An anti-ll37 antibody detection kit

Abstract

The application provides an anti-LL37 antibody detection kit, which comprises a magnetic particle-carbamylated LL37 protein complex suspension, a mixed solution of biotin-PEGn-carbamylated LL37 protein and alkaline phosphatase coupled with streptavidin and a chemiluminescent substrate solution, wherein the sequence of the carbamylated LL37 protein is: {L-HomoCit}LGDFFR{K-HomoCit}S{K-HomoCit}E{K-HomoCit}IGKEF{K-HomoCit}RIVQRI{K-HomoCit}DFLRNLVP RTES. The application is based on a double antigen sandwich method, and the capture efficiency of the anti-LL37 antibody in serum is improved by designing the sequences of the coated antigen and the labeled antigen, so that the LL37 antibody in serum can be detected rapidly and efficiently.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of in vitro diagnosis, and particularly relates to an anti-LL37 antibody detection kit. BACKGROUND

[0002] Psoriasis is a kind of immune-mediated chronic inflammatory skin disease mainly manifested as skin erythema and scales. The disease has the characteristics of long course, easy recurrence and lifelong non-healing in some patients, and has a great impact on the physical health and mental state of patients.

[0003] In clinical practice, there is also a lack of treatment plan for curing psoriasis. Although the conventional clinical treatment for psoriasis can relieve the symptoms of patients, long-term medication may also cause significant side effects. Therefore, how to better detect specific biological markers to realize the disease progression judgment, recurrence monitoring, and specific drug treatment effect evaluation for psoriasis patients has become a key requirement in clinical practice.

[0004] Existing studies have shown that LL37 has a pro-inflammatory effect in psoriasis, LL37 binds to nucleic acids and stimulates plasma cells / myeloid dendritic cells and secretes type I interferon (IFN-I) and pro-inflammatory factors, becomes one of the target antigens of psoriasis, and specific autoantibodies such as anti-LL-37 antibody have a high correlation with the onset of psoriasis.

[0005] For the clinical diagnosis of psoriasis, the characteristics of skin rash are mainly relied on, combined with medical history data, including the onset, evolution, and growth and decline rules, accompanying symptoms, treatment response, etc., and if necessary, skin lens, imaging technology and other auxiliary examinations are also needed to help confirm the diagnosis. At present, there is no commercialized clinical detection kit for anti-LL-37 antibody at home and abroad, so the practical application of this project in clinical practice is also limited. In the relevant reports of the existing clinical research on the correlation between anti-LL-37 antibody and psoriasis, the detection methods used are all traditional ELISA or indirect immunofluorescence method. For the LL37 antigen with low content in serum and difficult to detect, these methods all have shortcomings.

[0006] The basic principle of indirect immunofluorescence method is that specific antibodies in serum samples specifically bind to antigens in the section, then an indirect fluorescent antibody is used to bind to the antigen-antibody complex to form an antigen-antibody fluorescent complex. Under the fluorescence microscope, the detection result is determined according to the luminescence of the complex. Since the increase of the fluorescent antibody combined in the antigen-antibody complex, the fluorescence brightness is strong, and thus the sensitivity is strong. However, its disadvantages are also obvious: (1) false positives may occur when using this method; (2) the non-specific reaction cannot be distinguished according to the size of the molecular weight when analyzing the result; (3) the operation is relatively complex, and a relatively expensive fluorescence microscope is needed, which is difficult to popularize in many primary hospitals, and is not suitable for laboratories and diagnostic hospitals with a large sample size; (4) the background in fluorescence determination is high, and it is difficult to use fluorescence immunoassay for quantitative determination; (5) the result needs to be determined by experienced professionals, and the objectivity of the analysis result is insufficient.

