Resumo

Corynebacterium pseudotuberculosis, a gram-positive bacillus is the etiologic agent of chronic infections in several different species of mammals. Caseous lymphadenitis is one of the most important of those infections, leading to the appearance of granulomas in superficial and deep lymph nodes of goats and sheep. The aim of this study was to develop and standardize an indirect ELISA for detecting antibodies against the glycoprotein portion of cell membrane of C. pseudotuberculosis in mice sera. For the production of antigen, a wild strain of C. pseudotuberculosis (VD57) was grown in BHI (Brain-heart infusion) at 37°C for 72 hours; the bacterial mass was collected after centrifugation, washed in phosphate buffered saline (PBS) and sonicated at 60 Hz. Material was subjected to extraction with chloroform-methanol-water solution (5/10/4, v/v/v) in order to extracted the lipid fraction. Pellet was dried under N₂ gas and treated with a 9% solution of butanol. Supernatants were collected and evaporated. Protein concentration was determined by the Lowry method (Bio-Rad Laboratories, Richmond, California) using bovine serum albumin as standard. ELISA was standardized by the following parameters: concentration of antigen (0,5μg, 1μg, 2μg, 4μg), serum dilutions (1:25, 1:50, 1:100, 1:200 and 1:400) and conjugate dilution (1:5.000, 1:10.000, 1:20.000 and 1:40.000). All the steps were incubated for 15 minutes at room temperature. Optimum concentration of antigen was 2μg and the best sera and conjugate dilutions were 1:25 and 1:5.000, respectively. After standardization, the test was used in sera of 4 mice (Balb/c) immunized with glycoprotein antigen, and was able to detect the presence of specific antibodies against the antigen. Detection of antibodies in mice infected by a wild strain of C. pseudotuberculosis and evaluation of sensitivity and specificity of the ELISA test are still under study.