The Manufacture And Exploratory Development Of IRMA Kit Of Monoclonal Antibody Against Human Costimulatory Molecules B7-2

ObjectiveTo establish a useful and stable method for labeling monoclonal antibody 6C8 with 125I;Solid phase antibody 6A5 coating test tube method; To establish IRMA technology for analysis soluble B7-2 molecule in human serum and discussing clinical application.MethodsUsing chloramine T method for labeling monoclonal antibody 6C8 with 125I and purifying label antibody; Detecting the labeling efficiency and radiochemical purity with paper chromatography;activing test tube; To research fix method of monoclonal antibody for polystyrene test tube with physical adsorption and poly- lysine chemical conjugation procedure that activing tube;To find the best reaction conditions by adding different volument serum and 0.1% tween80 and react different times;To study double antibody method as separation technique; To establis IRMA technology for analysis soluble B7-2 molecule.ResultsBy experiment,we found the best reaction conditions labeling monoclonal antibody 6C8 with 125I: Na125I 7.4MBq,chloramine T 20μg,antibody 6C8 6μg,Na2S2O5100μg,reaction time 30 second,bulk volume 200～300μl;The best solid phase coating condition:After irradiating polystyrene test tube 12h withγray,we dilute antibody with 0.2mol/L phosphate buffer in PH7.4 until antibody 6A5 is 5μg/ml,and put the antibody solution in refrigerator 24h whose temperature is 4℃.Then we draw off the antibody solution and wash tube 5 times with 0.2mol/L phosphate buffer in PH7.4. At last we add 3%BSA 500μl to each tube sealing 6h, bloting solution and washing tube 5 times with 0.2mol/L phosphate buffer 0f PH7.4.The best reaction conditions is:Adding serum 200μl and 0.1% tween80 20μl and reacting 4h.To process standard curve with SPSS 11.5, Y= 787.484+42.86X,adjusted R2=0.969,P=0.0137(<0.05),the range of standard linear is 0.208～100ng/ml,NSB =4.35%,precision 5.8%。The method can successfully analyze soluble B7-2 molecule in serum.From the result,we know the patient of Graves disease is higher than healthy in B7-2 level, but the patient of tumor lower.ConclusionsIRMA technology the study establishing have significant value in clinical research for B7-2 molecule.