[0007] The detection of anti-LL37 antibody by enzyme-linked immunosorbent assay (ELISA) is limited due to the deficiencies of technology, tools or products, which mainly include the following aspects: (1) the detection reagent is in an open mode during the detection process, which is easily affected by the external environment, and is also easy to cause cross contamination between various reagents and affect the detection result; (2) the detection range and sensitivity of ELISA are relatively low; (3) the detection time of ELISA method is relatively long, and it generally takes more than 2 hours to complete a test, which cannot completely meet the needs of rapid diagnosis in clinic. SUMMARY

[0008] The technical problem solved by the present application is to overcome the deficiencies of the prior art, and to provide a kit for efficiently detecting anti-LL-37 antibody in a serum sample.

[0009] To solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0010] The present application provides a kind of anti-LL37 antibody detection kit, and the anti-LL37 antibody detection kit includes first reagent, second reagent and chemiluminescent substrate liquid.

[0011] The first reagent is a magnetic particle-carbamylated LL37 protein complex suspension, and the sequence of carbamylated LL37 protein in the complex is: {L-HomoCit}LGDFFR{K-HomoCit}S{K-HomoCit}E{K-HomoCit}IGKEF{K-HomoCit}RIVQRI{K-HomoCit}DFLRNLVPRTES.

[0012] The second reagent is a mixed solution of biotin-PEGn-carbamoylated LL37 protein and alkaline phosphatase coupled with streptavidin, and the sequence of the biotin-PEG6-carbamoylated LL37 protein is: Biotin-PEGn-{L-HomoCit}LGD FFR{K-HomoCit}S{K-HomoCit}E{K-HomoCit}IGKEF{K-HomoCit}RIVQRI{K-HomoCit}DFL RNLVPRTES.

[0013] The chemiluminescent substrate solution is an alkaline phosphatase chemiluminescent substrate solution.

[0014] Preferably, the PEGn in the biotin-PEGn-carbamoylated LL37 protein is PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9 or PEG10.

[0015] Preferably, the solvent of the first reagent is a buffer solution with a pH value of 5.5-6.5, which contains 40-60 mM MES, 100-200 mM NaCl, 0.05%-0.2% surfactant, 0.05%-0.2% preservative and 1%-3% bovine serum albumin.

[0016] Further preferably, the solvent of the first reagent is a buffer solution with a pH value of 5.8-6.2, which contains 45-55 mM MES, 120-170 mM NaCl, 0.08%-0.15% surfactant, 0.08%-0.15% preservative and 1.5%-2.5% bovine serum albumin.

[0017] Preferably, the solvent of the second reagent is a buffer solution with a pH value of 5.5-6.5, which contains 40-60 mM MES, 0.5-2 mM MgCl2, 0.01-0.1 mM ZnCl2, 100-200 mM NaCl, 0.05%-0.2% surfactant, 0.05%-0.2% preservative and 1%-3% bovine serum albumin.

[0018] Further preferably, the solvent of the second reagent is a buffer solution with a pH value of 5.8-6.2, which contains 45-55 mM MES, 0.8-1.5 mM MgCl2, 0.03-0.07 mM ZnCl2, 120-170 mM NaCl, 0.08%-0.15% surfactant, 0.08%-0.15% preservative and 1.5%-2.5% bovine serum albumin.

[0019] Preferably, the magnetic beads in the complex are carboxyl magnetic beads.

[0020] Preferably, the particle size of the magnetic beads in the complex is 0.5-5 μm, for example 0.5 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm.

[0021] Preferably, the concentration of the complex in the first reagent is 0.1-0.5 mg / mL, for example 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL.

[0022] Preferably, the concentration of the biotin-PEGn-carbamoylated LL37 protein in the second reagent is 0.04-0.1 μg / mL, for example 0.04 μg / mL, 0.05 μg / mL, 0.06 μg / mL, 0.07 μg / mL, 0.08 μg / mL, 0.09 μg / mL, 0.1 μg / mL.

[0023] Preferably, the concentration of the alkaline phosphatase coupled with streptavidin is 0.1-0.5 μg / mL, for example 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, 0.5 μg / mL.

