Test strip and kit for detecting lung cancer autoantibody and application

Abstract

The embodiment of the invention belongs to the technical field of detection, and relates to a test strip and a kit for detecting lung cancer autoantibodies and application, the test strip comprises a first colloidal gold test paper, a second colloidal gold test paper, a third colloidal gold test paper, a fourth colloidal gold test paper, a fifth colloidal gold test paper, a sixth colloidal gold test paper and a seventh colloidal gold test paper, a p53 protein gold marker and a p53 recombinant antigen are arranged on the first colloidal gold test paper; a PGP9.5 protein gold marker and a PGP9.5 recombinant antigen are arranged on the second colloidal gold test paper; an SOX2 protein gold marker and an SOX2 recombinant antigen are arranged on the third colloidal gold test paper; a GAGE7 protein gold marker and a GAGE7 recombinant antigen are arranged on the fourth colloidal gold test paper; a GBU4-5 protein gold marker and a GBU4-5 recombinant antigen are arranged on the fifth colloidal gold test paper; an MAGEA1 protein gold marker and an MAGEA1 recombinant antigen are arranged on the sixth colloidal gold test paper; and a CAGE protein gold marker and a CAGE recombinant antigen are arranged on the seventh colloidal gold test paper. According to the invention, instant detection is realized while the detection sensitivity is ensured.

Description

Technical Field

[0001] The present application relates to the field of detection technology, and in particular to a test strip, a kit and applications for detecting lung cancer autoantibodies. Background Art

[0002] Low-dose spiral CT is widely used to detect early lung nodules. However, the problem with low-dose spiral CT is the high false positive rate of its test results. Other early lung cancer detection methods, including PET, MRI, bronchial endoscopy, etc., have problems such as inability to screen early and high prices. Traditional conventional tumor markers are very low or even absent in the blood in the early stages of tumors.

[0003] The current method for measuring seven types of lung cancer autoantibodies is enzyme-linked immunosorbent assay. The enzyme-linked immunosorbent assay takes a long time to measure samples and is time-consuming and labor-intensive to operate. Although quantitative measurement can be achieved, it cannot achieve on-site testing of POCT reagents without interfering with each other.

[0004] With the rapid development of economy and technology, there is an urgent need to find a method for detecting lung cancer autoantibodies that can be detected immediately, has a short detection time and does not require large equipment. Summary of the invention

[0005] The purpose of the embodiments of the present application is to provide a test strip, a kit and an application for detecting autoantibodies of lung cancer, which can achieve real-time detection while ensuring detection sensitivity.

[0006] In order to solve the above technical problems, the present application embodiment provides a test strip for detecting autoantibodies for lung cancer, which adopts the following technical solution:

[0007] The test strips include a first colloidal gold test paper, a second colloidal gold test paper, a third colloidal gold test paper, a fourth colloidal gold test paper, a fifth colloidal gold test paper, a sixth colloidal gold test paper and a seventh colloidal gold test paper;

[0008] The first colloidal gold test paper, the second colloidal gold test paper, the third colloidal gold test paper, the fourth colloidal gold test paper, the fifth colloidal gold test paper, the sixth colloidal gold test paper and the seventh colloidal gold test paper all include a sample pad, a colloidal gold binding pad, an NC membrane and an absorption pad which are sequentially overlapped on a PVC bottom plate, and a quality control line is arranged on the NC membrane;

[0009] A p53 protein gold label is arranged on the colloidal gold binding pad of the first colloidal gold test paper, a first detection line is arranged on the NC membrane of the first colloidal gold test paper, and the first detection line is coated with a p53 recombinant antigen;

[0010] The colloidal gold binding pad of the second colloidal gold test paper is provided with a PGP9.5 protein gold label, and the NC membrane of the first colloidal gold test paper is provided with a first detection line, and the first detection line is coated with a PGP9.5 recombinant antigen;

[0011] The colloidal gold binding pad of the third colloidal gold test paper is provided with a SOX2 protein gold label, the NC membrane of the third colloidal gold test paper is provided with a third detection line, and the third detection line is coated with a SOX2 recombinant antigen;

[0012] The colloidal gold binding pad of the fourth colloidal gold test paper is provided with a GAGE7 protein gold label, the NC membrane of the fourth colloidal gold test paper is provided with a fourth detection line, and the fourth detection line is coated with a GAGE7 recombinant antigen;

[0013] The colloidal gold binding pad of the fifth colloidal gold test paper is provided with a GBU4-5 protein gold label, the NC membrane of the fifth colloidal gold test paper is provided with a fifth detection line, and the fifth detection line is coated with a GBU4-5 recombinant antigen;

[0014] The colloidal gold binding pad of the sixth colloidal gold test paper is provided with a MAGEA1 protein gold label, the NC membrane of the sixth colloidal gold test paper is provided with a sixth detection line, and the sixth detection line is coated with a MAGEA1 recombinant antigen;

[0015] The colloidal gold binding pad of the seventh colloidal gold test paper is provided with a CAGE protein gold label, the NC membrane of the seventh colloidal gold test paper is provided with a seventh detection line, and the seventh detection line is coated with a CAGE recombinant antigen.

