Development of a Kit for Rapid Immunochromatographic Detection of Sacbrood Virus Infecting <i>Apis cerana</i> (AcSBV) Based on Polyclonal and Monoclonal Antibodies Raised against Recombinant VP1 and VP2 Expressed in <i>Escherichia coli</i>
A Korean isolate of the sacbrood virus infecting <i>Apis cerana</i> (AcSBV-Kor) is the most destructive honeybee virus, causing serious economic damage losses in Korean apiculture. To address this, here, we attempted to develop an assay for the rapid detection of AcSBV-Kor based on immun...
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| Idioma: | English |
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MDPI AG
2021-12-01
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| coleção: | Viruses |
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| Acesso em linha: | https://www.mdpi.com/1999-4915/13/12/2439 |
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| author | Song Hee Lee Tae-Kyun Oh Sung Oh Seongdae Kim Han Byul Noh Nagarajan Vinod Ji Yoon Lee Eun Sun Moon Chang Won Choi |
| author_facet | Song Hee Lee Tae-Kyun Oh Sung Oh Seongdae Kim Han Byul Noh Nagarajan Vinod Ji Yoon Lee Eun Sun Moon Chang Won Choi |
| author_sort | Song Hee Lee |
| collection | DOAJ |
| description | A Korean isolate of the sacbrood virus infecting <i>Apis cerana</i> (AcSBV-Kor) is the most destructive honeybee virus, causing serious economic damage losses in Korean apiculture. To address this, here, we attempted to develop an assay for the rapid detection of AcSBV-Kor based on immunochromatographic detection of constituent viral proteins. Genes encoding VP1 and VP2 proteins of AcSBV-Kor were cloned into an expression vector (pET-28a) and expressed in <i>Escherichia coli</i> BL21(DE3). During purification, recombinant VP1 (rVP1) and VP2 (rVP2) proteins were found in the insoluble fraction, with a molecular size of 26.7 and 24.9 kDa, respectively. BALB/c mice immunized with the purified rVP1 and rVP2 produced polyclonal antibodies (pAbs) such as pAb-rVP1 and pAb-rVP2. Western blot analysis showed that pAb-rVP1 strongly reacted with the homologous rVP1 but weakly reacted with heterologous rVP2. However, pAb-rVP2 strongly reacted not only with the homologous rVP2 but also with the heterologous rVP1. Spleen cells of the immunized mice fused with SP2/0-Ag14 myeloma cells produced monoclonal antibodies (mAbs) such as mAb-rVP1-1 and mAb-rVP2-13. Western blot analysis indicated that pAb-rVP1, pAb-rVP2, mAb-rVP1-1, and mAb-rVP2-13 reacted with AcSBV-infected honeybees and larvae as well as the corresponding recombinant proteins. These antibodies were then used in the development of a rapid immunochromatography (IC) strip assay kit with colloidal gold coupled to pAb-rVP1 and pAb-rVP2 at the conjugate pad and mAb-rVP1-1 and mAb-rVP2-13 at the test line. One antibody pair, pAb-rVP1/mAb-VP1-1, showed positive reactivity as low as 1.38 × 10<sup>3</sup> copies, while the other pair, pAb-rVP2/mAb-VP2-13, showed positive reactivity as low as 1.38 × 10<sup>4</sup> copies. Therefore, the antibody pair pAb-rVP1/mAb-VP1-1 was selected as a final candidate for validation. To validate the detection of AcSBV, the IC strip tests were conducted with 50 positive and 50 negative samples and compared with real-time PCR tests. The results confirm that the developed IC assay is a sufficiently sensitive and specific detection method for user-friendly and rapid detection of AcSBV. |
| first_indexed | 2024-03-10T03:54:46Z |
| format | Article |
| id | doaj.art-0ac4c8f2ec154b51abda5bf69bea7f8d |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-10T03:54:46Z |
| publishDate | 2021-12-01 |
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| series | Viruses |
| spelling | doaj.art-0ac4c8f2ec154b51abda5bf69bea7f8d2023-11-23T10:57:56ZengMDPI AGViruses1999-49152021-12-011312243910.3390/v13122439Development of a Kit for Rapid Immunochromatographic Detection of Sacbrood Virus Infecting <i>Apis cerana</i> (AcSBV) Based on Polyclonal and Monoclonal Antibodies Raised against Recombinant VP1 and VP2 Expressed in <i>Escherichia coli</i>Song Hee Lee0Tae-Kyun Oh1Sung Oh2Seongdae Kim3Han Byul Noh4Nagarajan Vinod5Ji Yoon Lee6Eun Sun Moon7Chang Won Choi8Department of Biology & Medicinal Science, Pai Chai University, Daejeon 35345, KoreaGeNet Bio Company, Daejeon 305-500, KoreaDepartment of Biology & Medicinal Science, Pai Chai University, Daejeon 35345, KoreaDepartment of Biology & Medicinal Science, Pai Chai University, Daejeon 35345, KoreaDepartment of Biology & Medicinal Science, Pai Chai University, Daejeon 35345, KoreaDepartment of Biology & Medicinal Science, Pai Chai University, Daejeon 35345, KoreaDepartment of Biology & Medicinal Science, Pai Chai University, Daejeon 35345, KoreaDepartment of Biology & Medicinal Science, Pai Chai University, Daejeon 35345, KoreaDepartment of Biology & Medicinal Science, Pai Chai University, Daejeon 35345, KoreaA Korean