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Stable HRP (horse radish peroxidase) chemiluminescence substrate solution

An enzymatic chemiluminescence and substrate liquid technology, applied in the field of immunoassay, can solve the problems of inaccurate results, poor stability and high cost, and achieve the effects of high luminescence intensity value, good stability and low cost

Active Publication Date: 2017-07-28
武汉海吉力生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a stable HRP enzymatic chemiluminescent substrate solution to solve the problems of poor stability, high background, inaccurate results and high cost of existing chemiluminescent substrates.

Method used

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  • Stable HRP (horse radish peroxidase) chemiluminescence substrate solution
  • Stable HRP (horse radish peroxidase) chemiluminescence substrate solution
  • Stable HRP (horse radish peroxidase) chemiluminescence substrate solution

Examples

Experimental program
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Effect test

Embodiment 1

[0026] A stable HRP enzyme-catalyzed chemiluminescent substrate solution, respectively configured with 1000mL luminescent agent solution and oxidant solution, wherein the specific formula of luminescent agent solution is as follows:

[0027]

[0028] The specific formula of oxidant solution is as follows:

[0029]

[0030] In the above-mentioned luminescent agent solution, the buffer used may adopt other conventional buffer systems, such as phosphate buffer and the like.

[0031] Before use, the luminescent agent solution and the oxidant solution are mixed at a volume ratio of 1:1 before use. One day after the above-mentioned chemiluminescent substrate solution was mixed, the blank value did not increase, and the signal value was stable.

[0032] In order to ensure the stability of the above reagents after long-term storage, ProClin 300 with a mass concentration of 0.01-0.1% can be added to the oxidant solution. This mass concentration effectively guarantees the shelf l...

Embodiment 2

[0034] This embodiment configures the chemiluminescent substrate solution and the oxidizing agent solution on the basis of implementation 1, and the specific formula is as follows:

[0035] Luminescent agent solution in chemiluminescent substrate solution:

[0036] Luminol 1mmol / L

[0037] Phenolphthalein 0.1mmol / L

[0038] p-iodophenol 1.5mmol / L

[0039] 0.2mol / L boric acid-borax buffer solution, adjust the pH value to 9.6 with hydrochloric acid.

[0040] Oxidant solution in chemiluminescent substrate solution:

[0041]

[0042] 0.2mol / L citric acid buffer, adjust the pH value to 5.0 with hydrochloric acid

[0043] After mixing the above-mentioned luminescent agent solution and oxidant solution according to the volume ratio of 1:1, as a luminescent substrate solution and a commercially available imported ultra-sensitive luminescent substrate solution produced by ThermoFisher Pierce in the determination of 0.5 μg / mL HRP enzyme solution The signal-to-noise ratio (the ra...

Embodiment 3

[0049] The chemiluminescent substrate solution in Example 2 was used to detect γ-interferon by the double-antibody sandwich method, wherein the antibody was produced by Wuhan Haijili Biotechnology Co., Ltd.

[0050] The experimental steps are as follows:

[0051] 1. Add sample

[0052] (1) Use the calibrator diluent to dilute the γ-interferon calibrator reference substance to a total of 6 dilution concentrations of 12.5pg / mL, 25pg / mL, 50pg / mL, 100pg / mL, 200pg / mL, and 400pg / mL. Take 50 μL, add to the wells of the plate in order, and make two parallel wells; blank control 1 well, blank control plus 50 μL of calibrator dilution;

[0053] (2) Add 50 μL of enzyme-labeled reagent to each well;

[0054] 2. Incubation: Gently mix well and incubate in a constant temperature incubator at 37°C for 1 hour;

[0055] 3. Washing: Shake off the liquid in the wells, add 300 μL of 1× washing solution to each well for washing, pat dry, repeat 5 times;

[0056] 4. Luminescence: Add 100 μL of ...

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Abstract

The invention discloses stable HRP (horse radish peroxidase) chemiluminescence substrate solution which comprises luminescence agent solution and oxidant solution. The comprises luminescence agent solution is prepared by specific proportion of Luminol or Luminol derivative or Luminol sodium salt, p-Iodophenol, phenolphthalein and buffer solution, the p-Iodophenol serves as reinforcing agents, the phenolphthalein serves as synergistic reinforcing agents, the oxidant solution is prepared by specific proportion of urea peroxide, ethylene diamine tetraacetic acid, tween-20 and buffer solution, and the luminescence agent solution and the oxidant solution are mixed before use of the substrate solution. The HRP chemiluminescence substrate solution is simple to prepare, the p-Iodophenol serves as the reinforcing agents, the phenolphthalein serves as the synergistic reinforcing agents, the tween-20 serves as stabilizing agents, the stability of the chemiluminescence substrate solution is further improved, and the HRP chemiluminescence substrate solution is stable, high in signal-to-noise ratio and sensitivity, long in platform phase and purple red.

Description

technical field [0001] The invention relates to the field of immunoanalysis, in particular to a chemiluminescence substrate solution using HRP as an enzymatic reaction detection system. Background technique [0002] Chemiluminescence immunoassay (CLIA) is a new type of labeled immunoassay technology for the detection of trace antigens or antibodies established by combining luminescence analysis and immune response. It has both the specificity of immune response and the High sensitivity with chemiluminescence reaction. It overcomes the radioactive hazard and pollution problems caused by the use of radioactive isotopes in radioimmunoassay (RIA); it overcomes the shortcomings of complex instruments and large background interference in fluorescence immunoassay (FIA). With its unique advantages such as high sensitivity, low instrument price, easy to use, safety, and no radioactive pollution, it is favored by people and has become an important direction of marker immunization. A...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76
CPCG01N21/76
Inventor 黄俊路春桃赵平锋
Owner 武汉海吉力生物科技有限公司
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