[0016] Furthermore, the colloidal gold binding pad is provided with a mouse IgG gold label, and the quality control line is coated with a sheep anti-mouse IgG polyclonal antibody.

[0017] Furthermore, the coating concentration of the goat anti-mouse IgG polyclonal antibody was 1.0 mg / ml, and the coating volume was 1.0 μL / cm.

[0018] Furthermore, the coating concentrations of the p53 recombinant antigen, the PGP9.5 recombinant antigen, the SOX2 recombinant antigen, the GAGE7 recombinant antigen, the GBU4-5 recombinant antigen, the MAGEA1 recombinant antigen and the CAGE recombinant antigen are all 1.0-1.5 mg / ml, and the coating volumes are all 1.0 μL / cm.

[0019] Further, the p53 recombinant antigen, the PGP9.5 recombinant antigen, the SOX2 recombinant antigen, the GAGE7 recombinant antigen, the GBU4-5 recombinant antigen, the MAGEA1 recombinant antigen and the CAGE recombinant antigen are diluted with coating buffer;

[0020] Wherein, the coating buffer comprises, by mass fraction, 0.01-0.02 M phosphate buffer with a pH of 7.4 and 2% trehalose.

[0021] Furthermore, the conjugate pad is prepared by the following steps:

[0022] Preparing a target protein gold label according to a pre-prepared nano-colloidal gold solution, wherein the target protein gold label includes a p53 protein gold label, a PGP9.5 protein gold label, a SOX2 protein gold label, a GAGE7 protein gold label, a GBU4-5 protein gold label, a MAGEA1 protein gold label or a CAGE protein gold label;

[0023] Mouse IgG gold label was prepared according to the pre-prepared nano-colloidal gold solution;

[0024] The target protein gold label and the mouse IgG gold label are uniformly mixed in a colloidal gold diluent, sprayed on a glass cellulose membrane, and dried to obtain the binding pad;

[0025] Wherein, the colloidal gold diluent includes, by mass fraction, 0.01M Tris-HCl buffer with a pH of 8.5, 1% bovine serum albumin, 10% sucrose, 0.1% Tween-20 and 0.1% sodium azide.

[0026] Furthermore, the nano colloidal gold solution is prepared by the following steps:

[0027] Heat double distilled water to boiling, add 1% gold chloride aqueous solution, and continue boiling and heating;

[0028] Add 2% trisodium citrate solution and continue boiling and heating;

[0029] When the color of the solution changes to wine red, the heating is stopped to obtain the nano colloidal gold solution.

[0030] Furthermore, the step of preparing mouse IgG gold label according to the pre-prepared nano colloidal gold solution comprises:

[0031] Adding K2CO3 with a mass concentration of 0.2M to the nano-colloidal gold solution;

[0032] Add mouse IgG antibody, mix well, and add 10% BSA solution;

[0033] Centrifuge at a speed of 10000-12000 r / pm for 10-15 min, discard the supernatant, retain the precipitate, and obtain the mouse IgG gold label.

[0034] In order to solve the above technical problems, the present application also provides a kit for detecting autoantibodies for lung cancer, which adopts the following technical solution:

[0035] A kit for detecting autoantibodies for lung cancer, characterized in that the kit comprises a detection card, wherein the detection card comprises a card shell and a test strip for detecting autoantibodies for lung cancer according to any one of claims 1 to 8;

[0036] The test strip is placed in the card housing.

[0037] In order to solve the above technical problems, the present application also provides an application in detecting lung cancer autoantibodies for non-diagnostic purposes, using the following technical solutions:

[0038] The above kit is used for detecting lung cancer autoantibodies for non-diagnostic purposes.

[0039] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0040] Immunocolloidal gold rapid diagnostic technology is fast, easy to operate, highly specific, highly sensitive, low in detection cost, does not require complex techniques and special equipment, and is particularly suitable for real-time, rapid sampling and detection of a large number of samples on site. Therefore, it can simply and quickly achieve qualitative detection of human serum, plasma or whole blood samples. This detection method overcomes the defects of qualitative detection such as immunofluorescence technology that is prone to false negatives, simplifies the sample processing process, greatly shortens the detection time, reduces research costs and technical complexity; this application can be used for on-site detection through the colloidal gold method, without the need for large instruments and consumables, with strong anti-interference ability, convenient and fast, and can judge and monitor the health status of patients; this application is simple to operate and has a short detection time: only one sample is needed to obtain 7 test results, and after the sample is added, the results can be obtained in 10-15 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the scheme in the present application, a brief introduction is given below to the drawings required for use in the description of the embodiments of the present application. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0042] Figure 1 It is a schematic structural diagram of an embodiment of a test strip for detecting autoantibodies for lung cancer according to the present application;

[0043] Figure 2 It is a schematic diagram of the structure of an embodiment of a kit for detecting lung cancer autoantibodies according to the present application.

