Human anti-gluten antibody detection reagent, kit and preparation method

Abstract

The invention provides a human anti-gluten antibody detection reagent, a kit and a preparation method. The SELEX technology is utilized for screening for the first time to obtain two nucleic acid aptamers capable of specifically recognizing the anti-gluten antibody, the chromatographic technology-based detection test paper card is constructed on the basis of the nucleic acid aptamers, and the detection test paper card has excellent detection sensitivity and specificity, is suitable for large-scale detection, remarkably improves the detection efficiency, and has good application prospects. And the kit has important scientific significance and application value for early diagnosis of gliadiasis patients.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent immunoassay, and more specifically, to a human anti-gluten antibody detection reagent, a kit and a preparation method. Background Art

[0002] Anti-gluten antibodies (IgA-GA) are autoantibodies produced in patients with celiac disease and belong to IgA. Gluten contains gliadin, also known as alcohol-soluble protein, which is the main cause of celiac disease. This substance cannot be absorbed by patients and becomes a pathogenic factor, stimulating antibodies to produce anti-gluten antibodies.

[0003] In recent years, with the further development of immunochromatographic detection technology and nucleic acid aptamers, it is a popular trend to use nucleic acid aptamers to replace antibodies to construct new immunochromatographic test strips. However, there is no public immunochromatographic detection method for human anti-gluten antibodies in the prior art. In view of this, it is urgent to establish a new detection method for anti-gluten antibodies to provide powerful value information for early screening diagnosis, prognosis assessment or recurrence monitoring of clinical patients. Summary of the invention

[0004] In view of the technical problems existing in the prior art, the present invention provides a human anti-gluten antibody detection reagent, a kit and a preparation method. In this study, two nucleic acid aptamers that specifically recognize anti-gluten antibodies were screened using the SELEX technology, and a detection test paper card based on chromatography technology was constructed. The detection test paper card has excellent detection sensitivity and specificity, is suitable for large-scale detection, significantly improves the detection efficiency, and has important scientific significance and application value for the early diagnosis of patients with gluten enteropathy.

[0005] One of the purposes of the present invention is to provide a nucleic acid aptamer that specifically recognizes anti-gluten antibodies, wherein the sequence of the nucleic acid aptamer is shown in any one of SEQ ID NO.1-2.

[0006] Preferably, the present invention also provides use of nucleic acid aptamers in preparing a test card or a kit for detecting anti-gluten antibodies.

[0007] Furthermore, the present invention also provides a human anti-gluten antibody detection test paper card, which includes a base plate, a sample pad, a conjugation pad, a nitrocellulose membrane and a water absorbent pad adhered to the base plate and overlapped in sequence; a detection line is provided on the side of the nitrocellulose membrane close to the conjugation pad, and a quality control line is provided on the side of the nitrocellulose membrane close to the water absorbent pad; the conjugation pad is coated with Au@AP2; the detection line is coated with a complex of nucleic acid aptamer AP1 and streptavidin; the quality control line is coated with streptavidin; the nucleic acid aptamer AP1 and the nucleic acid aptamer AP2 are both biotin-labeled modified nucleic acid aptamers that can specifically recognize anti-gluten antibodies.

[0008] Preferably, the bottom plate is a PVC plate; Preferably, the sample pad is a polyacetic acid film; Preferably, the bonding pad is glass fiber; Preferably, the absorbent pad is absorbent filter paper; Preferably, the test paper card is packaged in a hard plastic card.

