Method for determining binding amount of biotinylated antibody of streptavidin labeled microspheres

Abstract

The invention provides a method for determining the binding amount of a biotinylated antibody of streptavidin labeled microspheres. The method comprises the following steps: 1) preparation of the biotinylated antibody: diluting 2mg of the antibody to 5mg / ml by using a PBS buffer solution, adding a certain amount of sulfo-NHS-LC-Biotin, uniformly shaking, reacting for 1 hour at room temperature, and dialyzing and purifying; 2) preparation of an acridinium ester-biotinylated antibody: diluting the biotinylated antibody in the step 1) to 2mg / ml, adding a certain amount of DMSO solution of acridinium ester, uniformly shaking, reacting for 2 hours in a dark place at room temperature, dialyzing and purifying in a dark place, and quantifying by using a BCA method; 3) drawing a standard curve, and reading a luminescence value RLU; and drawing an acridinium ester-biotinylated antibody addition mass-RLU relation curve. The method has the advantages that compared with a fluorescence method, the chemiluminescence method is higher in sensitivity and higher in anti-interference capability; and compared with a microsphere compound test method, the chemiluminescence method is simpler in operation and more accurate in result.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to a method for determining the binding amount of biotinylated antibodies on streptavidin-labeled microspheres. Background technique [0002] The streptavidin-biotin (SA-Biotin) system has a very high binding affinity and is widely used in the biological field. In recent years, streptavidin-labeled microspheres, including streptavidin-labeled latex microspheres, fluorescent microspheres, quantum dot microspheres, silica magnetic microspheres, polymer magnetic microspheres, etc., have been used in biological Applications in medicine, in vitro diagnostics and other fields are also becoming wider and wider. Streptavidin-labeled microspheres can be combined with biotinylated antibodies very conveniently and quickly, so as to obtain microspheres with specific antibodies on the surface, which can be used for subsequent immune reactions. [0003] Existing technical solutions: At present, ther...

AI Technical Summary

Problems solved by technology

[0004] Conventional organic small molecule fluorescent dyes (such as FITC, etc.) have a small Stokes shift, and the excitation wave overlaps with the emission wave to a certain extent, which is prone to interference and affects the accuracy of the measurement results.

In addition, it is generally believed that the sensitivity of fluorescence quantification is higher than that of ultraviolet-visible light, but lower than that of chemiluminescence, which limits its application in the direction of quantitative protein quantification.

[0005] On the other hand, through our experiments, we found that the microsphere and water system is a two-phase system, which is quite different from the test of the solution phase. Simply measuring the fluorescence value of the microsphere-antibody complex cannot accurately reflect the biotin The binding amount of the antibody is generally much lower than the actual binding amount, which may be due to the quenching of the fluorescence when it is transmitted between the two phases.