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Types of antibody testing

Last reviewed dd mmm yyyy. Last edited dd mmm yyyy

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HIV immunoassay technology

IV immunoassays based on different design principles are generally grouped into "generations":

  • 1st generation & 2 generation (indirect) enzyme linked immuno sorbent assay (ELISA)/enzyme immunoassays (EIAs)
    • plasma or serum is added to antigen coated wells containing viral lysate (first-generation assays) or recombinant HIV proteins or synthetic peptides (second-generation assays)
      • if anti HIV IgG is present in the sample it will bind to the antigens. An enzyme linked to anti-human IgG antibody is then added to the well (which binds to the anti-HIV IG). Finally a colour reagent is added.
        • a change in colour indicates the presence of anti-HIV IgG in the sample

  • 3rd generation "sandwich " ELISA
    • antigen coated well contains recombinant proteins or synthetic peptides. If the sample contains anti HIV IgG and IgM antibodies, it will bind with the antigen
      • an enzyme linked to HIV antigen is introduced (rather than to anti-IgG antibody) to the well which binds to anti HIV IgG and IgM
      • a colour reagent is added and if there is a change in colour it indicates the presence of anti-HIV IgG and IgM in the sample
    • useful in early detection of antibody response when compared to 1st and 2nd generation tests since they detect IgM as well as IgG

  • 4th generation "combination " ELISA
    • wells are coated with HIV antigen and p24 antibody
      • HIV antibodies in the sample binds to the antigen while anti p24 antibodies bind to the free p24
      • in the enzyme detection step, both enzyme-linked HIV antigen and enzyme-linked p24 antibody is added
      • a color change after addition of the color reagent indicates the presence of either anti-HIV antibody or p24 antigen
        • 2 different fluorescent labels can be used for independent detection of p24 antibody or HIV antigen.
    • when compared to previously available assays, 4th generation EIAs detect p24 antigens which enables even earlier detection of HIV infection (1)


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