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One-step chemiluminiscence quantitative detection kit for hyaluronic acid

A chemiluminescence reagent and hyaluronic acid technology, which is applied in the field of immunoanalysis medicine and chemiluminescence kits, can solve the problems of many interference factors, radioactive pollution, poor repeatability of reagents, etc.

Active Publication Date: 2012-09-26
BEIJING NORTH INST OF BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are also certain defects in the above method: one, the steps of adding samples are loaded down with trivial details
The whole experiment includes three steps of sample addition and centrifugation, and the total reaction time reaches more than 2 hours; 2. The automation of the kit operation is low, and the validity period of the kit is also very short
Only 30-45 days; 3. Use 125 I is used as a marker, causing radioactive pollution to the environment and potential hazards to the health of operators
[0004] Similarly, there are also some HA chemiluminescence and enzyme-linked detection reagents on the market. It is understood that most of them adopt a three-step reaction mode, and the immune reaction time generally takes about 1.5-2 hours. It can be said that the operation It's more cumbersome
The reason is that these kits all use the binding reaction between HA and HABP, and HABP is a protein with complex composition and structure, the purification process is complicated, the purity is not too high, and its binding force with HA is not high. The binding force between antigen and antibody is so strong, so, first, it cannot be directly coated with it, and there are many interference factors, resulting in poor repeatability of the reagent
2. It cannot be directly labeled with it. Some active groups of HABP are easily inactivated, resulting in poor stability of the reagent.

Method used

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  • One-step chemiluminiscence quantitative detection kit for hyaluronic acid
  • One-step chemiluminiscence quantitative detection kit for hyaluronic acid
  • One-step chemiluminiscence quantitative detection kit for hyaluronic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Preparation of the hyaluronic acid one-step chemiluminescence quantitative detection kit of the present invention

[0030] 1. Preparation of HA calibrator.

[0031] 1. 50mmol / L pH7.4 Phosphate Buffer (PB):

[0032] Na 2 HPO 4 12H 2 O 14.5 g

[0033] NaH 2 PO 4 2H 2 O 1.48 g

[0034] Dissolve in 1000ml deionized water, and measure its pH value to be 7.4.

[0035] Add 5% calf serum to the standard diluent.

[0036] 2. Accurately dilute the HA concentrated antigen to several concentrations of 25, 50, 100, 200, 400, 800 ng / ml, S0 is the calibrator diluent, a total of 7 bottles.

[0037] 2. Preparation of HA-coated plates

[0038] Buffer 1: Coating Solution

[0039] 50mmol / L pH7.4PB

[0040] Buffer 2: Blocking Solution

[0041] 50mmol / L pH7.4PB

[0042] 10% sucrose

[0043] 0.5%BSA

[0044] 0.1% preservatives.

[0045] Preparation of coated plate: Dilute HA-BSA to 50mmol / L pH7.4 PB coating solution in proportion, add 100μl / well to the microwell p...

Embodiment 2

[0052] Embodiment 2: The operation method of the hyaluronic acid one-step chemiluminescence quantitative detection kit of the present invention.

[0053] The specific operation method of the hyaluronic acid one-step chemiluminescence quantitative detection kit prepared in the above embodiment 1 is:

[0054] a. Add 50 μl of HA calibrator or sample to be tested to each well of the microplate.

[0055] b. Add 50 μl of HA enzyme conjugate to each well of the microplate.

[0056] c. Place the microplate on a shaker and shake it at room temperature for 60 minutes.

[0057] d. Discard the liquid in the microwell plate, add washing solution to each well, let it stand for 30 seconds, and then blot or spin dry. So repeated washing 5 times. Buckle the microplate dry on paper when finished.

[0058] e. Add 50 μl (1 drop) of substrate solution A and substrate solution B to each well, shake the microwell plate to mix after adding the samples.

[0059] f. Measure the luminescence value ...

Embodiment 3

[0060] Example 3 Methodological identification of the one-step chemiluminescence quantitative detection kit for hyaluronic acid of the present invention

[0061] Table 1.

[0062] Table 1 The methodological identification results of the kit

[0063]

[0064]

[0065] It shows that the accuracy, specificity, precision, sensitivity and stability of the kit of the present invention are fully qualified.

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Abstract

The invention relates to a chemiluminiscence kit for detecting content of hyaluronic acid in human serum, body fluid or tissue homogenate by one-step process, and belongs to the medical field of immunoassay. By solid coating and indirect enzyme labeling technology, the problem that a hyaluronic acid kit is unavailable for one-step detection is solved substantially, operation is more convenient, detection is more accurate, and reagents are more stable. The chemiluminiscence kit for detecting content of the hyaluronic acid in human serum, body fluid or tissue homogenate by one-step process comprises a calibrator, a coated board, an enzyme combination, chemiluminiscence substrate solution A and chemiluminiscence substrate solution B. The sample amount of each of the HA (hyaluronic acid) calibrator and the enzyme combination is 50 microliters in the kit, and reaction time of the one-step process is one hour. Scientific and reasonable range of normal values is determined by strict methodological appraisal, clinical measurement results are compared with a radioimmunoassay kit statistically, and accordingly the chemiluminiscence kit has the technical advantages of high sensitivity, accurate measurement, high repeatability, regent stability and the like and is worthy of vigorous popularization and application to market.

Description

technical field [0001] The invention relates to a chemiluminescent reagent kit for detecting the content of hyaluronic acid (Hyaluronic Acid, HA for short) in human serum, body fluid or tissue homogenate in one step. It belongs to the category of immunoanalysis medicine. Background technique [0002] In recent years, clinical medicine has successively found that the change of hyaluronic acid content is closely related to the growth, infiltration and spread of tumors. In particular, it has been found that the detection of serum HA has certain value in the diagnosis of tumors, and can be used as an important indicator to distinguish benign and malignant tumors. It also contributes to the diagnosis and differential diagnosis of liver diseases, condition judgment and prognosis evaluation, and is also a very important dynamic observation method for anti-fibrosis drug treatment. [0003] At present, the detection method of HA is mainly based on radioimmunoassay, and document CN10...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76G01N33/532
Inventor 白云鹏
Owner BEIJING NORTH INST OF BIOLOGICAL TECH
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