Design and applications of sensitive enzyme immunoassays specific for clostridial enoate reductases

Rabbit antisera raised against purified enoate reductase from Clostridium tyrobutyricum (DSM 1460) and horse-radish peroxidase-conjugated staphylococcal protein A or anti-rabbit immunoglobulin G, respectively, were used to develop enzyme immunoassays. Sensitivity limits of the assay are about 250 pg antigen if the enzyme immunoassays are performed on membrane filters and examined visually, and 20 pg for tests in aqueous solution with spectrophotometrical evaluation. The procedures were applied for dot and Western blots as well as colony lifts. Immunological distances between enoate reductases from different clostridia were determined and the amounts of antigen present in bacterial crude extracts were estimated. In crude extracts of C. thermoaceticum a protein of approximately the size of the enoate reductase and its subunits from C. tyrobutyricum was immunologically detected. Gel filtration chromatography of identically pretreated crude extracts from C. thermoaceticum and C. tyrobutyricum produced immunological signals at similar molecular weights and revealed a lesser tendency of the presumed thermophilic enoate reductase from C. thermoaceticum to disintegrate into its subunits and fragments as compared to its mesophilic counterpart.