Development and validation of a recombinant Rap1-based lateral flow immunoassay for rapid serodiagnosis of bovine babesiosis in Kazakhstan
Background and Aim: Bovine babesiosis, caused by Babesia bovis, poses significant economic challenges to Kazakhstan’s cattle industry. Early and accurate detection is crucial for interrupting transmission cycles, particularly in regions lacking advanced diagnostic infrastructure. This study aimed to...
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Veterinary World
2025-07-01
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| Series: | Veterinary World |
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| Online Access: | https://www.veterinaryworld.org/Vol.18/July-2025/9.pdf |
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| author | Kanatbek Mukantayev Zhansaya Adish Darkhan Kanayev Laura Tokhtarova Bisultan Abirbekov Yergali Abduraimov Aralbek Rsaliyev Kanat Tursunov |
| author_facet | Kanatbek Mukantayev Zhansaya Adish Darkhan Kanayev Laura Tokhtarova Bisultan Abirbekov Yergali Abduraimov Aralbek Rsaliyev Kanat Tursunov |
| author_sort | Kanatbek Mukantayev |
| collection | DOAJ |
| description | Background and Aim: Bovine babesiosis, caused by Babesia bovis, poses significant economic challenges to Kazakhstan’s cattle industry. Early and accurate detection is crucial for interrupting transmission cycles, particularly in regions lacking advanced diagnostic infrastructure. This study aimed to develop a rapid lateral flow immunoassay (LFIA) using a recombinant C-terminal fragment of the recombinant rhoptry-associated protein 1 (rRap1) antigen for the serodiagnosis of bovine babesiosis.
Materials and Methods: A C-terminal fragment (amino acids 345–480) of the B. bovis Rap1 gene was codon optimized and expressed in Escherichia coli. The recombinant protein was purified using metal-affinity chromatography and validated through sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and nanoflow liquid chromatography-tandem mass spectrometry. A diagnostic evaluation was performed using enzyme-linked immunosorbent assay (ELISA) and LFIA on sera from 102 uninfected and 15 infected cattle, all of which had been pre-tested using polymerase chain reaction. Colloidal gold-protein G conjugates were prepared for LFIA, and test conditions were optimized for antigen concentration and serum dilution. Assay performance was compared with previously published LFIAs.
Results: A 21-kDa rRap1 protein was successfully expressed and demonstrated high specificity to positive control sera. ELISA and LFIA both detected antibodies in 13 of 15 infected samples (sensitivity 86.6%). Specificity was 90.1% for ELISA and 88.2% for LFIA. Receiver operating characteristic analysis showed an area under the curve of 0.83, and Cohen’s Kappa indicated fair-to-moderate agreement between ELISA and LFIA. The LFIA exhibited comparable performance to assays based on merozoite surface antigen 1 or spherical body protein antigens, marking the first successful use of a B. bovis Rap1 C-terminal fragment for LFIA-based field diagnostics in Kazakhstan.
Conclusion: The developed rRap1-based LFIA is a promising, field-deployable diagnostic tool for bovine babesiosis, offering rapid results without the need for laboratory equipment. Despite slightly lower sensitivity than ELISA, its simplicity, cost-effectiveness, and specificity support its use in large-scale epidemiological surveillance. Further validation in diverse field conditions and cattle populations is recommended to refine sensitivity and broaden applicability. |
| format | Article |
| id | doaj-art-ebeaabd723994a7f8aa7ca2d16e47c01 |
| institution | Kabale University |
| issn | 0972-8988 2231-0916 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Veterinary World |
| record_format | Article |
| series | Veterinary World |
| spelling | doaj-art-ebeaabd723994a7f8aa7ca2d16e47c012025-08-20T03:33:53ZengVeterinary WorldVeterinary World0972-89882231-09162025-07-011871881189010.14202/vetworld.2025.1881-1890Development and validation of a recombinant Rap1-based lateral flow immunoassay for rapid serodiagnosis of bovine babesiosis in KazakhstanKanatbek Mukantayev0https://orcid.org/0000-0002-6048-0232Zhansaya Adish1https://orcid.org/0000-0001-9527-8774Darkhan Kanayev2https://orcid.org/0000-0001-9569-9034Laura Tokhtarova3https://orcid.org/0000-0003-4386-993XBisultan Abirbekov4https://orcid.org/0009-0002-0971-9962Yergali Abduraimov5https://orcid.org/0000-0002-4702-9471Aralbek Rsaliyev6https://orcid.org/0000-0002-9921-6076Kanat Tursunov7https://orcid.org/0000-0001-8260-2563Laboratory of Immunochemistry and Immunobiotechnology, National Center for Biotechnology, 010000, Astana, Kazakhstan.Laboratory of Immunochemistry and Immunobiotechnology, National Center for Biotechnology, 010000, Astana, Kazakhstan.Laboratory of Immunochemistry and Immunobiotechnology, National Center for