WO2018120855A1 - Time-resolved fluorescent immunochromatographic test strip and kit for detecting myo, and preparation method therefor - Google Patents

Abstract

A time-resolved immunochromatographic test strip and kit for detecting MYO, and a preparation method therefor. The test strip comprises a substrate, and a sample pad, a conjugating pad, a coated membrane and an absorbent paper provided on the substrate in sequence; the conjugating pad is coated with a fluorescent microsphere labeled MYO monoclonal detection antibody; the coated membrane comprises a detection area and a control area parallel to each other and arranged alternatingly; the detection area is coated with an MYO monoclonal capture antibody for identifying a single antigen epitope; the control area is coated with a goat anti mouse IgG antibody; the coated membrane is a nitrocellulose membrane conjugated with a polymer; the polymer is a material having 10% or less light transmittance at wavelengths of less than 450 nm and 95% or more light transmittance at wavelengths of 500 nm or more. The test strip can perform rapid and quantitative detection, provides accurate and reliable test results, has high sensitivity and a simple preparation method, is suitable for mass production, and has positive significance to MYO quantitative detection.

Description

Time-resolved fluorescent immunochromatographic test strip, kit and method for preparing MYO Technical field

The invention belongs to the technical field of medical testing, in particular to a time-resolved fluorescent immunochromatographic test strip, a kit and a preparation method thereof for quantitatively detecting MYO.

Background technique

Myoglobin (MYO) is an oxygen-binding heme protein mainly distributed in myocardium and skeletal muscle tissue, accounting for 0.1%-0.2% of total muscle. Diving mammals such as whales, seals and dolphins have so much muscle myoglobin content that their muscles are brownish red. The storage of oxygen by myoglobin allows these animals to potentially be underwater for long periods of time. In acute myocardial injury, MYO is first released into the blood. After about 2-3 hours of symptoms, Mb in the blood can exceed the upper limit of normal, peaks in 9-12 hours, and returns to normal after 24-36 hours. It can be increased 1.5h after infarction, but it will return to normal within 1~2d. Therefore, continuous sampling is recommended for patients suspected of ACS (Acute Coronary Syndrome) because there is a delay between the onset of symptoms and the release of protein markers into the blood. A large number of literature reports have been published on the clinical utility of early diagnosis and monitoring of ACS. MYO negative helps to rule out myocardial infarction.

Myoglobin is present in muscle and is a binding protein composed of a peptide chain and a heme prosthetic group. It is extremely rich in myocardium and is a protein that stores oxygen in muscle. Sperm whale myoglobin tertiary structure was elucidated in 1960 by Kendrew using X-ray diffraction, the world's first described protein tertiary junction. Since the tertiary structure is directly related to the biological function of the protein, and the analysis of the tertiary structure is very difficult, this work has been highly evaluated by the academic community.

Determination of serum myoglobin may be the most sensitive early indicator of acute myocardial infarction (AMI) diagnosis. However, poor specificity, skeletal muscle damage, trauma, kidney failure and other diseases can cause it to rise. MYO Yang Although it can not diagnose AMI, it can be used as an important indicator for early diagnosis of AMI. For example, if MYO is negative, myocardial infarction is basically excluded. It can also be used for the diagnosis of reinfarction. Combined with clinical, such as MYO re-raising, it should be considered as re-infarction or Infarction extends.

The measurement of myoglobin mainly uses the immunological method of double antibody sandwich, and the detection methods include:

1. Double-antibody sandwich immunochemiluminescence method - This method is simple in operation, strong in specificity and high in sensitivity.

2, gold standard method - the method has the characteristics of fast and easy, easy to observe, but qualitative detection, sensitivity is not high.

3, transmission immune turbidity method - the determination method is simple, rapid, and can be automated, suitable for batch detection, but the method and clinical application of immunoturbidimetric turbidity needs further verification.

The main feature of time-resolved immunochromatography POCT is to emphasize the rapid results and greatly shorten the turnaround time of the experimental results. For emergency treatment and rescue patients, these patients are often in critical condition and the cause is unknown, while the traditional clinical laboratory measurement time is generally more than 15 minutes, POCT generally can be completed within 5 minutes, according to the information provided by the doctor, The patient's timely diagnosis and preparation of a treatment plan will definitely reduce hospitalization time, reduce morbidity/mortality, and generate great social and economic benefits. At the same time, for some chronic diseases that require long-term monitoring, such as diabetes, patients can easily monitor blood sugar and urine sugar by the patient or family according to the doctor's request.

