Development and Evaluation of p16 based Double Antibody Sandwich ELISA for Detection of Cervical Precancer and Cancer.

Autor:	Bose M; Department of Molecular Oncology, Cancer Institute (WIA), Chennai, India., Sunder Singh S; Department of Onco-Pathology, Cancer Institute (WIA), 38, Sardar Patel Road, Guindy, Chennai, India., Ganesharaja S; Department of Radiation Oncology, Cancer Institute, Chennai, 600 036 Tamil Nadu, India., Chiwate AS; Cervical Cancer Prevention Programme, Nargis Dutt Memorial Cancer Hospital Barshi, Maharahstra, India., Hingmire SJ; Cervical Cancer Prevention Programme, Nargis Dutt Memorial Cancer Hospital Barshi, Maharahstra, India., Rajkumar T; Department of Molecular Oncology, Cancer Institute (WIA), Chennai, India.
Jazyk:	angličtina
Zdroj:	Asian Pacific journal of cancer prevention : APJCP [Asian Pac J Cancer Prev] 2023 Jul 01; Vol. 24 (7), pp. 2337-2346. Date of Electronic Publication: 2023 Jul 01.
DOI:	10.31557/APJCP.2023.24.7.2337
Abstrakt:	Objective: Cervical cancer is the third most common cancer in women, worldwide. This study was designed to develop an affordable, accurate and simpler screening test like Enzyme-linked immunosorbent assay (ELISA) which is low cost and will help in bringing down the disease burden in resource poor countries. Methods: In this study, we have raised and evaluated monoclonal antibodies against recombinant p16 using immunohistochemistry (IHC), western blot, immunoprecipitation and ELISA. Double antibody sandwich ELISA (DAS-ELISA) and cytokeratin ELISA was designed for screening women with cervical dysplasia and cancer. Results: Cloned, expressed and purified recombinant p16 were used for generation of monoclonal antibodies. After initial screening, six clones were selected, and affinity purified. Except 155D11G10, which was isotype Immunoglobulin (Ig) G1 all the others were found to be IgG2b. 133A6G5 and 151A7B9 were found to be best for p16 IHC, both showed 70 - 80% and 80 - 90% of nuclear staining respectively. All the antibodies positively detected p16 from the HeLa lysates in western blot except 133A6G5. Studies using immunoprecipitation showed 133A6G5, specifically detected p16. DAS-ELISA developed using a combination of our p16 monoclonal antibodies showed sensitivity of up to 2pg. A pilot study using DAS-ELISA and cytokeratin ELISA in cervical samples revealed the assay sensitivity and specificity as 100% and 80%, respectively. Conclusion: Using combination of DAS-ELISA and cytokeratin ELISA we have developed an accurate and reliable method for the early detection of cervical cancer in a subject, with minimal false results. In the future after large scale validation, p16 ELISA could be used as a reliable tool for diagnostic purposes.
Databáze:	MEDLINE
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