Rapid qualitative detection test strip based on capture method and competition method

Abstract

The invention discloses a rapid qualitative detection test strip based on a capture method and a competition method. The test strip comprises a long-strip-shaped sample pad, a combination pad, an NC membrane, absorbent paper and a PVC backboard, the conjugate pad is coated with a marked and sealed compound and a quality control material for marking a tracer, and the NC membrane is sequentially provided with a capture line, a detection line and a quality control line; the compound is obtained by carrying out blocking reaction on a competitive substance for marking a tracer and a competitive substance; the capture line coats a capture substance and is used for capturing a competed substance; the detection line is coated with a competitive substance and reacts with the competitive substance marking the tracer; the quality control line is coated with a quality control second antibody; the competed substance is an antigen, and the capturing substance is an antibody. A capture method and a competition method are combined, through combined use of a capture line and a detection line, color development inhibition through the competition method is converted into color development judgment, qualitative detection is achieved, a quality control system is introduced into a detection system, the reaction process is monitored, and whether the detection process is effective or not is judged.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical detection, and particularly relates to a rapid qualitative detection test strip based on a capture method and a competition method. BACKGROUND

[0002] Biological antigen-antibody and intermolecular interaction are reversible processes, and when multiple molecules can bind to the same site of another molecule at the same time, competition exists, and with different proportions of competitive molecules, the proportion of the complex formed is also different. Traditional competition method for detecting whether competitive molecules exist mainly uses a label to mark the competitive molecule substance or the molecule substance that competes with the to-be-detected substance. When there is no to-be-detected substance in the sample, the strongest reactivity can be detected, and when the to-be-detected substance exists in the sample, the to-be-detected substance competes with the labeled substance, so that the reactivity is weakened, and whether the to-be-detected substance exists is determined by judging whether the reactivity is weakened. This technology is widely used in the technical platforms of chemiluminescence, enzyme-linked immunoassay, chromatography and the like, and is used for qualitatively or quantitatively detecting small molecule antigens, haptens and detecting the interaction between specific sites of molecules in a sample.

[0003] However, when the competition method is used, the judgment of color development depth of the immunochromatography method is greatly influenced by subjectivity, and the judgment of whether the color development is weakened is not clear, which limits the application and popularization of the method. SUMMARY

[0004] The present application aims at the deficiencies in the prior art, and provides a rapid qualitative detection test strip based on a capture method and a competition method.

[0005] To achieve the above object, the technical scheme adopted by the present application is as follows:

[0006] The present application provides a rapid qualitative detection test strip based on a capture method and a competition method, which comprises a sample pad, a conjugate pad, an NC membrane, an absorbent paper and a PVC back plate. The NC membrane is adhered to the surface of the middle part of the PVC back plate, one end of the conjugate pad and one end of the absorbent paper are respectively laminated and jointed to adhere to both ends of the NC membrane, and one end of the sample pad is laminated and jointed to adhere to the other end of the conjugate pad. The conjugate pad is coated with a completed complex of a label and a blocking agent, and a quality control substance of a labeled tracer. The NC membrane is sequentially provided with a capture line, a detection line and a quality control line.

[0007] The complex is obtained by blocking reaction of a competitive substance of a labeled tracer and a competitive substance. The capture line is coated with a capture substance for capturing the competitive substance. The detection line is coated with the competitive substance for reacting with the competitive substance of the labeled tracer. The quality control line is coated with a quality control second antibody. The competitive substance is an antigen, and the capture substance is an antibody.

[0008] Further, the competitive substance is one of an antigen, an antibody, a protein, a polysaccharide, and a nucleic acid molecule.

[0009] Further, the tracer is one of colloidal gold, a colored microsphere, and a fluorescent microsphere.

[0010] Further, the material of the binding pad is glass fiber or polyester fiber.

[0011] Further, the NC membrane is a nitrocellulose membrane.

[0012] Further, the rapid qualitative detection test strip determines the positive / negative of the sample by whether the detection line develops color or not.

