Development Of ELISA Method For Erythropoietin And Study On The Clinical Application

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=1.75A₂₈₀-0.74A₂₆₀.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.