Improved Enzyme-Linked Immunosorbent Assay Using C-Terminal Truncated Recombinant Antigens of Babesia bovis Rhoptry-Associated Protein-1 for Detection of Specific Antibodies

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan.
Journal of Clinical Microbiology (Impact Factor: 3.99). 05/2004; 42(4):1601-4. DOI: 10.1128/JCM.42.4.1601-1604.2004
Source: PubMed


An enzyme-linked immunosorbent assay (ELISA) based on a recombinant rhoptry-associated protein-1 (RAP-1) of Babesia bovis has been previously developed, but it was imperfect because some cross-reactions were still present in Babesia bigemina-infected bovine sera. To improve its accuracy for the specific detection of the antibodies to B. bovis, we constructed three C-terminal truncated recombinant antigens of the RAP-1—rCT1 (amino acids [aa] 301 to 408), rCT2 (aa
388 to 490), and rCT3 (aa 466 to 565)—by using a baculovirus expression system and evaluated their diagnostic potentials using
ELISA. rCT1 and rCT2 were better diagnostic antigens in their sensitivities and diagnostic efficiencies than rCT3, although
none of the recombinant antigens showed any cross-reactivity to B. bigemina-infected bovine sera. These results confirmed that the N-terminal 300-aa region caused cross-reactivity of the entire RAP-1
antigen, and the C-terminal truncated recombinant antigens were shown to be useful reagents for species-specific serodiagnosis.