[0013] Compared with the prior art, the above technical solution has the following technical effects:

[0014] The present application combines the capture method and the competition method, and through the joint use of the capture line and the detection line, it converts the color development inhibition by the competition method into color development or not, realizes qualitative detection, and introduces a quality control system into the detection system to monitor the reaction process, so as to determine whether the detection process is effective. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is a schematic diagram of the rapid qualitative detection test strip of the present application;

[0016] Figure 2 is a legend and a legend explanation provided for the purpose of facilitating the description of the detection principle of the test strip of the present application;

[0017] Figure 3 shows the labeling process in the detection principle of the test strip of the present application;

[0018] Figure 4 is a schematic diagram of the coated reagent on the test strip of the present application;

[0019] Figure 5 is a schematic diagram of the positive detection result of the test strip of the present application;

[0020] Figure 6 is a schematic diagram of the negative detection result of the test strip of the present application;

[0021] Figure 7 shows the principle and result determination of the test strip of Example 1;

[0022] Figure 8 shows the results of the test strip of the present application in Example 1 in detecting 10 negative samples;

[0023] Figure 9Results of the test strip of the application in Example 1 for detecting 10 positive samples are shown;

[0024] Figure 10 Results of the test strip of the application in Example 1 for detecting 10 positive samples are shown;

[0025] Figure 11 Results of the test strip of the application in Example 1 for detecting 10 positive samples are shown;

[0026] The reference signs in the drawings are:

[0027] 1-sample pad; 2-binding pad; 3-NC membrane; 4-absorbent paper; 5-PVC backboard; 6-competitive substance; 7-substance to be detected; 8-tracer; 9-capture substance; 10-QC secondary antibody; 11-competitive substance; 12-QC antibody; 13-labeled competitive substance; 14-labeled competitive substance; 15-competitive reactant; 16-labeled QC substance. DETAILED DESCRIPTION

[0028] The application will be further described below in conjunction with the drawings and specific embodiments, but not as a limitation of the application. It should be noted that the embodiments in the application and the features in the embodiments can be combined with each other without conflict.

[0029] Reference Figures 1-4 The application provides a rapid qualitative detection test strip based on capture method and competition method, which comprises a sample pad 1, a binding pad 2, an NC membrane 3, absorbent paper 4 and a PVC backboard 5 in the form of a strip; the NC membrane 3 is adhered to the surface of the middle part of the PVC backboard 5, one end of the binding pad 2 and one end of the absorbent paper 4 are respectively laminated and adhered to the two ends of the NC membrane 3, and one end of the sample pad 1 is laminated and adhered to the other end of the binding pad 2; wherein the binding pad 2 is coated with a labeled and blocked compound (i.e. labeled competitive substance 14) and labeled tracer QC (i.e. labeled QC substance 16), and the NC membrane 3 is sequentially provided with a capture line T1, a detection line T2 and a QC line C.

[0030] The compound (14) is obtained by blocking reaction of the labeled tracer competitive substance (i.e. labeled competitive substance 13) and the competitive substance 11 (for reference Figure 3 ); the capture line T1 is coated with a capture substance 9 for capturing the competitive substance 11; the detection line T2 is coated with the competitive substance 11 and reacts with the labeled tracer competitive substance (i.e. labeled competitive substance 13); the QC line C is coated with a QC secondary antibody 10; wherein the competitive substance 11 is an antigen, and the capture substance 9 is an antibody.

[0031] In the application, the competitive substance 6 is one of an antigen, an antibody, a protein, a polysaccharide and a nucleic acid molecule.

[0032] In the present application, the tracer 8 is one of colloidal gold, colored microspheres, and fluorescent microspheres.

[0033] In a preferred embodiment, the material of the above-mentioned binding pad 2 is glass fiber or polyester fiber; and the above-mentioned NC membrane 3 is nitrocellulose membrane.

[0034] The rapid qualitative detection test strip of the present application judges the positivity / negativity of the sample by detecting whether the color of the detection line develops or not. The specific principle is as follows:

[0035] Positive detection result (reference Figure 5 ):

[0036] After adding the sample to be tested, when the molecule to be tested exists in the sample to be tested, the molecule to be tested competes with the labeled and blocked complex to be tested, so that the labeled competitive substance is freed, the complex of the molecule to be tested and the competitive substance is captured by the capture line, and the labeled competitive substance is captured by the detection line to gather and develop a color reaction.

[0037] Negative detection result (reference Figure 6 ):

[0038] After adding the sample to be tested, when the molecule to be tested does not exist in the sample to be tested, the labeled competitive substance will not be freed, and no color development reaction will occur.

[0039] Example 1: New coronavirus neutralizing antibody detection kit (colloidal gold method)

[0040] The kit is used for qualitatively detecting whether neutralizing antibodies exist in blood samples of new coronavirus infected persons and new coronavirus vaccine inoculated persons. In this example, ACE2 is a competitive substance, new coronavirus neutralizing antibodies are a substance to be detected, RBD / S protein is a competitive substance, and anti-human IgG is a capture substance. The specific method is as follows: (see Figure 7 )

[0041] 1.1 Colloidal gold labeling

[0042] ACE2 is labeled with colloidal gold (1-10 ug / mL colloidal gold). After labeling is completed, new coronavirus RBD / S protein is combined with the ACE2 colloidal gold complex. According to the size of the molecular weight, an excess of new coronavirus RBD / S protein is added, and after sufficient reaction, the excess unbound RBD / S protein is removed.

