Target protein-induced deoxyribonuclease (DNase) cycle generation-based homogeneous immunoassay method

Abstract

The invention relates to a target protein-induced deoxyribonuclease (DNase) cycle generation-based homogeneous immunoassay method. A detection solution of the immunoassay method is prepared from an antibody 1-DNA1 conjugate, an antibody 2-DNA2 conjugate, hemin, exonuclease III and DNA3 / DNA 4 double-stranded DNA. When a target protein exists, the target protein can be immunologically recognized by the antibody 1-DNA1 conjugate and the antibody 2-DNA2 conjugate at the same time; based on an ortho effect, a complex is formed by hybridizing DNA1 and DNA2 and then has a chain substitution reaction with DNA3 on the double-stranded DNA, so that DNA4 rich in a G4 sequence is released and combines with the hemin so as to form G-quadruple chain / hemin DNase; in addition, the DNA3 reacting with the DNA1 and the DNA2 can be recognized and cut by the exonuclease III, so that the DNA1 and the DNA2 can undergo a chain substitution reaction with another DNA3, and another DNA4 is accordingly released to produce DNase; therefore, if repeating in this way, a large amount of DNase can be produced. The DNase can catalyze the color development of tetramethylbenzidine (TMB) and the luminescence of luminol, and can be used for carrying out sensitive quantitative analysis on the target protein by means of color comparisons and chemiluminiscence. The immunoassay method is simple to operate, rapid, sensitive and high in universality, and has a certain clinical application value.

Description

[0001] 1. Technical field [0002] The invention is a homogeneous immunoassay method based on target protein-induced DNase cycle generation, which is used for simple, fast and sensitive detection of target protein. By preparing antibody-DNA conjugates, using the sandwich immune recognition reaction to generate the proximity effect, inducing nucleic acid hybridization, strand substitution, DNase generation and exonuclease III cycle amplification, so that a large number of DNase is generated in situ, through DNase Catalyzed by TMB chromogenic reagent or chemiluminescent substrate, the target protein can be sensitively and quantitatively analyzed by colorimetric or chemiluminescent imaging. [0003] 2. Background technology [0004] Immunoassay has been widely used in clinical disease diagnosis, food safety testing, environmental monitoring and other fields due to its good specificity. The most common immunoassay methods are enzyme-linked immunoassay, electrochemical immunoassay,...

AI Technical Summary

Problems solved by technology

Although these methods greatly improve the detection sensitivity, some physical or chemical factors can greatly affect the aggregation of nanoparticles and the activity of enzymes, such as pH value, ionic strength, temperature, other impurities, etc.

In addition, the process of labeling enzymes is quite complicated, which greatly reduces the catalytic activity of enzymes

The above factors inevitably affect the accuracy and reproducibility of colorimetric immunoassay, limiting its application in actual sample detection

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