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Neutrophil elastase assay reagent and preparation method thereof

An elastase and neutrophil technology, applied in the biological field, can solve the problems of no detection condition research, inability to accurately quantify, no unified formula and process, etc., and achieve the effects of good compliance, high accuracy and easy operation.

Inactive Publication Date: 2018-10-09
苏州普瑞斯生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, only chromatographic qualitative and enzyme immunoquantitative detection methods are available. Chromatographic qualitative can only be used to simply determine the presence or absence of samples after the concentration of the sample exceeds the detection limit, and cannot be accurately quantified. treatment, and there are large false negatives or false positives. The enzyme immunoassay method is mainly used by scientific researchers in scientific research. Due to the preparation of detection reagents, there is no unified formula and process, and there is no in-depth study of detection conditions, resulting in different results. The test results of batches of reagents vary greatly

Method used

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preparation example Construction

[0040] The preparation method of the R1 reagent comprises the following steps: mixing a buffer, a reaction accelerator, avidinated latex microspheres, a stabilizer, an inorganic salt, a preservative and water into a solution with a pH value of 7.4-8.0. ;

[0041] The preparation method of the R2 reagent comprises the following steps in turn:

[0042] S1. The anti-human neutrophil elastase antibody is dialyzed at least twice at 2-8 degrees Celsius in a sodium bicarbonate buffer solution with a pH of 8.0-9.0 to remove impurities that interfere with the labeling reaction;

[0043]S2. Dilute the anti-human neutrophil elastase antibody solution after dialysis to 1-2 mg / ml with phosphate buffer solution of pH 7.0-7.4, add excess biotin, and continue and gently mix for 1 hour at 25 degrees Celsius , carrying out a labeling reaction to prepare a biotinylated anti-human neutrophil elastase antibody solution;

[0044] S3, performing at least two dialysis on the solution obtained in st...

Embodiment 1

[0058] 1. Preparation of R2 reagent

[0059] Dialyze 10 mg anti-human neutrophil elastase antibody in 0.1 mol / L pH 8.5, 1 L sodium bicarbonate buffer solution at 2-8 degrees Celsius for 3 times, each time for 1 hour, to remove preservatives and other interfering markers Impurities in the reaction.

[0060] Dilute the dialyzed antibody solution to 1 mg / ml with 0.1 mol / L phosphate buffer at pH 7.2, dissolve 1 mg of activated biotin NHS-Biotin with 1 ml of ultrapure water, add it to the antibody solution, and keep at 25 degrees Celsius, The labeling reaction was performed with gentle mixing for 1 hour.

[0061] The biotinylated anti-human neutrophil elastase antibody solution was dialyzed three times in pH7.5, 0.1mol / L, 5L phosphate buffer at 2-8 degrees Celsius to remove unreacted biotin.

[0062] Dilute the dialyzed biotinylated antibody to 0.1 mg / ml with pH 7.5, 0.01 mol / L phosphate buffer, add 0.1 g gelatin, 0.1 g whey protein, and 1 g ascorbic acid to dissolve and mix well...

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Abstract

The invention discloses a neutrophil elastase assay reagent which comprises a reagent R1, a reagent R2 and a calibration reagent; the reagent R1 comprises a buffer agent, a reaction accelerator, an avidinized latex microsphere and a stabilizer; the diameter of latex microspheres of the avidinized latex microsphere is 50 to 100 nm; the reagent R2 comprises the buffer agent, a biotinylated anti-human neutrophil elastase antibody and the stabilizer; and the calibration reagent comprises a quantitative neutrophil elastase antigen. The invention further discloses a preparation method of the neutrophil elastase assay reagent. According to the invention, the biotinylated anti-human neutrophil elastase antibody is combined with the avidinized latex microsphere, and latex-enhanced immunoturbidimetry is adopted to determine the content of NE in human vaginal secretions and serum; and the reagent is convenient to operate, high in accuracy, good in repeatability, and suitable for high-throughput detection, can be used on automatic biochemical analyzers, and has good agreement with the results of an enzyme-free quantitative method.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a neutrophil elastase assay reagent capable of measuring neutrophil elastase content in human serum and vaginal secretions by a latex-enhanced immune turbidimetry method and a preparation method thereof. Background technique [0002] NE (neutrophil elastase) exists in neutrophil azurophilic blue granules. It is a single peptide chain composed of 218 amino acids, with a molecular weight of about 25.9kD and four disulfide bonds. It is a serine protease family Therefore, it has 30%-40% homology with other serine family proteases, and its optimum pH value is close to neutral. The HNE gene is located at the end of the short arm of chromosome 19. It is a 50kb DNA fragment. There is no HNE mRNA expression in mature neutrophils, indicating that this enzyme is only synthesized in immature bone marrow cells. The catalytic center of the enzyme is composed of three amino acid residues, His 41, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543
CPCG01N33/54313
Inventor 吴朝晖
Owner 苏州普瑞斯生物科技有限公司
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