OPTIMIZATION OF A PEPTIDE-BASED INDIRECT ELISA FOR THE DETECTION OF ANTIBODY IN THE SERUM OF HIV-1 SEROPOSITIVE PATIENTS

A modified peptide-based indirect ELISA technique for the detection of HIV-1 specific antibodies in the sera of HIV-1 seropositive individua ls is described. We found that the reduction of non-specific binding o f HIV-1 seropositive sera to the ELISA plate was essential for the rel iable detection of serum antibodies in the peptide based indirect ELIS A. Optimal results were obtained using Immulon microtitre plates, diff erent concentrations of denatured, purified grade of casein in the blo cking (1%) and washing (0.25%) solutions and by diluting HIV-1 seropos itive sera 1 in 1600. These conditions reduced non-specific binding an d improved assay sensitivity, We show that the inclusion of a control peptide is essential to reducing the incidence of false positive and f alse negative results. Taken together, the modifications described in this report improve reliability of the peptide-based indirect ELISA wi thout compromising its sensitivity and have particular relevance for t hose wishing to apply the peptide-based indirect ELISA technique to se rum samples which exhibit high levels of non-specific binding, To illu strate this, levels of antibody in the sera of HIV-1 seropositive and seronegative donors that are specific for peptides derived from a cons erved region of HIV-1 gp120 sharing homology with the FAS apoptosis an tigen were analysed using this technique.