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

Enzyme Linked Immunosorbent Assay (ELISA)

Spectrophotometer

NEGATIVE Inactivated HIV antigen Patient’s HIV antibody Anti-human immunoglobulin coupled to an enzyme alkaline phosphatase or horse radish peroxidase Chromogen or substrate para-nitrophenyl phosphate (PNPP) POSITIVE

REFERENCE VALUES (Optical Density at 450 nm) INTERPRETATION <0.300Negative >0.500Positive to 0.499Indeterminate ELISA Test Positive Control Negative Control Patient APatient BPatient C Assay Control

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)

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 TestsConfirmatory Test ELISARapidWestern Blot SensitiveSpecific measures /visualizes antibodies against whole virus visualizes antibodies against each viral protein glycoprotein 160 glycoprotein 120 protein 31 protein 24

Viral proteins that confirm HIV positivity gp160viral envelope precursor (env) gp120viral envelope protein (env) binds to CD4 p31Reverse Transcriptase (pol) p24viral core protein (gag)

Specific HIV viral proteins Patient’s antibody to specific HIV viral proteins Anti-human immunoglobulin conjugated with an enzyme Substrate solution BCIP+NBT gp160 viral envelope precursor (env) gp120 viral envelope protein (env) binds to CD4 p31 Reverse Transcriptase (pol) P24 viral core protein (gag) alkaline phosphatase or horse radish peroxidase 1 st Incubation (Overnight) 2 nd 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

gp160 viral envelope precursor (env) gp120 viral envelope protein (env) binds to CD4 p31 Reverse Transcriptase (pol) P24 viral core protein (gag) *Insoluble black-purple bands develop at the sites on the test strip occupied by antibodies Specific HIV viral proteins

No bands presentNEGATIVE 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 Criteria for the band pattern interpretation of HIV Western Blot test established by the Centers for Disease Control with several other organizations in 1987

What is an Indeterminate HIV Result? A neither positive nor negative result. ELISAWestern Blot Indeterminate INDETERMINATE RESULT ELISAWestern 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

PRE-TEST Counseling ELISA or HIV Rapid Test (Screening Test) POSITIVE test result NEGATIVE test result: POST-TEST Counseling for negative result POSITIVE 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, MI RMT, MA Theology Order of the Ministers of the Infirm (Camillians)