Quantitative determination method of cardiac myosin binding protein C and detection kit

A quantitative detection method, myosin technology, applied in the quantitative detection kit of cardiac myosin binding protein C, the field of quantitative detection of cardiac myosin binding protein C, can solve the problem of narrow linear range, long reaction time and sensitivity Low-level problems, achieve high correlation and coincidence rate, improve detection sensitivity and linear range

Inactive Publication Date: 2019-03-08
AUTOBIO DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the cMyBP-C ELISA kit is mostly used for the detection of cMyBP-C, bu

Method used

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  • Quantitative determination method of cardiac myosin binding protein C and detection kit
  • Quantitative determination method of cardiac myosin binding protein C and detection kit
  • Quantitative determination method of cardiac myosin binding protein C and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Preparation of a kit for quantitative detection of cardiac myosin binding protein C (enzyme conjugate using horseradish peroxidase)

[0025] 1. Preparation of antibody-coupled superparamagnetic particles

[0026] Take the superparamagnetic microparticles (1um~3um in diameter) whose surface active groups are carboxyl groups, wash them with coating buffer (50mmol / L, pH 7.6 phosphate buffer) for 5 times, add 10% glutaraldehyde Activation; after washing, coat with cMyBP-C antibody diluted to 100-500 μg / mL, shake and react for 2 hours; wash, then block with pH4.4, 0.02mol / L buffer containing 50mmol / L acetic acid for 1h ; After removing the blocking solution, add pH 7.4, 0.02 mol / L phosphate buffer containing 0.5% Tween-20, 1% BSA, 0.1% Procline300 for storage, and store at 2-8°C for later use.

[0027] 2. Preparation of antibody-conjugated horseradish peroxidase (HRP) conjugate

[0028] Weigh 5mg HRP and dissolve it in 1ml distilled water, add 0.06mol / L NaIO 4 A...

Embodiment 2

[0034] Embodiment 2 The detection method of kit of the present invention

[0035] A full-automatic chemiluminescence immunoassay analyzer was used, and the kit prepared in Example 1 was used as a detection tool: 25 μL of sample, 20 μL of cMyBP-C antibody-coated magnetic particles and 100 μL of horseradish peroxidase-labeled cMyBP-C antibody, after reacting for 15 minutes, carry out magnetic separation, the instrument sends the reaction mixture into the dark room, and sequentially add the luminescent substrate A solution (containing 0.1M luminol) and B solution (containing 0.5% H2O2) to carry out the luminescent reaction , and finally record the luminous intensity, and calculate the cMyBP-C content of the tested sample from the standard curve.

[0036] The cMyBP-C calibrator is detected by the above method, and the standard curve drawn is as follows figure 1 shown.

Embodiment 3

[0037] Embodiment 3 The performance test of kit of the present invention

[0038] 1. Sensitivity detection

[0039]The sensitivity of the cMyBP-C chemiluminescence immunoassay kit was calculated by referring to the recommended experimental scheme in the CLSI EP17-A document, and the obtained sensitivity was 0.05ng / mL.

[0040] 2. Linear detection

[0041] Perform linear analysis on standard substances with concentrations of 0ng / mL, 0.05ng / mL, 0.5ng / mL, 2ng / mL, 10ng / mL and 50ng / mL, and calculate the linear correlation coefficient, r 2 =0.99, the linear range of the kit for detection of cMyBP-C samples is 0.1-50ng / ml.

[0042] 3. Precision testing

[0043] Two cMyBP-C samples with a concentration of 0.1ng / mL and 20ng / mL were taken, and five parallel samples were made for each concentration of each sample. Three batches of kits were used for detection, and the intra-assay and inter-assay differences of the kits were calculated. The results See Table 1, Table 2, and Table 3 be...

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Abstract

The invention discloses a quantitative determination method of cardiac myosin binding protein C. According to the method, firstly, antibody coupling super paramagnetic micro particles and samples to be tested take specific combination reaction to obtain a first reactant, wherein the antibody coupling super paramagnetic micro particles are products obtained by coupling super paramagnetic micro particles and anti-cMyBP-C monoclonal antibodies A; then, the first reactant is taken to perform specific combination reaction with enzyme-labeled articles to obtain a second reactant, wherein the enzyme-labeled articles are products obtained by coupling enzymes and anti-cMyBP-C monoclonal antibodies B; finally, chemiluminescence substrates and the second reactant are taken to take enzymatic reaction;luminous signals are determined; the concentration value of cMyBP-C is obtained. The technology mode of the invention is a sandwich method. Compared with a cMyBP-C ELISA kit, the detection method andthe prepared kit have the advantages that the detection sensitivity and the linear range can be greatly improved. The technology evaluation on the prepared kit shows that the analysis sensitivity is0.05 ng/ml; the detection range is 0.1 to 50 ng/ml; high correlation and coincidence rate to clinic AMI patients are high.

Description

technical field [0001] The invention relates to biological immunological in vitro diagnostic technology, in particular to a quantitative detection method for cardiac myosin binding protein C, and also relates to a quantitative detection kit for cardiac myosin binding protein C. Background technique [0002] Acute myocardial infarction (AMI) is a serious cardiovascular disease with complex etiology. In recent years, its incidence has been increasing year by year, and it tends to become younger, which seriously endangers the health of patients. Therefore, early diagnosis and treatment of it is of great significance. The determination of serum myocardial necrosis markers plays an important role in the diagnosis of AMI. Cardiac troponin (cTn) has become the biomarker of choice for the diagnosis of AMI. However, for patients with myocarditis, pulmonary embolism, etc., cTn will also increase non-specifically, and for patients with onset time less than 6 hours or patients with rec...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/577G01N21/76
Inventor 李奎于林李双法董亚玲刘功成付光宇吴学炜苗拥军
Owner AUTOBIO DIAGNOSTICS CO LTD
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