CN111435136A - Time-resolved fluorescence immunochromatography kit for detecting novel coronavirus and preparation method thereof - Google Patents

Abstract

The invention discloses a time-resolved fluorescence immunochromatography kit for detecting novel coronavirus and a preparation method thereof. The kit includes a test strip and a fluorescence immunoassay analyzer, wherein the test strip includes a bottom plate, a and a sample filter pad, a microsphere marker pad, a detection pad and a water-absorbing pad located on the bottom plate; one end of the detection pad is stacked with one end of the microsphere marker pad, and the other end of the detection pad is overlapped with the One end of the absorbent paper is stacked, and one end of the sample filter pad is stacked with the other end of the microsphere marker pad. The invention can realize the qualitative and quantitative detection of the new coronavirus COVID-19 in various human samples, and can meet the needs of small batches and easy-to-operate grassroots community rapid early screening, as well as high-pass High-volume, high-accuracy clinical diagnosis in central hospitals.

Description

Time-resolved fluorescence immunochromatography kit for detection of novel coronavirus and the same Preparation

technical field

The invention relates to the technical field of biological detection, in particular to a time-resolved fluorescence immunochromatography kit for detecting novel coronavirus and a preparation method thereof.

Background technique

The new coronavirus, belonging to the genus of coronavirus beta, has an envelope, the particles are round or oval, often pleomorphic, with a diameter of 60-140nm. Its genetic characteristics are significantly different from SARSr-CoV and MERSr-Cov, and the homology with bat SARS-like coronavirus (bat-SL-CoVZC45) is more than 85%. On January 12, 2020, the World Health Organization tentatively named it "2019-nCoV" (2019 Novel Coronavirus). On February 11, WHO Director-General Tedros Adhanom Ghebreyesus announced at the Global Research and Innovation Forum press conference that the new coronavirus was officially named "COVID-19", where "Co" stands for "coronavirus" and "Vi". " stands for "virus" and "D" stands for "disease".

Coronaviruses belong to the order Nesteviridae, the family Coronaviridae, and the genus Coronaviridae. They are a class of RNA viruses with an envelope and a linear single-stranded positive-stranded genome. They are a large class of viruses that exist widely in nature. The viral genome has a methylated cap-like structure at the 5' end and a poly(A) tail at the 3' end. The full-length genome is 27-32 kb, and it is the virus with the largest genome among the known RNA viruses. The virus has four major structural proteins: spike protein (spike protein, S protein), nucleocapsid protein (nucleocapsid, N protein), membrane protein (membrane protein, M protein), envelope protein (envelope protein, E protein) ). While coronaviruses can cause colds and more serious diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS), the new coronavirus is a new strain of coronavirus that has never been found in humans before. Common signs of people infected with coronavirus include respiratory symptoms, fever, and cough. In severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, kidney failure, and even death. Severe symptoms include: acute respiratory distress syndrome, septic shock, metabolic acidosis that is difficult to correct, and coagulation dysfunction. It has a serious impact on patients with cardiopulmonary diseases and people with low immunity.

At present, the pneumonia epidemic caused by the new coronavirus is serious, and the confirmed and suspected cases are developing explosively. There is an urgent need for effective diagnosis and treatment and auxiliary diagnosis and treatment methods to improve the symptoms of patients, enhance the immunity of susceptible people, and reduce the harm of the virus. The methods used to detect the new coronavirus COVID-19 mainly include RT-PCR nucleic acid detection and other methods, but during the application of these methods, the requirements for detection equipment are high, the detection cost is high; there are many interference factors, the repeatability is not good, and the detection time is long .

SUMMARY OF THE INVENTION

To this end, the present invention provides a time-resolved fluorescence immunochromatography kit for detecting novel coronavirus and a preparation method thereof.

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

A time-resolved fluorescence immunochromatography kit for detecting novel coronavirus, the kit includes a test strip and a fluorescence immunoassay analyzer, wherein the test strip includes a bottom plate and a sample located on the bottom plate filter pad, microsphere marking pad, detection pad and absorbent pad; one end of the detection pad is stacked with one end of the microsphere marker pad, and the other end of the detection pad is stacked with one end of the absorbent paper, so One end of the sample filter pad is overlapped with the other end of the microsphere marker pad;

The microsphere marking pad is coated with a COVID-19 monoclonal antibody coating marked by microspheres, a detection line is provided on the side of the detection pad near the microsphere marking pad, and a layer near the water absorbing pad is provided with a detection line. The quality control line is coated with a COVID-19N protein monoclonal antibody coating.

