NEW, SENSITIVE AND SPECIFIC ELISA FOR THE DETECTION OF NEUROPEPTIDES IN CULTURE SUPERNATANTS

Accurate and sensitive sandwich ELISA has been developed for the detec tion and identification of each of the three neuropeptides, namely, Va soactive intestinal peptide, Somatostatin and Substance P. The neurope ptides conjugated with BSA and emulsified with Freund's adjuvant were used for immunisation of rabbits. Titres of polyclonal antibodies were checked by indirect immunofluorescence. The animals were bled when ti tres were high, sera separated, complement inactivated and IgG class o f antibodies were purified using a protein G column. Purified IgG anti bodies were used for coating the wells and for conjugation with HRPO a nd used for the detection of the synthetic neuropeptides in a standard solution or in the culture supernatant. The ELISA thus developed for the assay of each of the three neuropeptides had a sensitivity (0.01 n g - 12.8 ng / ml) equal to or better than that reported for these pept ides by radioimmunoassay. The assay was highly specific and did not re act with a panel of other neuropeptides tested. High level of sensitiv ity without compromising the specificity was achieved by using activat ed polyvinyl plates and using purified IgG from high titre rabbit anti -peptide sera. The non specific reaction was minimised by using 10,000 MW cut off amicon filtered supernatants.