阅读说明：本技术 一种比率荧光法免疫层析检测试条及其检测方法 (Immunochromatography detection test strip by ratio fluorescence method and detection method thereof ) 是由 段学军 张旭东 樊荣 李玉彬 常天亮 李竹青 陈春晓 何永平 于 2020-11-04 设计创作，主要内容包括：本发明提供了一种比率荧光法免疫层析检测试纸条,试纸条包括依次相连接的样品垫、标记垫、包被膜和吸水垫；所述包被膜上依次设置校准线C线、测试T1线和测试T2线,所述C线上包被独立质控抗体；所述标记垫上含有荧光标记的重组抗原和荧光标记的独立质控抗原；所述T1线上包被有重组抗原；T2线上包被有重组抗原配体。通过测定T1线和T2线上两者荧光检测信号的比率(T1/T2),该T1/T2比率与样本中的抗体浓度成正相关,建立相关校准曲线方程,从而建立定性或者定量检测的荧光免疫层析检测方法。本发明提供的试纸条及方法克服了荧光检测背景信号的干扰,提高荧光免疫层析的灵敏度、精确度及可信度。(The invention provides a test strip for immunochromatography detection by a ratiometric fluorescence method, which comprises a sample pad, a marking pad, a coating film and a water absorption pad which are sequentially connected; a calibration line C, a test T1 line and a test T2 line are sequentially arranged on the coating film, and the C line is coated with an independent quality control antibody; the marking pad contains a fluorescence-marked recombinant antigen and a fluorescence-marked independent quality control antigen; the T1 line is coated with recombinant antigen; the T2 line is coated with a recombinant antigen ligand. By measuring the ratio of fluorescence detection signals of the T1 line and the T2 line (T1/T2), the T1/T2 ratio is in positive correlation with the antibody concentration in a sample, and a correlation calibration curve equation is established, so that the fluorescence immunochromatography detection method for qualitative or quantitative detection is established. The test strip and the method provided by the invention overcome the interference of fluorescence detection background signals and improve the sensitivity, accuracy and reliability of fluorescence immunochromatography.)

1. A ratio fluorescence method immunochromatography detection test strip is characterized in that the test strip comprises a sample pad, a marking pad, a coating film and a water absorption pad which are connected in sequence; a calibration line C, a test T1 line and a test T2 line are sequentially arranged on the coating film, and the C line is coated with an independent quality control antibody; the marking pad contains a fluorescence-marked recombinant antigen and a fluorescence-marked independent quality control antigen; the T1 line is coated with recombinant antigen; the T2 line is coated with a recombinant antigen ligand.

2. The test strip of claim 1, wherein the ratio of the length of the sample pad, the length of the label pad, the length of the coating film, and the length of the absorbent pad are preferably (13-17) to (10-12) to (20-24).

3. The test strip of claim 1, wherein the quality control antibody coated on the C-line specifically binds to the fluorescently labeled independent quality control antigen on the label pad, but does not bind to the fluorescently labeled recombinant antigen.

4. The test strip of claim 3, wherein the quality control antibody is obtained by spraying and drying a quality control antibody solution at a spraying speed of 0.2-4 μ L/cm by a gold spraying and spotting machine.

5. The test strip of claim 1, wherein the T1 line is coated with a recombinant antigen, both the recombinant antigen and the fluorescently labeled recombinant antigen can bind to the antibody in the sample to form a double-antigen sandwich fluorescent immune complex, and the fluorescence detection signal on the T1 line is proportional to the concentration of the antibody in the sample.

6. The test strip of claim 5, wherein the recombinant antigen is obtained by spraying and drying a recombinant antigen solution at a spraying speed of 0.2-4 μ L/cm through a gold spraying and spotting machine.

7. The immunochromatographic test strip of any one of claims 1 to 6, wherein the recombinant antigen ligand coated on the T2 line can bind to a fluorescently labeled recombinant antigen; if the fluorescently labeled recombinant antigen is bound by the antibody in the sample, the recombinant antigen ligand on the T2 line will not bind to this portion of the fluorescently labeled recombinant antigen, and the fluorescence detection signal on the T2 line is inversely proportional to the concentration of the antibody in the sample.

