| Background The incidence of diabetes is increasing year by year because of the improvement of people’s living standards and the effect of the comprehensive factors, and the onset age tend to be younger, at the same time the incidence of complications also increased year by year. Diabetic retinopathy(DR) as one of the most commo n and severe complications of diabetes, with longer duration of diabetes, when DR develops into the late stage, which is proliferative diabetic retinopathy stage, it will cause irreversible damage to the eyesight.Diabetic retinopathy has seriously affected the quality of people’s lives, but its pathogenesis is not clear at present, the main pathological changes of diabetic retinopathy is that high blood glucose cause retinal capillary leakage, and then induce retinal edema, retinal tissue ischemia and hypoxia, eventually lead to the formation of new blood vessels. At present, research for Diabetic retinopathy is focused on the retina, study found that the retina edema, ischemia and hypoxia can change the expression level of aquaporin4 in the retina. In recent years, study found that aquaporin4 level of serum in patients with diabetic retinopathy is also changing, but it is not clear how aquaporin4 level changes in vitreous.Objective To establish enzyme- linked immunosorbent assay methods which are used for testing AQP4 antigen and antibody in vitreous of patients with diabet ic retinopathy, and using them to analyze the variation of AQP4 expression level.Methods Using AQP4 rabbit-anti- human antibody as coating antibody and goat-antirabbit Ig G-HRP as enzyme- labeled antibody, we set up ELISA methods for testing AQP4 antigen and antibody in human vitreous. And optimize the working conditions of the two methods, including antibody working concentration, closed temperature and time, incubation temperature and time, chromogenic temperature and time, etc. Then we evaluate the linear, detection range, lowest detection limit and repeatability. Finally we detect AQP4 antigen and antibody levels in normal persons and patients with diabetic retinopathy, and statistical methods to analyze the difference of them.Results This study initially established the double-antibody sandwich ELISA method for detection of AQP4 antigen and the competition ELISA method for the detection of AQP4 antibody. Double antibody sandwich ELISA working conditions were as follows: the best package concentration of rabbit anti-AQP4 antibody was 2.0μg/ml, the best closed condition was 37℃ 1h, sandwich primary antibody optimal working condition was 1:800 and reaction condition was 37℃ 1h, the best enzyme-labeled antibody dilution was 1:3500 and reaction condition was 37℃ 1h, optimal chromogenic condition was 37℃ 15 min. Standard curve y=0.0178x+0.1937, the coefficient of determination R2=0.9938. The linear range of detection was 3.125-100ng/ml and the lowest detection limit was 3.125 ng/ml. three intra-assays CV% were 3.80% 4.68% and 4.18%, respectively. inter-assay CV% was 3.01%.competition ELISA working conditions were as follows : the optimal package concentration of rabbit anti-AQP4 antibody was 1:500 and the optimal working condition of enzyme-labeled secondary antibody was 1:5000 1h,the best closed condition was 37℃ 1h, the optimal dilution of sand wich primary antibody was 1:400,the actual work concentration was 1:800,the best incubate condition was 37℃ 1h, the optimal chromogenic condition was 37℃ 15 min. standard curve y=-0.3937 lg X+1.4471, the coefficient of determination R2=0.9893. The linear range of detection was 2.5-80ng/ml and the lowest detection limit was 2.5 ng/ml. Three intra-assays CV% were 2.41%, 2.60% and 1.60%, respectively. Inter-assay CV% was 2.49%.The intra-assays CV% and the inter-assays CV% in the two methods was less than 5% and 10%, respectively. Difference of AQP4 antigen and antibody between normal persons and patients with diabetic retinopathy both have statistical significance(P<0.05).Conclusion1 The double antibodies sandwich ELISA methods of AQP4 antigen for human vitreous were preliminary established.2 The competitive ELISA methods of AQP4 antibody for human vitreous were preliminary established.3 AQP4 antigen and antibody expression levels were upregulated in patients with diabetic retinopathy. |
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