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    • "Babesia bovis remains a significant health and economic problem for the cattle industry, causing infections with significant mortality and morbidity rates in semi-tropical and tropical regions worldwide (Bock et al. 2004). Despite important research efforts in recent years focused on the development of improved methods of control, vaccination with live attenuated parasites is still the most effective method of prevention (Brown and Palmer, 1999; Bock et al. 2004; deWaal and Combrink, 2006). "
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    ABSTRACT: SUMMARYObjective. The Babesia bovis genome encodes a rap-1 related gene denominated RAP-1 related antigen (RRA). In this study, we analysed the pattern of expression, immunogenicity and functional relevance of RRA. Methods. Phylogenetic analysis was performed using the program Phylip. Expression of rra was analysed by Northern blots, RT-PCR, immunoprecipitation, Western blots and immunofluorescence. RRA antigenicity was tested by T-cell proliferation and Western blot analysis, and functional relevance was determined in an in vitro neutralization assay. Results. RRA is more closely related to RAP-1b of Babesia bigemina than to B. bovis RAP-1, and it is highly conserved among distinct strains. Transcriptional analysis suggests lower numbers of rra transcripts compared to rap-1. Immunoprecipitation of metabolically labelled B. bovis proteins with antibodies against synthetic peptides representing predicted antigenic regions of RRA confirmed the expression of a ∼43 kDa RRA in cultured merozoites. Antibodies present in B. bovis hyperimmune sera, but not in field-infected cattle sera, reacted weakly with recombinant RRA, and no significant stimulation was obtained using recombinant RRA as antigen in T-cell proliferation assays, indicating that RRA is a subdominant antigen. Antibodies against RRA synthetic peptides reacted with merozoites using immunofluorescence, and were able to significantly inhibit erythrocyte invasion in in vitro neutralization tests, suggesting functional relevance for parasite survival. Conclusion. B. bovis express a novel subdominant RAP-1-like molecule that may contribute to erythrocyte invasion and/or egression by the parasite.
    Full-text · Article · Apr 2011 · Parasitology
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    • "Because of its immunogenic properties and the lack of extensive differences in RAP-1 among geographically distinct isolates of B. bovis, RAP-1 was extensively used as a diagnostic tool. Thus, an antigen derived from the C-terminal region of RAP-1 was used for the development and validation of a competitive ELISA [13] [14] [15]. "
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    ABSTRACT: An immunosensor method for diagnosis of Babesia bovis in cattle based on impedance measurement is presented in this study. The method probes the interaction between antibodies present in serum of B. bovis infected cattle and a recombinant version of the C-terminal portion of RAP-1 obtained from the Portuguese B. bovis Santarém strain (rRAP-1/CT-STR). Following immobilization of rRAP-1/CT-STR on gold electrodes through the formation of a self-assembled layer, the alteration of the interface properties was traced by electrochemical impedance spectroscopy (EIS). The changes of the impedimetric properties of the deposited rRAP-1/CT-STR B. bovis protein layer, interaction with different concentrations of anti-rRAP-1/CT-STR antibodies, and the association constants of the immunoreactive molecules involved, were obtained using EIS. The results were compared with an enzyme-linked immunosorbent assay using the same antigen, and immunofluorescence. The results confirmed the potential for further developing of an immunosensor-based method for the detection and characterization of antibodies against B. bovis in cattle. In addition, such method would provide the advantage of determining the association constant between antibody–antigen interactions without the use of labelling molecules.
    Full-text · Article · Dec 2008 · Sensors and Actuators B Chemical
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    • "Common antigens between B. bovis and B. bigemina (Figueroa et al., 2006) with anti-erythrocyte antibodies (Góes et al., 2007) have been detected using WB and could explain, at least partially, the cross-reactions observed both in IFAT (Goldman et al., 1974) and ELISA, based on crude antigens (Waltisbuhl et al., 1987). Meanwhile, common epitopes between B. bovis and B. bigemina proteins have not only been identified (Suárez et al., 1991), but have also been involved in cross-reactions observed in ELISAs (Boonchit et al., 2004). "
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    ABSTRACT: Bovine babesiosis is caused by Babesia bovis and B. bigemina in Argentina. These protozoans are prevalent north of parallel 30 degrees S, where their natural vector Rhipicephalus (Boophilus) microplus is widespread. To prevent babesiosis outbreaks in endemic areas, an increasing population of 4-10-month-old calves are vaccinated with low virulence B. bovis R1A (BboR1A) and B. bigemina S1A (BbiS1A) strains. In non-endemic areas, an additional calf population is also vaccinated and boostered as adults, before they are relocated to R. microplus-endemic areas of the country. Serological tests are currently utilized not only to determine the status of natural Babesia spp. infections, but also to confirm the infection caused by vaccine strains. For this purpose, an indirect enzyme immunoassay (ELISA) based on the recombinant major surface antigen-2c (rMSA-2c) of B. bovis expressed in Escherichia coli, was standardized using sera from Babesia spp. experimentally infected cattle. ELISA(rMSA-2c) was validated using sera obtained weekly during 336 days from steers primed and boostered with BboR1A and/or BbiS1A on days 0 and 154, then compared with the immunofluorescent-antibody test (IFAT). Western blot (WB) protein analysis was used to confirm the specificity of the immune response to rMSA-2c. The sensitivity and specificity for ELISA(rMSA-2c) were 92 and 96% after the Babesia spp. priming and 88 and 73% after the boostering immunization, respectively. The sensitivity and specificity for IFAT were 99 and 90% after priming and 92 and 98% after boostering, respectively. Unlike IFAT, ELISA(rMSA-2c) detected a remarkable delayed booster response and a significant drop in specificity between 35 and 84 days after the booster immunization. Simultaneously, 87.5% of cattle boostered with B. bigemina showed cross-reactions in the ELISA(rMSA-2c), particularly between 63 and 77 days after the inoculation. A reaction against E. coli was observed, since bands of approximately 40 and/or 42kDa were detected using sera from cattle before and after Babesia spp. inoculations. ELISA(rMSA-2c) showed to be useful between 42 and 98 days after priming with Babesia spp. live vaccine to evaluate the success of infecting cattle. However, after boostering the test showed low specificity.
    Full-text · Article · Sep 2008 · Veterinary Parasitology
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