In an embodiment of the present invention, the quality control line is coated with a goat anti-mouse IgG coating.

In one embodiment of the present invention, the width of the microsphere marking pad is 6-8 mm;

The distance between the detection line and the microsphere marking pad is 4-6 mm, and the distance between the detection line and the quality control line is 4-6 mm.

In one embodiment of the present invention, the detection pad is a nitrocellulose membrane.

In one embodiment of the present invention, the test strip further includes an upper casing and a lower casing matched with the upper casing, the upper casing and the lower casing are formed to accommodate the test strip The upper casing is provided with a sample injection hole corresponding to the sample filter pad, and a viewing window is provided corresponding to the detection pad.

In an embodiment of the present invention, the detection kit further includes a standard curve card for calculating the detection concentration of the novel coronavirus.

The present invention also provides a method for preparing the time-resolved fluorescence immunochromatography kit for detecting novel coronavirus, comprising:

Label the COVID-19 monoclonal antibody with time-resolved fluorescent microspheres, and coat the labeled fluorescent microspheres on the microsphere labeling pad;

The COVID-19N protein monoclonal antibody and goat anti-mouse IgG were diluted with 10mmol/L PBS buffer containing 1% sucrose to a dilution concentration of 0.5-1.5mg/ml, respectively, and coated on the detection pad to form a detection line and a quality control line. ;

crimping the ends of the sample filter pad, the microsphere marker pad, the detection pad and the water-absorbing pad in sequence to form a test strip;

A fluorescence immunoassay analyzer is used in conjunction with the test strip to form the time-resolved fluorescence immunochromatography kit.

In one embodiment of the present invention, the method for labeling a COVID-19 monoclonal antibody with time-resolved fluorescent microspheres is:

1) Time-resolved fluorescent microsphere pre-labeling

Take 0.1 mg of time-resolved fluorescent microspheres, ultrasonically disperse them for 1-2 min, centrifuge at 18,000 rpm for 15 min, remove the supernatant, wash with MES buffer with pH 6.0 and a concentration of 50-100 mmol/L, and resuspend the precipitate; add 0.1 -0.2mg/ml carbodiimide and succinimide, react at room temperature for 15-30min, centrifuge at 18000rpm for 15min at low temperature, remove the supernatant, use pH 6.0, concentration 50-100 mmol/L MES buffer Wash and resuspend the pellet to 1 ml to obtain pre-labeled time-resolved fluorescent microspheres;

2) Coat the pre-labeled fluorescent microspheres on the microsphere labeling pad

Take 1ml of the pre-labeled time-resolved fluorescent microspheres, add 0.1-0.2mg of COVID-19 monoclonal antibody, and rotate at room temperature for 2-2.5h; then add pH 8.0, 50mmol/mL containing 1-2% BSA L of PBS blocking solution, rotating at room temperature for 2h; 18000rpm, low temperature centrifugation for 15min, removing the supernatant, using 10-50mmol/L containing 0.5-1% BSA, 0.2-0.3% Tween20, 0.1% NaN ₃ , pH 8.0 Wash and resuspend to 1 ml with PBS preservation solution, and store at 4°C in the dark to obtain time-resolved fluorescent microspheres labeled with COVID-19 monoclonal antibodies.

In one embodiment of the present invention, the preparation method of the microsphere marking pad includes:

The time-resolved fluorescent microspheres labeled with the COVID-19 monoclonal antibody were centrifuged at 18,000 rpm for 15 min at low temperature, and the supernatant was removed. 10-50 mmol/L of sucrose, pH 8.0 PBS microsphere resuspended, to obtain a time-resolved fluorescent microsphere resuspended with labeled COVID-19 monoclonal antibody, and the resuspended liquid was passed through a spraying device to After spraying at 3-5 μl/cm, drying at 35-38° C. in the dark to obtain a time-resolved fluorescent microsphere sprayed with COVID-19 monoclonal antibody.

In an embodiment of the present invention, the test strip is installed in the accommodating space formed by the upper casing and the lower casing.

In the present invention, nitrocellulose, also known as nitrocellulose and cellulose nitrate, is the product of the esterification reaction of cellulose and nitric acid. Nitrocellulose made from cotton fiber is called nitrocellulose. Nitrocellulose is a white fibrous polymer that is resistant to water, dilute acids, weak bases and various oils. It is especially suitable as the base film material of the detection pad of the present invention, and the theoretical chromatography speed of the obtained detection pad can reach about 15min.