[0044] Figure numerals: 1. sample pad; 2. colloidal gold binding pad; 3. first detection line; 4. NC membrane; 5. quality control line; 6. absorption pad; 7. PVC bottom plate; 8. card shell; 9. sample addition hole; 10. result display area. DETAILED DESCRIPTION

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by technicians in the technical field of the present application; the terms used in the specification of the application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application; the terms "including" and "having" and any variations thereof in the specification and claims of the present application and the above-mentioned drawings are intended to cover non-exclusive inclusions. The terms "first", "second", etc. in the specification and claims of the present application or the above-mentioned drawings are used to distinguish different objects, not to describe a specific order.

[0046] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0047] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0048] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0049] The present application provides a test strip for detecting autoantibodies for lung cancer, such as Figure 1 As shown, Figure 1 It is a schematic diagram of the structure of an embodiment of a test strip for detecting lung cancer autoantibodies according to the present application; specifically, it is a schematic diagram of the structure of the first colloidal gold test strip of the present application.

[0050] The test strips include a first colloidal gold test paper, a second colloidal gold test paper, a third colloidal gold test paper, a fourth colloidal gold test paper, a fifth colloidal gold test paper, a sixth colloidal gold test paper and a seventh colloidal gold test paper;

[0051] The first colloidal gold test paper, the second colloidal gold test paper, the third colloidal gold test paper, the fourth colloidal gold test paper, the fifth colloidal gold test paper, the sixth colloidal gold test paper and the seventh colloidal gold test paper all include a sample pad 1, a colloidal gold binding pad 2, an NC membrane 4 and an absorption pad 6 on a PVC base plate 7 and sequentially overlapped on the PVC base plate 7, and a quality control line 5 is provided on the NC membrane;

[0052] A p53 protein gold marker is arranged on the colloidal gold binding pad of the first colloidal gold test paper, a first detection line 3 is arranged on the NC membrane of the first colloidal gold test paper, and the first detection line 3 is coated with a p53 recombinant antigen;

[0053] The colloidal gold binding pad of the second colloidal gold test paper is provided with a PGP9.5 protein gold label, and the NC membrane of the first colloidal gold test paper is provided with a first detection line, and the first detection line is coated with a PGP9.5 recombinant antigen;

[0054] The colloidal gold binding pad of the third colloidal gold test paper is provided with a SOX2 protein gold label, the NC membrane of the third colloidal gold test paper is provided with a third detection line, and the third detection line is coated with a SOX2 recombinant antigen;

[0055] The colloidal gold binding pad of the fourth colloidal gold test paper is provided with a GAGE7 protein gold label, the NC membrane of the fourth colloidal gold test paper is provided with a fourth detection line, and the fourth detection line is coated with a GAGE7 recombinant antigen;

[0056] The colloidal gold binding pad of the fifth colloidal gold test paper is provided with a GBU4-5 protein gold label, the NC membrane of the fifth colloidal gold test paper is provided with a fifth detection line, and the fifth detection line is coated with a GBU4-5 recombinant antigen;

[0057] The colloidal gold binding pad of the sixth colloidal gold test paper is provided with a MAGEA1 protein gold label, the NC membrane of the sixth colloidal gold test paper is provided with a sixth detection line, and the sixth detection line is coated with a MAGEA1 recombinant antigen;

[0058] The colloidal gold binding pad of the seventh colloidal gold test paper is provided with a CAGE protein gold label, the NC membrane of the seventh colloidal gold test paper is provided with a seventh detection line, and the seventh detection line is coated with a CAGE recombinant antigen.

[0059] As an option in the present application, the colloidal gold binding pad is provided with a mouse IgG gold label, and the quality control line is coated with a sheep anti-mouse IgG polyclonal antibody.

[0060] In this embodiment, the sample pad, colloidal gold binding pad, NC membrane, and absorption pad are sequentially pasted on one surface of the PVC bottom plate, and the NC membrane is pasted in the middle of one surface of the PVC bottom plate, and the two ends of the NC membrane are overlapped with one end of the colloidal gold binding pad and one end of the absorption pad respectively, and the other end of the colloidal gold binding pad is overlapped with one end of the sample pad; the colloidal gold binding pad is provided with a nano-gold labeled antigen binding layer. In the test strip, the boundary distance between the absorption pad and one end of the NC membrane is 2mm, the boundary distance between the other end of the NC membrane and one end of the colloidal gold binding pad is 1-2mm, and the boundary distance between one end of the sample pad and the other end of the colloidal gold binding pad is 1-2mm, so as to obtain a better detection effect.

[0061] The width of the test strip is 3.0 mm, which is convenient for testing and cost control.

[0062] The test strips of this application are based on colloidal gold immunochromatography, which can realize the rapid detection of lung cancer autoantibodies and can detect seven types of lung cancer autoantibodies at the same time. It can perform qualitative detection of seven types of lung cancer autoantibodies at any time and place without the assistance of additional instruments. At the same time, colloidal gold and its gold label have excellent stability, accuracy, sensitivity and repeatability, which can make it widely used in rapid detection.

[0063] As an option in the present application, the coating concentration of the goat anti-mouse IgG polyclonal antibody is 1.0 mg / ml, and the coating volume is 1.0 μL / cm.

