Kit for detecting anti-LL37 IgG antibody and application thereof

Abstract

The invention relates to a kit for detecting an anti-LL37IgG antibody and application of the kit. The kit comprises a reagent A, a reagent B and a reagent C, wherein the reagent A is LL37 protein coated with magnetic particles; the reagent B is an IgG antibody marked by acridinium ester; and a diluent. The kit can be used for rapidly detecting the level condition of the anti-LL37 antibody in a psoriasis patient, and a novel detection method which is short in time consumption, low in cost and small in harm is provided for diagnosis of psoriasis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, in particular to a kit for detecting anti-LL37 IgG antibodies and application thereof. Background Art

[0002] Studies have found that the antimicrobial peptide (LL37) is a cationic peptide that plays a role in the antimicrobial defense of damaged skin. LL37 forms a complex with extracellular self-nucleic acids. LL37 molecules can bind to nucleic acid molecules and stimulate plasmacytoid / myeloid dendritic cells to secrete type I interferon (IFN-I) and other pro-inflammatory factors. At the same time, LL37 has also been confirmed to be an autoantigen that can activate psoriasis-specific T cell acquired immunity. Anti-LL37 antibodies (anti-LL37) are present in the plasma and synovial fluid of psoriasis patients and are highly correlated with the disease activity of psoriasis. Studies have found that anti-LL37 antibodies are highly correlated with the onset of psoriasis, and the level of LL37 autoantibodies is of great significance and value for the judgment of disease activity and the evaluation of therapeutic efficacy.

[0003] Chemiluminescence immunoassay (CLIA) is a technique that combines highly sensitive chemiluminescence assay technology with highly specific immune reactions and is used for the detection and analysis of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins and drugs. It is the latest immunoassay technology developed after radioimmunoassay, enzyme immunoassay, fluorescence immunoassay and time-resolved fluorescence immunoassay. At present, it has not been found that LL37 protein can be combined with chemiluminescence immunoassay technology (especially magnetic microparticle chemiluminescence technology) for the rapid detection of anti-LL37 antibodies in psoriasis patients. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a kit for detecting anti-LL37 IgG antibodies and its application. By combining LL37 protein with magnetic particle chemiluminescence technology, it is used to quickly detect the level of anti-LL37 antibodies in the serum of psoriasis patients, providing a time-saving, low-cost and less harmful detection method for the diagnosis of psoriasis.

[0005] To this end, the present invention adopts the following technical solution:

[0006] In one aspect, the present invention provides a kit for detecting anti-LL37 IgG antibodies, comprising:

[0007] Reagent A, which is LL37 protein coated with magnetic microparticles;

[0008] Reagent B, which is an acridinium ester-labeled IgG antibody;

[0009] diluent.

[0010] Preferably, the concentration of the reagent A is 10 μg / mL; the concentration of the reagent B is 0.02 μg / mL; and the volume ratio of the reagent A, reagent B and diluent is 1:5:5.

[0011] Preferably, the IgG antibody is Qrinia human IgG. Specifically, the Qrinia human IgG was purchased from Zhuhai Qinia Biotechnology Co., Ltd.; English name: Human-IgG; Product number: H01101; Type: Mab; Source: Mouse.

[0012] Preferably, the diluent contains components in the following proportions: 12.12 g of Tris (tromethamine), 7.6 mL of HCl, 18 g of NaCl, 2 mL of Tween-20 (Tween-20), 20 g of BSA (bovine serum albumin), and 2 mL of Proclin300.

[0013] In at least one embodiment, the preparation method of the reagent A comprises the following steps:

[0014] S11, take a centrifuge tube, add 900 μL magnetic bead coating solution and 100 μL magnetic bead stock solution, mix well and separate with a magnet, discard the supernatant for later use;

[0015] S12, add 900 μL of magnetic bead coating solution and 100 μL of DEC solution to the centrifuge tube, mix by ultrasonic at 18-28℃ for 0.5h-1h, separate with a magnet, and discard the supernatant for later use;

[0016] S13, add 900 μL magnetic bead coating solution and 25 μL pre-coated 4 mg / mL LL37 protein solution to the centrifuge tube, mix by ultrasonic at 18-28°C for 0.5h-1h, separate with a magnet, and discard the supernatant for later use;

[0017] S14, add 900 μL magnetic bead blocking solution to the centrifuge tube, mix by ultrasonic at 18-28°C for 0.5h-1h, and discard the supernatant for later use;

[0018] S15, after washing, separate with a magnet and discard the supernatant, repeat this step four times to obtain LL37 magnetic beads;

[0019] S16, diluting the LL37 magnetic beads with magnetic bead diluent to obtain the reagent A.