In the present invention, the detection principle of the kit for detecting novel coronavirus COVID-19 by time-resolved fluorescence immunochromatography is the double-antibody sandwich method. Among them, the microsphere labeling pads are labeled with time-resolved fluorescent microspheres with novel coronavirus COVID-19 monoclonal antibody; the detection line is coated with mouse anti-human novel coronavirus COVID-19 N protein monoclonal antibody; the quality control line is coated with Goat anti-mouse IgG antibody. During the test, drop the sample diluent to be tested on the sample filter pad, and through chromatography, the sample to be tested moves to the end of the water-absorbing pad, and when it flows through the microsphere labeling pad, the time-resolved fluorescent microsphere-labeled novel coronavirus COVID-19 19 The monoclonal antibody is redissolved. If the sample to be tested contains the antigen to be tested, a microsphere antibody-antigen complex is formed. When it is moved to the detection line, a microsphere antibody-antigen-antibody complex is formed, and the microsphere antibody is immobilized. Excess bead-labeled antibody was moved to the control line and captured by goat anti-mouse antibody. After the light-proof reaction, the quantitative detection kit was detected with a dry-type fluorescence immunoassay analyzer to obtain the intensity and ratio of the fluorescence intensities of the detection line and the quality control line. The concentration of the analyte in the sample can be analyzed from the set standard curve. On the other hand, after the end of the light-proof reaction, the qualitative detection kit is detected with a portable UV detection pen. Through the exposed part of the test paper strip in the detection window, observe the fluorescence display of the detection line and the control line under the UV detection pen, and qualitatively determine the detection. Results If the test line and the quality control line show fluorescence, the sample is judged to be positive. If the test line has no fluorescence display and the quality control line shows fluorescence, the sample is judged to be negative. If the quality control line has no fluorescence display, the test strip is invalid and needs to be retest.

In the present invention, time-resolved fluorescence immunoassay (time-resolved fluorescence immunoassay, TRFIA) uses lanthanide elements (trivalent rare earth ions and their chelates) as tracers to label antigens or antibodies, and when an immune reaction occurs, use The time-resolved fluorescence analyzer measures the fluorescence intensity of the final product of the immune reaction, and then judges the concentration of the analyte in the reaction system according to the ratio of the fluorescence intensity to the relative fluorescence intensity, so as to achieve the purpose of quantitative analysis. The sensitivity of TRFIA is as high as 10 ^-18 mol/L; the specificity is strong, and the specific fluorescence and non-specific fluorescence are distinguished by time delay, so that the theoretical background reaches 0; the fluorescence lifetime is extremely long, and the fluorescence decay time is extremely long. The luminous efficiency is high and very stable; the emission spectral band is narrow, the excitation spectral band is wide, and the Strokes shift is large, so there is less interference, high sensitivity, and high result accuracy.

The present invention has the following advantages:

The invention adopts a portable ultraviolet detection pen and a dry fluorometer respectively, which can realize the qualitative and quantitative detection of the new coronavirus COVID-19 in various human samples (respiratory tract, blood, cornea, stool and other samples), aiming at different applications. Scenarios and needs enable it to meet the needs of small batches and easy-to-operate rapid early screening in grassroots communities, as well as high-throughput and high-accuracy clinical diagnosis in central hospitals.

The invention utilizes the time-resolved fluorescence chromatography technology, which can avoid the fluorescence interference of the sample itself, and has the characteristics of strong specificity, high sensitivity, high accuracy, small amount of detection sample, convenient detection operation, accurate and reliable detection results and the like.

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required to be used in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only exemplary, and for those of ordinary skill in the art, other implementation drawings can also be obtained according to the extension of the drawings provided without creative efforts.

1 is a schematic structural diagram of a test strip in a time-resolved fluorescence immunochromatography kit for detecting novel coronavirus of the present invention, wherein: 1-bottom plate; 2-sample filter pad; 3-microsphere labeling pad; 4- -Absorptive pad; 5-Test pad; 6-Test line; 7-Quality control line;

Fig. 2 is the front structure schematic diagram of the test strip in the time-resolved fluorescence immunochromatography kit for detecting novel coronavirus of the present invention;

Fig. 3 is the standard curve diagram stored in the standard curve card for quantitative detection of novel coronavirus COVID-19 of the present invention;

FIG. 4 is a schematic diagram of the detection result of the qualitative detection of the novel coronavirus COVID-19 by the test strip of the present invention.

