Development of a Novel Recombinant Full-Length IgY Monoclonal Antibody against Human Thymidine Kinase 1 for Automatic Chemiluminescence Analysis on a Sandwich Biotin-Streptavidin Platform for Early Tumour Discovery
Serum thymidine kinase 1 protein (STK1p) concentration has been used successfully as a reliable proliferating serum biomarker in early tumour discovery and clinical settings. It is detected by an enhanced chemiluminescence (ECL) dot blot assay with the biotin-streptavidin (BSA) platform (a gold stan...
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| Format: | Article |
| Language: | English |
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Wiley
2023-01-01
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| Series: | Journal of Immunology Research |
| Online Access: | http://dx.doi.org/10.1155/2023/7612566 |
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| author | Xiangbin Wang Shan Li Rui Zhao Huijun Li Peng Gao Cuicui Jin Cong Fang Yongping Liu Ailian Hei Ji Zhou Jin Li Ellen He Sven Skog |
| author_facet | Xiangbin Wang Shan Li Rui Zhao Huijun Li Peng Gao Cuicui Jin Cong Fang Yongping Liu Ailian Hei Ji Zhou Jin Li Ellen He Sven Skog |
| author_sort | Xiangbin Wang |
| collection | DOAJ |
| description | Serum thymidine kinase 1 protein (STK1p) concentration has been used successfully as a reliable proliferating serum biomarker in early tumour discovery and clinical settings. It is detected by an enhanced chemiluminescence (ECL) dot blot assay with the biotin-streptavidin (BSA) platform (a gold standard) based on chicken anti-human thymidine kinase 1 IgY polyclonal antibody (hTK1-IgY-pAb). However, ECL dot blotting is a semiautomatic method that has been limited to large-scale applications due to the differences among batches of antibodies from individual hens, and the skill level of operation technicians sometimes results in unstable STK1p values. Therefore, a highly stable recombinant chicken full-length IgY monoclonal antibody in combination with a fully automated sandwich biotin-streptavidin (sandwich-BSA) platform was developed. Hens were immunized with 31-peptide, a key sequence of human TK1 (hTK1), before constructing an immune phage display scFv library. Finally, a recombinant full-length IgY monoclonal antibody (hTK1-IgY-rmAb#5) with high-affinity binding with human recombinant TK1 (rhTK1) (3.95×10−10 mol/L), high sensitivity with hTK1 calibrators (slope of linear curve: 89.98), and high specificity with low/elevated STK1p (r≈0.92-0.963) was identified. hTK1-IgY-rmAb#5 showed a specific immune response with thymidine kinase 1 (TK1) in TK1-positive/negative cell lysates by Western blotting and immunohistochemistry (IHC) in normal and cancer tissues. In particular, the detection of TK1 serum samples from health centres showed a high coincidence rate (r=0.988,n=90) between hTK1-IgY-rmAb#5 and hTK1-IgY-pAb and between the semiautomatic ECL dot blot BSA platform and the novel automatic chemiluminescence sandwich-BSA platform (r=0.857,n=292). hTK1-IgY-rmAb#5 is stable and highly sensitive for detecting the lowest STK1p value at 0.01 pmol/L (pM). The accuracy is high (SD<2.5%) between different batches. It is easy to use the novel hTK1-IgY-rmAb#5 on a new automatic chemiluminescence sandwich-BSA platform. It will be beneficial for large-scale health screenings. |
| format | Article |
| id | doaj-art-e23af5bca9b9483387f92f760cc53a83 |
| institution | Kabale University |
| issn | 2314-7156 |
| language | English |
| publishDate | 2023-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Journal of Immunology Research |
