Desenvolvimento e padronização de um teste ELISA para detecção de anticorpos (IgG) anti-Anaplasma marginale em bovinos submetidos ao processo de premunição

Abstract

An ELISA test using crude antigen was developed for detecting antibodies against A. marginale in bovine sera. The antigen was produced by inoculation of a splenectomized calf with approximately 5xl08 parasited erythrocytes stored in liquid nitrogen. During the peak of parasitaemia, blood was collected and after centrifugation the plasma and buffy coat were discarded. In half of the pellet the leukocytes were removed by filtration through a column containing sea sand. One part of the material was stored at 70°C with DMSO 30% within Phosphate Buffer Saline (PBS) and another was lysed with NH4Cl 0,9% and stored at -20°C. The other half of the pellet was divided into two parts. One part was stored at 70°C with DMSO 30% within PBS and another was lysedwith NH4C1 0,9% and stored at -20°C. Later all preparations were disrupted by sonication and the sediments were solubilized with Triton X-l0O. After each procedure, electron micrograph of the antigenic preparations were carried out. Typical Anaplasmal structures were seen only in the antigenic preparation from which the leukocytes had been removed and the erythrocytes had been stored with DMSO 30%. Optimal dilutions of the antigen, positive and negative reference sera were determined by chequerboard titrations and the cut off value was determined by the mean reading of 39 negative reference sera increased by two standard desviations. The ELISA sistem showed higher specificity (94,87%) than the IFAT (71,79%).