类风湿性关节炎特异性预警检测方法建立

发布时间：2018-06-13 15:24

本文选题：环瓜氨酸肽 + 抗环瓜氨酸肽抗体　；参考：《郑州大学》2016年硕士论文

【摘要】：目的:抗环瓜氨酸肽(cyclic citrullinated peptide,CCP)抗体由类风湿性关节炎(rheumatoid arthritis,RA)患者B细胞分泌,对RA早期诊断具有特异性,同时对预测骨损伤程度及RA临床预后评估有重要价值。本研究拟建立一种检测人血清中抗CCP抗体含量的胶体金定量检测方法,为该病的预警诊断及预后评估提供一种技术手段,以此来推动该疾病特异性检测方法的进步。方法:1.通过人工合成技术合成CCP多肽,经碳二亚胺偶联技术使CCP多肽与牛血清白蛋白(BSA)偶联,优化最佳偶联条件,对偶联产物应用紫外(UV)法、SDS-PAGE进行鉴定、利用间接酶联免疫吸附法(ELISA)进行生物活性检测,确定最适CCP多肽偶联物。2.研制用于定量检测血清中抗CCP抗体含量的胶体金试纸条。通过对胶体金颗粒、标记pH优化、标记最低抗体量优化、免疫金保护液pH值优化、试纸条材料的选择与处理、最佳包被量的确定等方面研究,对试纸条性能从线性范围、精密性、稳定性、回收率及临床样本的检测等方面进行评价。初步建立人血清抗CCP抗体胶体金定量检测方法。结果:1.确定CCP-BSA偶联12 h为最佳偶联用时,获得可与抗CCP抗体特异结合的CCP-BSA偶联物,经检测鉴定确定偶联成功、产物有效,可用于制备胶体金试纸条。2.本研究建立的间接胶体金定量检测方法的工艺技术条件为:氯金酸与柠檬酸三钠体积比1:1.5制备的胶体金效果最佳;胶体金溶液保存最佳条件为加入0.05%的叠氮钠;标记时1 mL胶体金溶液加入3μL 0.2 mol/mL K2CO3,最低标记抗体量为8.8μg;免疫金保护液最佳pH为8.8;质控线包被羊抗鼠IgG最佳浓度为1.0 mg/mL;检测线包被CCP-BSA偶联抗原最佳浓度为1.5 mg/mL;包被缓冲液中蔗糖含量0.4%;金垫铺金量为400μL/10cm,金标定量仪读数时间为试剂反应后14min。3.建立的胶体金定量检测方法性能指标为:线性范围50~500 RU/mL,R2=0.993,精密性质控品L1批内CV=2.6%,批间CV=4.52%;精密性质控品L2批内CV=2.5%,批间CV=4.28%,高值、中值、低值血清的回收率分别为100.96%、97.17%、91.46%,用40份临床样本与国外试剂盒平行测试,结果表明两者具有相关性,回归方程:Y=1.004X+7.968,R2=0.955。结论:1.利用碳化二亚胺偶联技术成功将CCP多肽与BSA大分子偶联,且偶联产物可与抗CCP抗体特异性结合。2.建立了一种人血清抗CCP抗体胶体金定量检测方法,可初步应用于临床对抗CCP抗体的定量检测,为RA的临床早期预警诊断及预后评估提供技术支撑。
[Abstract]:Objective: the anti-cyclic citrullinated peptide antibody secreted by B cells in patients with rheumatoid arthritis (RA) is specific to the early diagnosis of RA, and has important value in predicting the degree of bone injury and evaluating the clinical prognosis of RA. The aim of this study was to establish a colloidal gold quantitative assay for the detection of anti-CCP antibody in human serum in order to provide a technical means for the early warning diagnosis and prognosis evaluation of the disease, so as to promote the progress of the specific detection method for the disease. Method 1: 1. CCP peptide was synthesized by artificial synthesis and coupled with bovine serum albumin (BSA) by carbodiimide coupling technique. The optimum coupling conditions were optimized. The coupling product was identified by UV UV) method. The bioactivity of ELISAwas determined by indirect enzyme-linked immunosorbent assay (Elisa). A colloidal gold strip for quantitative determination of anti CCP antibody in serum was developed. Through the study of colloidal gold particles, labeling pH optimization, labeling minimum antibody quantity optimization, immune gold protection solution pH value optimization, selection and treatment of test strip material, determination of optimal coating amount, etc., the performance of test strip was linearly studied. Accuracy, stability, recovery and clinical sample detection were evaluated. A method for quantitative determination of colloidal gold with anti CCP antibody in human serum was established. The result is 1: 1. CCP-BSA conjugate was obtained when CCP-BSA was used for 12 h. The conjugate of CCP-BSA was successfully coupled with anti-CCP antibody. The product was effective and could be used to prepare colloidal gold strip. 2. The technological conditions of the indirect colloidal gold quantitative determination method are as follows: the colloidal gold prepared by the volume ratio of chlorgold acid and trisodium citrate 1: 1.5, the best condition of colloidal gold solution preservation is the addition of 0.05% sodium azide; When 1 mL colloidal gold solution was added 3 渭 L 0.2 mol / mL K _ 2CO _ 3, the lowest labeled antibody was 8.8 渭 g, the best pH of immunogold protection solution was 8.8, the best concentration of goat anti-mouse IgG was 1.0 mg / mL, the best concentration of CCP-BSA coupling antigen was 1.5 mg 路mL ~ (-1), the best concentration of goat anti-mouse IgG was 1.0 mg 路mL ~ (-1), the best concentration of CCP-BSA conjugated antigen was 1.5 mg 路mL ~ (-1). The content of sucrose in buffer solution was 0.4 and the amount of gold covered with gold was 400 渭 L / 10 cm. The reading time of gold calibrator was 14min. 3 after reagent reaction. The performance indexes of the established colloidal gold quantitative detection method are as follows: linear range 50 ~ 500 RUR / mL ~ (2 +) 0.993, precision property control L _ (1) C _ (2) C _ (2) C _ (2) C _ (2.5), interlot CVN _ (4.28), high value, median value, interlot CVN _ (4.52), precision property control L _ (2), CVI ~ (2) / R _ (2) = 0.993, precision property control L _ (1), The recoveries of low value serum were 100.96 and 97.177.171.46, respectively. 40 clinical samples were tested in parallel with foreign kits. The results showed that there was a correlation between the two. The regression equation was Y: 1.004X 7.298 R2O0.955. Conclusion 1. CCP peptide was successfully coupled with BSA macromolecule by carbonized diimide coupling technique, and the coupling product could specifically bind to anti-CCP antibody. A method for quantitative detection of anti-CCP antibody colloidal gold in human serum was established. It can be applied to the quantitative detection of anti-CCP antibody in clinic and provide technical support for clinical early warning diagnosis and prognosis evaluation of RA.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R593.22

【相似文献】