Proceeding2562

1566 การประชุมวิชาการระดับชาติมหาวิทยาลัยทักษิณ ครั้งที่ 29 ประจ�ำปี 2562 วิจัยและนวัตกรรมเพื่อการพัฒนาที่ยั่งยืน Development of enzyme-linked immunosorbent assay for anti-p16 INK4a antibody detection in serum from women with cervical lesions Piyawut Swangphon 1* , Lalita Munsamrith 2 , Sutanya Thanudaksorn 2 , Sasipha Chansuk 2 and Chayanit Noosanthad 2 Abstract Introduction: Infection with high-risk types of Human papillomavirus are associated with cervical carcinogenesis. Overexpression of p16 INK4a , which is tumor suppressor protein are presented in abnormal cells and using as a biomarker for HPV associated cervical neoplasia. Immune responses to self p16 INK4a protein was evaluated as antigen which can stimulates immune responses and associated with cervical lesion severity. High-throughput and more sensitivity detection method, such as ELISA method may develop and use for cervical cancer screening. Objective: To develop an ELISA method for detect anti-p16INK4a antibodies in serum collected from women with cervical lesions. Methods: Ninety-seven sera samples (positive 50 cases and negative 47 cases) from women with previously evaluated for anti-p16 INK4a antibodies with Western blot and used for ELISA optimization. Recombinant histidine-tagged human p16 INK4a protein was cloned into pTrcHisA plasmid, expressed in Escherichia coli BL21 strain and purified with affinity nickel column. ELISA plate was coated with purified p16 INK4a protein and used for anti-p16INK4a antibody detection. Cut-off value of anti-p16 INK4a antibody positive was calculated from 31 repeated values of pooled of 4 negative serum. Results: Cut-off value of this developed ELISA anti-p16 INK4a antibody is 0.908 at OD 450 . The sensitivity and spec- ificity are 94.0 % and 82.97 %, respectively. Data from this study show this ELISA gave more sensitivity, speci- ficity and suitable for antiboby detection than Western blot method. Conclusion: Data from this study show this ELISA gave high sensitivity and specificity Moreover, this ELISA condition for anti-p16 INK4a antibodies detection need to further evaluate to increase higher sensitivity and specificity. Keywords: human papillomavirus, p16 INK4a protein, enzyme-linked immunosorbent assay (ELISA), cervical lesion.