Competitive enzyme-linked immunosorbent assay based on a rhoptry-associated protein 1 epitope specifically identifies Babesia bovis-infected cattle

Abstract

The competitive enzyme-linked immunosorbent assay (cELISA) format has proven to be an accurate, reliable, easily standardized, and high-throughput method for detecting hemoparasite infections. In the present study, a species-specific, broadly conserved, and tandemly repeated B-cell epitope within the C terminus of the rhoptry-associated protein 1 of the hemoparasite Babesia bovis was cloned and expressed as a histidine-tagged thioredoxin fusion peptide and used as antigen in a cELISA. The assay was optimized with defined negative and positive bovine sera, where positive sera inhibited the binding of the epitope-specific monoclonal antibody BABB75A4. The cELISA accurately differentiated animals with B. bovis-specific antibodies from uninfected animals and from animals with antibodies against other tick-borne hemoparasites (98.7% specificity). In addition, B. bovis-specific sera from Australia, Argentina, Bolivia, Puerto Rico, and Morocco inhibited the binding of BABB75A4, confirming conservation of the epitope. The assay first detected experimentally infected animals between 13 and 17 days postinfection, and with sera from naturally infected carrier cattle, was comparable to indirect immunofluorescence (98.3% concordance). The assay appears to have the characteristics necessary for an epidemiologic and disease surveillance tool.

FIG. 1.

Western blot confirming the presence of the BABB75A4 epitope within the C-terminal RAP-1 recombinant construct rRCT. (A) MAb BABB75A4 was used as the probe. (B) MAb 23.53.156 was used as the probe. Lanes: 1, full-length rRAP-1 fusion protein; 2, rRCT fusion protein; 3, rRNT fusion protein.

FIG. 2.

ELISA block titration with the indicated concentrations of rRCT and MAb BABB75A4.

FIG. 3.

Effects of temperature and SDS treatment on the stability of rRCT. (A) Purified rRCT dried onto plates and stored for 48 h at the indicated temperatures before reaction with MAb BABB75A4. (B) Purified rRCT dried onto plates and used in a competitive format with MAb BABB75A4 and a known positive control bovine serum immediately (Fresh Antigen), stored at 50°C for 1 month without SDS treatment, and stored at 50°C for 1 month after SDS treatment.

FIG. 4.

Frequency distribution of percent inhibition of MAb BABB75A4 binding in the cELISA by samples from known B. bovis-infected and uninfected animals.

FIG. 5.

Kinetics of antibody detection by cELISA. (A) Samples obtained from a group of four cattle before and daily through 23 days after experimental intravenous inoculation of the T₂Bo isolate of B. bovis. (B) Samples obtained from a group of five cattle before and every other day beginning on day 13 and continuing through day 98 after experimental intravenous inoculation of the T₂Bo isolate of B. bovis. The horizontal dashed line indicates the threshold inhibition of 40%, above which samples are considered positive.