

Strips are
The membrane is incubated with
cut into strips patient serum
and antihuman
IgG conjugated
with an enzyme
(and chromagen)
HIV Western Blot Banding Pattern

env gp160
gp120
gp 41

gag p55
p18
p24

pol p65
p51
p31
 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
*

*
 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
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