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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)