Microballoon-based cup-type time resolution fluorescent troponin I analysis kit as well as preparation method and application thereof

A time-resolved fluorescence, troponin technology, applied in the field of clinical medical diagnosis, can solve the problems of high technical requirements of chemiluminescence, unsatisfactory detection precision, long detection time, etc. The effect of detection sensitivity

Inactive Publication Date: 2016-07-13
SHANGHAI UPPER BIO TECH PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, radioimmunoassay and enzyme-linked immunosorbent assay are complicated to operate and take a long time to detect, so they have almost been eliminated at present; chemiluminescence method has high technical requirements and is not easy to carry out routinely in clinical laboratories.
Although the colloidal gold immunochromatography method has the advantages of less sample consumption, simple, quick, and cheap, however, when the antigen or antibody content in some samples is extremely low, the color of the colloidal gold will be very light or even no color, and it is difficult to Judging the results with the naked eye is prone to misjudgment and low sensitivity
Although the sensitivity of fluorescence immunochromatography is higher than that of colloidal gold immunochromatography, its detection precision is not ideal, and the sensitivity is still far behind that of large-scale chemiluminescence or electrochemiluminescence instruments

Method used

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  • Microballoon-based cup-type time resolution fluorescent troponin I analysis kit as well as preparation method and application thereof
  • Microballoon-based cup-type time resolution fluorescent troponin I analysis kit as well as preparation method and application thereof
  • Microballoon-based cup-type time resolution fluorescent troponin I analysis kit as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] (1) Preparation of detection cuvette:

[0048] A. Detection cuvette coating: Troponin I antibody 8E10 (Hytest company, antigen recognition site 87-91) was diluted with 0.2mol / L phosphate buffer (pH7.8) to 10 μg / ml, 100 μL / well, Coating at 37°C for two hours and washing the plate.

[0049] B. Detection cuvette blocking: Add 200 μL / well of blocking buffer to the cuvette, block at 37°C for two hours, discard the blocking solution in the coated cuvette, and pat dry.

[0050] C. Drying of the test cuvette: place the sealed cuvette above in a 37° C. drying oven with a humidity lower than 30% for 4 hours, and store in a sealed and dry place.

[0051] (2) Preparation of fluorescently labeled antibodies:

[0052] A. Fluorescent particle activation:

[0053] Take 1mg of carboxyl time-resolved fluorescent microspheres (200nm, 0.1ml, 10mg / mL, Bangslab Company), add 60μL of 500mmol / L MES (2-(N-morpholine)ethanesulfonic acid) buffer, pH6.0, add 0.2 mg1-(3-dimethylaminopropyl)-3-e...

Embodiment 2

[0063] (1) Drawing of standard curve:

[0064] The reagents prepared in Example 1 were combined into a troponin I time-resolved fluorescence quantitative kit, and the calibrator was measured, and each concentration was repeated 10 times.

[0065] Add 10 μL of calibrator and 50 μL of fluorescently labeled antibody to each test, incubate at 37°C for 20 min, then wash the detection cup, and read on the VictorX4 fluorescence reader from PerkinElmer (excitation wavelength 340-380nm, detection wavelength 600-630nm). The data are shown in Table 1.

[0066] Table 1

[0067]

[0068] According to the data in Table 1, the concentration of the calibrator is taken as the abscissa, and the mean fluorescence signal is taken as the ordinate to draw a standard curve. standard curve as figure 2 shown. The standard curve has good linearity, and the concentration of troponin I contained in the sample can be quantitatively analyzed by the standard curve.

[0069] From the results in Tabl...

Embodiment 3

[0076] (1) Preparation of detection cuvette:

[0077] A. Detection cuvette coating: Troponin I antibody 8E10 (Hytest company, antigen recognition site 87-91) was diluted with 0.2mol / L phosphate buffer (pH7.8) to 10 μg / ml, 100 μL / well, Coating at 37°C for two hours and washing the plate.

[0078] B. Detection cuvette blocking: Add 200 μL / well of blocking buffer to the cuvette, block at 37°C for two hours, discard the blocking solution in the coated cuvette, and pat dry.

[0079] C. Drying of the test cuvette: place the sealed cuvette above in a 37° C. drying oven with a humidity lower than 30% for 4 hours, and store in a sealed and dry place.

[0080] (2) Preparation of fluorescently labeled antibodies:

[0081] A. Fluorescent particle activation:

[0082]Take 1mg of carboxyl time-resolved fluorescent microspheres (200nm, 0.1ml, 10mg / mL, Bangslab company), add 60ul 500mMMES (2-(N-morpholine)ethanesulfonic acid) buffer, pH 6.0, add 0.2mg1-(3 -Dimethylaminopropyl)-3-ethylcarb...

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Abstract

The invention belongs to the field of clinical medical diagnosis and especially relates to a microballoon-based cup-type time resolution fluorescent troponin I analysis kit as well as a preparation method and application thereof. The kit comprises a detection reaction cup, a fluorescence labeling antibody and a cleaning fluid. The method comprises the following steps of: in a specific detection process, firstly drawing a standard curve; adding a to-be-detected sample into the detection reaction cup; adding the fluorescence labeling antibody; incubating at 30-40 DEG C; washing with the cleaning fluid and removing the uncombined antibody and fluorescence labeling antibody; comparing a fluorescence signal with the standard curve, thereby acquiring the concentration of troponin I of the to-be-detected sample. The detection method for the troponin I is ultrahigh in sensitivity, so that the automatic operation can be easily realized; the reaction temperature is uniform; the method is not influenced by the environmental factor; the accuracy is better; the precision is excellent.

Description

technical field [0001] The invention belongs to the field of clinical medical diagnosis, and in particular relates to a cup-type time-resolved fluorescent troponin I analysis kit based on microspheres and a preparation method and application thereof. Background technique [0002] Cardiovascular disease is a serious disease that endangers human health and life, and has become the number one killer of human health in the 21st century. In August 2009, Bayer Pharmaceuticals reported at the annual meeting of the European Society of Cardiology that about 17.5 million people died of cardiovascular diseases in the world in 2005, and this number is expected to climb to 20 million in 2015, and about half of the cases occurred in the Asia-Pacific region. area. The improvement of living conditions and the change of lifestyle have made the risk of cardiovascular disease in Chinese people surpass that of developed countries such as the United States. [0003] In 1990, the mortality rate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N33/68G01N33/533
CPCG01N21/6486G01N21/6408G01N33/533G01N33/68
Inventor 石晓强李福刚徐建新周奕璇杨晶
Owner SHANGHAI UPPER BIO TECH PHARMA
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