[0024] Preferably, the alkaline phosphatase chemiluminescent substrate is AMPPD.

[0025] Preferably, the surfactant is one or more of PVA, Tween-20, Triton X-100.

[0026] Preferably, the preservative is one or more of Proclin-300, Proclin-950, BND-10.

[0027] Preferably, the anti-LL37 antibody detection kit further comprises an anti-LL37 antibody standard and / or an anti-LL37 antibody quality control.

[0028] Compared with the prior art, the present application has the following advantages:

[0029] The present application is based on a double antigen sandwich method, and through the design of the sequences of the coated antigen and the labeled antigen, the capture efficiency of the anti-LL37 antibody in the serum is improved, and the probability of positive missed detection is reduced. Further, through the optimization of the formulation of the reagent, the accuracy of the detection result is further improved. Compared with the existing detection method, the present application has better detection effect, is faster and lower in cost, is suitable for large-scale industrial production, and is applied to the detection of the anti-LL37 antibody in the serum of patients, and is expected to realize the judgment of the disease progression of psoriasis patients, the monitoring of the recurrence, the evaluation of the treatment effect of specific drugs, and the like, and has important significance and value for the judgment of the disease activity and the evaluation of the curative effect. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 A schematic diagram of the detection method of the kit of Example 1;

[0031] Figure 2 A HPLC purity detection result diagram of the chemically synthesized carbamoylated LL37 protein;

[0032] Figure 3 A HPLC purity detection result diagram of the chemically synthesized biotin-PEG6-carbamoylated LL37 protein;

[0033] Figure 4 A HPLC purity detection result diagram of the chemically synthesized citrullinated LL37 protein;

[0034] Figure 5 A HPLC purity detection result diagram of the chemically synthesized biotin-PEG6-citrullinated LL37 protein. DETAILED DESCRIPTION

[0035] Previous studies have shown that the existing method has the following problems in detecting LL37 antibodies in serum: (1) LL37 antigen contains only 37 amino acids, which is small in volume and not easy to be captured by antibodies after being coated on the carrier; (2) the content of anti-LL37 antibody in serum is low, and the affinity with LL37 antigen is not strong; (3) the existing technology uses mammalian cells to express LL37 antigen, which is high in cost, long in time, and high in risk of purification. In order to solve the above problems, the inventors of the present application, based on the chemiluminescence immunoassay (CLIA) method, started from the aspects of antigen treatment and reagent component adjustment, and after a large number of research and experimental verification, developed the kit of the present application.

[0036] Specifically, the principle of the detection method of the present application is shown in Figure 1 .

[0037] The present application adopts a chemical synthesis method to synthesize a LL37 protein with site-specific carbamoylation as a coated antigen, and the sequence is as follows:

[0038] {L-HomoCit}LGDFFR{K-HomoCit}S{K-HomoCit}E{K-HomoCit}IGKEF{K-HomoCit}RIVQRI{K-HomoCit}DFLRNLVPRTES;

[0039] The biotin-PEGn-carbamylated LL37 protein is mixed with alkaline phosphatase coupled with streptavidin in different proportions as a labeled antigen.

[0040] Compared with the prior art, the application has better detection effect, is faster and low in cost, is suitable for large-scale industrial production, and is applied to detection of anti-LL37 antibody in patient serum, so as to realize disease process judgment, recurrence monitoring and specific drug treatment effect evaluation for psoriasis patients, and has important significance and value for disease activity judgment and curative effect evaluation.

[0041] The application will be further described below in combination with examples. However, the application is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to different requirements of specific use, and the implementation conditions not mentioned are conventional conditions in the industry. The technical features involved in each embodiment of the application can be combined with each other as long as there is no conflict.

[0042] In the following examples and comparative examples, the implementation conditions not mentioned are conventional conditions in the industry, and the reagents not mentioned are commercial products. The instrument used for detection is a chemiluminescence immunoassay analyzer.

