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Microballoon-based cup type time resolution fluorescence myohemoglobin analysis kit, preparation method and application thereof

A time-resolved fluorescence and myoglobin technology, applied in the field of clinical medical diagnosis, can solve the problems of high technical requirements of chemiluminescence, unsatisfactory sensitivity and specificity, and unsatisfactory detection precision, so as to shorten the detection time and improve the detection Sensitive, easy to automate effects

Inactive Publication Date: 2016-08-17
SHANGHAI UPPER BIO TECH PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Latex turbidimetry generally uses polyclonal antibodies to coat latex microspheres, and the sensitivity and specificity are not ideal. If monoclonal antibodies are used to coat latex microspheres, the cost of reagents is very high
The chemiluminescence method has high technical requirements, the operation steps are relatively cumbersome, and a multi-step reagent addition process is required
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 fluorescence myohemoglobin analysis kit, preparation method and application thereof
  • Microballoon-based cup type time resolution fluorescence myohemoglobin analysis kit, preparation method and application thereof
  • Microballoon-based cup type time resolution fluorescence myohemoglobin analysis kit, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] (1) Preparation of detection cuvette:

[0046] A. Detection cuvette coating: Myoglobin antibody 7004 (Medix Company) was diluted to 20 μg / ml with 0.2mol / L phosphate buffer (pH7.8), 200 μL / well, coated at 37°C for 4 hours, and washed. .

[0047] B. Detection cuvette blocking: Add 300 μL / well of blocking buffer to the cuvette, block at 37°C for 4 hours, discard the blocking solution in the coated cuvette, and pat dry.

[0048] C. Detection of drying of the cuvette: place the sealed cuvette above in a drying oven at 37° C. with a humidity lower than 30% for 8 hours, and store in a sealed and dry place.

[0049] (2) Preparation of fluorescently labeled antibodies:

[0050] A. Fluorescent particle activation:

[0051] 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, pH 6.0, add Add purified water to 0.2mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiim...

Embodiment 2

[0061] (1) Drawing of standard curve:

[0062] The reagents prepared in Example 1 were combined into a myoglobin time-resolved fluorescence quantitative kit, and the calibrator was measured, and each concentration was repeated 10 times.

[0063] Add 5 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 Victor X4 fluorescence reader from PerkinElmer (excitation wavelength 340-380nm, detection wavelength 600-630nm) The specific data are shown in Table 1.

[0064] Table 1

[0065]

[0066] 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 myoglobin contained in the sample can be quantitatively analyzed by the standard curve.

[0067] It can be seen fro...

Embodiment 3

[0075] (1) Preparation of detection cuvette:

[0076] A. Detection cuvette coating: Myoglobin antibody 7004 (Medix Company) was diluted to 20 μg / ml with 0.2mol / L phosphate buffer (pH7.8), 200 μL / well, coated at 37°C for 4 hours, and washed. .

[0077] B. Detection cuvette blocking: Add 300 μL / well of blocking buffer to the cuvette, block at 37°C for 4 hours, discard the blocking solution in the coated cuvette, and pat dry.

[0078] C. Detection of drying of the cuvette: place the sealed cuvette above in a drying oven at 37° C. with a humidity lower than 30% for 8 hours, and store in a sealed and dry place.

[0079] (2) Preparation of fluorescently labeled antibodies:

[0080] A. Fluorescent particle activation:

[0081] Take 1mg of carboxyl time-resolved fluorescent microspheres (200nm, 0.1ml, 10mg / mL, Bangslab company), add 60ul 500mM MES (2-(N-morpholine)ethanesulfonic acid) buffer, pH 6.0, add 0.2mg 1 -(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), add...

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Abstract

The invention belongs to the field of clinical medical diagnosis, and in particular relates to a cup-type time-resolved fluorescent myoglobin analysis kit based on microspheres and its preparation method and application. The kit is composed of a detection reaction cup, a fluorescently labeled antibody and a cleaning solution. In the specific detection, first draw the standard curve, then add the sample to be tested into the detection cuvette, then add the fluorescently-labeled antibody, incubate at 30°C-40°C, wash with cleaning solution to remove unbound antigen and fluorescently-labeled antibody; then The fluorescence signal is compared with the standard curve to obtain the myoglobin concentration of the sample to be tested. The method for detecting myoglobin is easy to realize automatic operation due to its ultra-high sensitivity, and the reaction temperature is uniform, not affected by environmental factors, with better accuracy and excellent precision.

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 myoglobin 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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IPC IPC(8): G01N33/533G01N33/68
CPCG01N33/533G01N33/6887G01N2800/324
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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