[0009] Furthermore, the present invention also provides a method for preparing the test paper card: 1) Preparation of the conjugate pad: The conjugate pad was immersed in an aqueous solution containing 15wt% trehalose, 5wt% BSA and 0.5wt% Tween-20, and then dried for use; the Au@AP2 solution was diluted and evenly sprayed on the conjugate pad, and then dried to prepare the conjugate pad for use; 2) Preparation of sample pad: soak the sample pad in an aqueous solution containing 0.5 wt% Tween-20 and 5 wt% PEG30000, and dry it for later use; 3) Preparation of nitrocellulose membrane: Spray 5 mg / mL streptavidin onto the detection line and quality control line of the nitrocellulose membrane respectively. After drying, spray the nucleic acid aptamer AP1 onto the detection line of the nitrocellulose membrane. Then continue to dry the nitrocellulose membrane for later use. 4) Assembly of the test strip: Connect the sample pad, conjugate pad, chromatographic membrane and absorbent pad on the PVC base plate in sequence to obtain a test strip, with the detection line set at one end close to the conjugate pad and the quality control line set at the other end close to the absorbent pad.

[0010] Furthermore, the present invention also provides a detection method of the test paper card: The sample to be tested is added to the sample spotting hole, and the sample to be tested is chromatographed along the sample pad, the conjugate pad, the nitrocellulose membrane toward the absorbent filter paper end under the capillary action; During the chromatography process, the test substance in the sample to be tested first specifically binds to the Au@AP2 on the glass fiber binding pad and re-dissolves it, and the re-dissolved mixture continues to be chromatographed onto the nitrocellulose membrane; It reacts with the nucleic acid aptamer AP1 coated in the detection line area on the nitrocellulose membrane, and under the action of a certain concentration of the detection substance, a line of corresponding color appears; The excess Au@AP2 continues to move forward, and when the biotin labeled on AP2 reaches the quality control line area, it combines with the coated streptavidin, and the nanoparticles are enriched, showing the color line of the nanoparticles; If there is no analyte in the sample, Au@AP2 and aptamer AP1 will not react in the detection line area, but continue to chromatograph forward through the bio-strand enzyme avidin reaction, so the detection line is colorless, while the quality control line shows the color of the nanoparticles.

[0011] Preferably, the method comprises use for non-diagnostic purposes.

[0012] Another object of the present invention is to provide a human anti-gluten antibody detection kit, which comprises the above-mentioned test strip.

[0013] Another object of the present invention is to provide use of the test strip for detecting human anti-gluten antibodies or the human anti-gluten antibody kit in preparing an early diagnosis product for celiac disease.

[0014] In the present invention, the detection principle of the test paper card is as follows: during the chromatography process, the detection object in the sample to be tested first specifically binds to the Au@AP2 on the glass fiber binding pad and redissolves it. The redissolved mixture continues to be chromatographed onto the nitrocellulose membrane, and reacts with the nucleic acid aptamer AP1 coated in the detection line area on the nitrocellulose membrane. Under the action of a certain concentration of the detection object, a line of corresponding color is displayed; the excess Au@AP2 continues to move forward, and when the biotin labeled on AP2 reaches the quality control line area, it binds to the coated streptavidin, and the nanoparticles are enriched, showing the color line of the nanoparticles. If there is no analyte in the sample, Au@AP2 and the nucleic acid aptamer AP1 will not act in the detection line area, but continue to chromatograph forward through the biological streptavidin reaction. Therefore, the detection line is colorless, and the quality control line shows the color of the nanoparticles. The quality control line is used to judge the quality of the test kit: when the concentration of the test substance in the sample is too high, or there is high salt, Au@AP2 will be enriched in the test line, and there will be no excess colloidal gold to continue moving forward. The quality control line will not have nanoparticle gold enrichment and will appear colorless, indicating that the microenvironment of the test exceeds the applicable scope of this test kit and the result is invalid. When the nucleic acid aptamer is detached from the surface of the nanoparticle or degraded, or other reasons cause the quality of the test kit to change, nanoparticles will not appear on the test line and the quality control line. Both the test line and the quality control line are colorless, indicating that the test result is invalid. Therefore, in the test, the test line and the quality control line show the color of the nanoparticles at the same time, indicating that the test result is credible.