[0064] As an option in the present application, the coating concentrations of the p53 recombinant antigen, the PGP9.5 recombinant antigen, the SOX2 recombinant antigen, the GAGE7 recombinant antigen, the GBU4-5 recombinant antigen, the MAGEA1 recombinant antigen and the CAGE recombinant antigen are all 1.0-1.5 mg / ml, and the coating volumes are all 1.0 μL / cm.

[0065] As an option of the present application, the p53 recombinant antigen, the PGP9.5 recombinant antigen, the SOX2 recombinant antigen, the GAGE7 recombinant antigen, the GBU4-5 recombinant antigen, the MAGEA1 recombinant antigen and the CAGE recombinant antigen are diluted by coating buffer; wherein, by mass fraction, the coating buffer comprises 0.01-0.02 M phosphate buffer with a pH of 7.4 and 2% trehalose.

[0066] As an option of the present application, the sample pad is prepared by the following steps:

[0067] Soak the glass fiber membrane in the sample pad treatment solution;

[0068] The sample pad was dried overnight for 16-24 hours at 18-26° C. and relative humidity ≤30% to obtain the sample pad.

[0069] In this embodiment, the glass fiber membrane is soaked in the treatment solution, and the treated sample pad is taken out and placed on a clean, dry and flat drying rack. The drying rack is placed at a temperature of (18-26)°C and a relative humidity of ≤30% to dry overnight for (16-24) hours.

[0070] As an option of the present application, the sample pad treatment solution includes, by mass fraction, 0.01-0.02M phosphate buffer, 1-2% BSA, 0.1% Tween-20, and 2% trehalose, and the pH value of the sample pad treatment solution is 7.0-8.0.

[0071] In this embodiment, the sample treatment solution can increase the absorption capacity of the sample pad, control the release of the gold label, and promote the occurrence of chromatography.

[0072] As an option in this application, the conjugate pad is prepared by the following steps:

[0073] Preparing a target protein gold label according to a pre-prepared nano-colloidal gold solution, wherein the target protein gold label includes a p53 protein gold label, a PGP9.5 protein gold label, a SOX2 protein gold label, a GAGE7 protein gold label, a GBU4-5 protein gold label, a MAGEA1 protein gold label or a CAGE protein gold label;

[0074] Mouse IgG gold label was prepared according to the pre-prepared nano-colloidal gold solution;

[0075] The target protein gold label and the mouse IgG gold label are uniformly mixed in a colloidal gold diluent, sprayed on a glass cellulose membrane, and dried to obtain the binding pad;

[0076] Wherein, the colloidal gold diluent includes, by mass fraction, 0.01M Tris-HCl buffer with a pH of 8.5, 1% bovine serum albumin, 10% sucrose, 0.1% Tween-20 and 0.1% sodium azide.

[0077] As an option of the present application, the nano colloidal gold solution is prepared by the following steps:

[0078] Heat double distilled water to boiling, add 1% gold chloride aqueous solution, and continue boiling and heating;

[0079] Add 2% trisodium citrate solution and continue boiling and heating;

[0080] When the color of the solution changes to wine red, the heating is stopped to obtain the nano colloidal gold solution.

[0081] As an optional step of the present application, the step of preparing mouse IgG gold label according to the pre-prepared nano colloidal gold solution comprises:

[0082] Adding K2CO3 with a mass concentration of 0.2M to the nano-colloidal gold solution;

[0083] Add mouse IgG antibody, mix well, and add 10% BSA solution;

[0084] Centrifuge at a speed of 10000-12000 r / pm for 10-15 min, discard the supernatant, retain the precipitate, and obtain the mouse IgG gold label.

[0085] As an option in the present application, the material of the sample pad is glass cellulose membrane; the material of the colloidal gold binding pad is glass cellulose membrane; the material of the NC membrane is CN140 membrane; and the material of the absorption pad is filter paper fiber.

[0086] The present application also provides a kit for detecting autoantibodies of lung cancer, such as Figure 2 As shown, Figure 2 It is a schematic diagram of the structure of an embodiment of a kit for detecting lung cancer autoantibodies according to the present application, specifically a schematic diagram of the structure of the following detection card of the present application.

[0087] The test kit comprises a detection card, which comprises a card housing 8 and the above-mentioned test strip for detecting lung cancer autoantibodies; the test strip is placed in the card housing.

[0088] The card shell 8 is a shell made of hard plastic material with a receiving cavity (the receiving cavity is not shown in the figure), and the card shell 8 is provided with a sample addition hole 9 and a test result display area 10 that penetrate the receiving cavity; the specifications and sizes of the test strip and the card shell 8 are consistent; after the test strip is adapted to be placed in the receiving cavity of the card shell 8, the sample pad of the test strip is adapted to be correspondingly arranged with the sample addition hole 9, and the colloidal gold binding pad of the test strip is adapted to be correspondingly arranged with the test result display area 10. By detecting the test result display area 10, the changes of the quality control line (C line) and the test line (T line) can be observed, that is, the test result is displayed.

