Homogeneous chemiluminescence immune assay method based on adjacent position striking effect

A homogeneous chemiluminescence and immunoassay technology, which is applied in the field of homogeneous chemiluminescence immunoassay, can solve problems such as limited applications, and achieve the effects of improving detection sensitivity, simple equipment, and shortening analysis time

Inactive Publication Date: 2014-11-26
NANJING UNIV +1
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Problems solved by technology

However, the established proximity-strike immunoassay method mainly uses real-time PCR detection, which greatly limits the application

Method used

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  • Homogeneous chemiluminescence immune assay method based on adjacent position striking effect
  • Homogeneous chemiluminescence immune assay method based on adjacent position striking effect
  • Homogeneous chemiluminescence immune assay method based on adjacent position striking effect

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Effect test

Embodiment 1

[0027] Embodiment 1: in combination with figure 1 , illustrating the detection of the target protein (carcinoembryonic antigen) by a homogeneous chemiluminescent immunoassay method based on the proximity-striking effect

[0028] (1) Prepare detection solution: mix DNA1-antibody 1 conjugate, DNA2-antibody 2 conjugate, helper DNA3, helper DNA4, molecular beacon DNA5 and restriction endonuclease so that their final concentrations are 0.01 mg / mL, 0.01 mg / mL, 80 nM, 80 nM, 1 μM, and 70 U / mL.

[0029] (2) Mix 1 μL of standard solutions of different concentrations or a test solution containing target protein (carcinoembryonic antigen) with 29 μL of detection solution, and incubate at 37° C. for 30 minutes.

[0030] (3) Add 20 μL of chemiluminescence substrate to the incubated solution, and immediately detect the chemiluminescence signal of the solution, detection parameters: gain 2, voltage 950V. According to the recorded chemiluminescence value, the working curve for the detection...

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Abstract

The invention relates to a homogeneous chemiluminescence immune assay method based on an adjacent position striking effect. A detection solution of the method includes a DNA1-antibody 1 conjugate, a DNA2-antibody 2 conjugate, auxiliary DNA3, auxiliary DNA4, molecular beacon DNA5 and restrictive endonuclease. When a target protein exists, a DNA1-antibody 1 and a DNA2-antibody 2 form a sandwich immune complex to make DNA3 and DNA4 respectively hybridized with DNA1 and DNA2 close to each other in order to form an adjacent position strike complex, the adjacent position strike complex can be hybridized with the DNA5 to open its hairpin structure, and a dye Cy5 goes away from a quencher to generate chemiluminiscence. A double chain formed by the adjacent position strike complex and the DNA5 can be identified by endonuclease, new DNA5 is opened after the DNA5 is cut, and the cycle of above steps can realize onsite amplified luminescence in order to realize the highly-sensitive quantitative analysis of the target protein. The method has the advantages of realization of fast one-step protein detection, simple operation and high universality.

Description

1. Technical field [0001] The invention is a homogeneous chemiluminescence immunoassay method based on the proximity impact effect. Using the immune reaction to induce nucleic acid hybridization, resulting in a proximity strike effect, generating a sequence complementary to the hairpin molecular beacon switch, turning on the molecular beacon, and introducing an endonuclease cycle strategy to generate free chemiluminescent dyes, and in situ amplification chemistry Luminescence signals enable simple, rapid and highly sensitive determination of target proteins. 2. Background technology [0002] The determination of protein markers plays an important role in the research and diagnosis of cancer, and its bedside detection can be directly used for early screening, monitoring treatment and recurrence diagnosis, which is of great significance. Enzyme-linked immunoassay is one of the most common immunoassay methods. Although it has been popularized and applied, it is not suitable fo...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N21/76
CPCC12Q1/6804C12Q2521/301C12Q2525/301C12Q2563/107
Inventor 鞠熀先严枫宗晨吴洁
Owner NANJING UNIV
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