Test strip and test card for fluorescence immunochromatography of myeloperoxidase

A technology of fluorescence immunochromatography and myeloperoxidase, which is applied in the direction of analyzing materials, measuring devices, instruments, etc., can solve parameters such as unrecorded detection linear range and sensitivity, undisclosed test strip linear range, sensitivity and other parameters To achieve reliable diagnosis results, improve clinical diagnosis efficiency, and lower configuration requirements

Inactive Publication Date: 2018-08-14
河南省生物工程技术研究中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] At present, there have been reports on quantitative detection of myeloperoxidase using fluorescent microsphere immunochromatography technology. For example, patent application 201120513019.8 discloses a test paper for rapid detection of myeloperoxidase, which specifically discloses the use of fluorescent microspheres with a diameter of 210nm. Microspheres are used to label MPO monoclonal antibodies to prepare test strips. However, this document does not disclose parameters such as the linear range and sensitivity of the test strips; as another example, patent application 201611227045.8 discloses a human myeloperoxidase The preparation method of the immunochromatographic test strip, but the embodiment part only verified the qualitative detection of the test strip, and also did not record the detection linear range, sensitivity and other parameters

Method used

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  • Test strip and test card for fluorescence immunochromatography of myeloperoxidase
  • Test strip and test card for fluorescence immunochromatography of myeloperoxidase
  • Test strip and test card for fluorescence immunochromatography of myeloperoxidase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Prepare the MPO antibody-fluorescence immunochromatography quantitative detection test strip as follows:

[0039] a. Prepare the MPO antibody-fluorescent microsphere complex by EDC method:

[0040] Take 15ul fluorescent microspheres and add them to 0.6ml 0.05mol / l pH8.0 borate buffer solution, vortex and shake to mix; add 40ul 4mg / ml EDC, shake at room temperature for 30min; in the activated microsphere solution, add 125 μg of MPO-labeled antibody was coupled for 90 minutes; the coupled immune microsphere complex was reconstituted with a protective solution, the composition of which was 0.05M pH8.0 Tris-HCl buffer, 0.5% T-20 , 0.2% BSA, 0.5% casein, 4% trehalose and 0.05% NaN 3 , and then use blocking solution (containing 10% BSA, 0.05% NaN 3 ) to block for 2 hours to obtain the MPO antibody-fluorescent microsphere complex.

[0041] b. Prepare assembled test strips:

[0042] (1) Immerse the glass fiber member in the treatment solution, which includes Tris–HCl buffer...

Embodiment 2

[0049] Embodiment 2: Reaction condition optimization

[0050] Optimization of EDC amount:

[0051] Take 15ul fluorescent microspheres and add them to 0.6ml 0.05mol / l pH8.0 borate buffer solution, vortex and oscillate to mix; add 4mg / ml EDC 10, 20, 40, 60, 80ul in turn, shake at room temperature for 30min, press The method of embodiment 1 prepares 5 groups of test paper strips, add sample and detect respectively with linear standard substance, fit and obtain standard equation, detect linear range and R 2 value, the results are shown in Table 1:

[0052] Table 1

[0053]

[0054] As shown in Table 1, under the condition of adding 160 μg of EDC, the linearity is the best, the best linear range is 3.125~600 ng / ml, R 2 =0.9803.

[0055] Optimization of coupling protein amount and coupling time:

[0056] Add 75, 100, 125, and 150 μg of MPO-labeled antibody in sequence to the activated microsphere solution, and couple for 30 min, 60 min, 90 min, and 120 min, respectively. Pr...

Embodiment 3

[0069] Stability detection is carried out to the test strip prepared by the embodiment of the present invention 1:

[0070] The test strips prepared in Example 1 were stored at 37°C and 4°C respectively, and by using linear standards to detect the test strips stored at 37°C and 4°C, the linearity of each condition R 2 The results of the changes are shown in Table 3.

[0071] table 3

[0072]

[0073] As can be seen from Table 3, the test strips of the present invention can store 180 antennas in R under 4°C. 2Both can reach more than 0.97, and the stability is good. When the test strip is stored at 37°C for 10 days, R 2 As the value decreases to 0.96, the detection error increases. Storing at 37°C for 1 day is close to the effect of storing at 4°C for about 45 days. It is inferred that the test strips can be stored at 4°C for at least 8 months.

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Abstract

The invention discloses a test strip for fluorescence immunochromatography of myeloperoxidase. The test strip comprises a base plate, a sample pad, a binding pad, a nitrocellulose membrane and an absorbent pad, and the sample pad, the binding pad, the nitrocellulose membrane and the absorbent pad are assembled on the base plate in a sequential overlapping manner, wherein the absorbent pad and thebinding pad are respectively pressed on two ends of the nitrocellulose membrane in an overlapping manner, and a detection area is formed on the surface of the nitrocellulose membrane; the sample pad is pressed on the binding pad in an overlapping manner, and a myeloperoxidase antibody-fluorescent microsphere compound is immobilized on the binding pad; and the nitrocellulose membrane in the detection area is coated with a detection line formed by a monoclonal antibody for recognizing another epitope of myeloperoxidase and a control line formed by a goat anti-mouse IgG polyclonal antibody. The test strip has the advantages of high sensitivity, high stability, realization of the detection linearity being 3.125-600 ng/ml, no non-specificity, short detection time of 5 min, realization of bedside quick test, and great improvement of the clinical diagnosis efficiency.

Description

technical field [0001] The invention belongs to the technical field of POCT immunochromatography detection, and specifically relates to a myeloperoxidase fluorescence immunochromatography quantitative detection test strip, a test card containing the test strip, and their preparation methods. Background technique [0002] Coronary atherosclerotic heart disease is a heart disease caused by atherosclerotic lesions in the coronary arteries that cause stenosis or blockage of the vessel lumen, resulting in myocardial ischemia, hypoxia or necrosis, referred to as coronary heart disease (CHD) . At present, CHD has become the disease with the highest incidence rate among cardiovascular diseases in the world. Nearly 10 million people suffer from CHD in my country, and more than 800,000 people die from CHD every year, far exceeding the mortality rate of tumors, and the number of deaths increases with age. The occurrence of coronary heart disease is affected by many factors, such as s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577
CPCG01N33/577
Inventor 王云龙马雪虎李玉林王继创程蕾
Owner 河南省生物工程技术研究中心
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