Development and validation of an indirect enzyme immunoassay for detection of antibody to Anaplasma marginale in bovine sera.

An indirect enzyme immunoassay (IELISA) for detection of bovine antibody activity to Anaplasma marginale was developed. This assay used a crude antigen prepared from erythrocytes of infected calves, immobilized in a polystyrene matrix and a mouse monoclonal antibody to bovine IgG1, conjugated with horseradish peroxidase. Negative sera (n = 1842) were tested and the diagnostic specificity was 98.4 +/- 0.6% before retesting 29 positive samples. After retesting, eight samples remained positive and the specificity was calculated to be 99.6 +/- 0.3%. The diagnostic sensitivity, using 831 serum samples collected from naturally or experimentally infected cattle in Argentina, 370 from Mexico and 525 sera from experimentally vaccinated or infected cattle from Texas, was 87.3 +/- 1.6%.