Dot Blot Enzyme-Linked Immunosorbent Assay for Monitoring the Fate of Insecticidal Toxins from Bacillus thuringiensis in Soil

Abstract

The release of transgenic plants and microorganisms expressing truncated genes from Bacillus thuringiensis that code for active insecticidal toxins rather than for the inactive protoxins could result in the accumulation of these active proteins in soil, especially when bound on clay minerals and other soil particles. To monitor the fate of these toxins in soil, a dot blot enzyme-linked immunosorbent assay (ELISA) that detects free and particle-bound toxins from B. thuringiensis subsp. kurstaki and subsp. tenebrionis was developed. The lower limit of detection of the toxins, either free or adsorbed or bound on the clay minerals montmorillonite (M) or kaolinite (K) or on the clay-particle-size fraction separated from soil (by sedimentation according to Stokes' Law), was approximately 3 ng. Antibodies (Ab) to the toxins from B. thuringiensis subsp. kurstaki and from B. thuringiensis subsp. thuringiensis were raised in goats and rabbits, respectively, and each Ab was rendered specific by adsorption onto CNBr-activated Sepharose coupled with the other toxin. The preadsorbed Ab were specific for the toxins from 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 nonsterile soil amended with M or K or not amended were detected on the clay-particle-size fraction of the soil after various periods of incubation by the dot blot ELISA. No toxins were detected on the silt- and sand-particle-size fractions. Each dot blot, containing various amounts of toxins and/or clays, was applied to a polyvinylidene difluoride membrane 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 that when these toxins bind on clay minerals, they become resistant to utilization by microorganisms.