[0089] In this embodiment, the test card is based on the rapid diagnosis technology of colloidal gold immunochromatography, and qualitatively detects seven lung cancer autoantibodies (p53, PGP9.5, SOX2, GAGE7, GBU4-5, MAGEA1, CAGE) in human serum, plasma or whole blood samples, and has the advantages of simple detection operation, short detection time, rapid and accurate, high detection sensitivity, good specificity, etc. This kit adopts the point-of-care testing (POCT) method, and can achieve on-site detection through the colloidal gold method detection, without the need for large instruments and consumables, strong anti-interference ability, convenient and fast.

[0090] The present application also provides the use of the above-mentioned kit in detecting lung cancer autoantibodies for non-diagnostic purposes, comprising the following steps:

[0091] Place the test card flat on the workbench;

[0092] Add 100 μL of the sample to be tested to the sample well of the test card, and then add 4 drops of sample diluent (the sample diluent includes 0.01 M phosphate buffer);

[0093] Observe for 10 to 15 minutes, and observe the results displayed in the test result display area on the test card. The results displayed after 15 minutes will be invalid.

[0094] According to the displayed results, the detection effects of seven lung cancer autoantibodies were judged.

[0095] As an option in the present application, the sample to be tested includes human serum, human plasma or human whole blood.

[0096] In this embodiment, the serum tumor autoantibody spectrum test is non-invasive, has no radiation exposure risk, has high sensitivity, and has a positive accuracy rate of up to 90%. It can simultaneously detect early small cell lung cancer and non-small cell lung cancer for non-diagnostic purposes. Studies have shown that Chapman et al. used enzyme-linked immunosorbent assay to detect serum antibodies of seven tumors (p53, PGP9.5, SOX2, GAGE7, GBU4-5, MAGEA1, CAGE). The results showed that the positive detection rate of a single antibody was between 5% and 36%, and the diagnostic specificity reached 96% to 100%. The sensitivity of the combined detection of the seven antibodies can reach 76%, and the specificity is 92%. This fully demonstrates that the single detection of tumor markers has a high specificity, but low sensitivity and accuracy; after combined detection, the sensitivity and accuracy are greatly improved.

[0097] This application has the following advantages:

[0098] 1) Immunocolloidal gold rapid diagnostic technology is fast, easy to operate, highly specific, highly sensitive, low in detection cost, does not require complex techniques and special equipment, and is particularly suitable for real-time, rapid sampling and detection of a large number of samples on site. Therefore, it can simply and quickly achieve qualitative detection of human serum, plasma or whole blood samples. This detection method overcomes the defects of false negatives in qualitative detection such as immunofluorescence technology, simplifies the sample processing process, greatly shortens the detection time, and reduces research costs and technical complexity;

[0099] 2) This method can be tested on-site through the colloidal gold method, without the need for large instruments and consumables, has strong anti-interference ability, is convenient and fast, and can judge and monitor the health status of patients;

[0100] 3) This application is simple to operate and has a short detection time: you only need to add the sample once to get 7 test results. After adding the sample, the result can be obtained in 10-15 minutes, which is more suitable for grassroots applications

[0101] The above scheme is further described below in conjunction with specific implementation examples, but these embodiments are by no means limiting the present application. The preferred embodiments of the present application are described in detail as follows:

[0102] Example 1

[0103] The test strips for detecting lung cancer autoantibodies include the first colloidal gold test paper (unit colloidal gold A test paper), the second colloidal gold test paper (unit colloidal gold B test paper), the third colloidal gold test paper (unit colloidal gold C test paper), the fourth colloidal gold test paper (unit colloidal gold D test paper), the fifth colloidal gold test paper (unit colloidal gold E test paper), the sixth colloidal gold test paper (unit colloidal gold F test paper) and the seventh colloidal gold test paper (unit colloidal gold G test paper).

[0104] The NC membrane of the first colloidal gold test paper is provided with a first detection line and a quality control line which are parallel to each other; the NC membrane of the second colloidal gold test paper is provided with a second detection line and a quality control line which are parallel to each other; the NC membrane of the third colloidal gold test paper is provided with a third detection line and a quality control line which are parallel to each other; the NC membrane of the fourth colloidal gold test paper is provided with a fourth detection line and a quality control line which are parallel to each other; the NC membrane of the fifth colloidal gold test paper is provided with a fifth detection line and a quality control line which are parallel to each other; the NC membrane of the sixth colloidal gold test paper is provided with a sixth detection line and a quality control line which are parallel to each other; the NC membrane of the seventh colloidal gold test paper is provided with a seventh detection line and a quality control line which are parallel to each other.

[0105] Example 2

[0106] Preparation of colloidal gold conjugate pad

[0107] The colloidal gold binding pad of the first colloidal gold test paper is a p53 protein binding pad; the colloidal gold binding pad of the second colloidal gold test paper is a PGP9.5 protein binding pad; the colloidal gold binding pad of the third colloidal gold test paper is a SOX2 protein binding pad; the colloidal gold binding pad of the fourth colloidal gold test paper is a GAGE7 protein binding pad; the colloidal gold binding pad of the fifth colloidal gold test paper is a GBU4-5 recombinant antigen binding pad; the colloidal gold binding pad of the sixth colloidal gold test paper is a MAGEA1 protein binding pad; and the colloidal gold binding pad of the seventh colloidal gold test paper is a CAGE protein binding pad.