| spelling | doaj-art-e23af5bca9b9483387f92f760cc53a832025-08-20T03:25:15ZengWileyJournal of Immunology Research2314-71562023-01-01202310.1155/2023/7612566Development of a Novel Recombinant Full-Length IgY Monoclonal Antibody against Human Thymidine Kinase 1 for Automatic Chemiluminescence Analysis on a Sandwich Biotin-Streptavidin Platform for Early Tumour DiscoveryXiangbin Wang0Shan Li1Rui Zhao2Huijun Li3Peng Gao4Cuicui Jin5Cong Fang6Yongping Liu7Ailian Hei8Ji Zhou9Jin Li10Ellen He11Sven Skog12Genfine Basic Science Centre (GBSC)Department of MedicineGenfine Basic Science Centre (GBSC)Department of MedicineDepartment of MedicineDepartment of MedicineDepartment of MedicineChangzhou Cancer HospitalDepartment of MedicineDepartment of MedicineDepartment of MedicineDepartment of MedicineDepartment of MedicineSerum thymidine kinase 1 protein (STK1p) concentration has been used successfully as a reliable proliferating serum biomarker in early tumour discovery and clinical settings. It is detected by an enhanced chemiluminescence (ECL) dot blot assay with the biotin-streptavidin (BSA) platform (a gold standard) based on chicken anti-human thymidine kinase 1 IgY polyclonal antibody (hTK1-IgY-pAb). However, ECL dot blotting is a semiautomatic method that has been limited to large-scale applications due to the differences among batches of antibodies from individual hens, and the skill level of operation technicians sometimes results in unstable STK1p values. Therefore, a highly stable recombinant chicken full-length IgY monoclonal antibody in combination with a fully automated sandwich biotin-streptavidin (sandwich-BSA) platform was developed. Hens were immunized with 31-peptide, a key sequence of human TK1 (hTK1), before constructing an immune phage display scFv library. Finally, a recombinant full-length IgY monoclonal antibody (hTK1-IgY-rmAb#5) with high-affinity binding with human recombinant TK1 (rhTK1) (3.95×10−10 mol/L), high sensitivity with hTK1 calibrators (slope of linear curve: 89.98), and high specificity with low/elevated STK1p (r≈0.92-0.963) was identified. hTK1-IgY-rmAb#5 showed a specific immune response with thymidine kinase 1 (TK1) in TK1-positive/negative cell lysates by Western blotting and immunohistochemistry (IHC) in normal and cancer tissues. In particular, the detection of TK1 serum samples from health centres showed a high coincidence rate (r=0.988,n=90) between hTK1-IgY-rmAb#5 and hTK1-IgY-pAb and between the semiautomatic ECL dot blot BSA platform and the novel automatic chemiluminescence sandwich-BSA platform (r=0.857,n=292). hTK1-IgY-rmAb#5 is stable and highly sensitive for detecting the lowest STK1p value at 0.01 pmol/L (pM). The accuracy is high (SD<2.5%) between different batches. It is easy to use the novel hTK1-IgY-rmAb#5 on a new automatic chemiluminescence sandwich-BSA platform. It will be beneficial for large-scale health screenings.http://dx.doi.org/10.1155/2023/7612566 |
| spellingShingle | Xiangbin Wang Shan Li Rui Zhao Huijun Li Peng Gao Cuicui Jin Cong Fang Yongping Liu Ailian Hei Ji Zhou Jin Li Ellen He Sven Skog Development of a Novel Recombinant Full-Length IgY Monoclonal Antibody against Human Thymidine Kinase 1 for Automatic Chemiluminescence Analysis on a Sandwich Biotin-Streptavidin Platform for Early Tumour Discovery Journal of Immunology Research |
| title | Development of a Novel Recombinant Full-Length IgY Monoclonal Antibody against Human Thymidine Kinase 1 for Automatic Chemiluminescence Analysis on a Sandwich Biotin-Streptavidin Platform for Early Tumour Discovery |
| title_full | Development of a Novel Recombinant Full-Length IgY Monoclonal Antibody against Human Thymidine Kinase 1 for Automatic Chemiluminescence Analysis on a Sandwich Biotin-Streptavidin Platform for Early Tumour Discovery |
| title_fullStr | Development of a Novel Recombinant Full-Length IgY Monoclonal Antibody against Human Thymidine Kinase 1 for Automatic Chemiluminescence Analysis on a Sandwich Biotin-Streptavidin Platform for Early Tumour Discovery |
| title_full_unstemmed | Development of a Novel Recombinant Full-Length IgY Monoclonal Antibody against Human Thymidine Kinase 1 for Automatic Chemiluminescence Analysis on a Sandwich Biotin-Streptavidin Platform for Early Tumour Discovery |
| title_short | Development of a Novel Recombinant Full-Length IgY Monoclonal Antibody against Human Thymidine Kinase 1 for Automatic Chemiluminescence Analysis on a Sandwich Biotin-Streptavidin Platform for Early Tumour Discovery |
| title_sort | development of a novel recombinant full length igy monoclonal antibody against human thymidine kinase 1 for automatic chemiluminescence analysis on a sandwich biotin streptavidin platform for early tumour discovery |
| url | http://dx.doi.org/10.1155/2023/7612566 |
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