Proceeding2562

1568 การประชุมวิชาการระดับชาติมหาวิทยาลัยทักษิณ ครั้งที่ 29 ประจ�ำปี 2562 วิจัยและนวัตกรรมเพื่อการพัฒนาที่ยั่งยืน purification protein follow the manufacturer’s manuals. Purified histidine-tagged p16 INK4a protein concentration was measured by the Bradford protein microassay (Bio-Rad, Hercules, CA, USA) Serum samples and sample preparation Serum samples were obtained from 97 women with different cervical lesions including, anti-p16 INK4a antibodies were observed by Western blot analysis in previous study (5) including 50 cases of positive and 47 cases of negative. Serum samples were diluted to 1:200 in PBS with 0.01% sodium azide before further ELISA processes. To eliminate the non-specific background, serum were absorbed with formalin-fixed E. coli BL21 cells to final 2% cells (w/v) and 1:400 dilution at 37 °C for 1 hour(6). Absorbed serum was centrifuged at 13,000 rpm for 10 min and supernatant was collected for ELISA test. ELISA procedure Ninety-six well maxisorb immunoplate (Nunc, Thermo Fisher Scientific, USA) were coated with 100 ul of 2 ug/ml histidine-tagged p16 INK4a protein in bicarbonate buffer (pH 9.6) at 4 °C overnight. Plates were washed with 3 time with PBS containing 0.05% Tween-20 (PBST). Three-hundred microliters of 1% bovine serum albumin was used for blocking buffer, incubated at 37 °C for 2.5 hours and washed four times with PBST. Diluted and absorbed serum was added duplicated for 100 ul per well and incubated at room temperature in moisture chamber for 2 hours. Plates were washed four times with PBST, 100 µl diluted secondary antibody, 1:40,000 of HRP goat anti-human IgG (Immunology Consultants Laboratory, Inc.) was added and incubated with of at room temperature for 30 min. Washed five times with PBST, 100 µl of 3,3′,5,5′-tetramethylbenzidine (TMB) reaction solution were added to each well and incubated at room temperature for 30 min in complete darkness. Reaction was stopped with 100 µl of 1 M H 2 SO 4 . Developed color was measured at 450 nm with Infinite 200 PRO microplate reader (Tecan, Männedorf, Switzerland). Serum of 4 negative cases were pooled and tested for 31 repeats. OD 450 values were calculated for mean, standard derivative (SD) values and tested for cut-off value of positive result. Sensitivity and specificity of developed ELISA method was calculated compare with previous Western blot results Results and discussion Protein expression and purification The plasmids of pTrcHisA-p16 INK4a were transformed to E. coli BL21 strain for protein expression. Protein expression was induced by IPTG (optimal concentration = 1 mM). Purified histidine-tagged p16 INK4a protein was obtained up to 56 µg/ml. Determination of optimal condition for enzyme-linked immunosorbent assay Development of the indirect ELISA was simultaneously optimized the protein concentrations, antibody dilution, incubation temperature and incubation time to suitable ELISA condition. To define the optimal coating p16 INK4a protein concentration, purified protein was coat with 100 µl of 2 µg/ml at 4 ºC overnight which gave most suitable condition for coating step. For serum sample dilution step, serum were absorbed with 2% E. coli cells can reduce the background signal with 1:400 final dilution. The optimal dilution of HRP-conjugated goat anti-human IgG was defined at the dilution of 1:40,000. Color development from HRP-TMB reaction was 30 minutes followed with stop solution. Determination of cut-off value, sensitivity and specificity Mean of OD 450 pool negative serum sample was calculated from 31 repeated values is 0.69 with standard deviations (SD) of 0.054. Cut-off value was adjusted with mean+4SD (OD450>0.908, p<0.01). In this study, we opted for a slightly higher cut-off value to increase the sensitivity of the assay.