[0015] The advantages of the present invention are as follows: the present invention uses the SELEX technology for the first time to screen and obtain two nucleic acid aptamers that specifically recognize anti-gluten antibodies, and uses them to construct a detection test paper card based on chromatography technology. The detection test paper card has excellent detection sensitivity and specificity, is suitable for large-scale detection, and significantly improves the detection efficiency. It has important scientific significance and application value for the early diagnosis of patients with gluten enteropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 .Test strip card detection sensitivity analysis; Figure 2.Analysis of test strip card detection specificity. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0018] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0019] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0020] Example 1 The present invention provides a nucleic acid aptamer AP1 that specifically recognizes anti-gluten antibodies, and the sequence of the nucleic acid aptamer AP1 is shown in SEQ ID NO.1; 5'-tcatcaagcagtcgcggcccttcggataggttaccagcatccctaatcaatataaccgaagtgatcggggtaaacccaaatctggaa-3' (SEQ ID NO. 1), with Biotin modification at its 3' position; The present invention provides a nucleic acid aptamer AP2 for specific anti-gluten antibodies, wherein the sequence of the nucleic acid aptamer AP1 is shown in SEQ ID NO.2; 5'-aagtatgaagctcttccttccttagagtaatcgcgtatacagactcccgaaattaccggggctctgcatctatagcaccttcttagctgaagatg-3' (SEQ ID NO. 2), wherein there is a thiol modification at the 5' end and a biotin modification at the 3' end; The present invention also provides a nucleic acid aptamer AP2 labeled with colloidal gold Au@AP2 and a preparation method thereof, wherein the Au@AP2 preparation method comprises: 1) Take 100 mL of 0.01% HAuCl4 aqueous solution, add 3 mL of 1% trisodium citrate aqueous solution, heat and boil for 30 min, cool to 4°C, the solution turns red, and a 10 nm colloidal gold stock solution is obtained. Store at 4°C away from light.

[0021] 2) Add 5 μL of 100 μM aptamer AP2 to 5 μL of 5 mg / mL tri(2-carboxyethyl)phosphine hydrochloride solution and place in dark place for 2 h.

[0022] 3) Take 5 mL of 10 nm colloidal gold stock solution and centrifuge at 15,000 rpm for 10 min. Remove the supernatant and add 5 mL of ultrapure water to reconstitute.

[0023] 4) Then, the activated nucleic acid aptamer AP2 in step 2) was added to step 3), mixed and then allowed to react at room temperature for 16 hours to obtain the Au@AP2 solution for use.

[0024] Example 2 A test paper card for detecting anti-gluten antibodies by colloidal gold immunochromatography based on nucleic acid aptamers, the test paper card comprising a bottom plate, a sample pad, a conjugation pad, a nitrocellulose membrane and a water absorbent pad which are bonded to the bottom plate and overlapped in sequence; a detection line is provided on the side of the nitrocellulose membrane close to the conjugation pad, and a quality control line is provided on the side of the nitrocellulose membrane close to the water absorbent pad; the conjugation pad is coated with Au@AP2; the detection line is coated with a complex of nucleic acid aptamer AP1 and streptavidin; the quality control line is coated with streptavidin; the nucleic acid aptamer AP1 and the nucleic acid aptamer AP2 are both biotin-labeled and modified nucleic acid aptamers that can specifically recognize anti-gluten antibodies.

[0025] The bottom plate is a PVC plate; the sample pad is a polyacetic acid film; the binding pad is glass fiber; the water-absorbing pad is water-absorbing filter paper; and the test paper card is packaged by a hard plastic card.

