Production and characterization of monoclonal antibodies raised against the membrane ecdysone receptor from the anterior silk gland of the silkworm, Bombyx mori

Abstract

Several evidences for nongenomic action of ecdysteroids through membrane receptor (mEcR) in programmed cell death (PCD) of the Bombyx mori anterior silk glands (ASGs) have been reported. The isolation, characterization and sequencing of mEcR still remain elusive. In the present work, monoclonal antibodies against mEcR has been raised, for the first time, and expected to facilitate any further molecular characterization of that novel receptor. The solubilized ASGs membrane fractions were used for immunizing mice. Three out of 4 injected mice showed immune-reactive signal, as revealed by dot blot assay. Out of the 3 mice, only one mouse sera showed to react with [ 3 H]PonA binding protein, as revealed by double antibody capture assay (DACA). Out of 117 primary hybridoma cell lines prepared only 43 were selected by using dot blot assay, while 7 out of the 43 were selected by using DACA. From the 7 selected clones, clone (13-2) of the highest bound dpm in DACA was used for limiting dilution assay. The pattern of immune-reactivity of 7 out of 8 of the diluted cultures were very similar and showed two signal bands of 65 and 42 kDa, as revealed by western blot analysis. From which, a single clone designated (13-2-23) of the highest bound dpm in DACA was reacted with a single protein band of 42 kDa, suggesting its probable specificity to the extracellular binding domain of mEcR. This work will pave the way for more understanding of the molecular mechanism of ecdysteroid rapid actions during PCD of ASGs in Bombyx mori.