[0020] Furthermore, the magnetic bead coating solution is a PBS buffer solution containing 1% to 5% PEG (polyethylene glycol) by volume;

[0021] The magnetic bead stock solution is Agilent magnetic bead stock solution;

[0022] The magnetic bead blocking solution is a Tris buffer containing a protein stabilizer at a concentration of 1 to 5 mg / mL;

[0023] The cleaning solution used for the washing is morpholine sulfonic acid with a concentration of 0.05 mol / L and a pH of 6.0;

[0024] The magnetic bead diluent contains the following components: 0.05 mol / L Tris, 3% BSA, 0.05% Triton X-100, 5% trehalose, 1% NaCl, and 0.2% Proclin 300.

[0025] In at least one embodiment, the preparation method of the reagent B comprises the following steps:

[0026] S21, weighing IgG antibody and acridinium ester in a mass ratio of 2:1, mixing, oscillating and coupling for 0.5 h, removing free acridinium ester by dialysis, purifying by gel chromatography column, collecting effluent with high luminescence intensity, and obtaining a cross-linked luminescent reagent;

[0027] S22, diluting the cross-linked luminescent reagent with a luminescent diluent to obtain the reagent B.

[0028] Furthermore, the luminescent diluent comprises the following components: 0.01 mol / L PBS buffer, 0.05% polyethylene glycol octylphenyl ether, 0.2% casein, and 0.5% Proclin30.

[0029] On the other hand, the present invention also provides the use of the above-mentioned kit or the reagents contained therein in the preparation of a product for diagnosing psoriasis.

[0030] Compared with the prior art, the present invention achieves at least the following beneficial effects: the present invention can be used for auxiliary diagnosis of psoriasis by qualitatively detecting the level of serum anti-LL37 IgG antibodies, and the present invention can be used in the form of a kit, and can quickly perform chemiluminescent immunoassay, thereby reducing medical costs and improving the applicability of clinical testing, providing a new time-saving, low-cost, and less harmful inspection method for the screening of psoriasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Embodiments and / or experimental examples of the present invention will now be described by way of example only with reference to the accompanying drawings, in which:

[0032] Figure 1 The statistical results of luminescence values ​​obtained by using different IgG antibodies;

[0033] Figure 2The luminescence value statistics are obtained by using Qrinia human IgG antibody (Orinia IgG-2#). DETAILED DESCRIPTION

[0034] The present invention will be described in detail below with reference to the exemplary embodiments in the accompanying drawings. However, it should be understood that the present invention can be implemented in a variety of different forms and should not be construed as being limited to the embodiments described herein. These embodiments are provided herein to make the disclosure of the present invention more complete and to fully convey the concept of the present invention to those skilled in the art.

[0035] First, the present invention provides a kit for detecting anti-LL37 IgG antibodies, comprising:

[0036] Reagent A, which is LL37 protein coated with magnetic microparticles;

[0037] Reagent B, which is an acridinium ester-labeled IgG antibody;

[0038] diluent.

[0039] Among them, the concentration of the reagent A is 10 μg / mL; the concentration of the reagent B is 0.02 μg / mL; the volume ratio of the reagent A, reagent B and diluent is 1:5:5.

[0040] Wherein, the IgG antibody is Qrinia human IgG (Zhuhai Qinia Biotechnology Co., Ltd., product number H01101).

[0041] The diluent contains the following components: 12.12 g of Tris, 7.6 mL of HCl, 18 g of NaCl, 2 mL of Tween-20, 20 g of BSA, and 2 mL of Proclin300.

[0042] The methods of magnetic particle coating and acridinium ester labeling are both conventional techniques in the art. The present invention also provides two embodiments for specifically describing the preparation methods of reagent A and reagent B.

[0043] In one embodiment, the preparation method of the reagent A comprises the following steps:

[0044] S11, take a centrifuge tube, add 900 μL magnetic bead coating solution and 100 μL magnetic bead stock solution, mix well and separate with a magnet, discard the supernatant for later use;

[0045] S12, add 900 μL magnetic bead coating solution and 100 μL DEC solution to the centrifuge tube, mix by ultrasonic at 18-28℃ for 0.5h-1h, separate with a magnet, and discard the supernatant for later use;

[0046] S13, add 900 μL magnetic bead coating solution and 25 μL pre-coated 4 mg / mL LL37 protein solution to the centrifuge tube, mix by ultrasonic at 18-28°C for 0.5h-1h, separate with a magnet, and discard the supernatant for later use;

[0047] S14, add 900 μL magnetic bead blocking solution to the centrifuge tube, mix by ultrasonic at 18-28°C for 0.5h-1h, and discard the supernatant for later use;

[0048] S15, after washing, separate with a magnet and discard the supernatant, repeat this step four times to obtain LL37 magnetic beads;

[0049] S16, diluting the LL37 magnetic beads with magnetic bead diluent to obtain the reagent A.