[0108] (1) Preparation of p53 protein binding pad

[0109] a. Preparation of colloidal gold: colloidal gold nanoparticles were prepared by sodium citrate reduction method. 500 ml of double distilled water was placed in a 2000 ml beaker, heated to boiling on an electric stove, 60 ml of 1% gold chloride aqueous solution was added, and continued to heat to boiling, and then about 35 ml of 2% trisodium citrate solution was quickly added thereto, and continued to heat and boil for 5 minutes. The color of the colloidal gold solution changed from black to blue-purple to wine red, and the heating was stopped. When the prepared colloidal gold solution was naturally cooled to room temperature, the volume was adjusted to 1500 ml with purified water, and stored at room temperature for later use.

[0110] b. Preparation of p53 protein gold label: Take 1 mL of the colloidal gold solution prepared in step a, add 5-8 μl of 0.2 M K2CO3 to adjust the pH of the colloidal gold solution, mix well, add 5-10 μg of p53 recombinant antigen, mix well for 5 minutes, gradually add 100 μl of 10% BSA solution for blocking, mix well for about 10 minutes, centrifuge at a speed of 10000-12000 r / pm for 10-15 minutes, discard the supernatant, and precipitate to 100 μl. The precipitate is the p53 antigen gold label and is stored at 4°C for later use.

[0111] c. Preparation of mouse IgG gold label: Take 1 mL of the colloidal gold solution prepared in step a, add 5-8 μl of 0.2 M K2CO3 to adjust the pH of the colloidal gold solution, mix well, add 5-10 μg of mouse IgG antibody, mix well for 5 minutes, gradually add 100 μl of 10% BSA solution for blocking, mix well for about 10 minutes, centrifuge at a speed of 10000-12000 r / pm for 10-15 minutes, discard the supernatant, precipitate and make up to 100 μl, the precipitate is the mouse IgG gold label, and store at 4 ° C for later use;

[0112] d. Preparation method of colloidal gold diluent: Every 100 ml of colloidal gold is made of 0.01M Tris-HCl buffer with a pH of 8.5, 1% bovine serum albumin, 10% sucrose, 0.1% Tween-20 and 0.1% sodium azide, and ultrapure water.

[0113] e. The colloidal gold-antigen complex was re-dissolved with the colloidal gold diluent prepared in (d), and (b) and (c) were mixed and sprayed on the glass cellulose membrane. The membrane was fixed in a constant temperature drying oven at 37°C for 3 hours and then taken out for use.

[0114] (2) Preparation of PGP9.5 protein binding pad: PGP9.5 recombinant antigen was labeled in the same manner as in Example 2(1), and redissolved with colloidal gold diluent to obtain a PGP9.5 protein binding pad;

[0115] (3) Preparation of SOX2 protein binding pad: SOX2 recombinant antigen was labeled in the same manner as in Example 2(1), and redissolved with colloidal gold diluent to obtain a SOX2 protein binding pad;

[0116] (4) Preparation of GAGE7 protein binding pad: Label the GAGE7 recombinant antigen in the same manner as in Example 2(1), and redissolve it with colloidal gold diluent to obtain a GAGE7 protein binding pad;

[0117] (5) Preparation of GBU4-5 protein binding pad: Label the GBU4-5 recombinant antigen in the same manner as in Example 2(1), and redissolve it with colloidal gold diluent to obtain a GBU4-5 recombinant antigen binding pad;

[0118] (6) Preparation of MAGEA1 protein binding pad: The MAGEA1 recombinant antigen was labeled in the same manner as in Example 2(1), and redissolved with colloidal gold diluent to obtain a MAGEA1 protein binding pad;

[0119] (7) Preparation of CAGE protein binding pad: The CAGE recombinant antigen was labeled in the same manner as in Example 2(1), and redissolved with colloidal gold diluent to obtain a CAGE protein binding pad.

[0120] Example 3

[0121] NC membrane coating

[0122] (1) Preparation method of coating buffer: The coating buffer is made of 0.01-0.02M phosphate buffer with a pH of 7.4, 2% trehalose and purified water. The coating buffer is used to dilute the p53 recombinant antigen, the PGP9.5 recombinant antigen, the SOX2 recombinant antigen, the GAGE7 recombinant antigen, the GBU4-5 recombinant antigen, the MAGEA1 recombinant antigen and the CAGE recombinant antigen to the coating concentration, so as to be coated on the NC membrane.

[0123] (2) The NC140 membrane was adhered to the PVC base plate and lines were drawn at specific positions in sequence using a film drawing instrument; the coating concentration of the quality control line with goat anti-mouse IgG polyclonal antibody was 1.0 mg / ml and the coating volume was 1.0 μL / cm; the coating concentration of each test line was 1.0-1.5 mg / ml and the coating volume was 1.0 μL / cm. The membrane was fixed in a 37°C constant temperature drying oven for 16 h and then taken out for use.

