DOT-BLOT ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR MONITORING THE FATE OFINSECTICIDAL TOXINS FROM BACILLUS-THURINGIENSIS IN SOIL

The release of transgenic plants and microorganisms expressing truncat ed genes from Bacillus thuringiensis that code for active insecticidal toxins rather than for the inactive protoxins could result in the acc umulation of these active proteins in soil, especially when bound on c lay minerals and other soil particles. To monitor the fate of these to xins in soil, a dot blot enzyme-linked immunosorbent assay (ELISA) tha t detects free and particle-bound toxins from B. thuringiensis subsp. kurstaki and subsp. tenebrionis was developed. The lower limit of dete ction of the toxins, either free or adsorbed or bound on the clay mine rals montmorillonite (M) or kaolinite (K) or on the clay particle-size fraction separated from soil (by sedimentation according to Stokes' L aw), was approximately 3 ng. Antibodies (Ab) to the toxins from B. thu ringiensis subsp. kurstaki and from B. thuringiensis subsp. thuringien sis were raised in goats and rabbits, respectively, and each Ah was re ndered specific by adsorption onto CNBr-activated Sepharose coupled wi th the other toxin. The preadsorbed Ab were specific for the toxins fr om both subspecies, both free and bound on M, K, or the clay-particle- size fraction of soil. The toxins that were added to sterile and nonst erile soil amended with M or K or not amended were detected on the cla y-particle-size fraction of the soil after various periods of incubati on by the dot blot ELISA. No toxins were detected on the silt- and san d-particle-size fractions. Each dot blot, containing various amounts o f toxins and/or clays, was applied to a polyvinylidene difluoride memb rane in a dot blot vacuum system. The toxins were still detectable on the clay-particle-size fraction of nonsterile soil after 40 days. This agreed with preliminary results of other studies in this laboratory t hat when these toxins bind on clay minerals, they become resistant to utilization by microorganisms.