Biotechnology, 010000, Astana, Kazakhstan.Laboratory of Immunochemistry and Immunobiotechnology, National Center for Biotechnology, 010000, Astana, Kazakhstan.Laboratory of Immunochemistry and Immunobiotechnology, National Center for Biotechnology, 010000, Astana, Kazakhstan.Department of Science and Technology, National Holding Qazbiopharm, 010000, Astana, Kazakhstan.Department of Science and Technology, National Holding Qazbiopharm, 010000, Astana, Kazakhstan.Laboratory of Immunochemistry and Immunobiotechnology, National Center for Biotechnology, 010000, Astana, Kazakhstan.Background and Aim: Bovine babesiosis, caused by Babesia bovis, poses significant economic challenges to Kazakhstan’s cattle industry. Early and accurate detection is crucial for interrupting transmission cycles, particularly in regions lacking advanced diagnostic infrastructure. This study aimed to develop a rapid lateral flow immunoassay (LFIA) using a recombinant C-terminal fragment of the recombinant rhoptry-associated protein 1 (rRap1) antigen for the serodiagnosis of bovine babesiosis. Materials and Methods: A C-terminal fragment (amino acids 345–480) of the B. bovis Rap1 gene was codon optimized and expressed in Escherichia coli. The recombinant protein was purified using metal-affinity chromatography and validated through sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and nanoflow liquid chromatography-tandem mass spectrometry. A diagnostic evaluation was performed using enzyme-linked immunosorbent assay (ELISA) and LFIA on sera from 102 uninfected and 15 infected cattle, all of which had been pre-tested using polymerase chain reaction. Colloidal gold-protein G conjugates were prepared for LFIA, and test conditions were optimized for antigen concentration and serum dilution. Assay performance was compared with previously published LFIAs. Results: A 21-kDa rRap1 protein was successfully expressed and demonstrated high specificity to positive control sera. ELISA and LFIA both detected antibodies in 13 of 15 infected samples (sensitivity 86.6%). Specificity was 90.1% for ELISA and 88.2% for LFIA. Receiver operating characteristic analysis showed an area under the curve of 0.83, and Cohen’s Kappa indicated fair-to-moderate agreement between ELISA and LFIA. The LFIA exhibited comparable performance to assays based on merozoite surface antigen 1 or spherical body protein antigens, marking the first successful use of a B. bovis Rap1 C-terminal fragment for LFIA-based field diagnostics in Kazakhstan. Conclusion: The developed rRap1-based LFIA is a promising, field-deployable diagnostic tool for bovine babesiosis, offering rapid results without the need for laboratory equipment. Despite slightly lower sensitivity than ELISA, its simplicity, cost-effectiveness, and specificity support its use in large-scale epidemiological surveillance. Further validation in diverse field conditions and cattle populations is recommended to refine sensitivity and broaden applicability.https://www.veterinaryworld.org/Vol.18/July-2025/9.pdfbabesia bovisbovine babesiosisenzyme-linked immunosorbent assaylateral flow immunoassayrapid diagnosticsrecombinant rhoptry-associated protein 1serodiagnosis |
| spellingShingle | Kanatbek Mukantayev Zhansaya Adish Darkhan Kanayev Laura Tokhtarova Bisultan Abirbekov Yergali Abduraimov Aralbek Rsaliyev Kanat Tursunov Development and validation of a recombinant Rap1-based lateral flow immunoassay for rapid serodiagnosis of bovine babesiosis in Kazakhstan Veterinary World babesia bovis bovine babesiosis enzyme-linked immunosorbent assay lateral flow immunoassay rapid diagnostics recombinant rhoptry-associated protein 1 serodiagnosis |
| title | Development and validation of a recombinant Rap1-based lateral flow immunoassay for rapid serodiagnosis of bovine babesiosis in Kazakhstan |
| title_full | Development and validation of a recombinant Rap1-based lateral flow immunoassay for rapid serodiagnosis of bovine babesiosis in Kazakhstan |
| title_fullStr | Development and validation of a recombinant Rap1-based lateral flow immunoassay for rapid serodiagnosis of bovine babesiosis in Kazakhstan |
| title_full_unstemmed | Development and validation of a recombinant Rap1-based lateral flow immunoassay for rapid serodiagnosis of bovine babesiosis in Kazakhstan |
| title_short | Development and validation of a recombinant Rap1-based lateral flow immunoassay for rapid serodiagnosis of bovine babesiosis in Kazakhstan |
| title_sort | development and validation of a recombinant rap1 based lateral flow immunoassay for rapid serodiagnosis of bovine babesiosis in kazakhstan |
| topic | babesia bovis bovine babesiosis enzyme-linked immunosorbent assay lateral flow immunoassay rapid diagnostics recombinant rhoptry-associated protein 1 serodiagnosis |
| url | https://www.veterinaryworld.org/Vol.18/July-2025/9.pdf |
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