8. The test strip of claim 7, wherein the C-line coated independent quality control antibody is goat anti-chicken IgY antibody; the recombinant antigen containing the fluorescent marker on the marking pad is a rare earth element europium-embedded fluorescent microsphere marked recombinant new coronavirus S1-RBD antigen; the independent quality control antigen containing fluorescent markers on the marker pad is chicken IgY marked by fluorescent microspheres embedded with rare earth element europium; the recombinant antigen coated on the T1 line is a recombinant neocoronavirus S1-RBD antigen; the recombinant antigen ligand coated on the T2 line is angiotensin converting enzyme 2(ACE2) recombinant protein.

9. The immunochromatographic assay test strip according to any one of claims 1 to 8, wherein the analyte is a novel coronavirus S protein antibody.

10. The immunochromatographic assay method based on the strip of any one of claims 1 to 9, wherein the ratio of fluorescence detection signals on the T1 line and the T2 line (T1/T2) is determined, the ratio of T1/T2 is positively correlated with the antibody concentration in the sample, and a calibration curve equation is established, thereby establishing a qualitative or quantitative fluorescence immunochromatographic assay method.

Technical Field

The invention relates to the field of medical inspection, in particular to a ratio fluorescence immunochromatography test strip and a test method thereof.

Background

The fluorescence immunochromatographic rapid detection technology is a technology suitable for field detection and established based on the principle of immunochromatographic, and a typical fluorescence immunochromatographic test strip comprises a sample pad, a fluorescence label pad, a coating film and an absorption pad. The fluorescence immunochromatography technique takes a nitrocellulose membrane as a solid phase carrier, enables an antibody or an antigen in a sample to be detected to be combined with an antigen or an antibody coated on the membrane through the permeation and capillary siphon effects, and presents a reaction result through a marker on a fluorescence labeling pad. The technology has the advantages of simple and convenient operation, rapid detection and strong portability.

For the detection of infectious diseases, the requirements of repeatability, sensitivity and accuracy of detection are extremely high. For example, new coronavirus S protein antibody detection kits (fluoroimmunoassay) require a lower non-specific background for detection to reduce false positives, while also requiring good sensitivity to increase positive detection rates.

However, due to the complicated chromatographic process of the test strip, factors such as hydrophobic adsorption of the nitrocellulose membrane, electrostatic attraction of the coating substance and the like can cause a certain background of a fluorescence detection signal. If the fluorescence detection background signal is too high, a detection result with larger deviation can be caused, so that the content of a substance to be detected can not be accurately and truly reflected, and the detection sensitivity can be reduced. Therefore, how to overcome the interference of fluorescence detection background signals and improve the sensitivity, accuracy and reliability of fluorescence immunochromatography is a problem to be solved.

Disclosure of Invention

The invention aims to provide a ratio fluorescence immunochromatography detection test strip and a detection method thereof, which overcome the interference of fluorescence detection background signals and improve the sensitivity, accuracy and reliability of fluorescence immunochromatography.

In order to overcome the interference of fluorescence detection background signals and reduce false positives caused by sample characteristics and the like, a T1 line proportional to the concentration of an object to be detected is arranged, and a T2 line inversely proportional to the concentration of the object to be detected is introduced. When the signal intensity of the T1 line is increased, the signal intensity of the T2 line is reduced when the clinical sample is measured; when the signal intensity of the T1 line is reduced, the signal intensity of the T2 line is increased; after the signal characteristic requirements are met, the instrument can give effective measurement results, so that the error probability is reduced. On the other hand, through the setting of the signal rise and fall of the concentrations of the T1 and the T2 and the object to be detected, the calculated T1/T2 signal value can reflect the change of the concentration of the object to be detected more sensitively, thereby improving the sensitivity of the detection test strip.

