Production and Immunological Characterization of scFv Specific to Epitope of <i>Opisthorchis viverrini</i> Rhophilin-Associated Tail Protein 1-like (OvROPN1L)
(1) Background: <i>Opisthorchis viverrini</i> is a significant health problem in the Mekong subregion of Southeast Asia, causing aggressive cholangiocarcinoma. Current diagnostic procedures do not cover early diagnosis and low infection. Hence, an effective diagnostic tool is still requi...
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MDPI AG
2023-03-01
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| Series: | Tropical Medicine and Infectious Disease |
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| Online Access: | https://www.mdpi.com/2414-6366/8/3/160 |
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| author | Amornrat Geadkaew-Krenc Dawid Krenc Jeeraphong Thanongsaksrikul Rudi Grams Wansika Phadungsil Kittirat Glab-ampai Pathanin Chantree Pongsakorn Martviset |
| author_facet | Amornrat Geadkaew-Krenc Dawid Krenc Jeeraphong Thanongsaksrikul Rudi Grams Wansika Phadungsil Kittirat Glab-ampai Pathanin Chantree Pongsakorn Martviset |
| author_sort | Amornrat Geadkaew-Krenc |
| collection | DOAJ |
| description | (1) Background: <i>Opisthorchis viverrini</i> is a significant health problem in the Mekong subregion of Southeast Asia, causing aggressive cholangiocarcinoma. Current diagnostic procedures do not cover early diagnosis and low infection. Hence, an effective diagnostic tool is still required. Immunodiagnosis seems promising, but attempts to generate monoclonal antibodies have not yet been successful. This study aims to develop a single-chain variable antibody fragment (scFv) against Rhophilin-associated tail protein 1-like (ROPN1L), the sperm-specific antigen of adult <i>O. viverrini</i>, which has not been reported elsewhere. (2) Methods: The target epitope for phage screening was L3-Q13 of OvROPN1L, which showed the highest antigenicity to human opisthorchiasis analyzed in a previous study. This peptide was commercially synthesized and used for phage library screening. The isolated phage was produced in a bacterial expression system and tested for specificity in vitro and in silico. (3) Results: One of fourteen phages, named scFv anti-OvROPN1L-CL19, significantly bound to rOvROPN1L compared with non-infected hamster fecal extracts. This phage clone was successfully produced and purified using Ni-NTA chromatography. Indirect ELISA demonstrated that scFv anti-OvROPN1L-CL19 has a high reactivity with <i>O. viverrini</i>-infected hamster fecal extracts (12 wpi, <i>n =</i> 6) in comparison with non-infected hamster fecal extracts (0 wpi, <i>n</i> = 6), while the polyclonal rOvROPN1L antibodies did not show such a difference. Molecular modeling and docking confirmed our in vitro findings. (4) Conclusion: scFv anti-OvROPN1L-CL19 could be used as an effective material for developing <i>O. viverrini</i>-immunodiagnostic procedures in the future. |
| first_indexed | 2024-03-11T05:48:49Z |
| format | Article |
| id | doaj.art-f6faa05e1b604a958db7d57fa975c1ca |
| institution | Directory Open Access Journal |
| issn | 2414-6366 |
| language | English |
| last_indexed | 2024-03-11T05:48:49Z |
| publishDate | 2023-03-01 |
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| series | Tropical Medicine and Infectious Disease |
| spelling | doaj.art-f6faa05e1b604a958db7d57fa975c1ca2023-11-17T14:15:16ZengMDPI AGTropical Medicine and Infectious Disease2414-63662023-03-018316010.3390/tropicalmed8030160Production and Immunological Characterization of scFv Specific to Epitope of <i>Opisthorchis viverrini</i> Rhophilin-Associated Tail Protein 1-like (OvROPN1L)Amornrat Geadkaew-Krenc0Dawid Krenc1Jeeraphong Thanongsaksrikul2Rudi Grams3Wansika Phadungsil4Kittirat Glab-ampai5Pathanin Chantree6Pongsakorn Martviset7Graduate Program in Biomedical Sciences, Faculty of Allied Health Sciences, Thammasat University, Pathumthani 12120, ThailandChulabhorn International College of Medicine, Thammasat University, Pathumthani 12120, ThailandGraduate Program in Biomedical Sciences, Faculty of Allied Health Sciences, Thammasat University, Pathumthani 