Currently POCT has no reliable quality assurance. Each test unit of the POCT is independent, so there is no guarantee that the quality of each test unit is the same. Among them, the instrument for optical detection will be interfered with by hemolysis and chyle in the specimen, and the chemiluminescence method will be affected by exogenous redox species. Various test strips and instruments based on immunochromatography, chromatography and dry chemistry techniques affect the activity of microproteins in the matrix due to differences in temperature, humidity and pH, which in turn affects the results. Some POCT instrument methodological defects, sensitivity and reproducibility are not good, the linear range is narrow, only for emergency or urgent use for reference, if necessary, also need to be sent to the test Section for review.

The currently used immunofluorescence chromatography device uses a reflection method to detect the fluorescent signal on the porous membrane, and the fluorescence detector captures a specific antibody modified by the fluorescent dye on the surface of the porous membrane, and the fluorescent signal inside the porous membrane is not detected, resulting in detection. The sensitivity is reduced.

Summary of the invention

Based on this, in order to overcome the above drawbacks of the prior art, the present invention provides a time-resolved immunochromatographic test strip, a kit and a preparation method thereof for quantitatively detecting MYO, and the immunochromatographic test strip and kit can not only It provides high sensitivity and specificity, is easy to operate, meets the needs of clinical rapid testing, and reduces costs to meet the needs of the domestic market.

In order to achieve the above object of the invention, the present invention adopts the following technical solutions:

A time-resolved immunochromatographic test strip for detecting MYO, comprising a substrate, a sample pad, a bonding pad, a coating film and an absorbent paper sequentially disposed on the substrate, wherein the bonding pad is coated with fluorescent microspheres a labeled MYO monoclonal detection antibody comprising a detection zone and a control zone arranged in parallel and spaced apart from each other, the detection zone being coated with a MYO monoclonal capture antibody recognizing a single antigenic epitope, the control zone package A goat anti-mouse IgG antibody; the coating film is a chemically cross-linked nitrocellulose membrane having a transmittance of 10% or less at a wavelength of less than 450 nm and 95% or more at a wavelength of 500 nm or more Light transmittance of the material.

In some of these embodiments, the polymer is a mixture of polystyrene acrylonitrile and polycarbonate in one or a different ratio. This material allows most of the visible light to pass through, and the photodetector captures the fluorescent signals on the surface and inside of the multilayer porous membrane, making the detection results more accurate.

In some of the embodiments, the MYO monoclonal capture antibody and the goat anti-mouse IgG antibody that recognize a single epitope are at a concentration of 1-1.5 mg/ml and 0.3-0.5 mg/ml, respectively, on the coating film. Package The amount is 1-1.5 ul / cm.

In some of these embodiments, the bond pad is a nitrocellulose membrane that is capable of carrying a sufficient amount of fluorescent microspheres and that rapidly releases the microspheres upon encountering the sample.

In some embodiments, the fluorescent microspheres are selected from Eu ³⁺ lanthanide fluorescent microspheres for labeling antibodies, and the microspheres have active groups on the surface thereof, which can be linked to biological substances such as proteins and sugars. Contains fluorescein.

In some of these embodiments, the fluorescent microspheres have a diameter of from 290 nm to 350 nm.

In some of these embodiments, the detection zone is adjacent to the bond pad, the control zone is adjacent to the absorbent paper, and the detection zone and the control zone are spaced from 0.3 to 0.5 cm apart.

The invention also provides a preparation method of the above-mentioned time-resolved immunochromatographic test strip for detecting MYO, comprising the following steps:

(1) immobilizing a MYO monoclonal capture antibody and a goat anti-mouse IgG antibody that recognize a single epitope on the coating membrane to form a detection zone and a control zone;

(2) preparing a fluorescent microsphere-labeled MYO monoclonal detection antibody and spraying on the binding pad;

(3), lap the sample pad, the bonding pad, the coating film and the absorbent paper on the substrate to obtain.