Detailed ways

The embodiments of the present invention are described below by specific specific embodiments. Those who are familiar with the technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the present invention. , not all examples. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

In the present invention, the antigens and antibodies used were purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., wherein the new coronavirus antigen product number: 40588-V08B, and the COVID-19N protein antibody product number for coating was 40143-R019; The COVID-19 antibody catalog number is 40143-T62. Goat anti-mouse IgG product number is SSA021.

In the present invention, low temperature refers to a temperature between -4°C and 4°C.

In the present invention, the time-resolved fluorescent microspheres used were purchased from Bangs Lab Company in the United States.

Example 1. Preparation of time-resolved fluorescence immunochromatography kit for detection of novel coronavirus

A time-resolved fluorescence immunochromatography kit for the detection of coronavirus COVID-19. The test strip adopts the principle of double-antibody sandwich immunochromatography to detect the novel coronavirus in human samples (respiratory tract, blood, cornea, stool, etc.). The content of the virus COVID-19. The kit consists of test strips for the detection of novel coronavirus COVID-19 by time-resolved fluorescence immunochromatography, sample buffer and ID card containing the standard curve of novel coronavirus COVID-19.

Specifically, the test strip of the present invention is shown in Figures 1 and 2, and the PVC bottom plate 1 is sequentially overlapped with a sample filter pad 2, a microsphere marker pad 3, a detection pad 5 and a water absorption pad 4; the detection pad is nitrocellulose The membrane is used as the base membrane, and the microsphere labeling pad 3 is coated with microsphere-labeled COVID-19 monoclonal antibody; the detection pad 3 has the detection line (T) 6 and the quality control line (C) 7; the sample filter pad 2 is used as the The sample addition area is used to aspirate the sample to be tested. In this example, the mouse anti-human novel coronavirus COVID-19 monoclonal antibody coupled with time-resolved immunofluorescence microspheres is used on the microsphere labeling pad, and the COVID-19 monoclonal antibody is used as the labeling antibody. The concentration of the microspheres was 1 mg/ml, the average particle size of the flat microspheres was 200 nm, the excitation wavelength was 360 nm, and the emission wavelength was 615 nm; the coating concentration was antibody: fluorescent microspheres=1:10 (W:W). The microsphere marking pad was sprayed on the microsphere marking pad with a spray volume of 3 μl/cm using a gold-spraying stripper, and the microsphere marking pad was made of glass fiber membrane. The coating concentration of mouse anti-human novel coronavirus COVID-19 N protein monoclonal antibody in the detection line (T) is 1.5 mg/ml; the coating concentration of goat anti-mouse IgG antibody in the quality control line is 1 mg/ml. The test line and the quality control line were sprayed on the test pad with a spray volume of 1 μl/cm using a gold spray scriber.

The preparation method of the time-resolved fluorescence immunochromatography test strip for detecting the type coronavirus COVID-19 in the present invention is as follows:

(1) Pre-labeled time-resolved fluorescent microspheres

Take 0.1 mg of time-resolved fluorescent microspheres, ultrasonically disperse for 1 min, centrifuge at 18,000 rpm for 15 min, remove the supernatant, take 1 ml of 50 mmol/L (pH 6.0) MES buffer, wash and resuspend the precipitate; add carbodiimide and Succinimide, so that the final concentrations of both are 0.1 mg/ml. After rotating at room temperature for 30 min, centrifuge at 18,000 rpm for 15 min, remove the supernatant, wash with 50 mmol/L (pH 6.0) MES buffer, and resuspend the precipitate. to 1ml to obtain pre-labeled time-resolved fluorescent microspheres;

(2) Preparation of novel coronavirus COVID-19 monoclonal antibody labeled with time-resolved fluorescent microspheres

Take 1 ml of the above pre-labeled time-resolved fluorescent microsphere solution, add 0.1 mg of novel coronavirus COVID-19 monoclonal antibody, and rotate at room temperature for 2 h; then add 50 mmol/L (pH 8.0) PBS containing BSA blocking solution to BSA The mass concentration was 2%, and the reaction was rotated at room temperature for 2 hours; 18000rpm low temperature centrifugation for 5min, the supernatant was removed, and washed with 50mmol/ _L (pH 8.0) PBS preservation solution containing mass fraction 0.1%BSA, 0.2%Tween20, 0.1%NaN3 , and resuspended to 1ml, and stored at 4°C in the dark to obtain time-resolved fluorescent microsphere-labeled novel coronavirus COVID-19 monoclonal antibody;

