Double-antibody sandwich enzyme-linked immunosorbent assay for quantitation of endoglucanase I of Trichoderma reesei.
A sensitive and specific enzyme-liked immunosorbent assay for endoglucanase I (EG-I) has been developed. The monoclonal antibody a-EG-I 2, directed against an epitope on the core part of the enzyme, was used to capture the antigen in microtiter plate wells. A second, polyclonal antibody against the enzyme was then used to detect and quantitate the bound antigen. The test was specific for EG-I; neither endoglucanase II nor cellobiohydrolase I or II interfered. As little as 20 pg of EG-I protein could be detected. The coefficients of variation were 3.8% within plates and 6% between plates for a diluted Trichoderma reesei culture supernatant that contained 31 ng of EG-I per ml. Binding of the antigen to the monoclonal antibody was pH dependent and restricted to values between pH 6.5 and 10.5 with a maximum around pH 9. Standard solutions of EG-I were very stable at concentrations as low as 5 ng/ml when prepared in buffer that contained 1% bovine serum albumin and that was stored at -20 degrees C. After 37 weeks the antigenicity was still 97%. With this test it was possible to monitor the production of EG-I in a cellulase-producing strain of T. reesei and to demonstrate the apparent absence of the enzyme in a strain with the eglI gene deleted.
Bühler
R
R
Research Laboratories, Alko Ltd., Helsinki, Finland.
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D043366:Cellulose 1,4-beta-Cellobiosidase; D003470:Culture Media; D004795:Enzyme Stability; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D005656:Fungal Proteins / Q000032:analysis* / Q000276:immunology / Q000592:standards; D006026:Glycoside Hydrolases / Q000032:analysis* / Q000276:immunology / Q000592:standards; D011817:Rabbits; D012015:Reference Standards; D012996:Solutions / Q000592:standards; D014242:Trichoderma / Q000201:enzymology* / Q000254:growth & development
D000911:Antibodies, Monoclonal; D003470:Culture Media; D005656:Fungal Proteins; D012996:Solutions; D006026:Glycoside Hydrolases; D043366:Cellulose 1,4-beta-Cellobiosidase