A Simplified mAb-Based Antigen Detection Assay for Rapid Serotyping of Foot-and-Mouth Disease Virus
Foot-and-mouth disease (FMD) is a devastating infectious viral disease of cloven-hoofed animals. Differentiating FMD from other vesicular diseases is difficult based on only clinical symptoms, requiring an appropriate laboratory diagnostic test. The double-antibody sandwich (DAS)-ELISA is a reliable...
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| author | Mohammad A. Kashem Thanuja Ambagala Kate Hole Ming Yang Charles Nfon Shawn Babiuk |
| author_facet | Mohammad A. Kashem Thanuja Ambagala Kate Hole Ming Yang Charles Nfon Shawn Babiuk |
| author_sort | Mohammad A. Kashem |
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| description | Foot-and-mouth disease (FMD) is a devastating infectious viral disease of cloven-hoofed animals. Differentiating FMD from other vesicular diseases is difficult based on only clinical symptoms, requiring an appropriate laboratory diagnostic test. The double-antibody sandwich (DAS)-ELISA is a reliable diagnostic technique for antigen detection and serotyping of FMDV. However, classical DAS-ELISAs use polyclonal antibodies (pAbs), which are inconsistent in yields and limited in large-scale applications compared to hybridoma cell-secreted laboratory-made monoclonal antibodies (mAbs). Therefore, this study aimed to develop simplified and sensitive FMD serotype-specific DAS-ELISAs using HRP-conjugated mAbs and a TMB substrate. Six FMDV serotype-specific mAb-DAS-ELISAs were developed. All assays were optimized using BEI-inactivated FMD antigens. Real-time reverse-transcriptase PCR (RRT-PCR) was also used to verify the detection efficiency of all assays. Known negative and positive 10% tissue suspensions of different animal origins were examined to calculate the diagnostic specificity (DSp) and sensitivity (DSe). Serotype-specific mAb-DAS-ELISAs demonstrated 100%, 97%, 97%, 99%, 99%, and 94% DSp and 100%, 95%, 90%, 95%, 100%, and 100% DSe for serotypes O, A, Asia-1, SAT-1, SAT-2, and SAT-3, respectively. The detection efficiency of mAb-DAS-ELISAs was better than that of classical DAS-ELISAs. Also, all assays demonstrated minimal cross-reactivity and optimal reproducibility. Therefore, the mAb-DAS-ELISAs developed in this study could be useful for detecting and serotyping FMDV and ultimately replacing the classical DAS-ELISA. |
| format | Article |
| id | doaj-art-e26c267f07c54126b3f1e919bcf2dbde |
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| issn | 1999-4915 |
| language | English |
| publishDate | 2025-05-01 |
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| series | Viruses |
| spelling | doaj-art-e26c267f07c54126b3f1e919bcf2dbde2025-08-20T02:21:54ZengMDPI AGViruses1999-49152025-05-0117676110.3390/v17060761A Simplified mAb-Based Antigen Detection Assay for Rapid Serotyping of Foot-and-Mouth Disease VirusMohammad A. Kashem0Thanuja Ambagala1Kate Hole2Ming Yang3Charles Nfon4Shawn Babiuk5National Centre for Foreign Animal Disease, Canadian Food Inspection Agency, Winnipeg, MB R3E 3M4, CanadaNational Centre for Foreign Animal Disease, Canadian Food Inspection Agency, Winnipeg, MB R3E 3M4, CanadaNational Centre for Foreign Animal Disease, Canadian Food Inspection Agency, Winnipeg, MB R3E 3M4, CanadaNational Centre for Foreign Animal Disease, Canadian Food Inspection Agency, Winnipeg, MB R3E 3M4, CanadaNational Centre for Foreign Animal Disease, Canadian Food Inspection Agency, Winnipeg, MB R3E 3M4, CanadaNational Centre for Foreign Animal Disease, Canadian Food Inspection Agency, Winnipeg, MB R3E 3M4, CanadaFoot-and-mouth disease (FMD) is a devastating infectious viral disease of cloven-hoofed animals. Differentiating FMD from other vesicular diseases is difficult based on only clinical symptoms, requiring an appropriate laboratory diagnostic test. The double-antibody sandwich (DAS)-ELISA is a reliable diagnostic technique for antigen detection and serotyping of FMDV. However, classical DAS-ELISAs use polyclonal antibodies (pAbs), which are inconsistent in yields and limited in large-scale applications compared to hybridoma cell-secreted laboratory-made monoclonal antibodies (mAbs). Therefore, this study aimed to develop simplified and sensitive FMD serotype-specific DAS-ELISAs using HRP-conjugated mAbs and a TMB substrate. Six FMDV serotype-specific mAb-DAS-ELISAs were developed. All assays were optimized using BEI-inactivated FMD antigens. Real-time reverse-transcriptase PCR (RRT-PCR) was also used to verify the detection efficiency of all assays. Known negative and positive 10% tissue suspensions of different animal origins were examined to calculate the diagnostic specificity (DSp) and sensitivity (DSe). Serotype-specific mAb-DAS-ELISAs demonstrated 100%, 97%, 97%, 99%, 99%, and 94% DSp and 100%, 95%, 90%, 95%, 100%, and 100% DSe for serotypes O, A, Asia-1, SAT-1, SAT-2, and SAT-3, respectively. The detection efficiency of mAb-DAS-ELISAs was better than that of classical DAS-ELISAs. Also, all assays demonstrated minimal cross-reactivity and optimal reproducibility. Therefore, the mAb-DAS-ELISAs developed in this study could be useful for detecting and serotyping FMDV and ultimately replacing the classical DAS-ELISA.https://www.mdpi.com/1999-4915/17/6/761FMDVDAS-ELISAmAbsspecificitysensitivitycross-reactivity |
| spellingShingle | Mohammad A. Kashem Thanuja Ambagala Kate Hole Ming Yang Charles Nfon Shawn Babiuk A Simplified mAb-Based Antigen Detection Assay for Rapid Serotyping of Foot-and-Mouth Disease Virus Viruses FMDV DAS-ELISA mAbs specificity sensitivity cross-reactivity |
| title | A Simplified mAb-Based Antigen Detection Assay for Rapid Serotyping of Foot-and-Mouth Disease Virus |
| title_full | A Simplified mAb-Based Antigen Detection Assay for Rapid Serotyping of Foot-and-Mouth Disease Virus |
| title_fullStr | A Simplified mAb-Based Antigen Detection Assay for Rapid Serotyping of Foot-and-Mouth Disease Virus |
| title_full_unstemmed | A Simplified mAb-Based Antigen Detection Assay for Rapid Serotyping of Foot-and-Mouth Disease Virus |
| title_short | A Simplified mAb-Based Antigen Detection Assay for Rapid Serotyping of Foot-and-Mouth Disease Virus |
| title_sort | simplified mab based antigen detection assay for rapid serotyping of foot and mouth disease virus |
| topic | FMDV DAS-ELISA mAbs specificity sensitivity cross-reactivity |
| url | https://www.mdpi.com/1999-4915/17/6/761 |
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