[0043] The carbamylated LL37 protein used in the following examples and comparative examples is chemically synthesized by Gilsen Biochemical (Shanghai) Co., Ltd., and the HPLC purity detection result is shown in Table 1. Figure 2 The sequence is: {L-HomoCit}LGDFFR{K-HomoCit}S{K-HomoCit}E{K-HomoCit}IGKEF{K-HomoCit}RIVQRI{K-HomoCit}DFLRNLVPRTES.

[0044] The biotin-PEG6-carbamylated LL37 protein used is chemically synthesized by Gilsen Biochemical (Shanghai) Co., Ltd., and the HPLC purity detection result is shown in Table 2. Figure 3 The sequence is: Biotin-PEG6-{L-HomoCit}LGDFFR{K-HomoCit}S{K-HomoCit}E{K-HomoCit}IGKEF{K-HomoCit}RIVQRI{K-HomoCit}DFLRNLVPRTES.

[0045] The citrullinated LL37 protein used was chemically synthesized by GenScript (Shanghai) Co., Ltd., and the HPLC purity test results are shown in Table 1. Figure 4 The sequence is: LLGDFF(cit)KSKEKIGKEFK(cit)IVQ(cit)IKDFL(cit)NLVP(cit)TES.

[0046] The biotin-PEG6-citrullinated LL37 protein used was chemically synthesized by GenScript (Shanghai) Co., Ltd., and the HPLC purity test results are shown in Table 2. Figure 5 The sequence is: Bio-PEG6-LLGDFF(cit)KSKEKIGKEFK(cit)IVQ(cit)IKDFL(cit)NLVP(cit)TES.

[0047] Example 1

[0048] This example provides a magnetic microparticle chemiluminescence kit (double antigen sandwich method) for detecting anti-LL37 antibody, which comprises a first reagent and a second reagent, and the preparation method is as follows:

[0049] (1) Preparation of the first reagent:

[0050] The first reagent is a magnetic particle-carbamylated LL37 protein complex suspension liquid, which is prepared as follows: 10 mg of magnetic particles (particle size 1.5 μm, carboxyl magnetic beads) are taken in a 1.5 mL centrifuge tube, 1 mL of MES buffer (10 mM MES, pH 6.0) is added, and the mixture is mixed with a vortex mixer, and then the centrifuge tube is placed on a magnetic stand for about 1 minute to remove the supernatant; 1 mL of MES buffer is added for cleaning, and the mixture is mixed and shaken, then centrifuged and the supernatant is removed by magnetic separation, and the above steps are repeated three times; 850 μL of MES buffer (10 mM, pH 6.0) is added, and chemically synthesized carbamylated LL37 protein (sequence: {L-HomoCit}LGDFFR{K-HomoCit}S{K-HomoCit}E{K-HomoCit}IGKEF{K-HomoCit}RIVQRI{K-HomoCit}DFLRNLVPRTES) is added at an amount of 2 wt%, and then 50 μL of an EDTA solution (10 mg / mL) is added and mixed with a vortex mixer, and then the mixture is rotated and mixed at room temperature for 4 h; 1 mL of a Tris blocking agent solution (50 mM Tris, 150 mM NaCl, 0.1% Triton X100, 0.1% ProClin-300, 2% BSA, pH 7.8) is added and the mixture is rotated and mixed at room temperature for 2 h; after the reaction is completed, the centrifuge tube is placed on a magnetic stand for about 1 min to remove the supernatant; the supernatant is removed after the magnetic beads are resuspended in the Tris blocking agent solution, and the above steps are repeated four times; 1 mL of the Tris blocking agent solution is added to resuspend the magnetic beads, and the concentration of the resuspended magnetic beads is 10 mg / mL, thereby obtaining a magnetic particle suspension coated with carbamylated LL37 antigen, which is stored at 2-8°C, and diluted with a storage solution (50 mM MES, 150 mM NaCl, 0.1% Triton X-100, 0.1% ProClin-300, 2% BSA, pH 6.0) to a concentration of 0.25 mg / mL, thereby obtaining the first reagent.