In some of the embodiments, the method for preparing the fluorescently labeled MYO monoclonal detection antibody in the step (2) comprises the following steps:

(1) The MYO monoclonal detection antibody was dialyzed overnight at 4 ° C using a 0.02-0.05 mol/L phosphate buffer solution of pH 7.2-7.6, and the dialyzed MYO monoclonal detection antibody was adjusted to a concentration of 2-4mg/ml;

(2) Washing the microspheres with 0.01-0.05 mol/L MES activation buffer of pH 6.0, adding carbodiimide and N-hydroxysuccinimide to make the final concentration of the microspheres 0.2 mol/L, room temperature Reaction for 15-30 minutes, Wash the microspheres thoroughly and reconstitute with 0.02-0.05 mol/L boric acid buffer pH 7.4-7.6;

(3) According to the MYO monoclonal detection antibody and microsphere mass ratio of 1:5-6, MYO monoclonal antibody was added to the reconstituted microspheres, reacted at room temperature for 2 hours, and added with 5% BSA 0.02mol/L borate buffer pH 7.4-7.6, react at room temperature for 30 minutes, wash, and then use 0.02mol/L borate buffer pH 7.4-7.6 containing 0.5% BSA, 0.05% Tween-20 Reconstituted to the original volume, sprayed on the glass fiber membrane with a quantitative spray film sprayer at 4 ul / cm, protected from light, and dried.

The invention also provides a time-resolved fluorescent immunochromatography kit for detecting MYO, the kit comprising a plastic card case, the above-mentioned test strip, and a buffer bag disposed in the plastic card case.

In some of the embodiments, the buffer is a PBS buffer containing 0.5% BSA, 0.05% Tween 20, 0.1-1% reducing agent; the buffer is 0.01-0.1 based on the ordinary phosphate solution. % of a reducing agent used to reduce free peroxidase in the specimen.

In some of these embodiments, the reducing agent is reduced glutathione or ascorbic acid.

When the time-resolved fluorescent immunochromatography kit for detecting MYO of the present invention is used, the blood sample to be tested is added to the sample-adding hole, and the sample buffer and the blood sample are mixed and immersed into the sample pad by the needle-punching buffer bag. After the sample on the pad is saturated, the sample is delivered to the bond pad by capillary action. When the blood sample contains MYO, MYO forms an antigen-antibody complex with the antibody on the fluorescent microsphere. As the chromatography progresses, the complex moves forward to reach the MYO monoclonal capture antibody that coats a single epitope. At the detection zone T, an antibody-antigen-antibody sandwich complex is formed and aggregated at the detection zone T. Rare earth ion microspheres (Eu ^{3 +} lanthanide) that do not bind MYO monoclonal antibody continue to advance, and when the control region C is reached, the goat anti-mouse IgG antibody and the murine monoclonal antibody on the rare earth ion microsphere (ie, MYO single) The clone detection antibody binds, and aggregation of rare earth ion microspheres occurs at the C line. The entire reaction was completed in 10 minutes and the card was read on the machine. The intensity of the fluorescence generated under the excitation light source is proportional to the content of the conjugate on the test strip. When the light source is irradiated onto the detection zone and the control zone of the test strip, the attached fluorescent substance is excited, and the emitted light is collected and converted into an electrical signal. The strength of the electrical signal is related to the number of fluorescent molecules, and the detector calculates the content of the analyte in the sample.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The test strip of the invention adopts a special light-transmitting material, can not only achieve the quantitative analysis of the chemiluminescence method, but also achieve the rapid detection of the gold standard method, and ensures the accurate and reliable test results;

(2) The test strip of the invention introduces time-resolved immunochromatography technology into the quantitative detection of MYO, and combines the time-resolved fluorescence detector to realize the quantitative detection of single-part of MYO, and the sensitivity is high, and the difference between batch and batch is Small, providing great convenience for clinical use;

(3) The preparation method of the test strip of the invention is simple and suitable for large-scale production, and has positive significance for the quantitative detection of MYO.

detailed description

The invention will be described in detail below by way of specific examples.

The raw materials used in the following examples were all commercially available unless otherwise specified.

Example 1 Time-resolved immunochromatographic test strip for detecting MYO

The time-resolved immunochromatographic test strip for detecting MYO of the embodiment comprises a substrate, a sample pad, a bonding pad, a coating film and an absorbent paper which are sequentially disposed on the substrate, and the bonding pad is coated a fluorescent microsphere-labeled MYO monoclonal detection antibody (Raybiotech.), the coating membrane comprising a detection zone and a control zone disposed in parallel and spaced 0.5 cm apart from each other, the detection zone being adjacent to the binding pad, the control zone Adjacent to the blotting paper, the detection zone is coated with a MYO monoclonal capture antibody (produced, prepared by techniques well known in the art) that recognizes a single epitope, which is coated with a goat anti-mouse IgG antibody.

