Development of monoclonal antibody-based sandwich ELISA for the detection of nuclear polyhedra of nuclear polyhedrosis virus infection in Bombyx mori L.

Abstract

Murine monoclonal antibodies (MCAs) were produced against Borrelina bombycis nuclear polyhedra. In ELISA all five MCAs reacted with nuclear polyhedra of B. bombycis and to a variable extent with other strains of nuclear polyhedra, namely Amsaeta olbistriga, Heliothis armigera, and Spodoptera litura . These were devoid of reactivity with normal hemolymph proteins, Serratia marcescens, Nosema bombycis, group A streptococci, Staphylococcus aureus, and Escherichia coli, except Bacillus thuringiensis. However, MA-321 showed a low degree of reaction with B. thuringiensis. In Western blots, these recognized antigens of 31 (polyhedrin protein) and 67 kDa of nuclear polyhedra (MA-321 showed reduced binding). The MCAs specifically recognized nuclear polyhedra in either infected Bran cells or mid gut of Bombyx mori larvae. In sandwich enzyme immunoassay employing purified rabbit polyclonal antibodies and MA-321 as low as 100 nuclear polyhedra could be detected. The results obtained by sandwich ELISA on hemolymph samples of infected as well as normal larvae collected from field revealed 100% correlation with results by microscopic examination. Hemolymph samples collected on various days from II instar larvae infected in the laboratory revealed that the sandwich ELISA could detect infection as early as 96 hr after infection. These antibodies could be useful for detection of nuclear polyhedra infection at early stages, i.e., II to III instar of larvae.