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Kit for detecting interleukin-6 and method for detecting interleukin-6 for non-diagnostic purpose

An interleukin and kit technology, applied in the field of immunoassay, can solve the problems of unfavorable automatic detection instrument detection, long detection time, low sensitivity, etc. Effect

Active Publication Date: 2014-07-23
SHENZHEN GOLDSITE DIAGNOSTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] At present, the clinical detection of IL-6 basically adopts enzyme-linked immunosorbent assay (ELISA), which uses antibodies to coat the wells of solid-phase plates, and uses enzyme-catalyzed substrates to produce color changes for detection; The method requires multi-step operations, and the entire detection process takes 2 to 4 hours; and the method is usually only suitable for manual operation, which is not conducive to the detection of fully automatic detection instruments, and increases the error caused by human factors in the experimental results; and the ELISA method has The disadvantages of low sensitivity (usually only reaching the sensitivity level of ng / mL) and long detection time limit the further application of this method

Method used

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  • Kit for detecting interleukin-6 and method for detecting interleukin-6 for non-diagnostic purpose
  • Kit for detecting interleukin-6 and method for detecting interleukin-6 for non-diagnostic purpose
  • Kit for detecting interleukin-6 and method for detecting interleukin-6 for non-diagnostic purpose

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] The preparation of the superparamagnetic particle of embodiment 1 avidin coupling

[0069] Take 10 mg of superparamagnetic particles (500 nm to 5 μm in diameter) whose surface active groups are carboxyl groups, place them on a magnetic separator for 2 min, discard the supernatant; add 4 mL of 0.05 mol / mL PBS buffer, pipette evenly, Place on a magnetic separator for separation for 2 min, discard the supernatant, and repeat washing twice. After cleaning, add 50 mg / mL EDC solution (pH 7.0) and 1200 μL of activated superparamagnetic particles, pipette evenly, place on a shaker, and react for 1 hour to obtain activated superparamagnetic particles. Then add avidin to couple streptavidin (SA) protein, the specific operation is: SA is dissolved with 10mmol PBS buffer solution, 1500μL of 5mg / mL SA solution is added to the activated superparamagnetic particles, placed in React on the shaker for 3-6 hours. After the coupling is completed, add 3mL of 1% bovine serum albumin (Bovi...

Embodiment 2

[0070] The preparation of the superparamagnetic particle of embodiment 2 avidin coupling

[0071]Take 10 mg of superparamagnetic particles (1-3 μm in diameter) whose active groups are carboxyl groups on the surface, place them on a magnetic separator for 2 minutes, discard the supernatant; add 4 mL of 0.05 mol / mL PBS buffer solution, and pipette evenly , placed on a magnetic separator for separation for 2 min, the supernatant was discarded, and the washing was repeated twice. After cleaning, add 50 mg / mL EDC solution (pH 7.0) and 1200 μL of activated superparamagnetic particles, pipette evenly, place on a shaker, and react for 1 hour to obtain activated superparamagnetic particles. Then add avidin to couple streptavidin (SA) protein, the specific operation is: SA is dissolved with 10mmol PBS buffer solution, 1500μL of 5mg / mL SA solution is added to the activated superparamagnetic particles, placed in React on the shaker for 3-6 hours. After the coupling is completed, add 3mL...

Embodiment 3

[0072] Example 3 Preparation of Biotinylated Antibody

[0073] Take 1 mmol each of biotin, N-hydroxysuccinimide and dicyclohexylcarbodiimide, add them together to 6 mL of dimethylformamide solution, stir at room temperature (18-25 °C) for 18 h, filter, and decompress the filtrate The dry matter was washed repeatedly with ether, then redissolved in isopropanol, then concentrated to half of the original volume by vacuum drying, crystallized in a low-temperature refrigerator, filtered with filter paper, collected crystals, washed with ether and ready to use. Biotin N-hydroxysuccinimide ester (BNHS) was obtained.

[0074] With 0.1mol / L NaHCO 3 Solution Dilute anti-IL-6 monoclonal antibody A (from Thermo Fisher) to 2 mg / mL antibody solution, add 20 μL of BNHS (20 mg / mL) dissolved in dimethylformamide to each ml of antibody solution, mix well, and store at room temperature (22°C) to react for 1 hour, then dialyze in 0.1 mM PBS buffer solution for 24 hours at 4°C, and the reaction ...

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Abstract

The invention relates to the field of immunoassay and in particular relates to a kit for detecting interleukin-6 and a method for detecting interleukin-6 for non-diagnostic purpose. The kit comprises a biotinylation antibody, an enzyme-labeled antibody, a chemiluminescence substrate and avidin conjugated superparamagnetic particles, wherein the biotinylation antibody is a product of conjugating biotin N-hydroxy succinimide and an anti-interleukin-6 monoclonal antibody A; the enzyme-labeled antibody is a product of conjugating an enzyme and an anti-interleukin-6 monoclonal antibody B. The kit provided by the invention has the advantages of high detection sensitivity, high specificity, short detection time and simple detection process and can be conveniently used for full-automatic detection instruments.

Description

technical field [0001] The invention relates to the field of immune analysis, in particular to a kit for detecting interleukin-6 and a method for detecting interleukin-6 for non-diagnostic purposes. Background technique [0002] Interleukin-6 (IL-6) is a core member of cytokines. It was discovered in 1980 and named IL-6 in 1986. Its relative molecular weight is Mr26000. It is a multifunctional glycoprotein composed of 212 amino acids. Synthesized by monocyte-macrophages, T lymphocytes and fibroblasts, it can promote the synthesis of acute phase proteins in the liver, activate T lymphocytes, and induce the terminal differentiation of B lymphocytes, making them an immune system capable of secreting immunoglobulins living cells. IL-6 has a variety of biological functions and plays an important role in many aspects such as anti-infection, tumor, immune disease and immune regulation. Under abnormal circumstances, the increase of IL-6 may have adverse effects on the body, causin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N21/76
CPCG01N33/54333G01N33/6869G01N2333/5412
Inventor 陆春
Owner SHENZHEN GOLDSITE DIAGNOSTICS
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