In this embodiment, the coating film is a chemically crosslinked polycarbonate and polystyrene acrylonitrile (polymer) The nitrocellulose membrane, the polycarbonate and the polystyrene acrylonitrile polymer have a light transmittance of 10% or less at a wavelength of less than 450 nm, and a light transmittance of 95% or more at a wavelength of 500 nm or more. This material allows most of the visible light to pass through, and the photodetector captures the fluorescent signals on the surface and inside of the multilayer porous membrane, making the detection results more accurate.

In this embodiment, the bonding pad is a nitrocellulose membrane capable of carrying a sufficient amount of fluorescent microspheres and rapidly releasing the microspheres after encountering the sample.

In this embodiment, the fluorescent microspheres are selected from Eu ³⁺ lanthanide fluorescent microspheres for labeling antibodies, and the microspheres have reactive groups on the surface thereof, which can be linked to biological substances such as proteins and sugars. Contains fluorescein. The fluorescent microspheres have a diameter of 290 nm.

The method for preparing a time-resolved fluorescent immunochromatographic test strip for detecting MYO of the present embodiment comprises the following steps:

(1) Fixing a MYO monoclonal capture antibody and a goat anti-mouse IgG antibody that recognize a single epitope on the coating membrane to form a detection zone and a control zone; the specific method is: using 0.02 mol/L containing 5% sucrose Phosphate buffer pH 7.2-7.6, respectively, MYO monoclonal capture antibody and goat anti-mouse IgG antibody recognizing a single epitope were diluted to a concentration of 1.5 mg/ml and 0.3 mg/ml, respectively, using a quantitative spray film Spraying the two on the nitrocellulose membrane at an interval of 0.5 cm in an amount of 1 ul / cm, drying at 50 ° C for 5 h, adding a desiccant to seal for use;

(2) Preparing a fluorescent microsphere-labeled MYO monoclonal detection antibody and spraying it on the binding pad; the specific method is: (a) at 0.02-0.05 mol/L of pH 7.2-7.6 phosphoric acid at 4 ° C The MYO monoclonal detection antibody was dialyzed overnight in a salt buffer, and the dialyzed MYO monoclonal detection antibody was adjusted to a concentration of 2-4 mg/ml; (b) using 0.01-0.05 mol/L pH 6.0 MES activation. The microspheres were washed with buffer, and carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added, and the final concentration of the microspheres was 0.2 mol/L. The reaction was carried out for 15 minutes at room temperature, and the microspheres were thoroughly washed and reconstituted with 0.02 mol/L borate buffer of pH 7.4-7.6; (c), the ratio of the mass ratio of the antibody to the microspheres by the MYO monoclonal detection was 1:5, Add MYO monoclonal detection antibody to the reconstituted microspheres, react at room temperature for 2 hours, add 0.02 mol/L borate buffer containing pH 7.4-7.6 containing 5% BSA, react at room temperature for 30 minutes, wash, and then use 0.5% BSA, 0.05% Tween-20 0.02 mol/L borate buffer of pH 7.4-7.6 was reconstituted to the original volume, and sprayed on a glass fiber membrane at a dose of 4 ul/cm using a quantitative spray film, protected from light. , drying at 50 ° C for 6 hours, adding desiccant sealed for use;

(3) Laying the sample pad, the bonding pad, the coating film and the absorbent paper on the underlay, and cutting into a width of 0.42 cm, that is, obtained.

Example 2 Time-Resolved Immunochromatography Kit for Detection of MYO

The time-resolved fluorescent immunochromatography kit for detecting MYO of the present embodiment, the kit comprising: the test strip, the plastic card case and the buffer bag according to the embodiment 1; the test strip is mounted on the plastic card case The buffer bag is located at a corner of the plastic card, adjacent to the sample pad of the test strip, and the surface of the buffer bag is provided with a round hole for needling.

In this example, the reagent strip was soaked in PBS buffer containing 0.5% BSA, 0.05% Tween 20, 0.1-1% reducing agent. The buffer is a 0.01-0.1% reducing agent added to the ordinary phosphate solution for reducing the free peroxidase in the sample. The reducing agent is reduced glutathione or ascorbic acid.

