Anti-LL37 antibody detection kit

Abstract

The invention provides an anti-LL37 antibody detection kit, the kit comprises a magnetic particle-carbamoylated LL37 protein complex suspension, a mixed solution of biotin-PEGn-carbamoylated LL37 protein and alkaline phosphatase coupled with streptavidin, and a chemiluminescent substrate solution, the sequence of the carbamoylated LL37 protein is {L-HomoCit} LGDFFR {K-HomoCit} S {K-HomoCit} E {K-HomoCit} IGKEF {K-HomoCit} RIVQRI {K-HomoCit} DFLRNLVP RTES, and the magnetic particle-carbamoylated LL37 protein complex suspension is a magnetic particle-carbamoylated LL37 protein complex suspension. On the basis of a double-antigen sandwich method, the capture efficiency of the anti-LL37 antibody in the serum is improved by designing the sequences of the coating antigen and the labeled antigen, and the LL37 antibody in the serum is rapidly and efficiently detected.

Description

Technical Field

[0001] The invention belongs to the field of in vitro diagnosis, and specifically relates to an anti-LL37 antibody detection kit. Background Art

[0002] Psoriasis is a type of immune-mediated chronic inflammatory skin disease with clinical manifestations of skin erythema and scaling. The disease has the characteristics of long course, easy relapse and some patients may not be cured for life, which has a great impact on the physical health and mental state of patients.

[0003] In clinical practice, there is also a lack of treatment options to cure psoriasis. Although conventional clinical treatments for psoriasis can relieve patients' symptoms, long-term medication may also lead to significant side effects. Therefore, how to better detect specific biological markers to achieve disease progression judgment, relapse monitoring, and specific drug treatment effect evaluation for psoriasis patients has become a key clinical need.

[0004] Existing studies have shown that LL37 has a proinflammatory effect in lesional psoriasis. LL37 binds to nucleic acids and stimulates plasma cells / myeloid dendritic cells to secrete type I interferon (IFN-I) and proinflammatory factors, becoming one of the target antigens of psoriasis. Specific autoantibodies in the body, such as anti-LL-37 antibodies, are highly correlated with the onset of psoriasis.

[0005] The clinical diagnosis of psoriasis is mainly based on the characteristics of the rash, combined with medical history data, including the onset, evolution and growth and decline patterns, associated symptoms, treatment response, etc., and when necessary, auxiliary examinations such as dermatoscopes and imaging technology are also required to help confirm the diagnosis. At present, there are no commercial clinical detection kits for anti-LL-37 antibodies in China and internationally, which also limits the practical application of this project in clinical practice. In the relevant reports on clinical studies on the correlation between anti-LL-37 antibodies and psoriasis, the detection methods used are all traditional ELISA or indirect immunofluorescence. For the LL37 antigen, which is low in serum and difficult to detect, these methods have shortcomings.

[0006] The basic principle of indirect immunofluorescence is that after the specific antibody in the serum sample specifically binds to the antigen in the slice, an indirect fluorescent antibody is used to bind to the antigen-antibody complex to form an antigen-antibody fluorescent complex. Under a fluorescence microscope, the test result is determined based on the luminescence of the complex. As the number of fluorescent antibodies bound to the antigen-antibody complex increases, the fluorescence brightness is strong, so it has high sensitivity. However, its shortcomings are also obvious: (1) False positives may occur when using this method; (2) When analyzing the results, it is impossible to distinguish nonspecific reactions based on the molecular weight; (3) The operation is relatively complicated and requires an expensive fluorescence microscope, which is difficult to promote in many primary hospitals. At the same time, it is not suitable for laboratories and diagnostic hospitals with large sample volumes; (4) The background in fluorescence determination is high, and it is difficult to use fluorescence immunoassay technology for quantitative determination; (5) The result judgment requires experienced professionals, and the objectivity of the analysis results is insufficient.

[0007] The application of enzyme-linked immunosorbent assay (ELISA) for detecting anti-LL37 antibodies is limited due to many deficiencies in technology, tools or products. These deficiencies mainly include the following aspects: (1) The detection reagents are open during the detection process, which is not only easily affected by the external environment, but also easily causes cross-contamination between various reagents and affects the test results; (2) The detection range and sensitivity of ELISA are relatively low; (3) The detection time of the ELISA method is relatively long. It usually takes more than 2 hours to complete a test, which cannot fully meet the needs of rapid clinical diagnosis. Summary of the invention

[0008] The technical problem solved by the present invention is to overcome the deficiencies of the prior art and provide a kit capable of efficiently detecting anti-LL-37 antibodies in serum samples.

[0009] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0010] A first aspect of the present invention provides an anti-LL37 antibody detection kit, which comprises a first reagent, a second reagent and a chemiluminescent substrate solution.

