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HMGB1 detection kit and preparation method thereof

A technology for detection kits and reagents, applied in the field of medical detection, can solve the problems of uneven quality of detection kits, expensive imported reagents, complicated operation, etc., to speed up time and adsorption equilibrium stability, save reagent costs and sensitivity. high effect

Inactive Publication Date: 2018-12-18
长沙都正医学检验有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection of HMGB1 on the market mainly adopts ELISA (enzyme linked immunosorbent assay), which is cumbersome and time-consuming.
Moreover, the quality of domestic HMGB1ELISA detection kits is uneven, while imported reagents are generally expensive

Method used

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  • HMGB1 detection kit and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Preparation of HMGB1 antigen

[0059] Recombinant plasmids were constructed, positive recombinant plasmids were screened, and bacteria highly expressing His-SBP-HMGB1 fusion protein were cultivated. Under the conditions of 6000×g and 4°C, centrifuge the culture solution containing bacteria for 30 minutes, collect the bacteria and store them at -20°C for later use; take an appropriate amount of bacteria and resuspend them in the bacteriostasis buffer (containing 50mmol / L Tris-HCL, 250mmol / L Tris-HCL, 250mmol / L L NaCl and 10mmol / L imidazole, pH value is 8.0), sonicate in ice bath, centrifuge at 10000×g, 4℃ for 20min, and collect the supernatant for later use; The non-specific binding protein was washed with bacteria buffer, and then eluted with elution buffer (containing 50mmol / L Tris-HCl, 250mmol / L NaCl and 300mmol / L imidazole, pH value 8.0), and the target protein—HMGB1 was collected, Reserved as HMGB1 antigen. The collected protein concentration was tested ...

Embodiment 2

[0060] Example 2 Preparation of HMGB1 polyclonal antibody

[0061] Dilute the HMGB1 antigen with physiological saline to a concentration of 0.5mg / ml-2mg / ml, and mix it with Freund's adjuvant at a ratio of 1:1. The mixture was injected subcutaneously at multiple points and intramuscularly in the hind legs, and the dose for the first injection was 0.5 mg to 1 mg. Serum was collected every 1 to 2 weeks, and the titer of the serum was determined by agar diffusion test. If the titer did not reach the expected titer, a booster immunization was carried out, and the additional injection dose was 1 / 2 of the first injection. Generally, 2 to 4 booster immunizations were required. When the serum titer reaches the requirement, the rabbit serum is collected, and the collected serum is stored at -20°C for later use.

[0062] Add 100 mg of HMGB1 to 3 g of cyanogen bromide-activated agarose medium (CNBr-activated Sepharose 4B), prepare an HMGB1 affinity chromatography column with a volume of ...

Embodiment 3

[0063] Example 3 Labeling latex particles

[0064] a. Prepare latex solution

[0065] Prepare 1 mL latex solution (22 μL latex mother solution + 978 μL 50 mM MES buffer solution) with a final concentration of 0.11 v / v % with the latex mother solution (5 v / v%, 20 nm ~ 50 nm particle size), the pH value is 6.0.

[0066] b. Activate latex group

[0067] Add three solutions in order to 1mL latex solution: 4.5μL surfactant (5% stock solution prepared with 50mM MES, pH 6.0 solution or pure water); 4.5μL N-HS solution (concentration: 10mg / mL) ; 4.5 μL EDC solution (10 mg / ml concentration). After the solution was added, the latex solution was allowed to stand at 37°C for 1 hour.

[0068] c. Antibody Conjugation Labeling

[0069] 100 μg of HMGB1 antibody (20 μL of 5 mg / mL antibody stock solution) was added, reacted at 37° C. for 2 h, and stirred slowly.

[0070] d. Closed latex group

[0071] 100 μL of blocking solution (1M Tris-HCl, pH 8.0) was added and allowed to stand at 37° ...

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Abstract

The invention relates to an HMGB1 (High Mobility Group Box 1) detection kit and preparation method thereof. The HMGB1 detection kit comprises Reagent 1 and Reagent 2, wherein the Reagent 1 comprises HMGB1 antibody-labeled latex microspheres, a suspending agent, bovine serum albumin and a first buffer solution; the Reagent 2 comprises sodium chloride, a dispersing agent, a surfactant and a second buffer solution. According to the HMGB1 detection kit and preparation method thereof, the HMGB1 detection kit is a kit for detecting HMGB1 based on latex enhanced immunoturbidimetry; antigen and antibody reactions are carried out in a homogeneous reaction system when the HMGB1 detection kit is used; the absorbance value of a reaction solution is directly determined to reflect the concentration of ato-be-tested antigen after antigens bound to antibodies; and the HMGB1 detection kit has fast detection speed, low cost, time and labor saving, good stability and repeatability, and can truly reflectthe content of to-be-tested substances.

Description

technical field [0001] The invention relates to the technical field of medical detection, in particular to an HMGB1 detection kit and a preparation method thereof. Background technique [0002] High mobility group protein 1 (high mobility group protein, HMGB1) is a non-histone chromosome-binding protein present in the eukaryotic nucleus, named for its fast migration speed in polyacrylamide gel electrophoresis (PAGE). HMGB1 is ubiquitously present in mammalian tissue cells and is an important inflammatory factor. As a late-stage inflammatory factor, HMGB1 is expressed stably for a longer period of time than early-stage inflammatory factors, and can better reflect the occurrence of inflammation in the body, which has more important significance for the determination of clinical treatment plans. [0003] A large number of studies have shown that in inflammation caused by bacterial infection (such as endotoxemia and sepsis, especially acute inflammation), early inflammatory fac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/558
CPCG01N33/54313G01N33/558
Inventor 郭凡财赵洪斌郭飞余鹏李晓晖欧阳冬生
Owner 长沙都正医学检验有限责任公司
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