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Hyaluronic acid detection kit and detection method

A detection kit, a technology for hyaluronic acid, applied in the field of medical analysis, can solve the problems of measurement accuracy, poor sensitivity and precision, not wide enough linear range, unfavorable wide application, etc., and achieves simple operation, fast inspection speed, and convenient separation. Effect

Active Publication Date: 2020-03-24
AUTOBIO DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The Elisa assay method is generally used for semi-quantitative assays. The assay accuracy, sensitivity, and precision are relatively poor, and the operation is cumbersome, which is not conducive to wide application in clinical practice; the radioimmunoassay method is short-lived (half-life) because the reagent has radioactive contamination and the reagent is valid. ) and other defects are gradually being eliminated; chemiluminescence immunoassay (Chemiluminescence Immunoassay, CLIA) is a non-radioactive immunoassay that has developed very rapidly in the world in the past ten years, following radioimmunoassay (RIA) and enzyme immunoassay (EIA) An ultra-high-sensitivity micro-assay technology developed later has the advantages of high sensitivity, wide detection range, simple and fast operation, good stability of markers, no pollution, simple and economical instruments, etc. It is an ideal method for immunoquantitative analysis at present
However, the reported chemiluminescent kits for the detection of hyaluronic acid still have the problems of low accuracy and insufficient linear range.

Method used

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  • Hyaluronic acid detection kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The preparation of embodiment 1 kit of the present invention

[0035] 1. Preparation of HA derivatives

[0036] 1.1 Washing before BSA coupling: Add 2 times the volume of PBS buffer to the coupling container, then accurately weigh 3 mg of BSA, shake for 5 minutes, mix well, filter and discard the supernatant, and the first washing is over; repeat washing 5 times ;

[0037] 1.2 BSA surface activation: transfer the washed BSA into a small container, discard the supernatant and add 1.65 times the volume of EDC solution, then add 1.65 times the volume of NHS solution, shake and mix well, shake and react on a micro shaker at room temperature for 2 hours ;

[0038] 1.3 BSA surface activation: After the reaction, filter and discard the supernatant, transfer the activated BSA to a large container, add 33 times the volume of BSA in acetate buffer, shake for 5 minutes, mix well, filter and discard the supernatant solution, the end of one wash; repeat the wash 2 times;

[0039...

Embodiment 2

[0057] Embodiment 2 The using method of kit of the present invention

[0058] 1. Preparation before experiment

[0059] 1.1 Take the kit of Example 1 out of the refrigerated environment, and place it at room temperature (18-25°C) for 20-30 minutes to balance;

[0060] 1.2 Take a pack of lotion and dissolve it in 500ml purified water for later use;

[0061] 1.3 Adjust the temperature of the constant temperature water bath to 37°C.

[0062] 2. Experimental operation

[0063] 2.1 Add calibrator (for calibration) or sample into the reaction container (hereinafter referred to as "well") respectively, and the sample volume is 100 μl / well;

[0064] 2.2 Add 20 μl of magnetic particle suspension to each well;

[0065] 2.3 Add 20 μl of HA-binding protein solution to each well;

[0066] 2.4 Add 20 μl of enzyme conjugate to each well;

[0067] 2.5 After mixing, incubate at 37°C for 34 minutes;

[0068] 2.6 Wash with lotion 5 times;

[0069] 2.7 Add 50 μl each of luminescent substr...

Embodiment 3

[0071] The performance detection of embodiment 3 kits of the present invention

[0072] 1. Analytical specificity

[0073] Analytical specificity is mainly used to control that the detected substance of the kit is a specific substance rather than others, especially the test is aimed at substances with a similar structure to the tested substance, and the specificity of the antibody will be insufficient. error.

[0074] In order to avoid cross-reaction with the other three items of the liver fibrosis item, LN, PⅢNP, and CⅣ should be used as the indicators for detecting the specificity of HA. Since the reagents used are all from the same manufacturer, the test method is to use a batch of products to measure 1000ng / ml LN and CⅣ, and 100ng / ml PⅢNP respectively, and observe whether there is cross-reactivity. The experimental results are shown in Table 1 below.

[0075] Table 1 Specificity detection results

[0076]

[0077]

[0078] It can be seen from the test results in ...

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Abstract

The invention relates to the technical field of medical analysis, in particular to a hyaluronic acid detection kit and a detection method. The kit comprises a solid-phase carrier combined with an HA derivative, an HA binding protein and an enzyme-labeled HA binding protein antibody, wherein the HA derivative is formed by coupling the tail end of HA with a coupling protein. The kit disclosed by theinvention is good in accuracy, high in sensitivity, good in specificity and high in detection speed, and a result can be obtained within 40 minutes. The kit provided by the invention has extremely high conformity with liver biopsy results, overcomes many defects of a radioimmunoassay reagent and an enzyme immunoassay reagent, is suitable for the effective popularization and application in industry, has good stability, and can be stably stored for one year at 2-8 DEG C.

Description

technical field [0001] The invention relates to the technical field of medical analysis, in particular to a hyaluronic acid detection kit and detection method. Background technique [0002] Hyaluronic acid (HA) is widely distributed in the extracellular space and is a linear polymer composed of repeating disaccharide units with a molecular weight of 10-10000kD. [0003] HA is mainly synthesized by fibroblasts in tissues, enters the blood circulation through the lymphatic system, and is quickly cleared by the liver. Liver endothelial cells are the site of HA uptake and degradation. The half-life of HA in circulation is 2.5-5.5 minutes. Due to the effective HA clearance mechanism in the body, there is only a low level of HA in normal human serum. Liver diseases caused by various reasons can increase the level of serum HA, which is mainly related to the decrease of liver clearance function and the increase of synthesis of fibroblasts (hepatic stellate cells) in the liver. I...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/58G01N21/78
CPCG01N33/54326G01N33/581G01N21/78G01N2400/40
Inventor 谢茜尹伊许可
Owner AUTOBIO DIAGNOSTICS CO LTD
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