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Solid phase coating method for hyaluronic acid

A technology of hyaluronic acid and solid-phase carrier, applied in the field of immunoassay medicine, can solve the problems of unstable composition, affecting the accuracy and precision of test results, etc., to enhance stability, improve precision and reliability, and shorten the The effect of reaction time

Active Publication Date: 2014-12-24
BIOSCIENCE (TIANJIN) DIAGNOSTIC TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Solve the problem that when the biotin-labeled hyaluronic acid is coated on the solid phase carrier coupled with streptavidin, the composition is unstable and affects the accuracy and precision of the detection results

Method used

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  • Solid phase coating method for hyaluronic acid
  • Solid phase coating method for hyaluronic acid
  • Solid phase coating method for hyaluronic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] 1. Treatment of hyaluronic acid:

[0021] (1) Weigh 5mg hyaluronic acid and dissolve it in 1ml distilled water;

[0022] (2) Add 0.1ml of 1.5mol / L hydrochloric acid and react overnight at 2-8°C;

[0023] (3) Add 0.1ml1.5mol / L NaOH to neutralize to neutral;

[0024] (4) Borate buffer dialysis for 2 hours to remove excess salt ions;

[0025] 2. Reaction of NHS-activated biotin and treated hyaluronic acid:

[0026] (1) Dissolve 0.5 mg of NHS-LC-LC-Biotin in 0.5 ml of distilled water; add the Biotin solution into the hydrolyzed hyaluronic acid solution, and shake it in the dark for 4 hours to obtain the HA-Biotin complex and unbound NHS -LC-LC-Biotin mixed solution;

[0027] (2) Weigh 53.5mg NH 4 Dissolve Cl in 1ml of distilled water to prepare 1mol / LNH 4 Cl solution;

[0028] (3) Add the solution prepared in (2) to (1), and shake it for 4 hours at 2-8°C under dark conditions;

[0029] (4) Dialyze with phosphate buffer at 2-8° C. for 24 hours to obtain HA-biotin com...

Embodiment 2

[0038] Embodiment 2: Coated plate accelerated stability test

[0039] Use the method of the present invention to coat a 96-well luminescent plate, and coat three batches. Each batch is divided into two parts, one part is placed in a 4°C refrigerator, and the other part is placed in a 37°C refrigerator for accelerated stability. Take it out after 7 days, equilibrate to room temperature, and measure Calibrator, enterprise standard, quality control product, test the precision, sensitivity, and linearity of the coated plate to judge its stability. The experimental results are as follows:

[0040]

[0041]

[0042] It can be seen from the table that when the coated plate is placed at 37°C for 7 days, compared with 4°C, there is no significant difference in the sensitivity, precision and linearity of quality control products, and the stability is very good, and the precision is not affected .

Embodiment 3

[0043] Embodiment 3: Accuracy test of measured value of coated plate

[0044] Adopt conventional method and the method of the present invention to coat hyaluronic acid respectively, measure 9 samples, high value, medium value, low value respectively 3, compare the accuracy of the measured value of two groups of packs, obtain the following results:

[0045]

[0046] As can be seen from the table, the measurement accuracy of the coating method of the present invention is significantly improved compared with conventional methods.

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PUM

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Abstract

The invention provides a coating method for hyaluronic acid. The coating method comprises the following steps: 1, the hyaluronic acid is hydrolyzed by strong acid, wherein the used acid is excess; 2, redundant acid is neutralized with NaOH, wherein the concentration of the NaOH is 0.5 to 5 mol / L; 3, dialysis or chromatographic column desalination is performed with borate buffer solution; 4, shock reaction is performed to biotin activated by NHS and treated the hyaluronic acid in dark places at 2-8 DEG C; 5, uncombined biotin is dialyzed by a borate buffer solution or removed by a chromatographic column; 6, a solid phase carrier is coated with streptavidin; 7, the hyaluronic acid marked with biotin and the solid phase carrier coated with streptavidin are incubated together at 37 DEG C for 1h. The coating method has the advantages that biotinylated hyaluronic acid and the streptavidin solidify more stably; besides, the biotin is coated twice, so that the structure is more stable than that of the biotin added in the form of reagent, and the reaction time is shortened.

Description

technical field [0001] The invention relates to the field of immunoanalysis medicine, in particular, the invention provides a hyaluronic acid solid-phase coating method. Background technique [0002] Hyaluronic acid (Hyaluronan, HA) is an amino polysaccharide produced in interstitial cells, a linear polysaccharide structure composed of repeating structures of N-hexanoylglucosamine and D-glucuronic acid. Molecular formula: (C 14 h 20 NNaO 11 )n, with a molecular weight of 40-80 million, is taken up and degraded by liver endothelial cells. It is widely found in connective tissue, skin, joint fluid and vitreous humor. Good indicator of active fibrosis. [0003] The immunoreaction system of the hyaluronic acid (HA) quantitative detection kit adopts the principle of competition method, after coupling hyaluronic acid (HA) with biotin, it is coated on the solid phase carrier coupled with streptavidin On (solid phase includes microwell plate, magnetic particles, microplate, etc...

Claims

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Application Information

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IPC IPC(8): G01N33/531
CPCG01N33/531
Inventor 刘萍栾大伟张振斌陈露鸾侯玉文杨桂霞黄新城刘靳波
Owner BIOSCIENCE (TIANJIN) DIAGNOSTIC TECH CO LTD
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