[0051] (2) Preparation of the second reagent:

[0052] The second reagent contains both chemically synthesized biotin-PEG6-carbamoylated LL37 protein (sequence: Biotin-PEG6-{L-HomoCit}LGDFFR{K-HomoCit}S{K-HomoCit}E{K-HomoCit}IGKEF{K-HomoCit}RIVQRI{K-HomoCit}DFLRNLVPRTES) and alkaline phosphatase coupled with streptavidin (SA-AP) produced in the laboratory of the company. The biotin-PEG6-carbamoylated LL37 protein and SA-AP are added to the storage solution (50 mM MES, 1 mM MgCl2, 0.05 mM ZnCl2, 150 mM NaCl, 0.1% Triton X100, 0.1% ProClin-300, 2% BSA, pH 6.0) to control the concentration of the biotin-PEG6-carbamoylated LL37 protein at 0.06 μg / mL and the concentration of the SA-AP at 0.3 μg / mL, thus obtaining the second reagent.

[0053] (3) The chemiluminescent substrate solution is AMPPD substrate solution for chemiluminescent immunoassay analyzer, from Jiangsu Haobobio Biomedicine Co., Ltd.

[0054] (4) The cleaning solution used during washing is a cleaning solution for chemiluminescent immunoassay analyzer, from Jiangsu Haobobio Biomedicine Co., Ltd.

[0055] The on-machine reaction process of the magnetic microparticle chemiluminescence kit for detecting anti-LL37 antibodies of the present embodiment is shown in Table 1.

[0056] Table 1

[0057]

[0058] Comparative Example 1

[0059] The present comparative example provides another magnetic microparticle chemiluminescence kit for detecting anti-LL37 antibodies, which has the same composition and preparation method as those of Example 1, except that the raw material chemically synthesized carbamoylated LL37 protein in the first reagent is replaced by LL37 recombinant protein expressed by HEK293 cells and purchased, with the sequence being: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (SEQ ID No: 1).

[0060] The on-machine reaction process of the magnetic microparticle chemiluminescence kit for detecting anti-LL37 antibodies of the present comparative example is the same as that of Example 1.

[0061] Comparative Example 2

[0062] The comparative example 1 provides another magnetic microparticle chemiluminescence kit for detecting anti-LL37 antibody, which has the same composition and preparation method as the example 1, except that the second reagent is different. The second reagent in the comparative example 1 is prepared by coupling alkaline phosphatase (AP) and chemically synthesized carbamylated LL37 protein (sequence: {L-HomoCit}LGDFFR{K-HomoCit}S{K-HomoCit}E{K-HomoCit}IGKEF{K-HomoCit}RIVQRI{K-HomoCit}DFLRNLVPRTES) by a method conventional in the art, and then diluting the concentration of the coupled product to 0.06 μg / mL with a storage solution (50 mM MES, 1 mM MgCl2, 0.05 mM ZnCl2, 150 mM NaCl, 0.1% Triton X100, 0.1% ProClin-300, 2% BSA, pH 6.0).

[0063] The on-machine reaction procedure of the magnetic microparticle chemiluminescence kit for detecting anti-LL37 antibody of the comparative example 1 is the same as that of the example 1.

[0064] Comparative example 3

[0065] The comparative example 1 provides another magnetic microparticle chemiluminescence kit for detecting anti-LL37 antibody, which has the same composition and preparation method as the example 1, except that the second reagent is different. The second reagent in the comparative example 1 is prepared by coupling alkaline phosphatase (AP) and chemically synthesized carbamylated LL37 protein (sequence: {L-HomoCit}LGDFFR{K-HomoCit}S{K-HomoCit}E{K-HomoCit}IGKEF{K-HomoCit}RIVQRI{K-HomoCit}DFLRNLVPRTES) by a method conventional in the art, and then diluting the concentration of the coupled product to 0.06 μg / mL with a storage solution (50 mM MES, 1 mM MgCl2, 0.05 mM ZnCl2, 150 mM NaCl, 0.1% Triton X100, 0.1% ProClin-300, 2% BSA, pH 6.0).