[0043] Colloidal gold labeling quality control system: chicken IgY or rabbit IgG

[0044] 1.2 NC membrane coating

[0045] NC membrane scribe T1: 0.1-1 ug / 30 cm anti-human IgG / IgM; T2: 0.1-1 ug / 30 cm new crown RBD / S protein; C line: 0.1-1 ug / 30 cm quality control antibody anti-chicken IgY or anti-rabbit IgG

[0046] 1.3 Detection result judgment

[0047] The reagent detection result is divided into new crown neutralizing antibody positive and new crown neutralizing antibody negative.

[0048] T2 line color development indicates that there is new crown neutralizing antibody in the sample, and the color development depth is proportional to the amount of neutralizing antibody in the sample.

[0049] T2 line does not develop color, indicating that there is no new crown neutralizing antibody in the sample.

[0050] T1 line color development indicates that there is non-new crown neutralizing antibody that can bind to new crown RBD / S protein.

[0051] C line should develop color.

[0052] 1.4 Verification of the kit of the embodiment on clinical samples

[0053] 10 clinical negative samples and 10 clinical positive samples were detected using the kit, and the kit was used to detect new crown IgG antibody detection reagent (purchased from Huada GBI reagent) and new crown neutralizing antibody detection reagent (purchased from Hongwei Tes) for comparison. The detection results are shown in the following table Figures 8-11 .

[0054] Table 1

[0055]

[0056]

[0057] The negative detection rate of the kit is 10 / 10, and the positive detection rate is 10 / 10. The color depth of the positive sample detection is basically consistent with the result of the new crown IgG antibody detection reagent, and the T1 line of 4 samples develops color, indicating that there is antibody that can bind to other sites of RBD antigen in the sample, but the antibody cannot compete with RBD antigen to bind to ACE2.

[0058] Figure 11 In order to compare the color depth change with the reference of the quality control line to determine whether there is new crown neutralizing antibody to be detected, according to the detection result, 5 cases can accurately judge whether there is neutralizing antibody, and the rest cannot be determined whether there is neutralizing antibody, the detection rate is 5 / 10, and the sensitivity is low.

[0059] In summary, the kit can more accurately detect whether neutralizing antibodies that can block the binding site of the RBD of the virus to human ACE2 exist in the new crown sample.

[0060] The above merely describes preferred embodiments of the present application, and is not intended to limit the embodiments and protection scope of the present application. It should be understood by those skilled in the art that any equivalent substitution and obvious change made according to the content of the present application and the drawings should be included in the protection scope of the present application.

Claims

1. A rapid qualitative test strip based on capture and competition methods, characterized in that, The test strip comprises a sample pad, a conjugate pad, an NC membrane, an absorbent paper and a PVC back plate; the NC membrane is adhered to the surface of the middle part of the PVC back plate, one end of the conjugate pad and one end of the absorbent paper are respectively laminated and adhered to two ends of the NC membrane, and one end of the sample pad is laminated and adhered to the other end of the conjugate pad; wherein the conjugate pad is coated with a completed compound of a label and a blocking, and a quality control of a label tracer, and the NC membrane is sequentially provided with a capture line, a detection line and a quality control line; The compound is obtained by blocking reaction of a label tracer competitor and a competed substance; the capture line is coated with a capture substance for capturing the competed substance; the detection line is coated with the competed substance and reacts with the label tracer competitor; and the quality control line is coated with a quality control second antibody; wherein the competed substance is an antigen, and the capture substance is an antibody.

2. The rapid qualitative test strip based on capture and competition methods according to claim 1, characterized in that, The competitor is one of an antigen, an antibody, a protein, a polysaccharide and a nucleic acid molecule.

3. The rapid qualitative test strip based on capture and competition methods as claimed in claim 1, wherein, The tracer is one of colloidal gold, colored microspheres and fluorescent microspheres.

4. The rapid qualitative test strip based on capture and competition methods as claimed in claim 1, wherein, The material of the conjugate pad is glass fiber or polyester fiber.

5. The rapid qualitative test strip based on capture and competition methods as claimed in claim 1, wherein, The NC membrane is a nitrocellulose membrane.

6. The rapid qualitative test strip based on capture and competition methods as claimed in claim 1, wherein, The rapid qualitative detection test strip judges the positivity / negativity of a sample according to the color development of the detection line or not.

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