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C-reactive protein quantitative determination kit and preparation method thereof

A reactive protein and quantitative determination technology, applied in the field of quantitative detection of C-reactive protein in blood, can solve the problems of unfavorable high-throughput automatic detection, low sensitivity, poor specificity, etc., and achieve an optimized chemiluminescence enhancement system, high sensitivity, small variation effect

Active Publication Date: 2015-02-04
NANFANG HOSPITAL OF SOUTHERN MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This technique has low sensitivity, narrow linearity, poor specificity, and is not conducive to high-throughput automatic detection

Method used

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  • C-reactive protein quantitative determination kit and preparation method thereof
  • C-reactive protein quantitative determination kit and preparation method thereof
  • C-reactive protein quantitative determination kit and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: the configuration of various buffers, specifically as follows:

[0049] 1. Tris salt buffer

[0050] Add 12.12g of Tris and 5.82g of sodium chloride into 1L of purified water, stir well until completely dissolved, and adjust the final pH to 7.5 with hydrochloric acid.

[0051] 2. Preparation of calibrator buffer

[0052] Add 0.01g of tetracycline and 0.1g of neomycin sulfate to 1L of newborn bovine serum, fully dissolve and process through 0.22μm filter membrane to prepare.

[0053] 3. Anti-reagent buffer

[0054] Add 30.5mg of Tris, 0.03g of tetracycline, 3g of sheep serum, 8g of newborn bovine serum, and 3g of horse serum into 1L of purified water, stir well until completely dissolved;

[0055] 4. Magnetic particle buffer

[0056] Add 12.12mg of Tris, 5.82mg of sodium chloride, and 50g of methyl cellulose ether into 1L of purified water, stir well until completely dissolved.

[0057] 5. Luminescence substrate buffer

[0058] Add 50.21g of Tris, 5.8...

Embodiment 2

[0061] Embodiment 2: Preparation of C-reactive protein quantitative assay kit

[0062] 1. Preparation of calibrators and quality controls

[0063] Firstly, the C-reactive protein was dissolved in the standard buffer solution, and prepared as a calibrator and a quality control product with target concentrations as shown in Table 1; the C-reactive protein used in this example was purchased from the manufacturer Fitzgerald Company.

[0064] Table 1 Preparation of calibrators and quality controls

[0065]

[0066] 2. The preparation method of the anti-reagent is as follows:

[0067] 1) Coupling of fluorescein isothiocyanate and C-reactive protein antibody to obtain fluorescein isothiocyanate-labeled C-reactive protein-coated antibody:

[0068] First, use anti-reagent buffer to prepare FITC solution with a concentration of 2.5mg / mL, and then make C-reactive protein:FITC solution=1:1.1 according to the mass ratio Transfer the two to a brown glass bottle and stir at room temper...

Embodiment 3

[0079] Embodiment 3: The method for detecting C-reactive protein using the C-reactive protein quantitative assay kit described in Example 2, the method comprises the steps of:

[0080] 1) Take three test tubes and add 15 μL C-reactive protein calibrator, 15 μL LC-reactive protein quality control, and 15 μL sample to be tested;

[0081] 2) Add 60 μL of anti-reagent to each test tube, cover the test tube with a plastic film, shake the test tube gently for 30 seconds, and place it in a water bath at 37°C for 5 minutes;

[0082] 3) Add 30 μL of magnetic particle reagent to each test tube, cover the test tube with a plastic film, shake the test tube gently for 30 seconds, and place it in a water bath at 37°C for 5 minutes;

[0083] 4) Precipitate the test tube on the magnetic separator for 2 minutes, slowly invert the test tube and the magnetic separator, pour out the supernatant, put the inverted test tube together with the magnetic separator on the filter paper, and pat the botto...

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Abstract

The invention discloses a C-reactive protein quantitative determination kit, belonging to the field of biological immune in-vitro diagnosis of medical devices. The kit comprises a calibrator, a quality control product, an antibody reagent, a magnetic particle reagent and a luminescent substrate. The invention also discloses a preparation method of the kit and a method for detecting C-reactive protein by using the kit. According to the preparation method disclosed by the invention, the antibody reagent is prepared from a C-reactive protein-coated antibody labeled by fluorescein isothiocyanate and a C-reactive protein-labeled antibody labeled by alkaline phosphatase, and the magnetic particle reagent is prepared by coupling an anti-fluorescein-isothiocyanate antibody with carboxyl magnetic beads so as to ensure that uniform mixing and separation are easily carried out in immunoreaction, and the reaction speed can be greatly increased; and a novel chemical luminescent substrate ALPS is used as the substrate, so that the sensitivity and specific performances of the kit can be improved. The detection kit disclosed by the invention is reliable in performance, high in sensitivity and wide in linear range, and can be matched with semi-automatic and full-automatic instruments for use.

Description

technical field [0001] The invention relates to the field of in vitro diagnosis of biological immunity for medical devices, in particular to a kit for quantitatively detecting C-reactive protein in blood based on magnetic particle separation chemiluminescence method and a preparation method thereof, and the quantitative detection of C-reactive protein in blood by using the kit Methods. Background technique [0002] Serum CRP level is a sensitive and objective indicator of bacterial infection. During bacterial infection, the level of serum CRP can be moderately or significantly increased, and the positive rate can reach more than 90%. However, during viral infection, the level of CRP is mostly normal or slightly elevated, so it can help to distinguish bacterial infection from non-bacterial infection diagnosis. In addition, the quantitative measurement of CRP levels in cerebrospinal fluid and pleural effusion can also be of certain significance in the differential diagnosis ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577
CPCG01N33/6893
Inventor 郭志刚龚爱华
Owner NANFANG HOSPITAL OF SOUTHERN MEDICAL UNIV
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