(3) Preparation of Microsphere Labeling Pad

Take 0.1 ml of the above time-resolved fluorescent microsphere-labeled novel coronavirus COVID-19 monoclonal antibody solution, centrifuge at 18,000 rpm for 15 min, remove the supernatant, and use 0.1 ml containing 0.5% BSA, 0.2% Tween20, and 10% sucrose. 50 mmol/L (pH 8.0) of PBS microspheres resuspended, and sprayed the resuspended liquid with a spray volume of 3 μl/cm on the microsphere labeling pad with a new type of time-resolved fluorescent microsphere labeling Coronavirus COVID-19 monoclonal antibody coating, the sprayed microsphere labeling pad was placed in an oven, and dried at 35-38 °C in the dark to obtain time-resolved fluorescent microsphere-labeled novel coronavirus COVID-19 monoclonal antibody microspheres. Ball marking pad, microball marking pad is formed into a 4-6mm strip, and desiccant is added for storage.

(4) Preparation of detection pad

Mouse anti-human novel coronavirus COVID-19 N protein monoclonal antibody and goat anti-mouse IgG were diluted to 1.5mg/ml and 1.0mg/ml with 50mmol/L PBS (pH 8.0) buffer containing 1% sucrose, respectively ; Spray the quality control line coating and the detection line coating on the nitrocellulose membrane in parallel at an interval of 4 mm with a gold spray scribing instrument at an amount of 1 μl/cm to obtain a detection pad, put the detection pad into an oven, and avoid it at 35-38 °C. Light drying, add desiccant to seal for later use;

(4) Assembly of test strips

The sample filter pads are staggered 1.9-2.2mm on the PVC bottom plate in turn. The material of the sample filter pad is glass fiber, the material of the microsphere marking pad is glass fiber, and the material of the detection pad is nitrocellulose and water absorption. Pad, wherein the detection line is close to the side of the microsphere marking pad, and the quality control line is close to the side of the water-absorbing pad to obtain a test board, and further cut the test board into test strips with a width of 4 mm.

In this embodiment, the test strip for detecting the new coronavirus COVID-19 is assembled in a plastic case formed by a plastic upper case and a plastic lower case, and the upper case is provided with a sample hole and a viewing window, and the addition The sample hole corresponds to the position of the sample filter pad 2 of the new coronavirus COVID-19 test strip, and the window corresponds to the position of the test line 6 and the quality control line 7 of the new coronavirus COVID-19 test pad.

The sample buffer also included in the detection kit of this embodiment is 200 mmol/L NaCl, 0.5% BSA and 1% Tween20 dissolved in PBS buffer at pH 8.0, and 100 μl/tube are distributed into centrifuge tubes. This sample buffer is used for chromatographic samples.

Example 2. Time-resolved fluorescence immunochromatography kit for quantitative detection of novel coronavirus

Different concentrations of human novel coronavirus COVID-19 antigen calibrators (70ng/ml, 35ng/ml, 14ng/ml, 7ng/ml, 3.5ng/ml, 0.7ng/ml) were added to the test strips prepared according to Example 1. ml, 0.0ng/ml), a total of seven concentrations, each concentration set three replicates, all of which are prepared by diluting 0.5mg/ml human new coronavirus COVID-19N protein recombinant antigen with sample buffer, after 15min of chromatography, through The dry fluorescence immunoassay analyzer reads the fluorescence intensity ratio of the detection line and the quality control line, wherein the fluorescence intensity ratio=detected value of the detection line/detected value of the quality control line.

Taking the concentration of the new coronavirus COVID-19 calibrator as the X-axis, and the fluorescence intensity of the sample detection line/quality control line as the Y-axis, an equation was established and fitted to a standard curve. The standard curve is shown in Figure 3. The R value is 0.9974. As shown in Table 1, the concentration of the new coronavirus COVID-19N protein recombinant antigen calibrator was detected.