[0050] Wherein, the magnetic bead coating solution is a PBS buffer containing a volume fraction of 1% to 5% PEG. The magnetic bead stock solution is an Agilent magnetic bead stock solution. The magnetic bead blocking solution is a Tris buffer containing a protein stabilizer at a concentration of 1 to 5 mg / mL; the protein stabilizer contains one or more of BSA, TritonX-100, and Proclin300. In this embodiment, the weight ratio of the three is BSA:TritonX-100:Proclin300=2:0.05:0.5. The cleaning solution used for washing is morpholine sulfonic acid with a concentration of 0.05 mol / L and a pH of 6.0. The magnetic bead diluent contains the following components: 0.05 mol / L Tris, 3% BSA, 0.05% TritonX-100, 5% trehalose, 1% NaCl, and 0.2% Proclin300.

[0051] In one embodiment, the preparation method of the reagent B comprises the following steps:

[0052] S21, weighing IgG antibody and acridinium ester in a mass ratio of 2:1, mixing, oscillating and coupling for 0.5 h, removing free acridinium ester by dialysis, purifying by gel chromatography column, collecting effluent with high luminescence intensity, and obtaining a cross-linked luminescent reagent;

[0053] S22, diluting the cross-linked luminescent reagent with a luminescent diluent to obtain the reagent B.

[0054] The luminescent diluent contains the following components: 0.01 mol / L PBS buffer, 0.05% polyethylene glycol octylphenyl ether, 0.2% casein, and 0.5% Proclin30.

[0055] The above kit can be applied to the diagnosis of psoriasis. In order to verify its effect, the present invention also provides the following experimental examples:

[0056] 1. General Information

[0057] Serum samples from 33 healthy subjects and 33 psoriasis patients were collected from the Laboratory Department of Guangxi Zhuang Autonomous Region People's Hospital.

[0058] 2. Instruments and reagents

[0059] anti-H-IgG-8# (Holmes Biotechnology Co., Ltd., catalog number HM085L); anti-H-IgG-2# (Holmes Biotechnology Co., Ltd., catalog number HM090A); Orinia IgG-2# (Zhuhai Qiniya Biotechnology Co., Ltd., catalog number H01101); Orinia IgG-1# (Zhuhai Qiniya Biotechnology Co., Ltd., catalog number H01001); Tansheng mouse anti-human IgG (Chongqing Tansheng Technology Co., Ltd., catalog number FAB-S012-3B5); Shuojing anti-human IgG-1# (Qingdao Shuojing Biotechnology Co., Ltd., catalog number M1301); Shuojing anti-human IgG-2# (Qingdao Shuojing Biotechnology Co., Ltd., M1304); LL37 (Shanghai Hongpeptide Biotechnology Co., Ltd.); fully automatic chemiluminescence analyzer (Nanjing Renmai Biotechnology Co., Ltd., model MCL60).

[0060] 3. Detection method

[0061] 3.1. Preparation of Reagent A

[0062] The preparation of reagent A was carried out by referring to the above example, and finally 10 μg / mL of reagent A was obtained and stored in a refrigerator at 4°C for future use.

[0063] 3.2. Preparation of IgG antibody-acridinium ester luminescent reagent

[0064] The preparation of IgG antibody-acridinium ester luminescent reagent refers to the above example. Finally, different IgG antibodies labeled with acridinium ester (anti-H-IgG-8#0.05mg / mL, anti-H-IgG-2#0.05mg / mL, Orinia IgG-2#0.05mg / mL, Orinia IgG-1#0.05mg / mL, Tansheng mouse anti-human IgG 0.05mg / mL, Shuojing anti-human IgG-1#0.05mg / mL, Shuojing anti-human IgG-2#0.05mg / mL) were diluted to a concentration of 0.02μg / mL and stored in a 4°C refrigerator for later use.

[0065] 3.3 Detection steps

[0066] 1) Draw 3 mL of peripheral blood from healthy subjects and psoriasis patients, centrifuge to obtain serum, and freeze at -80°C for testing;

[0067] 2) Pick 3 healthy human sera and psoriasis sera respectively, take out the reaction cup and mark it, add 100 μL of diluent, 20 μL of serum and 20 μL of reagent A in sequence, mix well and incubate at 37°C for 14 minutes, discard the supernatant with a magnet, add 100 μL of IgG antibody-acridinium ester luminescent reagent, mix well and incubate for 14 minutes, discard the supernatant with a magnet, put the reaction cup into the fully automatic chemiluminescence analyzer to detect the luminescence value, the result is as follows Figure 1 , select suitable IgG antibodies to continue to expand sample testing;

[0068] 3) Reagent B prepared with Orinia IgG-2# (0.02 μg / mL) and reagent A, 33 healthy human sera and 33 psoriasis sera were used for testing. The testing process was referred to step 2). The results are as follows: Figure 2 .