In order to achieve the above object, a first aspect of the present disclosure provides an immunochromatographic strip comprising a sample pad, a label pad, a coating film and a water-absorbing pad connected in sequence; wherein the coating film is sequentially provided with a calibration line C line, a test T1 line and a test T2 line, and the C line is coated with an independent quality control antibody; the marking pad contains a fluorescence-marked recombinant antigen and a fluorescence-marked independent quality control antigen; the T1 line is coated with recombinant antigen; the T2 line is coated with a recombinant antigen ligand.

Preferably, the T1 line is coated with recombinant antigen, the recombinant antigen is obtained by spraying the recombinant antigen on the T1 line through a gold spraying film machine at a spraying speed of 0.2-4 muL/cm and drying, the recombinant antigen and the fluorescently-labeled recombinant antigen can be combined with the antibody in the sample to form a double-antigen sandwich fluorescent immune complex, and the fluorescent detection signal on the T1 line is in direct proportion to the concentration of the antibody in the sample.

Preferably, the T2 line is coated with a recombinant antigen ligand, and the recombinant antigen ligand is obtained by spraying the recombinant antigen ligand on the T1 line through a gold spraying film machine at a spraying speed of 0.2-4 muL/cm and drying the recombinant antigen ligand, and can be combined with a fluorescence labeled recombinant antigen; if the fluorescently-labeled recombinant antigen is bound by the antibody in the sample, the recombinant antigen ligand on the T2 line will not bind to this portion of the fluorescently-labeled recombinant antigen; therefore, the fluorescence detection signal on the T2 line is inversely proportional to the concentration of antibody in the sample.

By adopting the immunochromatographic strip provided by the disclosure, the ratio of fluorescence detection signals on a T1 line and a T2 line (T1/T2) can be obtained by a dry type fluorescence immunochromatographic instrument, the ratio of T1/T2 is in positive correlation with the antibody concentration in a sample, and a correlation calibration curve equation is established, so that the fluorescence immunochromatographic detection method for qualitative or quantitative detection is established. By utilizing linkage of a detection line T1 line and a T2 line, the interference of a fluorescence detection background signal is overcome, and the sensitivity, the accuracy and the reliability of fluorescence immunochromatography are improved.

In a second aspect, the present invention provides an immunochromatographic assay method, wherein the method comprises the steps of:

and (3) detection of the sample: detecting a sample to be detected by using the immunochromatographic test strip, collecting the peak integral area values of fluorescence curves at a T1 line, a T2 line and a C line on the immunochromatographic test strip, and substituting the ratio (T1/T2) of fluorescence detection signals of the T1 line and the T2 line into the calibration curve equation to obtain the detection concentration of an object to be detected in the sample to be detected;

optionally, the analyte is a new coronavirus S protein antibody.

Taking detection of a new coronavirus S protein antibody as an example, the test strip and the detection method provided by the disclosure coat a T1 line with a recombinant new coronavirus S1-RBD antigen, coat a T2 line with angiotensin converting enzyme 2(ACE2) recombinant protein, and coat a C line with a goat anti-chicken IgY antibody; meanwhile, marking the recombinant neocoronavirus S1-RBD antigen (S1-RBD @ EuNP) by using rare earth element europium-embedded fluorescent microspheres, and drying to prepare a marking pad; and assembling to prepare the new coronavirus S protein antibody detection kit.

In the detection process, the S1-RBD @ EuNP marker on the marker pad is combined with an S protein antibody in a sample and is chromatographed to a T1 line, the coated recombinant neocoronavirus S1-RBD antigen is combined to form a double-antigen sandwich recombinant neocoronavirus S1-RBD antigen-S protein antibody-S1-RBD @ EuNP compound, and a fluorescence detection signal on a T1 line is in direct proportion to the concentration of the antibody in the sample.

The S1-RBD @ EuNP marker, not captured by the T1 line, was continued to chromatography to T2, and the portion that did not bind to the S protein antibody in the sample would be captured by T2 coated ACE 2; the S1-RBD @ EuNP complex bound to the S protein antibody blocked the binding to ACE2 due to the RBD binding site being bound by the S protein antibody, so the fluorescence detection signal on the T2 line is inversely proportional to the concentration of antibody in the sample.