Table 1

The process specifically includes the following steps:

(1) Put the sample to be detected and the detection reagent to room temperature, and wait for the temperature to equilibrate;

(2) Take 100 μl of the sample to be tested and add it to 100 μl of sample buffer. As mentioned above, the sample buffer has been divided into 100 μl/tube and mixed well to obtain a mixed sample;

(3) Aspirate 100 μl of the above mixed sample, add it to the sample hole of the test strip, and react at room temperature for 15 minutes in the dark;

(4) Turn on the dry-type immunofluorescence analyzer, and insert the ID card corresponding to the detection of the new coronavirus COVID-19 after the initialization self-test is completed;

(5) Insert the test strip into the test strip socket of the instrument, run the instrument, and automatically calculate the concentration of the new coronavirus COVID-19 in the sample to be tested through the analysis software.

The detection kits in this example are all matched with an ID card containing the standard curve of the new coronavirus COVID-19. The standard curves of the same batch of products are the same, and different concentrations are determined by the new coronavirus COVID-19 test strips. The calibrator, take the concentration of the calibrator as the X-axis, the ratio of the fluorescence intensity of the detection line and the quality control line as the Y-axis, draw a standard curve, write and generate the corresponding barcode information, store it in the ID card, and print out the matching barcode. Paste on the test strip casing. When detecting the concentration, the ID card is inserted into the ID card socket of the instrument, and the dry-type fluorescence immunoassay analyzer reads the corresponding barcode information on the shell of the test strip to obtain the corresponding standard curve.

Example 3. Time-resolved fluorescence immunochromatography kit for qualitative detection of novel coronavirus

Test strips were prepared according to Example 1 of the present invention, and four different concentrations of human novel coronavirus COVID-19 antigen calibrators were set: 70ng/ml, 7ng/ml, 0.7ng/ml, and 0.0ng/ml.

Follow the steps below for qualitative detection:

(1) Put the sample to be detected and the detection reagent to room temperature, and wait for the temperature to equilibrate;

(2) Take 100 μl of the sample to be tested and add it to 100 μl of sample buffer, and mix well to obtain a mixed sample;

(3) Draw 100 μl of the above mixed sample, add it to the sample hole of the test strip, and react at room temperature for 10-15 minutes in the dark;

(4) Open the portable UV detection pen, irradiate the detection test strip through the exposed part of the detection window, observe the fluorescence display of the detection and control lines under the UV detection pen, and qualitatively determine the detection results.

The results are shown in Figure 4. For samples of 0ng/ml, the T line of the detection line has no fluorescence display, and the C line of the quality control line has fluorescence display; Line C lines all showed fluorescence, and with the increase of concentration, the fluorescence brightness of detection line T line increased in turn.

The fluorescence immunoassay analyzer of the present invention can be a portable ultraviolet detection pen or a dry-type fluorescence immunoassay analyzer.

Experimental example

Collect 10 positive samples for the detection of the new coronavirus COVID-19 in the hospital, and use the qualitative kit of the present invention to detect the new coronavirus COVID-19, and the results are all positive;

20 negative samples were collected and tested with the qualitative kit of the present invention for the detection of novel coronavirus COVID-19, all of which were negative. The clinical small sample size test showed that the qualitative coincidence rate was 100%.

Although the present invention has been described in detail above with general description and specific embodiments, some modifications or improvements can be made on the basis of the present invention, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

Claims (10)

1. A time-resolved fluoroimmunoassay kit for detecting a novel coronavirus is characterized by comprising a test strip and a fluoroimmunoassay analyzer, wherein the test strip comprises a bottom plate, and a sample filter pad, a microsphere marking pad, a detection pad and a water absorption pad which are positioned on the bottom plate; one end of the detection pad is laminated with one end of the microsphere marking pad, the other end of the detection pad is laminated with one end of the absorbent paper, and one end of the sample filter pad is laminated with the other end of the microsphere marking pad;

the COVID-19 monoclonal antibody coating of the microsphere marker is coated on the microsphere marker pad, a detection line is arranged on one side, close to the microsphere marker pad, of the detection pad, a quality control line is arranged on one layer, close to the water absorption pad, of the detection pad, and the COVID-19N protein monoclonal antibody coating is coated on the detection line.

2. The time-resolved fluoroimmunoassay kit for detecting a novel coronavirus according to claim 1,

the quality control line is coated with a goat anti-mouse IgG coating.

3. The time-resolved fluoroimmunoassay kit for detecting a novel coronavirus according to claim 1,

the width of the microsphere mark pad is 6-8 mm;

the distance between the detection line and the microsphere marking pad is 4-6mm, and the distance between the detection line and the quality control line is 4-6 mm.