[0069] 4. Results Analysis

[0070] Figure 1 It shows that compared with other IgG antibodies, the test results of Orinia IgG-2# (i.e. Qrinia human IgG, product number H01101) can better distinguish the autoantibody levels of psoriasis patients and healthy people;

[0071] Figure 2 The secondary antibody used was Orinia IgG-2# (i.e., Qrinia human IgG, catalog number H01101). The T test in GraphPad software was used for analysis, which showed that there was a significant difference in the anti-LL37 IgG antibody levels in the serum of healthy people and psoriasis patients, i.e., P<0.0001;

[0072] Result analysis: LL37 was combined with agilent magnetic particles and secondary antibodies to detect anti-LL37 IgG antibodies in human and psoriasis patient sera. The means of the two groups of data were compared by T test, and it was found that the level of anti-LL37 IgG antibodies in psoriasis patients was higher than that in healthy people, which can provide certain value and significance for the diagnosis of psoriasis and the evaluation of therapeutic effect.

[0073] It should be understood that all the above embodiments are illustrative rather than restrictive, and various modifications or variations made by those skilled in the art to the specific embodiments described above under the concept of the present invention should be within the protection scope of the present invention.

Claims

1. A kit for detecting anti-LL37 IgG antibodies, characterized in that: include: Reagent A, which is LL37 protein coated with magnetic microparticles; Reagent B, which is an acridinium ester-labeled IgG antibody; diluent.

2. The kit according to claim 1, characterized in that: The concentration of the reagent A is 10 μg / mL; The concentration of the reagent B is 0.02 μg / mL; The volume ratio of the reagent A, reagent B and diluent is 1:5:

5.

3. The kit according to claim 1, characterized in that: The IgG antibody is Qrinia human IgG.

4. The kit according to claim 1, characterized in that The diluent contains the following components: 12.12 g of Tris, 7.6 mL of HCl, 18 g of NaCl, 2 mL of Tween-20, 20 g of BSA, and 2 mL of Proclin300.

5. The kit according to claim 1, characterized in that: The preparation method of the reagent A comprises the following steps: S11, take a centrifuge tube, add 900 μL magnetic bead coating solution and 100 μL magnetic bead stock solution, mix well and separate with a magnet, discard the supernatant for later use; S12, add 900 μL of magnetic bead coating solution and 100 μL of DEC solution to the centrifuge tube, mix by ultrasonic at 18-28℃ for 0.5h-1h, separate with a magnet, and discard the supernatant for later use; S13, add 900 μL magnetic bead coating solution and 25 μL pre-coated 4 mg / mL LL37 protein solution to the centrifuge tube, mix by ultrasonic at 18-28°C for 0.5h-1h, separate with a magnet, and discard the supernatant for later use; S14, add 900 μL magnetic bead blocking solution to the centrifuge tube, mix by ultrasonic at 18-28°C for 0.5h-1h, and discard the supernatant for later use; S15, after washing, separate with a magnet and discard the supernatant, repeat this step four times to obtain LL37 magnetic beads; S16, diluting the LL37 magnetic beads with magnetic bead diluent to obtain the reagent A.

6. The kit according to claim 5, characterized in that: The magnetic bead coating solution is a PBS buffer solution containing 1% to 5% PEG by volume; The magnetic bead stock solution is Agilent magnetic bead stock solution; The magnetic bead blocking solution is a Tris buffer containing a protein stabilizer at a concentration of 1 to 5 mg / mL.

7. The kit according to claim 5, characterized in that: The cleaning solution used for the washing is morpholine sulfonic acid with a concentration of 0.05 mol / L and a pH of 6.0; The magnetic bead diluent contains the following components: 0.05 mol / L Tris, 3% BSA, 0.05% TritonX-100, 5% trehalose, 1% NaCl, and 0.2% Proclin300.

8. The kit according to claim 1, characterized in that The preparation method of the reagent B comprises the following steps: S21, weighing IgG antibody and acridinium ester in a mass ratio of 2:1, mixing, oscillating and coupling for 0.5 h, removing free acridinium ester by dialysis, purifying by gel chromatography column, collecting effluent with high luminescence intensity, and obtaining a cross-linked luminescent reagent; S22, diluting the cross-linked luminescent reagent with a luminescent diluent to obtain the reagent B.

9. The kit according to claim 8, characterized in that: The luminescent diluent comprises the following components: 0.01 mol / L PBS buffer, 0.05% polyethylene glycol octylphenyl ether, 0.2% casein, 0.5% Proclin30.

10. Use of the kit according to any one of claims 1 to 9 or the reagents contained therein in the preparation of a product for diagnosing psoriasis.

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