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Evaluation and improvement of an immunoenzimatic assay (ELISA) for diagnosis of bovine anaplasmosis, using recombinant MSP5 as antigen

Abstract:

Anaplasmosis, is a disease produced by Anaplasma marginale widely distributed in Venezuela, causing negative effects on the health and productivity of bovine herds. Until now, 6 constitutive Anaplasma marginals Mayor Surface Proteins (MSPs) have been characterized, including MSP5 which appears to be an excellent polypeptide for the diagnosis of this disease since it is highly conserved and immunogenic. This has motivated its cloning and insertion into a plasmid in E. coli and the use of the purified antigen in immunoenzymatic assays. However, subsequent indicated that E. coli recombinant proteins, that copurify with MSP5, interfere with the ELISA giving rise to false positives. In the present study, it was accomplished the standardization of an indirect ELISA, using MSP5 as the antigen and it was also diminishing the non-specific unions to the contaminating proteins of E. coli by adding anti-E. coli rabbit serum that blocks the epitopes of these proteins. With the use of a double entry contingency table, the parameters of validation of the ELISA were determined, comparing it to the acridine orange-ethidium bromide technique. The result indicate that the improve MSP5 indirect ELISA has a 96.1% sensitivity, 9% specificity and a pbkp_redictive value of 88.6%. It was also studied a bovine population of 48 cattle from the Experimental Station "La Iguana" (Guaa7acute;rico State), using both techniques, obtaining a seroprevalence of 93.7% with ELISA and a prevalence of 54.1% with orange acridine-ethidium bromide. These results show that the blockage of the contaminating E. coli protein epitopes using an anti-E. coli rabbit serum permits the diminishment of false negatives when using recombinant proteins.