Detection of the Bacillus thuringiensis serovar japonensis strain Buibui protoxin with enzyme-linked immunosorbent assay and its application to detection of the protoxin in soil.

Abstract

Antiserum raised against protoxin from parasporal crystals of Bacillus thuringiensis serovar japonensis strain Buibui was used to develop a method for detecting its presence and assessing its specificity against several other B. thuringiensis strains. Three kinds of ELISA reactions, i.e., avidin-biotin complex system (ABC-ELISA), antigen captured (AC-ELISA) and double antibody sandwich (DAS-ELISA), were equally effective in determining the concentration of the Buibui protoxin. The AC-ELISA was employed for further experiments because it required the least labor. Protoxins from B. thuringiensis serovar kurstaki HD-1, HD-73, strain KB500 and var. tenebrionis did not react with the Buibui antibody. When crystals from strain Buibui were placed in Gley soils (I and II), Regosol or sea sand and then solubilized with an alkali treatment, the concentration of the protoxins could be determined using the ELISA(s). The protoxins, however, were strongly adsorbed to soil and recover was only 7%. The amount adsorbed did not appear to depend on the clay content or cation exchange capacity of the soils. When the crystals added to the soils were separated prior to the solubilization, the recovery of the protoxins reached 80% in Gley soil I and 60% in Gley soil II and Regosol. This separation method is considered to be applicable to various soil samples as long as B. thuringiensis pesticides are applied as a crystal formulation.