Elisa immuno maxisorp plates

Fifteen amino acid long linear peptides with 11 amino acid overlap, spanning the entire length of gp160 of the Indian subtype C virus, 93IN101, were synthesized commercially (Infinity Biotech andResource Inc., PA). The peptides were adsorbed onto 96-well ELISA immuno maxisorp plates (Thermo Fisher) at a concentration of 5 μg/ml in 100 mM NAHCO₃, pH 9.6, by overnight incubation at 4°C and ELISA was performed as described previously (14 (link)). Briefly each peptide was coated in individual wells of 96-well ELISA plates overnight at a concentration of 5 μg/ml. Plates were blocked for 1 h at room temperature. Plasma sample diluted 1:50 in diluent and was added to respective wells of the peptide coated 96-well plates. After incubation at 37°C for 1 h, the plates were washed four times with PBST buffer. Secondary incubation was performed in the presence of HRP-conjugated anti-human antibody. The wells were then washed with PBST four times followed by addition of 100 μl/well aqueous 3,3′,5,5′-tetramethylbenzidine substrate. Color development was stopped using 2 n sulfuric acid after 30 min. The absorbance was measured at 450 nm. Plasma samples were tested in duplicate and each experiment was performed on two independent occasions. Healthy Human Plasma pool (HHP) was included as the negative control in all experiments.

In order to determine binding reactivity of the antibodies in the plasma to whole conformational proteins, plasma were tested in an ELISA with HIV-1 subtype C (C.1086), D7gp120 monomer (Cat No.12582) and gp140C trimer (Cat.No. 12581), and its mutant forms C.1086 D7gp120K160N (12579) and C.1086 gp140C K160N (12580) as well as V1V2 tag (Cat.No. 12568), all obtained from the NIH AIDS Research Reagent Program, Division of AIDS, NIAID, NIH. The proteins were adsorbed onto 96-well ELISA immunomaxisorp plates (Thermo Fisher) at a concentration of 2 μg/mL in 100 mM NaHCO₃, pH 9.6, by overnight incubation at 4°C and tested using the ELISA protocol described above. Plasma samples were tested in triplicate at a dilution of 1:200. HHP used as the negative control. The experiment was performed on two independent occasions and mean values were calculated.