Determination of the absence or presence of
Disclosed is a method for the determination of the absence or presence of bacillus cereus enterotoxin in a protein sample via a western blot.
1. A method for determining the absence or presence of one or more components of Bacillus Diarrheal Enterotoxin (BDE) in a pancreatin Active Pharmaceutical Ingredient (API) sample comprising separating the one or more components of BDE from the pancreatin API sample by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and identifying one or more components of BDE by using at least one probe antibody specific for at least one or more components of BDE; wherein the one or more components of BDE are selected from the group consisting of Hemolysin BL (Hbl), Nonhemolytic Enterotoxin (Nhe), Cytotoxin K (CytK) and Enterotoxin T (BcET).
2. The method according to claim 1 wherein identifying the one or more components further comprises
transferring the separated components to a nitrocellulose membrane and sequentially exposing the components to solutions containing primary antibodies, and
soaking the membrane in a substrate solution to develop a colored reaction of the bound primary antibody to the component, resulting in identifying the component as a visible band on the membrane.
3. The method according to claim 2 wherein the one or more components is Nhe.
4. The method according to claim 3 wherein the Nhe component is represented by a NheA subunit.
5. The method according to claim 1 wherein the ratio of sodium dodecyl sulfate to pancreatin sample in the SDS-PAGE is about 1.4 to 1.
6. The method according to claim 2 wherein the SDS-PAGE is run with a matrix positive control and a blank positive control to determine the detection of BDE.
7. The method according to claim 1 wherein the SDS-PAGE is run on a Bis-Tris or Tris-Glycine SDS-polyacrylamide gel.
8. The method according to claim 2 wherein the components are exposed to solutions containing secondary antibodies after exposure to the primary antibodies.
9. The method according to claim 6 wherein the membrane is stained to detect the primary antibody bound component.
10. The method according to claim 9 wherein the stain is selected from the group consisting of chloronapthol and diaminobenzidine, nitro-blue tetrazolium and 5-bromo-4-chloro-3′-indolphosphate.