Enzyme Linked Immunosorbent Assay (ELISA) Rapid Test Western Blot Assay.
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Transcript of Enzyme Linked Immunosorbent Assay (ELISA) Rapid Test Western Blot Assay.
HIVINFECTION
is diagnosed largely by the detection of antibodies
against HIV in the blood of infected patients
Screening Test
Confirmatory Test
Enzyme Linked Immunosorbent Assay (ELISA)
Rapid Test
Western Blot Assay
elisa
NEGATIVE
Inactivated HIV antigen
Patient’s HIV antibody
Anti-human immunoglobulin coupled to an enzyme
alkaline phosphatase or horse radish peroxidase
Chromogen or substratepara-nitrophenyl phosphate
(PNPP)
POSITIVE
REFERENCE VALUES (Optical Density at 450 nm)
INTERPRETATION
<0.300 Negative
>0.500 Positive
0.300 to 0.499 Indeterminate
ELISA Test
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
●
False Negatives
●
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
Window period (seroconversion)
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
Why is Western Blot a Confirmatory Test?Screening Tests Confirmatory Test
ELISA Rapid Western Blot
Sensitive Specific
measures /visualizes antibodies against
whole virus
visualizes antibodies against
each viral protein
glycoprotein 160glycoprotein 120
protein 31protein 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)
Western blot
Specific HIV viral proteins
Patient’s antibody to specific HIV viral proteins
Anti-human immunoglobulin conjugated with an enzyme
Substrate solutionBCIP+NBT
gp160viral envelope
precursor (env)
gp120viral envelopeprotein (env)binds to CD4
p31Reverse
Transcriptase (pol)
P24viral core
protein (gag)
alkaline phosphatase or horse radish peroxidase
1st Incubation(Overnight)
2nd Incubation(1 hour)
Color Reaction(10-15 minutes incubation)
Wash 3 times for 5 minutes each
Wash 4 times for 5 minutes each
Strips are washed with distilled water
5-Bromo-4-Chloro-3'-Indolyphosphate p-Toluidine Salt and
Nitro-Blue Tetrazolium Chloride
gp160viral envelope
precursor (env)
gp120viral envelopeprotein (env)binds to CD4
p31Reverse
Transcriptase (pol)
P24viral core
protein (gag)
*Insoluble black-purple bands develop at the sites on the test strip
occupied by antibodies
Specific HIV viral proteins
Western blot
No bands present NEGATIVE
Bands at either p31 OR p24AND
bands present at either gp160 OR gp120POSITIVE
Bands present, but pattern does not meet criteria for positivity
INDETERMINATE
Criteria for the band pattern interpretation of HIV Western Blot testestablished by the
Centers for Disease Control with several other
organizations in 1987
What is an Indeterminate HIV Result?
A neither positive nor negative result. ELISA Western Blot
Indeterminate Indeterminate
INDETERMINATE RESULT
ELISA Western Blot
Positive Negativeor
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
PRE-TEST Counseling
ELISA or HIV Rapid Test (Screening Test)
POSITIVE test resultNEGATIVE test result: POST-TEST Counseling
for negative resultPOSITIVE result
WESTERN BLOT(Confirmatory Test)
POSITIVE test result: POST-TEST Counseling
for positive result
NEGATIVE test result: POST-TEST Counseling
for negative result
Algorithm for HIV Antibody Testing and Counseling Services
“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, MIRMT, MA TheologyOrder of the Ministers of the Infirm (Camillians)