The invention is arranged on a dry type fluorescence immunochromatographic instrument: the instrument can give effective measurement results after the signal characteristic requirements of mutual linkage of the T1 line and the T2 line are met. On the other hand, through the setting of the signal rise and fall of the concentrations of the T1 and the T2 and the object to be detected, the calculated T1/T2 signal value can reflect the change of the concentration of the object to be detected more sensitively, thereby improving the sensitivity of the detection test strip.

By adopting the ratio fluorescence method new coronavirus S protein antibody detection test strip provided by the invention, the ratio of fluorescence detection signals on a T1 line and a T2 line (T1/T2) can be obtained by a dry fluorescence immunochromatographic instrument, the ratio of T1/T2 is in positive correlation with the antibody concentration in a sample, and a correlation calibration curve equation is established, so that the qualitative or quantitative detection new coronavirus S protein antibody fluorescence immunochromatographic detection method is established. By using the linkage of the detection line T1 line and the detection line T2 line, the interference of fluorescence detection background signals is overcome, and the sensitivity, the accuracy and the reliability of the new coronavirus S protein antibody are improved.

The invention provides an immunochromatographic test strip, which comprises a sample pad, a marking pad, a coating film and a water absorption pad which are connected in sequence; wherein the coating film is sequentially provided with a calibration line C line, a test T1 line and a test T2 line, and the C line is coated with an independent quality control antibody; the marking pad contains a fluorescence-marked recombinant antigen and a fluorescence-marked independent quality control antigen; the T1 line is coated with recombinant antigen; the T2 line is coated with a recombinant antigen ligand.

Wherein the length ratio of the sample pad, the marking pad, the coating film and the absorbent pad is preferably (13-17) to (10-12) to (20-24).

In the test strip provided by the invention, the line C, the line T1 and the line T2 are arranged in parallel in the length direction of the test strip, and the widths of the lines are equal. The distances between the C line and the T1 line and the T2 line can be the same or different, and preferably, the distances between the C line and the T1 line and between the T1 line and the T2 line can be 3-4mm in order to obtain more accurate quantitative detection data.

The independent quality control antibody coated on the C line is obtained by spraying an independent quality control antibody solution with the concentration of 10-40ng/mL on the C line through a gold spraying film spotting machine at the spraying speed of 0.2-4 muL/cm and drying.

The recombinant antigen coated on the T1 line is obtained by spraying 0.2-2mg/mL recombinant antigen solution on a T1 line through a gold spraying film spotting machine at the spraying speed of 0.2-4 muL/cm and drying.

The recombinant antigen ligand coated on the T2 line is obtained by spraying 0.2-2mg/mL recombinant antigen ligand solution on a T2 line through a gold spraying film spotting machine at the spraying speed of 0.2-4 muL/cm and drying.

In a specific embodiment of the invention, the recombinant antigen coated by the T1 line is recombinant neocoronaviruse S1-RBD antigen, the ligand of the recombinant antigen coated by the T2 line is ACE2, the independent quality control antibody coated by the C line is goat anti-chicken IgY antibody, and the independent quality control antigen is chicken IgY.

In the preparation process of the test strip provided by the invention, rare earth element europium-embedded fluorescent microsphere labeled recombinant neocoronavirus S1-RBD antigen (S1-RBD antigen @ EuNP) and rare earth element europium-embedded fluorescent microsphere labeled chicken IgY (IgY @ EuNP) are dissolved in a spraying buffer solution at the concentration of 2-3mg/mL, and are sprayed on a marking pad by a gold spraying film machine at the spraying speed of 3-5 muL/cm for drying;

wherein the spraying buffer solution is a phosphate buffer solution which contains BSA with the mass volume ratio of 0.3-0.7% and Tween-20 with the mass volume ratio of 0.2-0.3% and has the concentration of 0.01-0.03M, and the pH value of the spraying buffer solution is 7.2-7.6.