[0011] The first reagent is a suspension of magnetic microparticle-carbamylated LL37 protein complex, 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.

[0012] The second reagent is a mixed solution of biotin-PEGn-carbamylated LL37 protein and alkaline phosphatase coupled with streptavidin. The sequence of biotin-PEG6-carbamylated 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-carbamylated 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 to 6.2, which contains 45 to 55 mM MES, 0.8 to 1.5 mM MgCl2, 0.03 to 0.07 mM ZnCl2, 120 to 170 mM NaCl, 0.08% to 0.15% surfactant, 0.08% to 0.15% preservative and 1.5% to 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 to 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, or 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 biotin-PEGn-carbamylated 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, and Triton X-100.

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

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

[0029] The present invention is based on the double antigen sandwich method. By designing the coating antigen and the labeling antigen sequence, the capture efficiency of the anti-LL37 antibody in the serum is improved, the probability of positive missed detection is reduced, and the accuracy of the detection result is further improved by further optimizing the formulation of the reagent. Compared with the existing detection method, the present invention has better detection effect, is faster and has low cost, and is suitable for large-scale industrial production. It is applied to the detection of anti-LL37 antibodies in the serum of patients, and is expected to achieve disease progression judgment, relapse monitoring, specific drug treatment effect evaluation, etc. for psoriasis patients, which is of great significance and value for the judgment of disease activity and the evaluation of efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0031] Figure 2 This is a graph showing the HPLC purity test results of the chemically synthesized carbamylated LL37 protein;

[0032] Figure 3 This is a graph showing the HPLC purity test results of the chemically synthesized biotin-PEG6-carbamylated LL37 protein;

[0033] Figure 4 This is a graph showing the HPLC purity test results of the chemically synthesized citrullinated LL37 protein;

[0034] Figure 5 This is a graph showing the HPLC purity test results of the chemically synthesized biotin-PEG6-citrullinated LL37 protein. DETAILED DESCRIPTION

[0035] Previous studies have shown that the existing methods have the following problems when detecting LL37 antibodies in serum: (1) LL37 antigen contains only 37 amino acids and is small in size, so it is not easy to be captured by antibodies after being coated on a carrier; (2) The content of anti-LL37 antibodies 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 costly, time-consuming, and has high purification risks. In order to solve the above problems, the inventors of this application developed the kit of the present invention based on the chemiluminescent immunoassay (CLIA) method, starting from antigen processing, reagent component adjustment, etc., after a lot of research and experimental verification.

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

[0037] The present invention adopts a chemical synthesis method to synthesize a site-specific aminoformylated LL37 protein as a coating 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 present invention adopts a chemical synthesis method to synthesize a biotin-PEGn-carbamylated LL37 protein, the sequence of which is as follows: Biotin-PEGn-{L-HomoCit}LGDFFR{K-HomoCit}S{K-HomoCit}E{K-HomoCit}IGKEF{K-HomoCit}RIVQRI{K-HomoCit}DFLRNLVPRTES. The biotin-PEGn-carbamylated LL37 protein and alkaline phosphatase coupled with streptavidin are mixed in different proportions as a labeled antigen.

[0040] Compared with the existing technology, the present invention has better detection effect, is faster and has low cost, and is suitable for large-scale industrial production. It is applied to the detection of anti-LL37 antibodies in patient serum, and is expected to achieve disease progression judgment, relapse monitoring, and specific drug treatment effect evaluation for psoriasis patients. It has important significance and value for the judgment of disease activity and the evaluation of efficacy.

[0041] The present invention is further described below in conjunction with the examples. However, the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific use, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.

[0042] In the following examples and comparative examples, the unspecified implementation conditions are conventional conditions in the industry, and the unspecified reagents are all commercially available products. The instrument used for detection is a chemiluminescent immunoassay analyzer.

[0043] The carbamylated LL37 protein used in the following examples and comparative examples was chemically synthesized by Gill Biochemical (Shanghai) Co., Ltd. The HPLC purity test results are shown in Figure 2 , and its 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 was chemically synthesized by Gill Biochemical (Shanghai) Co., Ltd. The HPLC purity test results are shown in Figure 3 , whose 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 Gill Biochemical (Shanghai) Co., Ltd. The HPLC purity test results are shown in Figure 4 , whose 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 Gill Biochemical (Shanghai) Co., Ltd. The HP LC purity test results are shown in Figure 5 , whose sequence is: Bio-PEG6-LLGDFF(cit)KSKEKIGKEFK(cit)IVQ(cit)IKD FL(cit)NLVP(cit)TES.

