Improvement of western blot test specificity for detecting equine serum antibodies to Sarcocystis neurona.

This research endeavoured to improve the specificity of the Western blot test, which is used to detect antibodies of Sarcocystis neurona, a parasite causing equine neurological disease. The researchers used bovine antibodies to block proteins unlinked to S.neurona and deemed the 30- and 16-kD bands as positive test markers, which led to a high specificity rate in their tests.

Research Objectives and Methodology

• The main objective was to develop stringent criteria for a positive Western blot test result to detect antibodies to Sarcocystis neurona in equine serum.
• The study assessed the ability of bovine antibodies to Sarcocystis cruzi to act as a blocking agent to minimize false-positive results.
• The methodology involved harvesting Sarcocystis neurona merozoites from equine dermal cell culture, heat denaturing, and separating the proteins via sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
• These separated proteins were transferred to polyvinylidene fluoride membranes and blocked in a mixture of 1% bovine serum albumin and 0.5% Tween-Tris-buffered saline.
• Serum samples from horses with confirmed S. neurona infections and horses without infections were tested using Western blot.

Findings and Conclusion

• Reactivity to the 62-, 30-, 16-, 13-, 11-, 10.5-, and 10-kD bands was observed in horses from both infection and non-infections groups.
• The tests were repeated by treating blots with bovine S. cruzi antibodies before loading the equine samples. After this modification, positive infection status was significantly associated with reactivity to the 30- and 16-kD bands (P<0.001, Fisher's exact test).
• When these new criteria were applied, the Western blot test had a sample sensitivity of 100% and sample specificity of 98%.
• Thus, it was concluded that the specificity of the Western blot test for detecting S. neurona in equine serum is greatly improved by blocking proteins irrelevant to the parasite and using reactivity to the 30- and 16-kD bands as indicators of a positive test.