In the detection process, the S1-RBD @ EuNP marker on the marker pad in the test strip provided by the invention is combined with an S protein antibody in a sample, chromatography is carried out to a T1 line, the coated recombinant neo-coronavirus S1-RBD antigen is combined to form a double-antigen sandwich recombinant neo-coronavirus S1-RBD antigen-S protein antibody-S1-RBD @ EuNP compound, and a fluorescence detection signal on a T1 line is in direct proportion to the antibody concentration in the sample.

The S1-RBD @ EuNP marker, not captured by the T1 line, was continued to chromatography to T2, and the portion that did not bind to the S protein antibody in the sample would be captured by T2 coated ACE 2; the S1-RBD @ EuNP complex bound to the S protein antibody blocked the binding to ACE2 due to the RBD binding site being bound by the S protein antibody, so the fluorescence detection signal on the T2 line is inversely proportional to the concentration of antibody in the sample.

The fluorescent microspheres are polystyrene-embedded lanthanide microspheres, and preferably rare earth element europium-embedded fluorescent microspheres.

Wherein, the size of the fluorescent microsphere is preferably 50-300nm, preferably 100 nm.

In a second aspect, the present invention provides an immunochromatographic detection method comprising the steps of:

by adopting the ratio fluorescence method detection test strip provided by the invention, the ratio of fluorescence detection signals on a T1 line and a T2 line (T1/T2) can be obtained by a dry type fluorescence immunochromatographic instrument, the ratio of T1/T2 is in positive correlation with the antibody concentration in a sample, and a correlation calibration curve equation is established, so that the fluorescence immunochromatographic detection method for qualitative or quantitative detection is established.

And (3) detection of the sample: detecting a sample to be detected by using the immunochromatographic test strip, collecting the peak integral area values of fluorescence curves at a T1 line, a T2 line and a C line on the immunochromatographic test strip, and substituting the ratio (T1/T2) of fluorescence detection signals of the T1 line and the T2 line into the calibration curve equation to obtain the detection concentration of an object to be detected in the sample to be detected;

in the method of the present invention, the integrated area values of the fluorescence curve peak at the C line, the T1 line and the T2 line can be collected by using an apparatus for collecting a detection signal of a fluorescence immunochromatographic strip, which is conventional in the art. For example, a dry fluorescent immunoassay can be used.

In the invention, the collection, processing, calculation and storage of data can be completed by using computer software programmed according to the technical scheme disclosed by the invention, so that the automatic output of the detection result is realized, and the detection labor amount is saved.

In a preferred embodiment of the present invention, the test substance is a novel coronavirus S protein antibody.

The detection principle of the new coronavirus S protein antibody immunochromatographic test strip may include: dropping a sample to be detected into a sample pad, if a sample to be detected has a new coronavirus S protein antibody to be detected, combining the new coronavirus S protein antibody to be detected in the sample to be detected with the marked S1-RBD @ EuNP to form a complex, wherein the complex formed by the antibody to be detected and the marked S1-RBD @ EuNP swims forwards along a coating film due to capillary action, and meets a recombinant new coronavirus S1-RBD antigen coated on a nitrocellulose-based film when reaching a detection line (T1 line), and the recombinant new coronavirus S1-RBD antigen and the S1-RBD @ EuNP can be respectively combined with different antibody epitopes of the antibody to be detected, so that a 'recombinant new coronavirus S1-RBD antigen-antibody to be detected-S1-RBD @ NP' complex is formed, and a specific layer separation line is formed at the T1 line; the marked S1-RBD @ EuNP which is not combined with the antibody to be detected can directly pass through a T1 line, and is captured by the ACE2 on the T2 line, so that a chromatography line is formed on the T2 line, and a positive result is judged; if the sample does not contain the antibody to be detected, a 'recombinant new coronavirus S1-RBD antigen-antibody to be detected-S1-RBD @ EuNP' complex cannot be formed on a T1 line, so that no chromatographic line is generated on the T1 line, and the labeled S1-RBD @ EuNP which is not combined with the antibody to be detected directly passes through a T1 line and is captured by ACE2 after reaching a T2 line, so that the antibody to be detected is enriched on the T2 line, namely, the result is judged to be negative.

