Method of extraction of an enzyme from plant or animal tissue
This invention provides a method for the extraction and detection of a peptide from transgenic plant tissues wherein a non-immunogenic solubility-promoting compound is used to release the enzyme into the solution fraction during the purification process. In some embodiments, this invention provides a method for the extraction and detection of the enzyme Amy797E, which is a heterologous thermo-tolerant α-amylase, from the tissues of corn event 3272 using a non-immunogenic amylase during the purification process. This invention allows for a limit of detection of 1:1000 of Amy797E in an enzyme-linked immunosorbent assay (ELISA).
1. An improved method of extraction and detection of the enzyme Amy797E from a biological sample, wherein the biological sample comprises maize transgenic event 3272, comprising:
a) homogenizing the biological sample in the presence of extraction buffer and a solubility-promoting compound, wherein the solubility-promoting compound is an amylolytic enzyme;
b) incubating the sample to permit solubilization of the enzyme Amy797E; and
c) centrifuging the sample to separate the soluble and insoluble fractions, wherein the enzyme Amy797E fractionates into the soluble fraction, and
d) detecting the enzyme Amy797E in the soluble fraction by immunoassay,
wherein the enzyme Amy797E extracted from the biological sample can be detected with a limit of detection of no greater than 1:1000 maize kernels.