DEVELOPMENT OF A HIGHLY SENSITIVE ELISA FOR THE DETERMINATION OF PBANAND ITS APPLICATION TO THE ANALYSIS OF HEMOLYMPH IN SPODOPTERA-LITTORALIS

A highly sensitive enzyme linked immunosorbent assay (ELISA) for the d etermination of the pheromone biosynthesis activating neuropeptide (PB AN) 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 specif icity, using as haptens PBAN or PBAN(20-33) with a Cys residue attache d to their amino-terminal side. The Cys thiol group has been used to c ovalently bind the peptide to keyhole limpet hemocyanin (KLH) by using N-succinimidyl-4-(maleidimidomethyl) cyclohexane carboxylate (SMCC) a s a convenient heterobifunctional cross-linker. Several usable competi tive immunoassays have been obtained by synthesizing eight different c oating antigens and screening the sera against all of them. The best a ssay was obtained with antibody 4 using Cys-Hez-PBAN(20-33) coupled to bovine serum albumin (BSA) through the Lys groups by using the homobi functional cross-linker dimethylpimelidate dihydrochloride (DMP) as th e coating antigen. The optimized assay allows to detect PBAN at concen trations as low as 1 fmol/well (I-50 = 2.5 fmol/well). An extraction p rocedure for the hemolymph has been developed that allows to perform P BAN measurements in this tissue even after a tenfold dilution. In thes e conditions matrix effect is negligible. Preliminary results on the p resence of PBAN-like immunoreactivity (PBAN-IR) in the hemolymph of Sp odoptera littoralis females are reported. (C) 1995 Wiley-Liss, Inc.