Parasitol

Parasitol. 148: 137C143 [PubMed] [Google Scholar] 27. this cELISA were reevaluated and adjusted when necessary, and a cutoff value was determined by receiver operator characteristic (ROC) curve analysis of a total of 357 bovine sera of known reactivity, as assessed by indirect immunofluorescence assay (IFAT). The established rMSA-2c cELISA demonstrated a specificity of 98% and a sensitivity of 96.2%. An additional set of 303 field bovine sera from regions where ticks are endemic and tick-free regions of Argentina was tested by both rMSA-2c cELISA and IFAT, and the results were shown to be in very good agreement (kappa index, 0.8325). The performance shown by rMSA-2c cELISA in the detection of severely limits cattle breeding in vast tropical and subtropical areas of the world, where its tick vectors, belonging to the family antibodies is periodically performed in regions of enzootic instability to decide the application of control measures, such as vaccination with live attenuated vaccines (2, 24, 25). Merozoite surface antigen 2c (MSA-2c) is one of the five variable merozoite surface antigens (VMSAs) that are encoded in the same genomic region (17, 34). Antibodies recognizing recombinant forms of all VMSA members (MSA-1, MSA-2a1, MSA-2a2, MSA-2b, and MSA-2c) have been demonstrated in calves infected with a homologous Mexican strain of (17, 34). MSA-2c is a species-specific, immunodominant antigen and the most conserved member of this family, showing very high amino acid sequence identity among strains from Argentina, the United States, Mexico, and Australia (12, 19, Diethylstilbestrol 38). These features encouraged the use of MSA-2c for the development of serological tests, like an indirect enzyme-linked immunosorbent assay (ELISA) and a rapid immunochromatographic diagnostic test (6, 26). A competitive ELISA (cELISA) is an adequate serological tool for the epidemiological surveillance of the spread of bovine babesiosis, as it can be easily standardized, is less laborious and less time-consuming than the traditionally used indirect immunofluorescence assay (IFAT) (immunofluorescence antibody test), and, in addition, has the potential to display higher specificity than an indirect ELISA. In a previous work, a monoclonal antibody (MAb) against recombinant MSA-2c (rMSA-2c) was generated which showed competitive binding for this antigen with antisera of in Argentina (22, 32). Esr1 MATERIALS AND METHODS Production and purification of recombinant antigen and monoclonal antibody. Recombinant expression of MSA-2c with an N-terminal histidine tag and subsequent purification by affinity chromatography in Ni-agarose was carried out as described previously (13, 38). Validation and quality assessment of expression were analyzed by Western blotting. To Diethylstilbestrol this end, a sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis (SDS-PAGE) was run, protein transfer was carried out, and the resulting blot was probed using either an anti-histidine antibody (GE Healthcare, Chalfont, United Kingdom) or the MAb H9P2C2 (20 g/ml) as the primary antibody (see below). Anti-mouse alkaline phosphatase-conjugated IgG (KPL, Gaithersburg, MD; 1/1,500) was used as the secondary antibody, and immunodetection was carried out using nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP) (Promega, Fitchburg, WI) as the substrate. Quantity assessment of rMSA-2c expression was carried out by comparison of band sizes with known amounts of bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO) after SDS-PAGE and Coomassie blue staining. The H9P2C2 hybridoma cell line producing the anti-rMSA-2c MAb H9P2C2 was cultured (13). Subsequently, the culture supernatant was collected, and the MAb was purified by affinity chromatography using the Affi-Gel Protein A MAPS II Kit (Bio-Rad, Hercules, CA). After protein quantification with a BCA colorimetric kit (Pierce, Rockford, IL), the Diethylstilbestrol MAb was aliquoted and stored at ?20C until it was used. Serum samples. Bovine blood samples were aseptically collected without anticoagulants from different geographical regions of Argentina as indicated below. Serum was separated by centrifugation, aliquoted, and stored at ?20C until it was used. For Diethylstilbestrol calculation of the cutoff value Diethylstilbestrol by receiver operator characteristic (ROC) analysis, a set of known-positive and known-negative sera was used. The known-positive sera (= 104) originated from (i) animals from regions of endemicity in the provinces of Salta and Chaco that tested positive by diagnostic nested PCR, as reported by Figueroa et al. (15) (= 27), and (ii) experimentally = 77). In each case, establishment of infection was verified by observation of = 253) originated from (i) animals from tick-free regions (= 200), (ii) animals that had been experimentally infected with (= 28) after confirmation of their hemoparasite-free status by IFAT and nested PCR (18), and (iii) animals from tick-free regions that were naturally infected.