【中文摘要】目的:建立一种针对促红细胞天生素(EPO)的酶联免疫学检测(enzyme linkedimmunosorbent assay,ELISA)方法,建立相关的试剂盒。应用直接包被与间接包被方法。并运用此试剂盒对临床上相关疾病进行检测,了解其临床应用价值,指导相关疾病的诊治。方法:1.免疫层析法将购买的重组人类红细胞天生素(rhEPO)抗原纯化,紫外分光光度计法测定蛋白含量。2.抗体的纯化。用金黄色葡萄球菌蛋白A(SPA)亲和层析法纯化EPO抗体。3.用辣根过氧化物酶(HRP)标记EPO抗体。4.用酶联免疫检测(ELISA)的双抗体夹心法制备直接包被的试剂盒,并对板、试剂、各种反应条件进行优化探讨。5.利用生物素—亲和素系统的优点,将酶标板包被BBC,与生物素化的抗体反应,组盒。6.将以上的直接包被法进行灵敏性、重复性、稳定性等的方法学评价。7.用试剂盒检测相关的肿瘤、贫血标本,并进行正常血清的对照试验。与医院常用的放射性免疫检测结果对比。结果:1.将抗体连接在凝胶上,装在柱子中,制成免疫层析柱,依次过以下液体:平衡液,洗脱往带分离抗原蛋白中的杂质蛋白;高盐溶液,洗往可以与凝胶柱的非特异结合蛋白;洗脱液,根据PH值的变化或者离子强度的改变,将于凝胶上的抗体结合的抗原蛋白洗脱下来。收集洗脱峰的各个部分,分装到很多小瓶中,每个小瓶1ml。测定蛋白浓度,根据经验公式:蛋白浓度=1.750D_(280)-0.74OD_(260),用进口标准试剂盒测定各个收集组分,确定确为EPO抗原纯化产物。2.酶标抗体的制备采用过碘酸钠法。把HRP溶于乙酸钠,与NaIO_4室温避光搅拌30min,用乙二醇终止上述氧化反应。再透析脱盐,上述酶液与抗体反应,结束后,透析离心,取上清,过Sephadex G200层析柱。得到的即为标记后的纯化酶。测定酶的量:1.2mg/ml,IgG为3.2mg/ml。3.ELISA最适条件的选择如下:对比美国COSTAR板与国产板,前者在吸附性能、灵敏性及本底方面均优于后者,选择包被COSTAR酶标板;对酶标板经过异丙醇活化和紫外照射后明显可以降低本底的干扰,并且使板条的吸附性能有所加强;反应时间最适为30min;温度37℃;直接包被的抗体浓度在以纯化的抗体的基础之上,1:600为最合适,抗原标准品包含正常范围的一系列浓度为0mU/mL、5mU/mL、10mU/mL、20mU/mL、40mU/mL、80mU/mL、160mU/mL,抗原制备的160点相当于制备纯化抗原的1:400稀释,酶标抗体的最适合浓度为:1:3000稀释。本试剂盒的范围为5～160 mU/mL,足以包含正常范围6～25mU/mL;灵敏度为0.46mU/mL;高浓度样品均匀回收率为106.74%,低浓度样品均匀回收率为104.78%;正确度为38.982mU/mL;稳定性良好;精密度实验结果显示,批内变异＜10%,批间变异＜15%。ELISA方法测定的标本值与放射免疫检测符合性良好。结论:本研究对ELISA反应体系可能碰到的题目加以改良,对反应温度、时间均加以比较探索直至最合适,采用高纯度的抗体对板条进行包被,板条经过紫外照射,通过棋盘滴定法确定了抗原、抗体及酶的最佳工作浓度。灵敏度、回收率、批内变异、批间变异、稳定性及特异性等方法学评价指标均良好。同生物素间接包被相比较,后者会提供更加灵敏、及本底更好的方法,因此,本方法提供了临床上快速、正确、方便检测EPO的工具,很有发展前途。');
【Abstract】 Objective:Set up an enzyme linked immunosorbent assay(ELISA) method for detecting erythropoietin(EPO) and establish the relative kit.We want to use the direct and indirect coating ways.The kit is to be used to detece the diseases,evaluate its clinical value to to direct the diagnosis and therapy.Methods:1.Purify the antigen of rhEPO with the immunochromatographic method,and determine protein content with the luminance meter.2.Purification of the EPO antibody.Staphylococcus aureus can be used to purify the EPO antibody.3.Label the EPO antibody with HRP.4.The kit should be made with the method of ELISA,and it may be improved by modifing the plate,reagent and conditions,ect.5.The biotin-avidin system has many merits.The kit should be coated by biotinylated bovine serum albumin(BBS).6.Estimate the sensitivity,reproducibility and stability of the technologies about the direct coating method.7.Detect the blood preparations like oncoma and anaemia with the kit developed by us.The results should be compared with normal blood serum.Also the method should be in contrast with radioimmunity.Results:1.To link the antibody and gel carefully and then fill them into the chromatographic column,so the system is available.Now the experiment should bagain with rinse solutions like balanced solution,salt solution and elutriant.They have the functions respectively as follows:wash the impurity,non-special binding proteins off,then the elutriant can purify the antigen with washing the them off according to the variation of ionic strength and PH.Gather eluting peaks separately, fill;the solutions into flaskets,1ml per flasket.Use the following formula to calculate the protein concentration:Value=1.75A_(280)-0.74A_(260).It can be confimed that the collections are all we want.2.Heptaiodic acid should be used to make the enzyme conjugation.The HRP, dissolved in sodium acetate,responses with NaIO4 thoroughly,being stird in 30 minutes.The reaction can be stopped by ethylene alcohol.Then dialyse and centrifuge them.To get the above solution and make the gel chromatography,and we can get the pure enzyme.The concentrations is 1.2mg/ml,and IgG is 3.2mg/ml.3.Choose the optimum condition:the adsorption capability,sensitivity and background of the costar plate are much better than the national plate.So the costar plate is chosen and,it also can be improved by exposured under ultraviolet rays or be washed by dimethyl carbinol.We foud the proper time and temperate.They are 30min and 37℃.The concentration of antibody is 1:600,the 1:400 dilution of antigen is 160 mU/mL,the suitable resule of enzyme is 1:3000.The range of the kit is 5～160 mU/mL,sensitivity is 0.46mU/mL,recovery of the high and low concentration are 106.74%and 104.78%,accuracy rating is 38.982mU/mL,stability is perfect, variation is less than 15%.The result of ELISA is good in comparation with radiological survey.Conclusions:The research aims to modify the conclusions of ELISA,such as temperature, time,etc.The proper condition of the test is found.The antibody coated is pure,the plate is illuminated with ultraviolet ray.And the suitable qualification of antigen and enzyme can be explored with crossed match.The index of evaluation like sensitivity, recovery rate,variation,stability are all perfect.Indirect coating method with biotin is much better.This experiment offers a good method to test EPO,which is promising.
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