Examples were tested in duplicate in serial dilutions ranging from 1:2 to 1 1:200. Ultimately, can invade the central nervous system of the infected horse, causing focal or multifocal inflammation and EPM. infection in horses is assessed by the detection of antibodies against the parasite in either the serum or cerebrospinal fluid (CSF); however, not all horses that seroconvert to will develop EPM (9, 27). The seroprevalence of infection in horses in the United States ranges between 0 and 89.2%, depending upon geographic locale (1-3, 10, 34, 37, 39, 40). In contrast, the incidence of clinical EPM has been estimated at <1% (28). It is not well understood what factors are responsible for the dichotomy between inapparent infection and clinical disease, but this ambiguity creates a major hindrance to EPM diagnosis and disease control. Current technologies for detecting antibodies in equine serum and CSF samples include Western blotting (17), a modified version of Western blotting (35), an direct-agglutination test (SAT) (25), GSK189254A and an indirect fluorescent-antibody test (5). Each of these current serodiagnostic assays utilizes complete merozoite preparations as the antigen source, which has several drawbacks. Specifically, propagation of parasite cultures is relatively time-consuming and expensive, and the use of whole-parasite preparations can increase the risk of false-positive results due to cross-reactivity with closely related pathogens, such as (11, 38). Additionally, the current assays are not very amenable to quantitation, and their results can be subject to interpretation (16, 32). Given these shortcomings, a detailed and in-depth characterization of equine humoral responses to infection is not feasible with the existing serologic tests. Four related surface antigens have been identified in merozoites, and these have been designated SnSAG1, SnSAG2, SnSAG3, and SnSAG4 (13, 20). To develop better tools for analyzing antibody responses to infection, antibody capture enzyme-linked immunosorbent assays (ELISAs) were designed to utilize recombinant forms of the four surface antigens (rSnSAGs). Comparison of the rSnSAG ELISAs with Western blot analysis of merozoites confirmed that three of these assays are highly accurate and reliable. These ELISAs will serve as valuable tools for the SMARCA4 evaluation of the equine humoral immune response to infection, which may in turn allow discrimination between horses with EPM and those with asymptomatic infections. MATERIALS AND METHODS Parasite GSK189254A culture. The SN3 strain of and the Oregon strain of (7, 18) were maintained by serial passage in bovine turbinate cell monolayers. Upon lysis of the host cell monolayer, zoites were passed twice through 20-gauge (20-G), 22-G, GSK189254A and 25-G needles and filtered through a 3.0-m Nucleopore (Whatman) membrane to remove host cell debris. The harvested parasites were counted with a hemocytometer, washed with phosphate-buffered saline (PBS), and stored at ?20C. Recombinant-protein preparation. The four SnSAGs were expressed as recombinant proteins and purified by nickel column chromatography, as described previously (20). The concentration of the purified protein was determined by a colorimetric assay (Coomassie Plus Protein Assay Reagent; Pierce). Purified rSnSAG1, rSnSAG2, rSnSAG3, and rSnSAG4 were each diluted in elute buffer (0.5 M NaCl and 20 mM Tris-HCl) without urea to final protein concentrations of 8.15 g/ml, 23.0 g/ml, 14.56 g/ml, and 10.3 g/ml, respectively. Serum and CSF samples. GSK189254A The positive control serum samples were from two clinically affected horses that had histologically confirmed EPM. The negative control sample for all assays was a preinfection serum sample from a weanling used in an infection trial (14). Thirty-six equine sera submitted to Equine Biodiagnostics (EBI)/IDEXX for serology testing were used for standardization of the rSnSAG ELISAs. These samples had been classified as positive or negative in Western blots using criteria established by EBI/IDEXX (9, 10). Samples from 27 EPM-confirmed horses were from a collection compiled at the University of Kentucky Gluck Equine Research Center. All cases were confirmed by histological examination of central nervous system tissues for the presence of lesions consistent with EPM, and prior Western blot analyses had demonstrated that all 27 horses had CSF antibodies against challenge trial (38) were used to examine assay cross-reactivity. An positive control serum sample was taken from a horse that was naturally infected with (30) and was provided by A. E. Marsh, Ohio State University. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting..