tion was used as a control in the SDS-PAGE assessment; this offers useful information about potential binding to non-Bt proteins. A set of monoclonal (or polyclonal) antibodies with less cross-reactivity might prove useful in future tests. In general, good analytical tools to monitor Bt toxins and their concentrations are necessary for reliable human health and environmental testing.
Glycosylation appeared to be absent in all the Bt proteins on the basis of standard tests, and this result seems reasonably straightforward.
Because the DNA sequences of the Bt proteins were modified substantially in order to increase plant expression, there is always a question of whether the final amino acid sequences are identical (that is, cloning artifacts could lead to some amino acid substitutions). The sequencing of some of the terminal and internal amino acids cannot itself prove identity; however, combined with results of other tests, the finding that these sequences were identical offers additional support of equivalence.
Tests showing similar toxicity of the proteins to one or two insect species offer some support of equivalence. If such tests demonstrated substantial differences in toxicity, there would be strong evidence of nonequivalence. The finding of similar toxicity, however, does not prove equivalence. In the current studies, when two insect species were used, they were taxonomically closely related. Future tests with less related species would be useful. There is a question of how many biochemically distinct insect species should be tested in this kind of study.
The digestibility tests for each of the toxins were similar and reasonably straight forward. Microbially-produced Bt toxins were incubated with simulated gastric fluids and separately with simulated intestinal fluids. The gastric fluids caused the proteins to lose their biological activity against insect species and to lose their potential for binding with an antibody used for protein detection. In contrast, the intestinal fluids only truncated the full-length proteins to their active forms and did not affect the pretruncated proteins. Because the proteins reach the gastric fluids first, the lack of degradation by intestinal fluids would be unimportant unless an individual lacked active gastric digestion.
Mice were given oral doses of the microbially-produced Bt toxins about 100-1000 times the acute dose that they would encounter in consuming one-tenth of their body weight in plant material. Ten 7-week-old