isolate of the sacbrood virus infecting <i>Apis cerana</i> (AcSBV-Kor) is the most destructive honeybee virus, causing serious economic damage losses in Korean apiculture. To address this, here, we attempted to develop an assay for the rapid detection of AcSBV-Kor based on immunochromatographic detection of constituent viral proteins. Genes encoding VP1 and VP2 proteins of AcSBV-Kor were cloned into an expression vector (pET-28a) and expressed in <i>Escherichia coli</i> BL21(DE3). During purification, recombinant VP1 (rVP1) and VP2 (rVP2) proteins were found in the insoluble fraction, with a molecular size of 26.7 and 24.9 kDa, respectively. BALB/c mice immunized with the purified rVP1 and rVP2 produced polyclonal antibodies (pAbs) such as pAb-rVP1 and pAb-rVP2. Western blot analysis showed that pAb-rVP1 strongly reacted with the homologous rVP1 but weakly reacted with heterologous rVP2. However, pAb-rVP2 strongly reacted not only with the homologous rVP2 but also with the heterologous rVP1. Spleen cells of the immunized mice fused with SP2/0-Ag14 myeloma cells produced monoclonal antibodies (mAbs) such as mAb-rVP1-1 and mAb-rVP2-13. Western blot analysis indicated that pAb-rVP1, pAb-rVP2, mAb-rVP1-1, and mAb-rVP2-13 reacted with AcSBV-infected honeybees and larvae as well as the corresponding recombinant proteins. These antibodies were then used in the development of a rapid immunochromatography (IC) strip assay kit with colloidal gold coupled to pAb-rVP1 and pAb-rVP2 at the conjugate pad and mAb-rVP1-1 and mAb-rVP2-13 at the test line. One antibody pair, pAb-rVP1/mAb-VP1-1, showed positive reactivity as low as 1.38 × 10<sup>3</sup> copies, while the other pair, pAb-rVP2/mAb-VP2-13, showed positive reactivity as low as 1.38 × 10<sup>4</sup> copies. Therefore, the antibody pair pAb-rVP1/mAb-VP1-1 was selected as a final candidate for validation. To validate the detection of AcSBV, the IC strip tests were conducted with 50 positive and 50 negative samples and compared with real-time PCR tests. The results confirm that the developed IC assay is a sufficiently sensitive and specific detection method for user-friendly and rapid detection of AcSBV.https://www.mdpi.com/1999-4915/13/12/2439sacbrood virus infecting <i>Apis cerana</i> (AcSBV)recombinant VP1 (rVP1) and VP2 (rVP2) proteinspolyclonal antibodies (pAb-rVP1 and pAb-rVP2)monoclonal antibodies (mAb-rVP1 and mAb-rVP2)immunochromatography (IC) strip assay |
| spellingShingle | Song Hee Lee Tae-Kyun Oh Sung Oh Seongdae Kim Han Byul Noh Nagarajan Vinod Ji Yoon Lee Eun Sun Moon Chang Won Choi Development of a Kit for Rapid Immunochromatographic Detection of Sacbrood Virus Infecting <i>Apis cerana</i> (AcSBV) Based on Polyclonal and Monoclonal Antibodies Raised against Recombinant VP1 and VP2 Expressed in <i>Escherichia coli</i> Viruses sacbrood virus infecting <i>Apis cerana</i> (AcSBV) recombinant VP1 (rVP1) and VP2 (rVP2) proteins polyclonal antibodies (pAb-rVP1 and pAb-rVP2) monoclonal antibodies (mAb-rVP1 and mAb-rVP2) immunochromatography (IC) strip assay |
| title | Development of a Kit for Rapid Immunochromatographic Detection of Sacbrood Virus Infecting <i>Apis cerana</i> (AcSBV) Based on Polyclonal and Monoclonal Antibodies Raised against Recombinant VP1 and VP2 Expressed in <i>Escherichia coli</i> |
| title_full | Development of a Kit for Rapid Immunochromatographic Detection of Sacbrood Virus Infecting <i>Apis cerana</i> (AcSBV) Based on Polyclonal and Monoclonal Antibodies Raised against Recombinant VP1 and VP2 Expressed in <i>Escherichia coli</i> |
| title_fullStr | Development of a Kit for Rapid Immunochromatographic Detection of Sacbrood Virus Infecting <i>Apis cerana</i> (AcSBV) Based on Polyclonal and Monoclonal Antibodies Raised against Recombinant VP1 and VP2 Expressed in <i>Escherichia coli</i> |
| title_full_unstemmed | Development of a Kit for Rapid Immunochromatographic Detection of Sacbrood Virus Infecting <i>Apis cerana</i> (AcSBV) Based on Polyclonal and Monoclonal Antibodies Raised against Recombinant VP1 and VP2 Expressed in <i>Escherichia coli</i> |
| title_short | Development of a Kit for Rapid Immunochromatographic Detection of Sacbrood Virus Infecting <i>Apis cerana</i> (AcSBV) Based on Polyclonal and Monoclonal Antibodies Raised against Recombinant VP1 and VP2 Expressed in <i>Escherichia coli</i> |
| title_sort | development of a kit for rapid immunochromatographic detection of sacbrood virus infecting i apis cerana i acsbv based on polyclonal and monoclonal antibodies raised against recombinant vp1 and vp2 expressed in i escherichia coli i |
| topic | sacbrood virus infecting <i>Apis cerana</i> (AcSBV) recombinant VP1 (rVP1) and VP2 (rVP2) proteins polyclonal antibodies (pAb-rVP1 and pAb-rVP2) monoclonal antibodies (mAb-rVP1 and mAb-rVP2) immunochromatography (IC) strip assay |
| url | https://www.mdpi.com/1999-4915/13/12/2439 |
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