[0124] Among them, the NC membrane of the first colloidal gold test paper is coated with p53 recombinant antigen, and the coating concentration is 1.0-1.5 mg / ml; the NC membrane of the second colloidal gold test paper is coated with PGP9.5, recombinant antigen, and the coating concentration is 1.0-1.5 mg / ml; the NC membrane of the third colloidal gold test paper is coated with SOX2 recombinant antigen, and the coating concentration is 1.0-1.5 mg / ml; the NC membrane of the fourth colloidal gold test paper is coated with GAGE7 recombinant antigen, and the coating concentration is 1.0-1.5 mg / ml; the NC membrane of the fifth colloidal gold test paper is coated with GBU4-5 recombinant antigen, and the coating concentration is 1.0-1.5 mg / ml; the NC membrane of the sixth colloidal gold test paper is coated with MAGEA1 recombinant antigen, and the coating concentration is 1.0-1.5 mg / ml; the NC membrane of the seventh colloidal gold test paper is coated with CAGE recombinant antigen, and the coating concentration is 1.0-1.5 mg / ml.

[0125] Example 4

[0126] Test strip assembly

[0127] First, the sample pad 1, the colloidal gold binding pad 2, the NC membrane 3, and the absorption pad 6 are sequentially pasted on one surface of the PVC bottom plate 7 to obtain a sample test strip; wherein, the boundary distance between the absorption pad 6 and one end of the NC membrane 4 laminated and overlapped is 2 mm; the boundary distance between the other end of the NC membrane 4 and one end of the colloidal gold binding pad 2 laminated and overlapped is 1 to 2 mm; the boundary distance between one end of the sample pad 1 and the other end of the colloidal gold binding pad 2 laminated and overlapped is 1 to 2 mm.

[0128] Secondly, use a strip cutter to cut the sample test strips into 3.0 mm wide test strips, then pack them in aluminum foil bags, add desiccant, and seal them for storage.

[0129] Example 5

[0130] Test card assembly

[0131] The detection card comprises a test strip and a card shell. The first colloidal gold test paper, the second colloidal gold test paper, the third colloidal gold test paper, the fourth colloidal gold test paper, the fifth colloidal gold test paper, the sixth colloidal gold test paper and the seventh colloidal gold test paper are respectively installed in the card shell 8. The card shell 8 is provided with a sample addition hole 9 and a test result display area 10.

[0132] It should be understood that, although the steps in the flowchart of the accompanying drawings are displayed in sequence as indicated by the arrows, these steps are not necessarily executed in sequence in the order indicated by the arrows. Unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in other orders. Moreover, at least a part of the steps in the flowchart of the accompanying drawings may include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn or alternately with other steps or at least a part of the sub-steps or stages of other steps.

[0133] Example 6

[0134] Clinically relevant tests:

[0135] To illustrate the test results, the present application tested 50 clinically relevant samples with the kit obtained in Example 5, and the obtained data are shown in the following table:

[0136] Table 1 Clinical relevance test results

[0137]

[0138]

[0139]

[0140]

[0141] Further, in order to illustrate the test results, the present application simultaneously tested the above 50 relevant samples with the reagents of Hangzhou Kepaobao Biotechnology Co., Ltd., and the obtained data are shown in the following table:

[0142] Table 2 Test results of the above 50 related samples tested simultaneously by the kit of Hangzhou Kepaobao Biotechnology Co., Ltd.

[0143]

[0144]

[0145]

[0146] Furthermore, in order to illustrate the relevance of the test results, the present application conducted a statistical analysis on the 50 clinical samples tested simultaneously above and calculated the positive compliance rate.

[0147] Table 3 Summary of the test results of the applied reagents and the state Kepao biological reagents

[0148]

[0149] Positive coincidence rate = 9 / (9+1)×100% = 90.00%. Compared with the kit of Hangzhou Kepaobao Biological, the positive accuracy of the kit of the present application obtained in Example 5 is 90%.

[0150] Obviously, the embodiments described above are only some embodiments of the present application, rather than all embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application is described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions recorded in the aforementioned specific implementation methods, or to perform equivalent replacement of some of the technical features therein. Any equivalent structure made using the contents of the specification and drawings of this application, directly or indirectly used in other related technical fields, is similarly within the scope of patent protection of this application.

