Enzyme Linked Immunosorbent Assay (ELISA) Rapid Test Western Blot Assay.

HIV ANTIBODY TESTING

HIVINFECTION

triggers an HIV antibody production in an HIV-infected

person’s blood

HIVINFECTION

is diagnosed largely by the detection of antibodies

against HIV in the blood of infected patients

HIVANTIBODY TESTis a blood test which looks for

HIV antibodies

MEDICAL tECHNOLOGISTS

cLINICAL lABORATORY

vENIPUNCTURE

Screening Test

Confirmatory Test

Enzyme Linked Immunosorbent Assay (ELISA)

Rapid Test

Western Blot Assay

Enzyme Linked Immunosorbent Assay (ELISA)

Screening Test

Spectrophotometer Spectrophotometer

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)

Screening Test

Rapid Test

Rapid test

Inactivated HIV antigen

Patient’s HIV antibody

Conjugate

Color development reagent

Rapid test

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

confirmatory Test

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)

THANK YOU PO!