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Homogeneous fluorescence immunoassay reagent for rapid and quantitative detection of troponin T and preparation and detection methods thereof

A technology for quantitative detection and troponin, which is applied in the field of medical testing, can solve the problems of long continuous rise time and poor repeatability of nitrocellulose membrane detection, and achieve the effect of sufficient reaction, elimination of matrix effect, and high cost performance

Inactive Publication Date: 2017-05-10
SHANDONG UNIV QILU HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

(3) It lasts longer in the blood
However, both methods suffer from poor reproducibility due to the heterogeneity of the nitrocellulose membrane itself.

Method used

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  • Homogeneous fluorescence immunoassay reagent for rapid and quantitative detection of troponin T and preparation and detection methods thereof
  • Homogeneous fluorescence immunoassay reagent for rapid and quantitative detection of troponin T and preparation and detection methods thereof
  • Homogeneous fluorescence immunoassay reagent for rapid and quantitative detection of troponin T and preparation and detection methods thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0041] The preparation method of quantitatively detecting troponin T (cTnT) homogeneous fluorescent immunological reagent comprises the steps:

[0042] 1. Preparation of anticTnT for labeling:

[0043] Purified anti-cTnT monoclonal antibody expressed by genetic engineering is used. Eu 3+ The product code of anti-cTnT monoclonal antibody for labeling is 19C7; the product code of anti-cTnT monoclonal antibody for fluorescein labeling is 16A11 and 560.

[0044] 2. Preparation of rare earth element chelate labeled anti-cTnT:

[0045] The mouse anti-human cTnT monoclonal antibody 19C7 solution (3 mg / mL) was dialyzed twice at 4° C. with 3 L of 0.9% NaCl, 24 hr each time. Add water to adjust the concentration to 1.5mg / mL. Take 0.6mL of the antibody solution, add 1mL NaHCO 3 (0.2mol / L), and adjust the pH to 9.1 with 1mol / L NaOH. 20 μL of BHHCT methanol solution (30 μg / mL) was added dropwise into the antibody solution under stirring, and the stirring reaction was continued for 1 ...

Embodiment 2

[0051] The preparation method of the present embodiment is basically the same as that of Example 1, the difference is:

[0052] 2. The preparation method of rare earth element chelate labeled anti-cTnT is:

[0053] The mouse anti-human cTnT solution (3 mg / mL) was dialyzed twice at 4° C. with 3 L of 0.9% NaCl, 24 hr each time. Add water to adjust the concentration to 1.5mg / mL. Take 0.6mL of the antibody solution, add 1mL NaHCO 3(0.2mol / L), and adjust the pH to 9.1 with 1mol / L NaOH. 20 μL of BHHBCB methanol solution (30 μg / mL) was added dropwise to the antibody solution under stirring, and the stirring reaction was continued for 1 hr. After centrifugation (10000rpm, 10min) to remove insoluble matter, put on SephadexG-25 column, use 0.05mol / L NH 4 HCO 3 (pH=8.0) to separate the labeled protein and free label. UV / visible spectrophotometer detects the A of each collection liquid 330 value, pool the solutions containing the labeled antibodies. Add final concentrations of 0.1...

Embodiment 3

[0055] The preparation method of the present embodiment is basically the same as that of Example 1, the difference is:

[0056] 3. Dilute anti-cTnT monoclonal antibodies 16A11 and 560 with 0.1M sodium bicarbonate solution to 1 mg / mL respectively, take 5 mL of antibody solution, add 40 mg of fluorescein DyLight-DY647 solution, stir well, and incubate at room temperature for 1.5 hours , and mix every 15 minutes. Finally, the G25 gel column was used for column separation and purification, and the labeled fluorescein-labeled antibody was collected and diluted with 0.01mol / L phosphate buffer containing 0.05% PEG600, 3.5% BSA, 10% glycerol, and 0.05% surfactant. Seal the package with a plastic bottle and store at 4°C.

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Abstract

The invention discloses a homogeneous fluorescence immunoassay reagent for rapid and quantitative detection of troponin T and preparation and detection methods thereof. The homogeneous fluorescence immunoassay reagent for rapid and quantitative detection of troponin T comprises anti-cTnT (cardiac troponin) labeled with rare earth element chelate, another anti-cTnT labeled with a near infrared fluorescent compound and cTnT calibrators with a series concentrations. With the adoption of a rapid homogeneous fluorescence detection technology as well as the high sensitivity characteristic of fluorescence, adverse effects on detection accuracy and repeatability due to the non-uniform characteristics of pores of a nitrocellulose membrane in colloidal gold or fluorescent cTnT dry immune test paper are also prevented, and the detection sensitivity and the linear range can be greatly improved. The reagent has the characteristics of being easy to operate, rapid, sensitive, good in specificity and the like when applied to detection of cTnT in human blood, serum or plasma, and has good clinical application prospect.

Description

technical field [0001] The invention belongs to the field of medical testing, and in particular relates to a homogeneous fluorescent immunological reagent for rapid quantitative detection of troponin T and a preparation and detection method thereof. Background technique [0002] Coronary heart disease has become a major disease affecting the health of the population. Acute myocardial infarction is the main cause of death in coronary heart disease patients. How to timely discover patients with acute myocardial infarction and corresponding treatment is the key to saving the lives of patients with myocardial infarction. Traditional diagnosis is mainly based on typical clinical manifestations, ECG changes and laboratory enzyme examination. However, a considerable number of patients with myocardial infarction have no obvious clinical manifestations, and there is no obvious change in the early ECG. In this case, the application of specific markers of myocardial injury plays a k...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/536G01N33/533
CPCG01N33/6887G01N33/533G01N33/536G01N2333/4712G01N2800/324
Inventor 黄亚丽李新华魏芳赵珂谢爱武
Owner SHANDONG UNIV QILU HOSPITAL
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