Types of antibody testing

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 colour change after addition of the colour 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)

Reference:

1. Centers for Disease Control and Prevention. Laboratory testing for the diagnosis of HIV infection: updated recommendations. Jun 2014 [internet publication].