A New Test Strip For Rapid And Sensitive Detection Of NT-proBNP And Its Preparation Method

Fluorescence immunochromatography test is a rapid detection method based on antigen-antibody reaction.It has the advantages of simple operation,rapid detection,high accuracy,easy to carry and store.This article uses the NT-proBNP as the research object.Based on the conventional immunochromatography method,the biotin-streptavidin system was added to establish a new type of immunochromatographic test strip to further improve the sensitivity of NT-proBNP detection.The main findings are as follows:1.Using conventional immunochromatographic techniques,different combinations of NT-proBNP antibodies were used to perform immunochromatographic assays,and fluorescence intensity curves were plotted to compare T/C values.Comprehensive consideration of the results of each experimental group,the detection of antibody 5B6-15F11 has the highest T/C value and the most obvious advantage.Therefore,antibody 5B6 was selected as the labeled antibody for fluorescent microspheres,and antibody 15F11 was selected as the monoclonal antibody combined with activated biotin.To lay the foundation for the preparation of new immunochromatographic test strips.2.Optimize important parameters in the preparation of novel immunochromatographic strips:the amount of conjugated antibody and biotinylated antibody,the ionic strength of PB chromatography buffer,the amount of fluorescent microspheres conjugated to antibodies,and streptavidin concentration.The experiments were performed on the five parameters.The experimental results show that:When the amount of conjugated antibody and the amount of mixture were optimized,the nonspecificity of NT-proBNP was lower and the linear relationship was better under the experimental conditions of 50μg of antibody 5B6 and 2μl of biotinylated antibody;When the ionic strength of PB chromatography buffer was optimized,although the T/C value was the highest under the condition of 500 m M,200 m M PB buffer was selected as the chromatographic fluid because the higher concentration of 500 m M was not good for chromatography;When optimizing the amount of fluorescent microspheres,0.125μL was finally selected as the dosage parameter of the fluorescent microspheres in the novel immunochromatography technique;In optimizing streptavidin concentrations,the experimental results showed that 2.5μg/μL was the best SA lineage concentration.3.Based on the experimental results of the first two parts,a novel immunochromatographic assay was established.Based on the experimental results of the first two parts,a new immunochromatographic assay was established,and conventional immunochromatographic assays and novel immunochromatographic assays were compared.The results showed that:The new immunochromatography method still has good discrimination at low concentrations of NT-proBNP,and the new detection method has higher sensitivity.The novel immunochromatographic strip was used to detect the NT-proBNP concentration in different whole blood samples and compared with the results of the Radiometer test.The new immunochromatographic test strip has better advantages,and it still has good discrimination at low concentrations of NT-proBNP(0.02-0.2μg/L).the T/C value showed a good linear relationship with NT-proBNP concentration(R~2=0.9992).