Optimisation 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 individuals is described. We found that the reduction of non-specific binding of HIV-1 seropositive sera to the ELISA plate was essential for the reliable detection of serum antibodies in the peptide based indirect ELISA. Optimal results were obtained using Immulon microtitre plates, different concentrations of denatured. purified grade of casein in the blocking (1%) and washing (0.25%) solutions and by diluting HIV-1 seropositive sera 1 in 1600. These conditions reduced non-specific binding and improved assay sensitivity. We show that the inclusion of a control peptide is essential to reducing the incidence of false positive and false negative results. Taken together, the modifications described in this report improve reliability of the peptide-based indirect ELISA without compromising its sensitivity and have particular relevance for those wishing to apply the peptide-based indirect ELISA technique to serum samples which exhibit high levels of non-specific binding. To illustrate this, levels of antibody in the sera of HIV-1 seropositive and seronegative donors that are specific for peptides derived from a conserved region of HIV-1 gp120 sharing homology with the FAS apoptosis antigen were analysed using this technique.
Brown
L
L
Division of Oncology, St. George's Hospital Medical School, London, UK.
Westby
M
M
Souberbielle
B E
BE
Szawlowski
P W
PW
Kemp
G
G
Hay
P
P
Dalgleish
A G
AG
D000595:Amino Acid Sequence; D000818:Animals; D003951:Diagnostic Errors; D004797:Enzyme-Linked Immunosorbent Assay; D015483:HIV Antibodies / Q000097:blood*; D006679:HIV Seropositivity / Q000097:blood*; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D011817:Rabbits
D015483:HIV Antibodies