Claims

1. A test strip for detecting autoantibodies for lung cancer, characterized in that: The test strips include a first colloidal gold test paper, a second colloidal gold test paper, a third colloidal gold test paper, a fourth colloidal gold test paper, a fifth colloidal gold test paper, a sixth colloidal gold test paper and a seventh colloidal gold test paper; The first colloidal gold test paper, the second colloidal gold test paper, the third colloidal gold test paper, the fourth colloidal gold test paper, the fifth colloidal gold test paper, the sixth colloidal gold test paper and the seventh colloidal gold test paper all include a sample pad, a colloidal gold binding pad, an NC membrane and an absorption pad which are sequentially overlapped on a PVC bottom plate, and a quality control line is arranged on the NC membrane; A p53 protein gold label is arranged on the colloidal gold binding pad of the first colloidal gold test paper, a first detection line is arranged on the NC membrane of the first colloidal gold test paper, and the first detection line is coated with a p53 recombinant antigen; The colloidal gold binding pad of the second colloidal gold test paper is provided with a PGP9.5 protein gold label, and the NC membrane of the first colloidal gold test paper is provided with a first detection line, and the first detection line is coated with a PGP9.5 recombinant antigen; The colloidal gold binding pad of the third colloidal gold test paper is provided with a SOX2 protein gold label, the NC membrane of the third colloidal gold test paper is provided with a third detection line, and the third detection line is coated with a SOX2 recombinant antigen; The colloidal gold binding pad of the fourth colloidal gold test paper is provided with a GAGE7 protein gold label, the NC membrane of the fourth colloidal gold test paper is provided with a fourth detection line, and the fourth detection line is coated with a GAGE7 recombinant antigen; The colloidal gold binding pad of the fifth colloidal gold test paper is provided with a GBU4-5 protein gold label, the NC membrane of the fifth colloidal gold test paper is provided with a fifth detection line, and the fifth detection line is coated with a GBU4-5 recombinant antigen; The colloidal gold binding pad of the sixth colloidal gold test paper is provided with a MAGEA1 protein gold label, the NC membrane of the sixth colloidal gold test paper is provided with a sixth detection line, and the sixth detection line is coated with a MAGEA1 recombinant antigen; The colloidal gold binding pad of the seventh colloidal gold test paper is provided with a CAGE protein gold label, the NC membrane of the seventh colloidal gold test paper is provided with a seventh detection line, and the seventh detection line is coated with a CAGE recombinant antigen.

2. The test strip for detecting lung cancer autoantibodies according to claim 1, characterized in that: The colloidal gold binding pad is provided with a mouse IgG gold label, and the quality control line is coated with a sheep anti-mouse IgG polyclonal antibody.

3. The test strip for detecting lung cancer autoantibodies according to claim 2, characterized in that: The coating concentration of the goat anti-mouse IgG polyclonal antibody was 1.0 mg / ml, and the coating volume was 1.0 μL / cm.

4. The test strip for detecting lung cancer autoantibodies according to claim 1, characterized in that: The coating concentrations of the p53 recombinant antigen, the PGP9.5 recombinant antigen, the SOX2 recombinant antigen, the GAGE7 recombinant antigen, the GBU4-5 recombinant antigen, the MAGEA1 recombinant antigen and the CAGE recombinant antigen were all 1.0-1.5 mg / ml, and the coating volumes were all 1.0 μL / cm.

5. The test strip for detecting lung cancer autoantibodies according to claim 1, characterized in that: The p53 recombinant antigen, the PGP9.5 recombinant antigen, the SOX2 recombinant antigen, the GAGE7 recombinant antigen, the GBU4-5 recombinant antigen, the MAGEA1 recombinant antigen and the CAGE recombinant antigen are diluted with coating buffer; Wherein, the coating buffer comprises, by mass fraction, 0.01-0.02 M phosphate buffer with a pH of 7.4 and 2% trehalose.

6. The test strip for detecting lung cancer autoantibodies according to claim 1, characterized in that: The conjugate pad is prepared by the following steps: Preparing a target protein gold label according to a pre-prepared nano-colloidal gold solution, wherein the target protein gold label includes a p53 protein gold label, a PGP9.5 protein gold label, a SOX2 protein gold label, a GAGE7 protein gold label, a GBU4-5 protein gold label, a MAGEA1 protein gold label or a CAGE protein gold label; Mouse IgG gold label was prepared according to the pre-prepared nano-colloidal gold solution; The target protein gold label and the mouse IgG gold label are uniformly mixed in a colloidal gold diluent, sprayed on a glass cellulose membrane, and dried to obtain the binding pad; Wherein, the colloidal gold diluent includes, by mass fraction, 0.01M Tris-HCl buffer with a pH of 8.5, 1% bovine serum albumin, 10% sucrose, 0.1% Tween-20 and 0.1% sodium azide.

7. The test strip for detecting lung cancer autoantibodies according to claim 6, characterized in that: The nano colloidal gold solution is prepared by the following steps: Heat double distilled water to boiling, add 1% gold chloride aqueous solution, and continue boiling and heating; Add 2% trisodium citrate solution and continue boiling and heating; When the color of the solution changes to wine red, the heating is stopped to obtain the nano colloidal gold solution.

8. The test strip for detecting lung cancer autoantibodies according to claim 6, characterized in that: The step of preparing mouse IgG gold label according to the pre-prepared nano colloidal gold solution comprises: Adding K2CO3 with a mass concentration of 0.2M to the nano-colloidal gold solution; Add mouse IgG antibody, mix well, and add 10% BSA solution; Centrifuge at a speed of 10000-12000 r / pm for 10-15 min, discard the supernatant, retain the precipitate, and obtain the mouse IgG gold label.

9. A kit for detecting autoantibodies for lung cancer, characterized in that: The kit comprises a test card, wherein the test card comprises a card shell and a test strip for detecting lung cancer autoantibodies according to any one of claims 1 to 8; The test strip is placed in the card housing.

10. Use of the kit according to claim 9 in detecting autoantibodies for lung cancer for non-diagnostic purposes.

Images