[0047] Example 1

[0048] This embodiment provides a magnetic particle chemiluminescence kit (double antigen sandwich method) for detecting anti-LL37 antibodies, which includes 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 suspension of magnetic microparticles-carbamylated LL37 protein complex, and the preparation method is as follows: take 10 mg of magnetic microparticles (particle size 1.5 μm, carboxyl magnetic beads) in a 1.5 mL centrifuge tube, add 1 mL of MES buffer (10 mM MES, pH 6.0), mix with a vortex mixer, let stand on a magnetic stand for about 1 minute, and remove the supernatant; add 1 mL of MES buffer to wash, oscillate to mix, centrifuge, remove the supernatant by magnetic separation, and repeat three times; add 850 μL of MES buffer (10 mM, pH 6.0), add the chemically synthesized carbamylated LL37 protein (sequence: {L-HomoCit}LGDFFR{K-HomoCit}S{K-HomoCit}E{K-HomoCit}IGKEF{K-HomoCit}RIVQRI{K-HomoCit}DFLRNLVPRTES) at an addition amount of 2wt%, add 50μL EDC solution (10mg / mL) and mix with a mixer, rotate and mix at room temperature, and react for 4h; add 1mL of Tris blocking agent solution (50mM Tris, 150mM NaCl, 0.1% TritonX100, 0.1% ProClin-300, 2% BSA, pH7.8) was rotated evenly at room temperature and reacted for 2 hours; after the reaction, the centrifuge tube was placed on a magnetic stand for about 1 minute, and the supernatant was removed; after adding a Tris blocking agent solution to resuspend the magnetic beads, the supernatant was removed, and repeated 4 times; 1 mL of Tris blocking agent solution was added to resuspend the magnetic beads, and the concentration of the magnetic beads after resuspension was 10 mg / mL, to obtain a magnetic microparticle suspension coated with carbamylated LL37 antigen, which was stored at 2-8°C and diluted to 0.25 mg / mL with a storage solution (50 mM MES, 150 mM Na Cl, 0.1% Triton X-100, 0.1% ProClin-300, 2% BSA, pH6.0), to obtain the first reagent.

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

[0052] The second reagent contains both chemically synthesized biotin-PEG6-carbamylated LL37 protein (sequence: Biotin-PEG6-{L-HomoCit}LGDFFR{K-HomoCit}S{K-HomoCit}E{K-HomoCit}IGKEF{K-HomoCit}RIVQRI{K-HomoCit}DFLRNLVPRTES) and streptavidin-coupled alkaline phosphatase (SA-AP) produced in our laboratory. Add biotin-PEG6-carbamylated LL37 protein and SA-AP to the preservation solution (50mM MES, 1mM MgCl2, 0.05mM ZnCl2, 150mM NaCl, 0.1% TritonX100, 0.1% ProClin-300, 2% BSA, pH6.0), and control the concentration of biotin-PEG6-carbamylated LL37 protein to 0.06μg / mL and the concentration of SA-AP to 0.3μg / mL to obtain the second reagent.

[0053] (3) Chemiluminescent substrate solution is AMPPD substrate solution for chemiluminescent immunoassay analyzer, which comes from Jiangsu Haooubo Biopharmaceutical Co., Ltd.

[0054] (4) The cleaning solution used for washing is the cleaning solution for the chemiluminescent immunoassay analyzer, which is from Jiangsu Hao Ou Bo Biotechnology Co., Ltd.

[0055] The reaction flow of the magnetic microparticle chemiluminescence kit for detecting anti-LL37 antibodies in this example is shown in Table 1.

[0056] Table 1

[0057]

[0058] Comparative Example 1

[0059] This comparative example provides another magnetic particle chemiluminescent kit for detecting anti-LL37 antibodies, whose composition and preparation method are basically the same as those in Example 1, except that the aminoformylated LL37 protein chemically synthesized as the raw material in the first reagent is replaced with a purchased LL37 recombinant protein expressed by HEK293 cells, the sequence of which is: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (SEQ ID No: 1).

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

[0061] Comparative Example 2

[0062] This comparative example provides another magnetic particle chemiluminescent kit for detecting anti-LL37 antibodies. Its composition and preparation method are basically the same as those in Example 1, except that the second reagent is different. The second reagent in this comparative example couples alkaline phosphatase AP with chemically synthesized aminoformylated LL37 protein (sequence: {L-HomoCit}LGDFFR{K-HomoCit}S{K-HomoCit}E{K-HomoCit}IGKEF{K-HomoCit}RIVQRI{K-HomoCit}DFLRNLVPRTES) by conventional methods in the art, and then dilutes the conjugate to a concentration of 0.06 μg / mL using a preservation solution (50 mM MES, 1 mM MgCl2, 0.05 mM ZnCl2, 150 mM NaCl, 0.1% TritonX100, 0.1% ProClin-300, 2% BSA, pH 6.0).

