Premium
Development of a highly sensitive ELISA for the determination of PBAN and its application to the analysis of hemolymph in Spodoptera littoralis
Author(s) -
Marco M.Pilar,
Fabriàs Gemma,
Camps Francisco
Publication year - 1995
Publication title -
archives of insect biochemistry and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.576
H-Index - 66
eISSN - 1520-6327
pISSN - 0739-4462
DOI - 10.1002/arch.940300406
Subject(s) - keyhole limpet hemocyanin , hapten , hemolymph , bovine serum albumin , biology , biochemistry , antiserum , peptide , hemocyanin , antigen , chromatography , chemistry , immunology
A highly sensitive enzyme linked immunosorbent assay (ELISA) for the determination of the pheromone biosynthesis activating neuropeptide (PBAN) has been developed. Six antisera have been obtained that recognize the carboxyl terminal side of this peptide. Two immunogens have been rationally designed and synthesized in order to direct antibody specificity, using as haptens PBAN or PBAN(20‐33) with a Cys residue attached to their amino‐terminal side. The Cys thiol group has been used to covalently bind the peptide to keyhole limpet hemocyanin (KLH) by using N‐succinimidyl‐4‐(maleidimidomethyl) cyclohexane carboxylate (SMCC) as a convenient heterobifunctional cross‐linker. Several usable competitive immunoassays have been obtained by synthesizing eight different coating antigens and screening the sera against all of them. The best assay was obtained with antibody 4 using Cys‐Hez‐PBAN(20‐33) coupled to bovine serum albumin (BSA) through the Lys groups by using the homobifunctional cross‐linker dimethylpimelidate dihydrochloride (DMP) as the coating antigen. The optimized assay allows to detect PBAN at concentrations as low as 1 fmol/well (l 50 = 2.5 fmol/well). An extraction procedure for the hemolymph has been developed that allows to perform PBAN measurements in this tissue even after a tenfold dilution. In these conditions matrix effect is negligible. Preliminary results on the presence of PBAN like immunoreactivity (PBAN‐IR) in the hemolymph of Spodoptera littoralis females are reported.© 1995 Wiley‐Liss, Inc.