Establishment of the Detection Method of Serum IL-6 Level and Its Preliminary Application Evaluation based on Acridine Ester Chemiluminescence Immune Quantitative Analysis Technology
Establishment of the Detection Method of Serum IL-6 Level and Its Preliminary Application Evaluation based on Acridine Ester Chemiluminescence Immune Quantitative Analysis Technology
Objective To establish a method for the detection of serum interleukin(IL)-6 based on acridine ester chemiluminescence immune quantitative analysis.Methods Double-antibody sandwich method was applied,acridine ester was used to label complete antibody,and biotin was used to label enzyme-cut fragment antibody.Next,then they formed a sandwich complex with tested substance IL-6,in which biotin and streptavidin-coated magnetic solid particles reacted specifically to luminescence.Quantitative analysis was conducted by measuring the luminescence signal value,and the conditions for labeling antibody dilution buffer,concentration of labeled antibody and sample addition amount were optimized.The performance indexes such as limit of blank(LOB),limit of detection(LOD),intermediate precision,reportable range,interference test and HOOK effect were evaluated.Meanwhile,145 serum samples were selected and compared with Roche electrochemiluminescence by linear regression analysis.Results A method for the detection of serum IL-6 based on acridine ester chemiluminescence immune quantitative analysis was established successfully.MES-BSA was selected as the dilution buffer of labeled antibody,0.2μg/ml was selected as the concentration of labeled antibody,and 50 μl was selected as the sample addition amount.The LOB and LOD of the method were 0.2 pg/ml and 0.5 pg/ml,respectively,and the reportable range and the intermediate precision were 0.5～3 0000 pg/ml and less than 5.3％,respectively.When IL-6 concentration reached 200 000 pg/ml,HOOK effect did not appear.In the concentration range of triglyceride(TG)30 000 μg/ml,hemoglobin(HGB)9 000 μg/ml,bilirubin(TBIL)330μg/ml and rheumatoid factor(RF)1 500 IU/ml,the interfering substances did not affect on the detection results.Compared with the electrochemiluminescence method for IL-6 determination,the linear regression equation was Y=0.980 2X-3.487 9,r=0.997 7,and the results of reagent determination by the two methods were highly correlated(P＜0.05).Conclusion A method for the detection of serum IL-6 based on acridine ester luminescence is established.All the indexes meet the requirements of clinical application,making them suitable for promotion and application in clinical laboratories.
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