4. The time-resolved fluoroimmunoassay kit for detecting a novel coronavirus according to claim 1,

the detection pad is a nitrocellulose membrane.

5. The time-resolved fluoroimmunoassay kit for detecting a novel coronavirus according to claim 1,

the test paper strip still include the casing and with go up casing complex lower casing, go up the casing with lower casing form can hold the space of test paper strip, go up to correspond on the casing the sample filter pad is equipped with the application of sample hole, corresponds detection pad department is equipped with the window.

6. The time-resolved fluoroimmunoassay kit for detecting a novel coronavirus according to claim 1,

the detection kit also comprises a standard curve card used for calculating the detection concentration of the novel coronavirus.

7. A method for preparing the time-resolved fluoroimmunoassay kit for detecting a novel coronavirus according to claim 1, comprising:

marking the COVID-19 monoclonal antibody by using a time-resolved fluorescent microsphere, and coating the marked fluorescent microsphere on a microsphere marking pad;

the COVID-19N protein monoclonal antibody and goat anti-mouse IgG are respectively diluted to 0.5-1.5mg/ml by 10 mmol/L PBS buffer solution containing 1% sucrose, and are respectively coated on a detection pad to form a detection line and a quality control line;

sequentially crimping the end parts of the sample filter pad, the microsphere marking pad, the detection pad and the water absorption pad to form a test strip;

and the fluorescence immunoassay instrument is matched with the test strip for use to form the time-resolved fluorescence immunochromatography kit.

8. The method of claim 7,

the method for labeling the COVID-19 monoclonal antibody by using the time-resolved fluorescent microspheres comprises the following steps:

1) time-resolved fluorescent microsphere pre-labeling

Taking 0.1mg of time-resolved fluorescent microspheres, carrying out ultrasonic dispersion for 1-2min, carrying out low-temperature centrifugation at 18000rpm for 15min, removing supernatant, washing with MES buffer solution with pH of 6.0 and concentration of 50-100 mmol/L, re-suspending precipitate, adding 0.1-0.2mg/ml of carbodiimide and succinimide, reacting at room temperature for 15-30min, carrying out low-temperature centrifugation for 15min, removing supernatant, washing with MES buffer solution with pH of 6.0 and concentration of 50-100 mmol/L, and re-suspending precipitate to 1ml to obtain pre-labeled time-resolved fluorescent microspheres;

2) coating the pre-marked fluorescent microspheres on a microsphere marking pad

Adding 0.1-0.2mg of COVID-19 monoclonal antibody into 1ml of the pre-labeled time-resolved fluorescent microspheres, carrying out room-temperature rotation reaction for 2-2.5h, then adding PBS blocking solution which has the pH of 8.0 and contains 50 mmol/L of 1-2% BSA by mass fraction, carrying out room-temperature rotation reaction for 2h, 18000rpm, carrying out low-temperature centrifugation for 15min, removing supernatant, and carrying out low-temperature centrifugation for 15min by using the mixture containing 0.5-1% BSA, 0.2-0.3% Tween20 and 0.1% NaN₃And washing the washed PBS storage solution with the concentration of 10-50 mmol/L and the pH value of 8.0, suspending the washed PBS storage solution to 1ml, and storing the PBS storage solution at 4 ℃ in a dark place to obtain the time-resolved fluorescent microspheres labeled with the COVID-19 monoclonal antibody.

9. The method of claim 8,

the preparation method of the microsphere marking pad comprises the following steps:

and (2) centrifuging the time-resolved fluorescent microspheres labeled with the COVID-19 monoclonal antibody at 18000rpm for 15min at low temperature, removing supernatant, re-suspending with 0.1-0.2ml of PBS microsphere heavy suspension containing 0.5-1% of BSA, 0.2-0.3% of Tween20, 10% of sucrose and 10-50 mmol/L of pH 8.0 to obtain time-resolved fluorescent microsphere heavy suspension labeled with the COVID-19 monoclonal antibody, spraying the heavy suspension by a spraying device at 3-5 mu l/cm, and drying in the dark at 35-38 ℃ to obtain the time-resolved fluorescent microsphere sprayed with the COVID-19 monoclonal antibody.

10. The method of claim 7,

the test paper strip is arranged in the containing space formed by the upper shell and the lower shell.

Images