[0066] The on-machine reaction procedure of the magnetic microparticle chemiluminescence kit for detecting anti-LL37 antibody of the comparative example 1 is the same as that of the example 1.

[0067] Comparative example 4

[0068] The comparative example 1 provides another magnetic microparticle chemiluminescence kit for detecting anti-LL37 antibody, which has the same composition and preparation method as the example 1, except that the second reagent is different. The second reagent in the comparative example 1 is prepared by coupling alkaline phosphatase (AP) and chemically synthesized carbamylated LL37 protein (sequence: {L-HomoCit}LGDFFR{K-HomoCit}S{K-HomoCit}E{K-HomoCit}IGKEF{K-HomoCit}RIVQRI{K-HomoCit}DFLRNLVPRTES) by a method conventional in the art, and then diluting the concentration of the coupled product to 0.06 μg / mL with a storage solution (50 mM MES, 1 mM MgCl2, 0.05 mM ZnCl2, 150 mM NaCl, 0.1% Triton X100, 0.1% ProClin-300, 2% BSA, pH 6.0).

[0069] The reaction procedure of the magnetic particle chemiluminescence kit for detecting anti-LL37 antibody of the present comparative example is the same as that of Example 1.

[0070] Comparative Example 5

[0071] The present comparative example provides another magnetic particle chemiluminescence kit (indirect method) for detecting anti-LL37 antibody, which comprises a first reagent (magnetic particle- carbamylated LL37 protein complex suspension), a second reagent (anti-human IgG antibody solution labeled with alkaline phosphatase AP), and a chemiluminescence substrate solution (the same as that of Example 1).

[0072] The preparation method of the first reagent is basically the same as that of the first reagent of Example 1, except that the storage solution is different. The storage solution used in the present comparative example has the following formula: 50 mM Tris, 150 mM NaCl, 0.1% Triton X-100, 0.1% ProClin-300, 2% BSA, pH 7.8.

[0073] The preparation method of the second reagent is as follows: alkaline phosphatase AP-labeled goat anti-human IgG antibody is diluted to a concentration of 0.1 μg / mL using another storage solution (50 mM Tris, 150 mM NaCl, 0.1% Triton X-100, 0.1% ProClin-300, 2% BSA, 1 mM MgCl2, 0.05 mM ZnCl2, pH 7.8).

[0074] The reaction procedure of the magnetic particle chemiluminescence kit for detecting anti-LL37 antibody of the present comparative example is shown in Table 2.

[0075] Table 2

[0076]

[0077] Comparative Example 6

[0078] The present comparative example provides another magnetic particle chemiluminescence kit for detecting anti-LL37 antibody, which has basically the same composition and preparation method as those of Example 1, except that the storage solutions used in the preparation of the first reagent and the second reagent are different.

[0079] The storage solution used in the preparation of the first reagent is as follows: 50 mM Tris, 150 mM NaCl, 0.1% Triton X-100, 0.1% ProClin-300, 2% BSA, pH 7.8.

[0080] The storage solution used by the second reagent is: 50 mM Tris, 150 mM NaCl, 0.1% Triton X-100, 0.1% ProClin-300, 2% BSA, 1 mM MgCl2, 0.05 mM ZnCl2, pH 7.8.

[0081] The on-machine reaction process of the magnetic microparticle chemiluminescence kit for detecting anti-LL37 antibody of the comparative example is the same as that of Example 1.

[0082] The kits of Example 1 and Comparative Examples 1-6 were respectively used to detect 60 clinical positive serum samples and 30 serum samples of apparently healthy persons.

[0083] The experimental results were interpreted as follows: the maximum value of the luminescence value of the apparently healthy persons was selected as the critical value, and then the test value of the above-mentioned clinical positive samples was compared with the critical value as a semi-quantitative result interpretation. If the test value is greater than the critical value, it is interpreted as positive. The results are shown in Table 3.