[0026] The preparation method of the test paper card: 1) Preparation of the conjugate pad: The conjugate pad was immersed in an aqueous solution containing 15wt% trehalose, 5wt% BSA and 0.5wt% Tween-20, and then dried for use; the Au@AP2 solution was diluted and evenly sprayed on the conjugate pad, and then dried to prepare the conjugate pad for use; 2) Preparation of sample pad: soak the sample pad in an aqueous solution containing 0.5 wt% Tween-20 and 5 wt% PEG30000, and dry it for later use; 3) Preparation of nitrocellulose membrane: Spray 5 mg / mL streptavidin onto the detection line and quality control line of the nitrocellulose membrane respectively. After drying, spray the nucleic acid aptamer AP1 onto the detection line of the nitrocellulose membrane. Then continue to dry the nitrocellulose membrane for later use. 4) Assembly of the test strip: Connect the sample pad, conjugate pad, chromatographic membrane and absorbent pad on the PVC base plate in sequence to obtain a test strip, with the detection line set at one end close to the conjugate pad and the quality control line set at the other end close to the absorbent pad.

[0027] The detection method of the test paper card: The sample to be tested is added to the sample spotting hole, and the sample to be tested is chromatographed along the sample pad, the conjugate pad, the nitrocellulose membrane toward the absorbent filter paper end under the capillary action; During the chromatography process, the test substance in the sample to be tested first specifically binds to the Au@AP2 on the glass fiber binding pad and re-dissolves it, and the re-dissolved mixture continues to be chromatographed onto the nitrocellulose membrane; It reacts with the nucleic acid aptamer AP1 coated in the detection line area on the nitrocellulose membrane, and under the action of a certain concentration of the detection substance, a line of corresponding color appears; The excess Au@AP2 continues to move forward, and when the biotin labeled on AP2 reaches the quality control line area, it combines with the coated streptavidin, and the nanoparticles are enriched, showing the color line of the nanoparticles; If there is no analyte in the sample, Au@AP2 and aptamer AP1 will not react in the detection line area, but continue to chromatograph forward through the bio-strand enzyme avidin reaction, so the detection line is colorless, while the quality control line shows the color of the nanoparticles.

[0028] Example 3 1) Sensitivity detection: Determination of anti-gluten antibody standard solution on test paper: Anti-gluten antibodies can be detected from the blood of patients with gluten enteropathy, and 1×PBS, pH 7.0 is used to dilute the final concentrations to 1, 5, 10, 15, 20, 25 and 50 ng / mL. The above 20 μL of different concentrations of anti-gluten antibodies are added to the test paper described in Example 2 in sequence, and the relative signal intensity (T / C) of colloidal gold is measured after reacting for 15 minutes, and a standard curve of the corresponding relationship between the relative signal intensity (T / C) and different anti-gluten antibody concentrations is established, wherein the blank group is replaced by an equal amount of 1×PBS.

[0029] The results showed that when the anti-gluten antibody was diluted to 1 ng / mL, only the C line appeared, and no T line appeared, indicating a negative result; when the anti-gluten antibody was diluted to 5 ng / mL, both the C line and the T line appeared at the same time, indicating a positive result. It can be seen that the detection line can reach 5 ng / mL, and has good detection results between 5-20 ng / mL, indicating that the prepared test paper card has a high sensitivity.

[0030] 2) Specificity detection 10 ng / mL of human anti-gluten antibody, human IgG, human IgM, human IgE and human IgD were dripped onto the sample pad of the test strip in sequence.

[0031] The test results showed that only the test strip with anti-gluten antibodies had both the test line and the quality control line appear, indicating a positive result, while for the rest, only the quality control line appeared, indicating a negative result. This test result indicated that the prepared test strip had good specificity.

[0032] It is necessary to point out that the above embodiments are limited to further elaboration and explanation of the technical solution of the present invention, and are not further limitations of the technical solution of the present invention. The method of the present invention is only a preferred implementation scheme, and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A nucleic acid aptamer that specifically recognizes anti-gluten antibodies, characterized in that: The sequence of the nucleic acid aptamer is shown in any one of SEQ ID NO.1-2.

2. Use of the nucleic acid aptamer according to claim 1 in preparing a test card or a kit for detecting anti-gluten antibodies.

