人玻璃体中AQP4的ELISA方法建立及在糖尿病视网膜病变中的初步应用

发布时间：2018-10-23 13:28

【摘要】：背景随着人们生活水平的提高以及一些综合因素的作用,糖尿病的发病率也逐渐上升,并趋向年轻化,与此同时其并发症的发病率也逐年升高。作为糖尿病患者最常见和最严重的并发症之一,糖尿病性视网膜病变(diabetic retinopathy,DR)随着糖尿病病程的延长,如果发展到视网膜病变的后期即增殖型糖尿病视网膜病变期,将会对视力造成不可逆转的损害。糖尿病性视网膜病变已严重影响了人们的生活质量,但目前其发病机制并不完全明确,糖尿病性视网膜病变的主要病理改变是由于高血糖引起视网膜毛细血管渗漏造成视网膜水肿,继而引起视网膜组织缺血缺氧,最终引发新生血管的生成。目前糖尿病性视网膜病变中研究较多的是视网膜,研究发现视网膜水肿、缺血和缺氧等条件均会引起视网膜内水通道蛋白4(aquaporin4,AQP4)表达水平的改变,而近年的研究发现糖尿病性视网膜病变患者血清中水通道蛋白的水平也发生改变,但玻璃体内水通道蛋白4的表达水平如何目前尚不明确。目的建立检测糖尿病性视网膜病变患者玻璃体内水通道蛋白4抗原及抗体的酶联免疫检测方法,并用本试验建立的方法分析糖尿病性视网膜病变患者玻璃体内AQP4表达水平的变化。方法本研究以兔抗人水通道蛋白4抗体为包被抗体,羊抗兔Ig G-HRP为酶标抗体,分别建立检测人眼玻璃体内水通道蛋白4抗原抗体的ELISA检测方法;并对两种方法的工作条件进行优化,包括抗体的工作浓度,封闭温度和时间,孵育温度和时间,显色温度和时间等。随后对两种方法的线性、检测范围、最低检测限以及重复性等性能进行评价。最后用这两种方法分别测定正常人和糖尿病性视网膜病变患者玻璃体内水通道蛋白4抗原抗体的水平,统计学方法分析正常人和糖尿病性视网膜病变患者玻璃体内水通道蛋白4抗原抗体表达水平的差异。结果初步建立了检测人眼玻璃体内水通道蛋白4抗原的双抗体夹心ELISA方法和检测水通道蛋白4抗体的竞争ELISA方法。双抗体夹心ELISA的工作条件:最佳包被抗体浓度为2.0μg/ml;最佳封闭温度和时间为37℃封闭1h,夹心一抗最佳工作浓度为1:800,最佳孵育温度和时间为37℃1h,酶标抗体最适浓度为1:3500,最佳显色条件为时间为37℃显色15min。标准曲线y=0.0178x+0.1937,决定系数R2=0.9938,线性范围为3.125-100ng/ml,最低检测限为3.125ng/ml,3次批内CV%分别为3.80%、4.68%、4.18%,批间CV%为3.01%。竞争法ELISA工作条件:包被抗体和酶标抗体的最适浓度分别为1:500和1:5000,最佳封闭温度和时间为37℃封闭1h,夹心一抗的最佳稀释度为1:400,实际工作浓度是1:800,最佳孵育条件为37℃孵育1h,最佳显色条件为37℃显色15min。标准曲线y=-0.3937lg X+1.4471,决定系数R2=0.9893,线性范围为2.5-80ng/ml,最低检测限为2.5 ng/ml,三批试验测得的批内CV%分别为2.41%,2.60%,1.60%,批间CV%为2.49%。两种ELISA方法的批内变异系数均小于5%,批间CV%小于10%。正常人与糖尿病性视网膜病变患者玻璃体内水通道蛋白4抗原水平比较有显著性差异(P0.05),而两组间的抗体水平比较也有统计学意义(P0.05)。结论1初步建立了检测人眼玻璃体内AQP4抗原的双抗体夹心ELISA检测方法。2初步建立了检测人眼玻璃体内AQP4抗体的竞争法ELISA检测方法。3糖尿病性视网膜病变患者玻璃体内AQP4抗原及抗体的表达水平均上调。
[Abstract]:Background With the improvement of people's living standard and the effect of some comprehensive factors, the incidence of diabetes is increasing gradually, and the incidence of complications is increasing year by year. As one of the most common and most serious complications of diabetes mellitus, diabetic retinopathy (DR) increases with the course of diabetes, and will cause irreversible damage to vision if it develops to the late stage of retinopathy, namely proliferative diabetic retinopathy. Diabetic retinopathy has seriously affected people's quality of life, but the pathogenesis of diabetic retinopathy is not completely clear, and the main pathological changes of diabetic retinopathy are retinal edema due to blood capillary leakage caused by hyperglycemia, In turn causes retinal tissue ischemia and hypoxia, resulting in the formation of new blood vessels. At present, there are many studies in diabetic retinopathy, and studies have found that retinal edema, ischemia and hypoxia can cause changes in the expression level of aquaporin 4 (AQP4) in the retina. In recent years, it has been found that the level of water channel protein in serum of diabetic retinopathy is also changed, but the expression level of water channel protein 4 in the vitreous body is unclear at present. Objective To establish an enzyme-linked immunosorbent assay (ELISA) for detecting aquaporin 4 antigen and antibody in vitreous of diabetic retinopathy patients, and to analyze the changes of AQP4 expression level in diabetic retinopathy patients. Methods Using rabbit anti-human aquaporin 4 antibody as coated antibody, goat anti-rabbit Ig G-HRP as enzyme-labeled antibody, we established ELISA detection method for detecting water channel protein 4 antigen antibody in human eye glass body, and optimized the working conditions of two methods, including the working concentration of antibody. Close temperature and time, incubation temperature and time, color temperature and time, etc. The linearity, detection range, minimum detection limit and repeatability of the two methods were evaluated. Finally, the levels of water channel protein 4 antigen in the vitreous body of normal and diabetic retinopathy patients were determined by the two methods. The difference of the expression level of water channel protein 4 antigen was analyzed in normal subjects and diabetic retinopathy patients. Results The double antibody sandwich ELISA for detecting the water channel protein 4 antigen in human eyes was established and the competitive ELISA for detecting the water channel protein 4 antibody was established. The optimal concentration of sandwich antibody was 1: 800, the optimal incubation temperature and time was 37 鈩，

本文编号：2289396