When the time-resolved fluorescent immunochromatography kit for detecting MYO of the present invention is used, the blood sample to be tested is added to the sample-adding hole, and the sample buffer and the blood sample are mixed and immersed into the sample pad by the needle-punching buffer bag. After the sample on the pad is saturated, the sample is delivered to the bond pad by capillary action. When the blood sample contains MYO, MYO forms an antigen-antibody complex with the antibody on the fluorescent microsphere. As the chromatography progresses, the complex moves forward to reach the MYO monoclonal capture antibody that coats a single epitope. At the detection zone T, an antibody-antigen-antibody sandwich complex is formed and aggregated at the detection zone T. Rare earth ion microspheres (Eu ^{3 +} lanthanide) not bound to MYO monoclonal antibody continue to advance, and when the control region C is reached, the goat anti-mouse IgG antibody and the murine monoclonal antibody on the rare earth ion microsphere (MYO monoclonal) The detection antibody binds, and aggregation of rare earth ion microspheres occurs at the C line. The entire reaction was completed in 10 minutes and the card was read on the machine. The intensity of the fluorescence generated under the excitation light source is proportional to the content of the conjugate on the test strip. When the light source is irradiated onto the detection zone and the control zone of the test strip, the attached fluorescent substance is excited, and the emitted light is collected and converted into an electrical signal. The strength of the electrical signal is related to the number of fluorescent molecules, and the detector calculates the content of the analyte in the sample.

Test Example 1 MYO was detected using the time-resolved immunochromatographic kit of Example 2.

A, fitting the standard curve

Different concentrations of MYO standards were added to the sample addition zone of the time-resolved immunochromatographic test strip of Example 1 (take 7 different concentrations, respectively 0 ng/mL, 5 mg/mL, 10 ng/mL, 50 ng/mL, 100ng/mL, 200ng/mL, 400ng/mL, 5 parallel samples for each concentration), after 10 minutes of membrane chromatography reaction, the instrument reads the quality control line C and the detection line T signal to detect the detection line of the sample. The ratio of the fluorescence value to the fluorescence value of the quality control line is the abscissa, and the MYO standard concentration is the ordinate. The equation is established and fitted to the standard curve y=0.9133x+5.5891.

The standard curve R ² =0.9957, which is linear, can be quantitatively analyzed by the standard curve for the MYO concentration contained in the sample.

B, sample testing

The sample to be tested was added to the sample application area of the fluorescent immunochromatographic test strip of MYO, and the membrane was subjected to a reaction for 10 minutes. Turn on the fluorescence detection device, read the standard curve in the IC card, and insert the test strip into the fluorescence detection device. The prepared card slot runs the instrument, and the instrument automatically calculates the MYO concentration in the sample to be tested through the corresponding analysis software, and takes the actual detection value into the preset standard curve according to the information on the calibration card to calculate the quantitative result.

Test Example 2 Determination of the performance of the kit of Example 2

The kit was tested for performance, including minimum detection limits, precision, sensitivity, specificity, and more.

1. Linear range: Take the same batch of time-resolved immunochromatographic kits for six concentrations of myoglobin reference products (0ng/mL, 5mg/mL, 10ng/mL, 50ng/mL, 100ng/mL, 400ng/ mL, 5 concentrations of each concentration were tested. The detection range was 0 ng/mL to 400 ng/mL, and the correlation coefficient r was calculated, where r should be ≥0.99.

2. Intra-assay precision: 10 time-resolved immunochromatographic kits of the same batch number were randomly selected, and the same concentration of myoglobin reference material was detected, and the coefficient of variation CV (%) value was ≤15%.

3. Inter-assay precision: Randomly extract three consecutive batches of time-resolved immunochromatographic kits. Each batch of 3 batches was tested for the same concentration of myoglobin reference material, and its coefficient of variation CV (%) value ≤ 15%.

4. Accuracy: Three levels of myoglobin reference products were determined by time-resolved immunochromatographic kits of the same batch number, and the mean and relative deviations of the sample determination results were calculated, wherein the relative deviation (B%) was ±15%. Inside.

5. Minimum detection limit: Take 10 copies of the same batch of time-resolved immunochromatography kit, test the matrix of the reference material, and calculate the average value of the sample.

And standard deviation SD, where

6. Analytical specificity: Select the same concentration of myoglobin reference product to add cholesterol, triglyceride and bilirubin respectively, so that the final concentration of interfering substances is cholesterol 60mg/ml, triglyceride 40mg/ml, bilirubin 2mg/ml. The interference samples were repeatedly tested 3 times, and the mean and relative deviation of the sample detection results were calculated, wherein the relative deviation (B%) was within ±15%.

The technical features of the above-described embodiments may be arbitrarily combined. For the sake of brevity of description, all possible combinations of the technical features in the above embodiments are not described. However, as long as there is no contradiction between the combinations of these technical features, All should be considered as the scope of this manual.