3. A human anti-gluten antibody detection test paper card, characterized in that: The test paper card comprises a bottom plate, a sample pad, a binding pad, a nitrocellulose membrane and a water absorbent pad which are bonded to the bottom plate and overlapped in sequence; a detection line is provided on the side of the nitrocellulose membrane close to the binding pad, and a quality control line is provided on the side of the nitrocellulose membrane close to the water absorbent pad; the binding pad is coated with Au@AP2; the detection line is coated with a complex of a nucleic acid aptamer AP1 and streptavidin; the quality control line is coated with streptavidin; the nucleic acid aptamer AP1 and the nucleic acid aptamer AP2 are both biotin-labeled modified nucleic acid aptamers that can specifically recognize anti-gluten antibodies, wherein the nucleic acid sequences of the nucleic acid aptamer AP1 and the nucleic acid aptamer AP2 are shown in SEQ ID NO.1-2, respectively.

4. The test paper card as claimed in claim 3, characterized in that: The bottom plate is a PVC plate, and the sample pad is a polyacetic acid film.

5. The test paper card as claimed in claim 3, characterized in that: The combination pad is glass fiber, and the water-absorbing pad is water-absorbing filter paper.

6. The test paper card as claimed in claim 3, characterized in that: The test paper card is packaged by a hard plastic card.

7. The method for preparing the test paper card according to any one of claims 3 to 6, characterized in that: The method comprises the following steps: 1) Preparation of the conjugate pad: The conjugate pad was immersed in an aqueous solution containing 15wt% trehalose, 5wt% BSA and 0.5wt% Tween-20, and then dried for use; the Au@AP2 solution was diluted and evenly sprayed on the conjugate pad, and then dried to prepare the conjugate pad for use; 2) Preparation of sample pad: soak the sample pad in an aqueous solution containing 0.5 wt% Tween-20 and 5 wt% PEG30000, and dry it for later use; 3) Preparation of nitrocellulose membrane: Spray 5 mg / mL streptavidin onto the detection line and quality control line of the nitrocellulose membrane respectively. After drying, spray the nucleic acid aptamer AP1 onto the detection line of the nitrocellulose membrane. Then continue to dry the nitrocellulose membrane for later use. 4) Assembly of the test strip: Connect the sample pad, conjugate pad, chromatographic membrane and absorbent pad on the PVC base plate in sequence to obtain a test strip, with the detection line set at one end close to the conjugate pad and the quality control line set at the other end close to the absorbent pad.

8. The detection method of the test paper card according to any one of claims 3 to 6, characterized in that: The detection method comprises the following steps: The sample to be tested is added to the sample spotting hole, and the sample to be tested is chromatographed along the sample pad, the conjugate pad, the nitrocellulose membrane toward the absorbent filter paper end under the capillary action; During the chromatography process, the test substance in the sample to be tested first specifically binds to the Au@AP2 on the glass fiber binding pad and re-dissolves it, and the re-dissolved mixture continues to be chromatographed onto the nitrocellulose membrane; It reacts with the nucleic acid aptamer AP1 coated in the detection line area on the nitrocellulose membrane, and under the action of a certain concentration of the detection substance, a line of corresponding color appears; The excess Au@AP2 continues to move forward, and when the biotin labeled on AP2 reaches the quality control line area, it combines with the coated streptavidin, and the nanoparticles are enriched, showing the color line of the nanoparticles; If the analyte is not present in the sample, Au@AP2 and the aptamer AP1 will not react in the detection line area, but continue to chromatograph forward through the bio-streptavidin reaction, so the detection line is colorless, while the quality control line shows the color of the nanoparticles; the method includes applications for non-diagnostic purposes.

9. A human anti-gluten antibody detection kit, characterized in that: The kit comprises the test paper card according to any one of claims 3 to 6.

10. Use of the test card according to any one of claims 3 to 6, the test strip for detecting human anti-gluten antibodies, or the human anti-gluten antibody kit in the preparation of an early diagnosis product for celiac disease.

Images