Conjugation of HIV gag p24 to LTB was performed by using the bifunctional crosslinker, SPDP. (A) The conjugate was dialyzed against PBS pH 7.4, loaded and run on 10% protein gel under non-reducing condition. The gel shows 2 bands, a lower band of approximately 100 KD and a higher band on the top of the gel of approximately 200 KD. (B) A GM1-ELISA was performed for the analysis of the conjugate. The conjugate was probed on GM1-coated wells. The conjugate was serially diluted (2x) and probed with mouse anti-gag p24 and goat anti-mouse alkaline phosphatase. The positive control consisted of mouse anti-gag p24 and goat anti-mouse alkaline phosphatase incubated in wells coated with gag p24 (p24 control). The negative control consisted of GM1 wells incubated with LTB-gag p24 conjugate and goat anti-mouse alkaline phosphatase in the absence of the primary antibody. The reaction was developed by addition of alkaline phosphatase substrate and read at 405 nm.