The above-described embodiments are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be determined by the appended claims.

Claims (10)

1. A time-resolved immunochromatographic test strip for detecting MYO, comprising: a substrate, and a sample pad, a bonding pad, a coating film and an absorbent paper which are sequentially disposed on the substrate, and the bonding pad is coated a fluorescent microsphere-labeled MYO monoclonal detection antibody comprising a detection zone and a control zone arranged in parallel and spaced apart from each other, the detection zone being coated with a MYO monoclonal capture antibody that recognizes a single antigenic epitope, The control region is coated with a goat anti-mouse IgG antibody; the coating film is a polymer-bound nitrocellulose membrane having a light transmittance of 10% or less at a wavelength of less than 450 nm and 95% or more at a wavelength of 500 nm or more. Light transmittance of the material.
2. The time-resolved immunochromatographic test strip for detecting MYO according to claim 1, wherein the polymer is one or both of polystyrene acrylonitrile and polycarbonate.
3. The time-resolved immunochromatographic test strip for detecting MYO according to claim 1 or 2, wherein the concentration of the MYO monoclonal capture antibody and the goat anti-mouse IgG antibody which recognize a single antigen epitope is 1.5 mg, respectively. /ml and 0.3 mg/ml, the amount of coating on the coating film was 1 ul/cm.
4. The time-resolved immunochromatographic test strip for detecting MYO according to claim 1 or 2, wherein the binding pad is a nitrocellulose membrane, and the fluorescent microspheres are Eu ³⁺ lanthanide fluorescent microspheres.
5. The time-resolved immunochromatographic test strip for detecting MYO according to claim 4, wherein the fluorescent microspheres have a diameter of from 290 nm to 350 nm.
6. The time-resolved immunochromatographic test strip for detecting MYO according to claim 1 or 2, wherein the detection zone is adjacent to the bonding pad, and the control zone is adjacent to the absorbent paper, the detection zone and The control zone has an interval of 0.3-0.5 cm.
7. The method for preparing a time-resolved immunochromatographic test strip for detecting MYO according to any one of claims 1 to 6, characterized in that it comprises the following steps:

   (1) immobilizing a MYO monoclonal capture antibody and a goat anti-mouse IgG antibody that recognize a single epitope on the coating membrane to form a detection zone and a control zone;

   (2) preparing a fluorescent microsphere-labeled MYO monoclonal detection antibody and spraying on the binding pad;

   (3), lap the sample pad, the bonding pad, the coating film and the absorbent paper on the substrate to obtain.
8. The method for preparing a time-resolved immunochromatographic test strip for detecting MYO according to claim 7, wherein the method for preparing the fluorescently labeled MYO monoclonal detection antibody in the step (2) comprises the following steps:

   (1) The MYO monoclonal detection antibody was dialyzed overnight at 4 ° C using a 0.02-0.05 mol/L phosphate buffer solution of pH 7.2-7.6, and the dialyzed MYO monoclonal detection antibody was adjusted to a concentration of 2-4mg/ml;

   (2) Washing the microspheres with 0.01-0.05 mol/L MES activation buffer of pH 6.0, adding carbodiimide and N-hydroxysuccinimide to make the final concentration of the microspheres 0.2 mol/L, room temperature The reaction is carried out for 15-30 minutes, the microspheres are thoroughly washed, and reconstituted with 0.02-0.05 mol/L boric acid buffer pH 7.4-7.6;

   (3) According to the MYO monoclonal detection antibody and microsphere mass ratio of 1:5-6, MYO monoclonal antibody was added to the reconstituted microspheres, reacted at room temperature for 2 hours, and added with 5% BSA 0.02mol/L borate buffer pH 7.4-7.6, react at room temperature for 30 minutes, wash, and then use 0.02mol/L borate buffer pH 7.4-7.6 containing 0.5% BSA, 0.05% Tween-20 Reconstituted to the original volume, sprayed on the glass fiber membrane with a quantitative spray film sprayer at 4 ul / cm, protected from light, and dried.
9. A time-resolved fluorescent immunochromatography kit for detecting MYO, characterized in that the kit comprises a plastic card holder, the test strip according to any one of claims 1 to 6, and a buffer solution disposed in the plastic card case bag.
10. A time-resolved fluorescent immunochromatography kit for detecting MYO according to claim 9, The buffer is a PBS buffer containing 0.5% BSA, 0.05% Tween 20, 0.1-1% reducing agent; the reducing agent is reduced glutathione or ascorbic acid.