| D-dimer is a specific product produced by cross-linking activated coagulation factor XIII(FXIIIa)when plasmin cleaves fibrin.It is widely used in the diagnosis and monitoring of deep vein thrombosis,pulmonary embolism,DIC and other diseases in clinical practice.In this study,hybridoma cell technology was used to develop monoclonal antibodies against human D-dimers and applied to develop D-dimer fluorescence immunochromatographic assay kits.The advantages of this kit are the new type,rapidity,and easy operation,etc.It is hoped that it can effectively meet the testing needs of D-dimer in small and medium primary hospitals and promote the balance of medical resources.Using natural D-dimer protein as immunogen,16 strains of hybridoma cells secreting anti-human D-dimer monoclonal antibody were successfully developed with hybridoma technology,named 4B6,4A6,1E12,4D3,2A7,3C4,4E12,2C8,2C3,3H10,2F9,4H3,1C11,1B12,3E5,4H9.The subtypes of all these antibodies are Ig G1.The ascites fluid is prepared by in vivo induction method,and the antibodies were purified by saturated ammonium sulfate precipitation method,with the purity of 70%～80%.Then antibodies were further purified by Protein G column to make the antibody purity more than 90%.The indirect ELISA method showed that the antibody titer of 16 strains was all more than 10~5.Using the initially established design scheme of fluorescence immunochromatography products,the optimal antibody pair 2F9 and 3C4 was selected from the 10 monoclonal antibodies with the highest titer Based on the antibody combination,the reaction system of the kit was further optimized.According to the parameters of detection limit,sensitivity and linear range,it is determined that the concentration of 16#antibody spray pad is 0.1mg/m L,the coating concentration of 4#antibody is 1.5mg/m L,the sample volume is 100μL,and the test time is 10 minutes.By evaluating the analysis performance,the precision of the kit can be controlled within 10%,the detection limit is less than 0.100mg/L,the linear range can reach 0.100～5.000mg/L,and the test is not affected by hemoglobin,triglyceride,free bilirubin and other interfering substances.The clinical performance of the self-made kits was evaluated with the kits that have been on the market with the same methodology.The results showed that the positive coincidence rate of the self-made kits was 100.00%,the negative coincidence rate was95.36%,and the total coincidence rate was 98.56%.The statistical software SPSS22.0 was used to further analyze the test results of 278 valid samples.The result shows that more than95%of the samples were within the consistency limit,and the correlation coefficient of the two kits was r=0.996>0.975.The analysis results of linear regression and paired test indicate that the test results of the two kits have a significant correlation,and the linear relationship is established and there is no significant difference.The anti-human D-dimer monoclonal antibody developed in this subject has successfully developed a D-dimer fluorescence immunochromatographic kit.The analytical performance of the kit meets the design requirements,and is equivalent to the clinical application of the marketed kit,which can meet the clinical testing needs. |
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