[0084] Table 3

[0085]

[0086]

[0087] Table 3 shows that the kit of Example 1 detects the largest number of positive samples, and the detection time is shorter than that of ELISA detection, and the detection cost is lower than that of indirect immunofluorescence method. The kit of Example 1 is more suitable for large-scale sample rapid detection.

[0088] The above has described the present application in detail, which is intended to enable those skilled in the art to understand the content of the present application and implement it, and cannot limit the protection scope of the present application. Any equivalent changes or modifications made according to the spirit and principle of the present application should be covered within the protection scope of the present application.

Claims

1. An anti-LL37 antibody detection kit, characterized by: The anti-LL37 antibody detection kit comprises a first reagent, a second reagent and a chemiluminescent substrate solution, The first reagent is a magnetic particle-carbamylated LL37 protein complex suspension, and the sequence of the carbamylated LL37 protein in the complex is: {L-HomoCit}LGDFFR{K-HomoCit}S{K-HomoCit}E{K-HomoCit}IGKEF{K-HomoCit}RIVQRI{K-HomoCit}DFLRNLVPRTES, The second reagent is a mixed solution of biotin-PEGn-carbamylated LL37 protein and alkaline phosphatase coupled with streptavidin, and the sequence of the biotin-PEG6-carbamylated LL37 protein is: Biotin-PEGn-{L-HomoCit}LGDFFR{K-HomoCit}S{K-HomoCit}E{K-HomoCit}IGKEF{K-HomoCit}RIVQRI{K-HomoCit}DFLRNLVPRTES, The chemiluminescent substrate solution is an alkaline phosphatase chemiluminescent substrate solution.

2. The anti-LL37 antibody test kit according to claim 1, characterized in that: The PEGn in the biotin-PEGn-carbamylated LL37 protein is PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9 or PEG10.

3. The anti-LL37 antibody test kit according to claim 1, characterized by: The solvent of the first reagent is a buffer solution with a pH value of 5.5-6.5, which contains 40-60 mM MES, 100-200 mM NaCl, 0.05%-0.2% surfactant, 0.05%-0.2% preservative and 1%-3% bovine serum albumin.

4. The anti-LL37 antibody test kit according to claim 1, characterized by: The solvent of the second reagent is a buffer solution with a pH value of 5.5-6.5, which contains 40-60 mM MES, 0.5-2 mM MgCl2, 0.01-0.1 mM ZnCl2, 100-200 mM NaCl, 0.05%-0.2% surfactant, 0.05%-0.2% preservative and 1%-3% bovine serum albumin.

5. The anti-LL37 antibody test kit according to claim 1, characterized by: The magnetic beads in the complex are carboxyl magnetic beads; and / or, the particle size of the magnetic beads in the complex is 0.5-5 μm.

6. The anti-LL37 antibody test kit according to claim 1, characterized by: The concentration of the complex in the first reagent is 0.1-0.5 mg / mL.

7. The anti-LL37 antibody test kit according to claim 1, characterized by: The concentration of the biotin-PEGn-carbamylated LL37 protein in the second reagent is 0.04-0.1 μg / mL, and the concentration of the alkaline phosphatase coupled with streptavidin is 0.1-0.5 μg / mL.

8. The anti-LL37 antibody detection kit according to claim 1, characterized by: The alkaline phosphatase chemiluminescent substrate is AMPPD.

9. The anti-LL37 antibody test kit according to claim 3 or 4, characterized in that: The surfactant is one or more of PVA, Tween-20 and Triton X-100; And / or, the preservative is one or more of Proclin-300, Proclin-950 and BND-10.

10. The anti-LL37 antibody detection kit according to claim 1, characterized in that: The anti-LL37 antibody detection kit further comprises an anti-LL37 antibody standard and / or an anti-LL37 antibody quality control.

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