Development of an antigen specific colloidal gold immunochromatographic assay for detection of antibody to M. wenyonii in bovine sera.

The goal of this research was to develop a colloidal gold immunochromatographic strip test for detection of antibody to Mycoplasma wenyonii (M. wenyonii) in bovine using specific antigen. M. wenyonii was isolated from blood samples from the spontaneously infected cattle in Hebei province, China. Suspensions of the M. wenyonii antigenic proteins were prepared by freeze-thaw cycles and ultrasonication. Candidate antigens were screened with sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The specific bands of the most antigenic proteins were excised from the gel and were purified by using a gel extraction kit. A colloidal gold immunochromatographic assay using the purified specific proteins as the coating antigen (sp-GICA) was developed for detection of antibody to M. wenyonii. Blood samples from cows in the field were tested for antibody to M. wenyonii by the sp-GICA strip and enzyme-linked immunosorbent assay (ELISA) simultaneously to compare the specificity, sensitivity and accuracy. The results showed that the specific proteins bands with sufficient immunoreactivity have been identified. The apparent molecular weights of the proteins were 115 kDa and 60 kDa, respectively. The stability and reproducibility were quite excellent after the storage of the strip at room temperature for 5 months. This sp-GICA showed 95.48% (148/155), 92.86% (39/42) and 94.92% (187/197) in terms of specificity, sensitivity and accuracy compared to ELISA. The sp-GICA described here shows excellent agreement with ELISA and it is shown to be a simple, convenient, specific and highly sensitive assay for detection of serum antibodies to M. wenyonii.