[0063] The reaction process of the magnetic microparticle chemiluminescence kit for detecting anti-LL37 antibodies in this comparative example is the same as that in Example 1.

[0064] Comparative Example 3

[0065] This comparative example provides another magnetic particle chemiluminescent kit for detecting anti-LL37 antibodies, whose composition and preparation method are basically the same as those in Example 1, with the only difference being that the chemically synthesized aminoformylated LL37 protein as the raw material in the first reagent is replaced with a chemically synthesized unmodified LL37 protein, the sequence of which is: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES.

[0066] The reaction process of the magnetic microparticle chemiluminescence kit for detecting anti-LL37 antibodies in this comparative example is the same as that in Example 1.

[0067] Comparative Example 4

[0068] This comparative example provides another magnetic particle chemiluminescent kit for detecting anti-LL37 antibodies, whose composition and preparation method are basically the same as those in Example 1, except that the chemically synthesized carbamylated LL37 protein as a raw material in the first reagent is replaced by a chemically synthesized citrullinated LL37 protein, the sequence of which is: LLGDFF(cit)KSKEKIGKEFK(cit)IVQ(cit)IKDFL(cit)NLVP(cit)TES, and at the same time, the chemically synthesized biotin-PEG6-carbamylated LL37 protein as a raw material in the second reagent is replaced by a chemically synthesized biotin-PEG6-citrullinated LL37 protein, the sequence of which is: Bio-PEG6-LLGDFF(cit)KSKEKIGKEFK(cit)IVQ(cit)IKDFL(cit)NLVP(cit)TES.

[0069] The reaction process of the magnetic microparticle chemiluminescence kit for detecting anti-LL37 antibodies in this comparative example is the same as that in Example 1.

[0070] Comparative Example 5

[0071] This comparative example provides another magnetic particle chemiluminescence kit (indirect method) for detecting anti-LL37 antibodies, which includes 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 chemiluminescent substrate solution (the same as the chemiluminescent substrate solution in Example 1).

[0072] The preparation method of the first reagent is basically the same as the first reagent in Example 1, the only difference is the preservation solution. The preservation solution formula used in this comparative example is: 50mM Tris, 150mM NaCl, 0.1% Triton X-100, 0.1% ProClin-300, 2% BSA, pH7.8.

[0073] The preparation method of the second reagent is: use another preservation solution (50mM Tris, 150mM NaCl, 0.1% TritonX-100, 0.1% ProClin-300, 2% BSA, 1mM MgCl2, 0.05mM ZnCl2, pH7.8) to dilute the alkaline phosphatase AP-labeled goat anti-human IgG antibody to a concentration of 0.1μg / mL.

[0074] The reaction process of the magnetic microparticle chemiluminescence kit for detecting anti-LL37 antibodies in this comparative example is shown in Table 2.

[0075] Table 2

[0076]

[0077] Comparative Example 6

[0078] This comparative example provides another magnetic particle chemiluminescent kit for detecting anti-LL37 antibodies, and its composition and preparation method are basically the same as those in Example 1, except that different storage solutions are used in preparing the first reagent and the second reagent.

[0079] The storage solution used for the first reagent is: 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 for 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 reaction process of the magnetic microparticle chemiluminescence kit for detecting anti-LL37 antibodies in this comparative example is the same as that in Example 1.

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

[0083] The method of interpreting the experimental results is to select the maximum value as the critical value through the luminescence value of the observed healthy people, and then compare the test value of the above clinical positive samples with the critical value as a semi-quantitative result. The value greater than the critical value is interpreted as positive. The results are statistically 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 rapid detection of large-scale samples.

[0088] The above detailed description of the present invention is intended to enable persons familiar with the art to understand the contents of the present invention and implement them. It does not limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. An anti-LL37 antibody detection kit, characterized in that: The anti-LL37 antibody detection kit comprises a first reagent, a second reagent and a chemiluminescent substrate solution, The first reagent is a suspension of magnetic microparticle-carbamylated LL37 protein complex, wherein 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, The second reagent is a mixed solution of biotin-PEGn-carbamylated LL37 protein and alkaline phosphatase coupled with streptavidin, and the sequence of biotin-PEG6-carbamylated 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, The chemiluminescent substrate solution is an alkaline phosphatase chemiluminescent substrate solution.

2. The anti-LL37 antibody detection 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 detection kit according to claim 1, characterized in that: 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 detection kit according to claim 1, characterized in that: 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 detection kit according to claim 1, characterized in that: 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 to 5 μm.

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

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

9. The anti-LL37 antibody detection kit according to claim 3, 4 or 8, 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 also includes an anti-LL37 antibody standard product and / or an anti-LL37 antibody quality control product.

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