triggers an HIV antibody
production in an HIV-infected
person’s blood
is diagnosed largely by the
detection of antibodies
against HIV in the blood of
infected patients
is a blood test which looks for
HIV antibodies
Enzyme Linked Immunosorbent Assay
(ELISA)
Rapid Test
Western Blot Assay
Enzyme Linked Immunosorbent Assay
(ELISA)
Spectrophotometer
Chromogen or substrate
para-nitrophenyl phosphate
(PNPP)
Anti-human immunoglobulin
coupled to an enzyme
alkaline phosphatase or
horse radish peroxidase
Patient’s HIV antibody
Inactivated HIV antigen
POSITIVE
NEGATIVE
ELISA Test
REFERENCE VALUES
INTERPRETATION
(Optical Density at 450 nm)
<0.300
Negative
>0.500
Positive
0.300 to 0.499
Indeterminate
Positive
Control
Negative
Control
Patient A
Patient B
Patient C
Assay
Control
1.689
0.153
0.055
0.412
1.999
0.123
False Positives
● People (especially women who have had multiple
pregnancies) may possess antibodies directed
against human leukocyte antigens (HLA)
 HLA are present on the host cells used to propagate HIV.
As HIV buds from the surface of the host cell, it incorporates some
of the host cell HLA into its envelope
False Negatives
● Window period (seroconversion)
Rapid Test
Inactivated HIV antigen
Patient’s HIV antibody
Conjugate
Color development reagent
Advantages
 quicker to perform
 do not require batching
 do not require specialized equipment
 results delivered on the same day
*Only ‘WHO recommended’ rapid HIV antibody tests should be used to ensure quality
Western Blot Assay
Why is Western Blot a Confirmatory Test?
Screening Tests
ELISA
Rapid
Sensitive
measures /visualizes
antibodies against
whole virus
Confirmatory Test
Western Blot
Specific
visualizes antibodies
against
each viral protein
glycoprotein 160
glycoprotein 120
protein 31
protein 24
Viral proteins that confirm
HIV positivity
gp160
viral envelope precursor (env)
gp120
viral envelope protein (env) binds to CD4
p31
Reverse Transcriptase (pol)
p24
viral core protein (gag)
Substrate solution
BCIP+NBT
 Strips are washed with
distilled water
5-Bromo-4-Chloro-3'-Indolyphosphate p-Toluidine Salt
and
Nitro-Blue Tetrazolium Chloride
Color
Reaction
(10-15 minutes
incubation)
Anti-human immunoglobulin
conjugated with an enzyme
alkaline phosphatase or
horse radish peroxidase
 Wash 4 times for
5 minutes each
2nd Incubation
(1 hour)
Patient’s antibody to
specific HIV viral proteins
 Wash 3 times for
5 minutes each
1st Incubation
(Overnight)
Specific HIV viral proteins
gp160
gp120
p31
P24
viral envelope
precursor (env)
viral envelope
protein (env)
binds to CD4
Reverse
Transcriptase (pol)
viral core
protein (gag)
*Insoluble black-purple bands
develop at the sites on the test strip
occupied by antibodies
Specific HIV viral proteins
gp160
gp120
p31
P24
viral envelope
precursor (env)
viral envelope
protein (env)
binds to CD4
Reverse
Transcriptase (pol)
viral core
protein (gag)
Criteria for the band pattern interpretation of HIV Western Blot test
established by the
Centers for Disease Control
with several other
organizations in 1987
No bands present
NEGATIVE
Bands at either p31 OR p24
AND
bands present at either gp160 OR gp120
POSITIVE
Bands present, but pattern does not
meet criteria for positivity
INDETERMINATE
What is an Indeterminate HIV Result?
A neither positive nor negative result.
ELISA
Western Blot
Indeterminate
Indeterminate
INDETERMINATE RESULT
ELISA
Western Blot
Positive
Negative
or
only reactive to a couple of the
HIV antibodies
Triggered by
● a number of naturally occurring antibodies that may be present in one’s
blood
● recent influenza vaccination
● other viral infections
● window period
What happens if one has an
Indeterminate HIV Result?
● return for follow up testing
● counsellors can serve as one’s support in this stressful period
Algorithm for HIV Antibody Testing and Counseling Services
PRE-TEST Counseling
ELISA or
HIV Rapid Test
(Screening Test)
POSITIVE
result
POSITIVE
test
result
NEGATIVE test result:
POST-TEST Counseling
for negative result
WESTERN BLOT
(Confirmatory Test)
POSITIVE test result:
POST-TEST Counseling
for positive result
NEGATIVE test result:
POST-TEST Counseling
for negative result
“A person infected by HIV/AIDS
is Jesus among us. How can we
say “no” to Him? ”
- Mother Teresa of Calcutta
Bro. John Jay C. Magpusao, MI
RMT, MA Theology
Order of the Ministers of the Infirm (Camillians)