New, sensitive and specific ELISA for the detection of neuropeptides in culture supernatants.
Accurate and sensitive sandwich ELISA has been developed for the detection and identification of each of the three neuropeptides, namely, Vasoactive intestinal peptide, Somatostatin and Substance P. The neuropeptides 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 titres were high, sera separated, complement inactivated and IgG class of antibodies were purified using a protein G column. Purified IgG antibodies were used for coating the wells and for conjugation with HRPO and 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 ng - 12.8 ng/ml) equal to or better than that reported for these peptides by radioimmunoassay. The assay was highly specific and did not react with a panel of other neuropeptides tested. High level of sensitivity without compromising the specificity was achieved by using activated 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.
Microbiology Dept. National Institute of Immunology, New Delhi, India.
M
M
Jaggi
R
R
Mukherjee
D000818:Animals; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007106:Immune Sera; D051379:Mice; D008807:Mice, Inbred BALB C; D009479:Neuropeptides / Q000032:analysis*; D011817:Rabbits; D051381:Rats; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D017077:Culture Media, Conditioned; D007106:Immune Sera; D009479:Neuropeptides