12120, ThailandGraduate Program in Biomedical Sciences, Faculty of Allied Health Sciences, Thammasat University, Pathumthani 12120, ThailandGraduate Program in Biomedical Sciences, Faculty of Allied Health Sciences, Thammasat University, Pathumthani 12120, ThailandCenter of Research Excellence in Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, ThailandDepartment of Preclinical Science, Faculty of Medicine, Thammasat University, Pathumthani 12120, ThailandDepartment of Preclinical Science, Faculty of Medicine, Thammasat University, Pathumthani 12120, Thailand(1) Background: <i>Opisthorchis viverrini</i> is a significant health problem in the Mekong subregion of Southeast Asia, causing aggressive cholangiocarcinoma. Current diagnostic procedures do not cover early diagnosis and low infection. Hence, an effective diagnostic tool is still required. Immunodiagnosis seems promising, but attempts to generate monoclonal antibodies have not yet been successful. This study aims to develop a single-chain variable antibody fragment (scFv) against Rhophilin-associated tail protein 1-like (ROPN1L), the sperm-specific antigen of adult <i>O. viverrini</i>, which has not been reported elsewhere. (2) Methods: The target epitope for phage screening was L3-Q13 of OvROPN1L, which showed the highest antigenicity to human opisthorchiasis analyzed in a previous study. This peptide was commercially synthesized and used for phage library screening. The isolated phage was produced in a bacterial expression system and tested for specificity in vitro and in silico. (3) Results: One of fourteen phages, named scFv anti-OvROPN1L-CL19, significantly bound to rOvROPN1L compared with non-infected hamster fecal extracts. This phage clone was successfully produced and purified using Ni-NTA chromatography. Indirect ELISA demonstrated that scFv anti-OvROPN1L-CL19 has a high reactivity with <i>O. viverrini</i>-infected hamster fecal extracts (12 wpi, <i>n =</i> 6) in comparison with non-infected hamster fecal extracts (0 wpi, <i>n</i> = 6), while the polyclonal rOvROPN1L antibodies did not show such a difference. Molecular modeling and docking confirmed our in vitro findings. (4) Conclusion: scFv anti-OvROPN1L-CL19 could be used as an effective material for developing <i>O. viverrini</i>-immunodiagnostic procedures in the future.https://www.mdpi.com/2414-6366/8/3/160<i>Opisthorchis viverrini</i>OvROPN1Lsingle-chain variable fragment (scFv)phage display |
| spellingShingle | Amornrat Geadkaew-Krenc Dawid Krenc Jeeraphong Thanongsaksrikul Rudi Grams Wansika Phadungsil Kittirat Glab-ampai Pathanin Chantree Pongsakorn Martviset Production and Immunological Characterization of scFv Specific to Epitope of <i>Opisthorchis viverrini</i> Rhophilin-Associated Tail Protein 1-like (OvROPN1L) Tropical Medicine and Infectious Disease <i>Opisthorchis viverrini</i> OvROPN1L single-chain variable fragment (scFv) phage display |
| title | Production and Immunological Characterization of scFv Specific to Epitope of <i>Opisthorchis viverrini</i> Rhophilin-Associated Tail Protein 1-like (OvROPN1L) |
| title_full | Production and Immunological Characterization of scFv Specific to Epitope of <i>Opisthorchis viverrini</i> Rhophilin-Associated Tail Protein 1-like (OvROPN1L) |
| title_fullStr | Production and Immunological Characterization of scFv Specific to Epitope of <i>Opisthorchis viverrini</i> Rhophilin-Associated Tail Protein 1-like (OvROPN1L) |
| title_full_unstemmed | Production and Immunological Characterization of scFv Specific to Epitope of <i>Opisthorchis viverrini</i> Rhophilin-Associated Tail Protein 1-like (OvROPN1L) |
| title_short | Production and Immunological Characterization of scFv Specific to Epitope of <i>Opisthorchis viverrini</i> Rhophilin-Associated Tail Protein 1-like (OvROPN1L) |
| title_sort | production and immunological characterization of scfv specific to epitope of i opisthorchis viverrini i rhophilin associated tail protein 1 like ovropn1l |
| topic | <i>Opisthorchis viverrini</i> OvROPN1L single-chain variable fragment (scFv) phage display |
| url | https://www.mdpi.com/2414-6366/8/3/160 |
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