Method for marking protein by carboxyl microsphere

Abstract

The invention provides a method for marking protein by a carboxyl microsphere, belonging to the field of preparation of immunologic diagnosis kits. According to invention, in a system for marking the protein by the carboxyl microsphere, according to the content of carboxyls on the surface of the microsphere, the molar ratio of cross-linking agents EDC to -COOH is set to be (0.8-2) to 1, and the molar ratio of EDC to NHS or Sulfo to NHS is (1-3) to 1, so that the optimal ratio of the number of the carboxyls on the surface of the microsphere to the number of activated carboxyls is achieved; meanwhile, the pH is regulated, so that the reaction system is rapidly converted from condition beneficial for activation into state beneficial for antibody connection, the step of centrifugalization or tangential flow filtering is saved, and finally, the purposes of simplifying marking steps, saving cost, and marking protein by the carboxyl microsphere efficiently are achieved. The method provided by the invention can be applied to preparation of a detection kit taking the latex microsphere or the magnetic microsphere as a carrier, and has good market application prospect and economic value.

Description

technical field [0001] The invention relates to the technical field of preparation of immunodiagnostic kits, in particular to a novel, low-cost and easy-to-operate carboxyl microsphere-labeled protein method. Background technique [0002] Nano-microspheres are widely used in in vitro diagnostic reagents, which greatly improve the sensitivity of the reagents and can detect substances with minimal sample content. Nano-microspheres are used as carriers in diagnostic reagents with uniform size. The surface of the microspheres is coated with protein molecules such as antibodies and antigens. There are two methods for protein molecules to adsorb onto nano-microspheres: physical adsorption and chemical coupling. Physical adsorption is a relatively traditional and effective method, but it also has the disadvantages of being sensitive to system changes and poor stability; chemical coupling can avoid these defects. In a narrow sense, chemical coupling refers to the use of commerciall...

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Problems solved by technology

[0004] The advantage of the one-step method is that it is easy to operate. Although it is relatively simple to use EDC as a cross-linking agent, this method is uncontrollable. In addition to the target product, the final product may also have a large number of self-ligated products of multiple antibodies, which not only wastes antibodies. , also greatly reduces the reaction rate

The two-step method uses EDC and NHS or Sulfo-NHS as a cross-linking agent, because the reaction is carried out step by step, the first step is to activate the carboxyl group, and then add the antibody after washing the microspheres, so the direction of the reaction is certain and controllable There are two most commonly used methods for washing microspheres after activation. One is centrifugation. Even precipitation seriously affects the efficiency of coupling; the second is tangential flow filtration, which will not cause latex precipitation, but it takes a long time and affects the efficiency of the second step reaction; if it is magnetic carboxyl microspheres, it is relatively Convenience, beads can be washed using a magnetic separation system

The common disadvantage of the two chemical coupling methods is that the requirements for the reaction conditions are relatively harsh. For example, the one-step method must be reacted in an acidic environment, the first step of the two-step method must be in an acidic environment, and the second step is preferably in an acidic environment. In an alkaline environment

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