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Home / Equine Research Bank / J Clin Microbiol / Detection of equine antibodies to babesia caballi by recombi...
Journal of clinical microbiology1999; 37(7); 2285-2290; doi: 10.1128/JCM.37.7.2285-2290.1999

Detection of equine antibodies to babesia caballi by recombinant B. caballi rhoptry-associated protein 1 in a competitive-inhibition enzyme-linked immunosorbent assay.

Abstract: A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was developed for detection of equine antibodies specific for Babesia caballi. The assay used recombinant B. caballi rhoptry-associated protein 1 (RAP-1) and monoclonal antibody (MAb) 79/17.18.5, which is reactive with a peptide epitope of a native 60-kDa B. caballi antigen. The gene encoding the recombinant antigen was sequenced, and database analysis revealed that the gene product is a rhoptry-associated protein. Cloning and expression of a truncated copy of the gene demonstrated that MAb 79/17.18.5 reacts with the C-terminal repeat region of the protein. The cELISA was used to evaluate 302 equine serum samples previously tested for antibodies to B. caballi by a standardized complement fixation test (CFT). The results of cELISA and CFT were 73% concordant. Seventy-two of the 77 serum samples with discordant results were CFT negative and cELISA positive. Further evaluation of the serum samples with discordant results by indirect immunofluorescence assay (IFA) demonstrated that at a serum dilution of 1:200, 48 of the CFT-negative and cELISA-positive serum samples contained antibodies reactive with B. caballi RAP-1. Four of five CFT-positive and cELISA-negative serum samples contained antibodies reactive with B. caballi when they were tested by IFA. These data indicate that following infection with B. caballi, horses consistently produce antibody to the RAP-1 epitope defined by MAb 79/17.18.5, and when used in the cELISA format, recombinant RAP-1 is a useful antigen for the serologic detection of anti-B. caballi antibodies.
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Publication Date: 1999-06-12 PubMed ID: 10364599PubMed Central: PMC85139DOI: 10.1128/JCM.37.7.2285-2290.1999Google Scholar: Lookup
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  • Journal Article
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  • U.S. Gov't
  • Non-P.H.S.

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research deals with the development of a new testing method for Babesia caballi antibodies in horses, using a competitive-inhibition enzyme-linked immunosorbent assay (cELISA) technique, which proved to be more accurate than the standardized method due to the use of recombinant B. caballi rhoptry-associated protein 1 (RAP-1) and a specific monoclonal antibody.

Research Method

  • The study began with the development of a cELISA for the detection of equine antibodies specifically for Babesia caballi – a parasite that affects horses.
  • This process utilized a recombinant (synthetically created) protein from B. caballi known as rhoptry-associated protein 1 (RAP-1), along with monoclonal antibody (MAb) 79/17.18.5, known to react with a peptide epitope of a native 60-kDA B. caballi antigen.
  • The gene encoding the recombinant antigen was sequenced and database analysis revealed that the gene product is a rhoptry-associated protein.

Cloning and Expression of the cELISA

  • The researchers cloned and expressed a truncated copy of the gene, which showed that MAb 79/17.18.5 reacts with the C-terminal repeat region of the protein.
  • To evaluate the cELISA, they used 302 equine serum samples previously tested for anti-B. caballi antibodies using a standardized complement fixation test (CFT).
  • They found that the results of the cELISA and the CFT were in agreement in 73% of the cases.

Validation of cELISA

  • When further tested using indirect immunofluorescence assay (IFA) with discordant samples, 48 CFT-negative and cELISA-positive samples contained antibodies reactive with B. caballi RAP-1.
  • Moreover, 4 out of 5 CFT-positive and cELISA-negative samples contained antibodies reactive with B. caballi when tested by IFA.
  • This shows that horses infected with B. caballi consistently produce antibodies to the RAP-1 epitope defined by MAb 79/17.18.5.
  • This demonstrates that when used in a cELISA format, recombinant RAP-1 is a beneficial antigen for detecting anti-B. caballi antibodies, showing an enhanced sensitivity and specificity compared to the standard CFT test.

Cite This Article

APA
Kappmeyer LS, Perryman LE, Hines SA, Baszler TV, Katz JB, Hennager SG, Knowles DP. (1999). Detection of equine antibodies to babesia caballi by recombinant B. caballi rhoptry-associated protein 1 in a competitive-inhibition enzyme-linked immunosorbent assay. J Clin Microbiol, 37(7), 2285-2290. https://doi.org/10.1128/JCM.37.7.2285-2290.1999

Publication

Journal of clinical microbiology
ISSN: 0095-1137
NlmUniqueID: 7505564
Country: United States
Language: English
Volume: 37
Issue: 7
Pages: 2285-2290

Researcher Affiliations

Kappmeyer, L S
  • Animal Disease Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Pullman, Washington 99164-7030, USA.
Perryman, L E
    Hines, S A
      Baszler, T V
        Katz, J B
          Hennager, S G
            Knowles, D P

              MeSH Terms

              • Amino Acid Sequence
              • Animals
              • Antibodies, Monoclonal
              • Antibodies, Protozoan / blood
              • Antigens, Protozoan / genetics
              • Antigens, Protozoan / immunology
              • Babesia / genetics
              • Babesia / immunology
              • Babesiosis / diagnosis
              • Babesiosis / immunology
              • Enzyme-Linked Immunosorbent Assay
              • Epitopes / analysis
              • Horses
              • Molecular Sequence Data
              • Protozoan Proteins / chemistry
              • Protozoan Proteins / genetics
              • Protozoan Proteins / immunology
              • Recombinant Proteins / immunology
              • Repetitive Sequences, Amino Acid

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