Simplified approaches for the development of an ELISA to detect circulating autoantibodies to p53 in cancer patients |
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Authors: | Ratchada Cressey Saranya Pimpa Busyamas Chewaskulyong Nirush Lertprasertsuke Somchareon Saeteng Chatchai Tayapiwatana Watchara Kasinrerk |
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Institution: | (1) Division of Clinical Chemistry, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, 50200, Thailand;(2) Department of Internal Medicine, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand;(3) Department of Pathology, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand;(4) Department of Surgery, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand;(5) Division of Clinical Immunology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, 50200, Thailand;(6) Biomedical Technology Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Chiang Mai, Thailand |
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Abstract: | Background The recognition that human tumors stimulate the production of autoantibodies has initiated the use of this immune response
as serological markers for the early diagnosis and management of cancer. The enzyme-linked immunosorbent assay (ELISA) is
the most common method used in detecting autoantibodies, which involves coating the microtiter plate with the tumor associated
antigen (TAA) of interest and allowing serum antibodies to bind. The patient's sample is directly in contact with the coating
antigen so the protein used for coating must be pure to avoid non-specific binding. In this study, a simplified method to
selectively and specifically immobilize TAAs onto microtiter plates in order to detect circulating autoantibodies in cancer
patients without prior purification process was described. Wild type full-length p53 protein was produced in fusion with biotin
carboxyl carrier peptide (BCCP) or hexahistidine (His)6] using pAK400 and pET15b(+) vectors, respectively. The recombinant
p53 fusion protein produced was then subjected to react with either a commercial p53 monoclonal antibody (mAb) or sera from
lung cancer patients and healthy volunteers in an enzyme-linked immunosorbent assay (ELISA) format. |
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