Diagnostic Performance of Competitive ELISA and Western Blot Methods for the Detection of Antibodies against Theileria equi and Babesia caballi

Abstract

Theileria equi (T. equi) and Babesia caballi (B. caballi) are the causative pathogens of Equine piroplasmosis (EP), a disease that has brought huge economic losses and great restrictions to the global equine industry. Rapid and accurate diagnostic methods are critical for the effective monitoring of the disease. In this study, we developed novel competitive ELISA methods and western blot assays based on the EMA1 or Bc48 proteins to detect antibodies against T. equi or B. caballi, respectively. In the novel cELISA, horseradish peroxidase (HRP)-labeled monoclonal antibodies are used in place of enzyme-conjugated secondary antibodies, in order to speed up the entire procedure. These methods have high sensitivity and no cross-reactivity with antibodies against other equine diseases. In the newly developed western blot assays, we optimized the dilution of T. equi or B. caballi positive serum samples to 1:200. Compared with the commercially available kit, both the novel cELISA assay and the western blot assay showed high coincidence rates in detecting antibodies against T. equi and B. caballi. Taken together, the novel cELISA and the western blot assays for detecting antibodies against T. equi or B. caballi have the potential to rapidly test for T. equi or B. caballi and to contribute to the surveillance and control of this disease.

Diagnostic performance of a rapid immunochromatographic test for the simultaneous detection of antibodies to Theileria equi and Babesia caballi in horses and donkeys

“…ELISA kits for equine piroplasmosis are available commercially but require multiple steps, including dilution of serum, incubation of primary antibody, and incubation of secondary antibody, as well as washing at each step. Therefore, a competitive ELISA with an improved protocol was developed by introducing a direct HRP conjugation [17]. The coincidence rates between this novel ELISA and the commercial cELISA assays were 97.5% for T. equi and 98% for B. caballi.…”

“…Plates were read on a portable microplate reader at an optical density of 450 mm (The Absorbance 96, Byonoy GmbH, Hamburg, Germany). The percentage inhibition equal to or greater than 40% identi ed an antibody-positive sample, and the sample was regarded as antibody-negative below < 40% [17].…”

“…As a next step, a novel competitive ELISA was developed using recombinant proteins EMA1 and BC48, wherein the corresponding monoclonal antibodies were directly conjugated to horseradish peroxidase (HRP) [17]. The HRP-labelled monoclonal antibodies instead of enzyme-conjugated secondary antibodies signi cantly shortened the ELISA procedure for diagnosing antibodies against both parasites in equine blood samples.…”

“…This novel cELISA protocol was also compared with the commercially available cELISA kit from VMRD (Pullman, WA, USA). The coincidence rates between the novel ELISA and the commercial cELISA assays were based on 200 equine serum samples collected from horses in Inner Mongolia, 97.5% for T.equi and 98% for B.caballi [17]. This paper discusses the diagnostic performance of the rapid immunochromatographic test for the simultaneous detection of antibodies to T.equi and B. caballi in horses and donkeys.…”

Development of a promising antigenic cocktail for the global detection of Babesia caballi in horse by ELISA

“…This indicated that the host has strong humoral immunity against these proteins. Bc48 is one rhoptry protein of merozoites of B. caballi, which was previously evaluated as a promising antigen for the serological detection of antibodies of B. caballi [12]. Moreover, BC134 protein was developed and proved to be highly specific for the detection of B. caballispecific equine antibodies [13].…”