Drawings

FIG. 1 is a schematic diagram of the detection principle of a test strip for detecting the new coronavirus S protein antibody by a ratiometric fluorescence method.

FIG. 2 is a calibration curve for the detection of antibodies to the S protein of the novel coronavirus by ratiometric fluorescence.

Detailed Description

The present disclosure is further illustrated by the following examples:

in the following examples, unless otherwise specified, the borate buffer was 0.03-0.07M at pH 8.0-8.5, and the phosphate buffer was 0.01-0.03M at pH 7.2-7.6.

The recombinant neocoronavirus S1-RBD antigen, angiotensin converting enzyme 2(ACE2) recombinant protein and an anti-neocoronavirus RBD single-domain antibody are commercially available; the FIC-H dry-type fluorescence immunoassay instrument is produced by domestic manufacturers.

EXAMPLE 1 preparation of immunochromatographic strip

Preparing a recombinant new coronavirus S1-RBD antigen solution: carrying out ultrafiltration on the recombinant new coronavirus S1-RBD antigen by using borate buffer solution to obtain a recombinant new coronavirus S1-RBD antigen solution; the concentration of the recombinant new coronavirus S1-RBD antigen solution is adjusted to 1.0 mg/mL.

Preparing angiotensin converting enzyme 2(ACE2) recombinant protein solution: ultrafiltering the recombinant angiotensin converting enzyme 2(ACE2) recombinant protein with borate buffer solution to obtain angiotensin converting enzyme 2(ACE2) recombinant protein solution; the concentration of the recombinant protein solution of angiotensin-converting enzyme 2(ACE2) was adjusted to 2.0 mg/mL.

Respectively spraying the recombinant new coronavirus S1-RBD antigen solution and angiotensin converting enzyme 2(ACE2) recombinant protein solution onto a nitrocellulose membrane by using a gold spraying and film spotting machine to form a detection T1 line and a detection T2 line which are parallel to each other; drying; dissolving rare earth element europium-embedded fluorescent microsphere labeled recombinant neocoronavirus S1-RBD antigen (S1-RBD antigen @ EuNP) in a spraying buffer solution at the concentration of 2-3mg/mL, and spraying the solution on a labeling pad by a gold spraying film spotting machine at the spraying speed of 3-5 mu L/cm for drying; the spraying buffer solution is a phosphate buffer solution with the concentration of 0.01-0.03M and containing BSA with the mass-volume ratio of 0.3-0.7% and Tween-20 with the mass-volume ratio of 0.2-0.3%, and the pH value of the spraying buffer solution is 7.2-7.6.

Then the sample pad, the marking pad, the coating film (nitrocellulose membrane) sprayed with T1 line and T2 line and the absorbent paper are connected and assembled in sequence, and the length ratio of the sample pad, the marking pad, the coating film and the absorbent pad is 15: 10: 20: 24. And cutting the package for later use to obtain the immunochromatographic test strip.

Example 2 detection of samples to be tested

(1) Preparation of sample dilutions

Tween 20 at a final concentration of 1% (v/v), sodium chloride at a final concentration of 0.9%% and proclin300 at a final concentration of 0.03%, Biosaccharade guide-1 at a final concentration of 0.2% (v/v) were added to a boric acid buffer solution at a pH of 8.0 to 8.5.

(2) Sample preparation

The anti-neocoronaviruse RBD single domain antibody is subjected to gradient dilution by using normal human serum subjected to 2018 physical examination, and a series of anti-neocoronaviruse RBD single domain antibody calibration solutions with the concentrations of 0mg/mL, 0.039mg/mL, 0.078mg/mL, 0.156mg/mL, 0.313mg/mL, 0.625mg/mL and 1.25mg/mL are respectively obtained.

(3) Plotting of calibration curves

Taking the test strip, balancing for 10 minutes at room temperature, opening the package and taking out the test strip; and (3) uniformly mixing 50 mu L of anti-new coronavirus RBD single-domain antibody solution calibration solution and 50 mu L of sample diluent, adding 90 mu L of mixed solution onto a test strip sample pad, standing for 15 minutes, inserting the test strip into a dry-type fluorescence immunoassay instrument according to the arrow direction, and detecting the calibration solution with each concentration. Collecting the peak integral area values of the fluorescence curves at the T1 line, the T2 line and the C line on the immunochromatographic test paper, taking the ratio of the fluorescence detection signals of the T1 line and the T2 line (T1/T2) and the concentration value of the anti-neocoronavirus RBD single-domain antibody of each calibration solution as a calibration curve, and obtaining a calibration curve equation, wherein the curve equation is as follows: y 3.190x +0.040 as shown in fig. 2.

(4) Comparative example test

The immunochromatographic test strip prepared by the method of example 1 is different in that the test strip only has a T line and a C line, the spraying speed of a gold spraying film spraying machine is 1 muL/cm, the T line is obtained by coating and drying recombinant new coronavirus S1-RBD antigen solution with the concentration of 1mg/mL, and the C line is obtained by coating and drying goat anti-chicken IgY antibody solution with the concentration of 1mg/mL, so that the conventional comparative fluorescence immunochromatographic test strip is obtained.

The comparison test strip of comparative example 1 is used for detecting the anti-new coronavirus RBD single domain antibody calibration solution and a prepared sample, a dry-type fluorescence immunoassay instrument is used for collecting the detection results of the peak integral area values of the fluorescence curves of a T line and a C line, the ratio (T/C) of the detection signal of the T line to the detection signal of the C line is taken as an ordinate y, the concentration value of the calibrator solution is taken as an abscissa x, and a calibration curve is obtained, wherein the curve equation is as follows: y is 1.309x + 0.030.

(5) Test example detection

The immunochromatographic test strip of the example 1 is used for detecting a sample to be detected (anti-new coronavirus RBD single domain antibody solutions with the preparation concentrations of 0.020mg/mL and 0.04 mg/mL) respectively, a FIC-H type dry fluorescence immunoassay instrument is used for obtaining the ratio of fluorescence detection signals of the two on a T1 line and a T2 line (T1/T2), and the detection concentration of the object to be detected in the sample to be detected is calculated through the calibration curve equation; the results are shown in Table 1.

The immunochromatographic test strip of comparative example 1 was used to test a sample to be tested (prepared anti-neocoronavirus RBD single domain antibody solutions with concentrations of 0.020mg/mL and 0.04 mg/mL), and according to a conventional method, a dry-type fluorescence immunoassay instrument was used to collect the peak integrated area value detection results of the fluorescence curves of the T line and the C line, and the ratio (T/C) of the detection signal of the T line to the detection signal of the C line was substituted into the calibration curve to calculate the anti-neocoronavirus RBD single domain antibody concentration of the sample, and the detection results are shown in Table 1.

TABLE 1 test results of test examples and comparative examples

As can be seen from the results in table 1, in example 1, when the ratio fluorescence immunochromatography test strip and the detection method thereof provided by the present disclosure are used for detecting a sample to be detected, the detection signals T1/T2 of the 0.020mg/mL anti-neocoronavirus RBD single domain antibody solution and the 0mg/mL anti-neocoronavirus RBD single domain antibody solution are clearly distinguished; comparative example the case of the conventional fluorescence immunochromatographic strip is as follows: the detection signal T1/T2 of the 0.04mg/mL anti-neocoronavirus RBD single domain antibody solution is obviously distinguished from the detection signal T1/T2 of the 0mg/mL anti-neocoronavirus RBD single domain antibody solution. The result shows that compared with the traditional common fluorescence immunochromatographic test strip and the detection method, the ratio fluorescence immunochromatographic test strip and the detection method thereof can obtain more sensitive detection data.

The preferred embodiments of the present disclosure are described in detail above with reference to the accompanying drawings, and the technical concept and scheme of the present disclosure are illustrated by a specific fluorescence method novel coronavirus S protein antibody test strip. However, the present disclosure is not limited to the specific details of the above-described embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical spirit of the present disclosure, and these simple modifications all fall within the scope of protection of the present disclosure.

It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again. In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.