Eosinophil cationic protein antigen epitope peptide, application and kit

Abstract

The present invention discloses an eosinophil cationic protein antigen epitope peptide, application and kit, and relates to the technical field of polypeptide and immunology. An eosinophil cationic protein antigen epitope peptide, the eosinophil cationic protein antigen epitope peptide is a polypeptide shown by the amino acid sequence of SEQ ID No.1 or SEQ ID No.2. The present invention solves the problem of insufficient specificity of existing detection methods.

Description

Technical Field

[0001] The invention relates to the technical field of polypeptides and immunology, and in particular to eosinophil cationic protein antigen epitope peptides, applications and kits. Background Art

[0002] Eosinophil cationic protein (ECP) is an alkaline protein released by eosinophils (EOS), also known as ribonuclease 3 (RNAse 3). It is encoded by the RNAse 3 gene and is a member of the RNAse A protein superfamily. ECP is an important component of the strongly alkaline protein released by activated EOS. It can directly kill pathogens, participate in immune regulation, promote inflammatory responses and the aggregation of immune cells. At the same time, it is an important marker of EOS activation, reflecting the degree of EOS activation. In clinical diseases related to eosinophil activation, such as allergic asthma, allergic rhinitis, atopic dermatitis or autoimmune diseases of the joints, the ECP level will increase. The ECP content is also related to the severity of asthma. During the asthma attack, the ECP content is high, and when the inflammation is reduced to the remission period, the ECP content decreases. Therefore, the increase in ECP content can indicate the occurrence of related diseases such as airway inflammation, reflect the severity of the clinical condition, and can be used for auxiliary diagnosis of diseases such as allergic asthma, rhinitis, atopic dermatitis, etc.

[0003] The ideal method for detecting ECP in clinical and research is immunoassay, which usually requires an antigen-antibody reaction based on ECP antibodies, so specific ECP antigens and antibodies are required.

[0004] ECP has a high sequence similarity with other members of the RNAse A protein superfamily. In particular, eosinophil-derived neurotoxin (EDN / RNAse 2), another protein released by eosinophils, has an amino acid sequence similarity of 71.64% between ECP and EDN. In addition, ECP has three natural glycosylation forms (molecular weights of 18, 20, and 22 kda, respectively). Therefore, antibodies prepared by conventional methods can easily produce immune cross-reactions with ECP analogs, or fail to recognize all glycosylated and non-glycosylated ECP types, resulting in insufficient specificity of the detection method. Summary of the invention

[0005] The technical problem to be solved by the present invention is that antibodies prepared by conventional methods are prone to immune cross-reaction with ECP analogs, or cannot recognize all glycosylated and non-glycosylated ECP types, resulting in insufficient specificity of the detection method. The purpose is to provide an eosinophil cationic protein antigen epitope peptide, application and kit to solve the problem of insufficient specificity of the detection method.

[0006] The present invention is achieved through the following technical solutions:

[0007] In a first aspect, the present invention provides an eosinophil cationic protein antigen epitope peptide, wherein the eosinophil cationic protein antigen epitope peptide is a combination of polypeptides represented by the amino acid sequences of SEQ ID No. 1 and SEQ ID No. 2.

[0008] Epitope peptide ① is a segment from 16 to 25 of the N-terminal of the ECP protein, containing a peptide of 10 amino acids, plus two amino acids, lysine (K) and serine (s), and the amino acid sequence is SEQ ID No. 1; epitope peptide ② is a segment from 112 to 123 of the N-terminal of the ECP protein, containing a peptide of 12 amino acids, plus two amino acids, K and S, and the amino acid sequence is SEQ ID No. 2. Lysine has a long side chain and a primary amine, which is convenient for coupling in subsequent steps; serine contains a polar hydroxyl group (OH), which is hydrophilic and provides conformational flexibility, making it easier for the epitope peptide to form an antigen binding site. The above-mentioned epitope peptides have good antigenicity through bioinformatics analysis and experimental confirmation, and have a large difference in structure from the corresponding local peptide segment of the ECP homologous protein-RNAse A protein superfamily member, and do not contain the natural glycosylation site of ECP.

[0009] ①SEQ ID No.1: KSISLNPPRCTI

[0010] ②SEQ ID No.2: KSDNRDPRDSPRYP

[0011] In a second aspect, the present invention provides an eosinophil cationic protein antigen, comprising the above-mentioned eosinophil cationic protein antigen epitope peptide.

[0012] As a possible design, the method for preparing the above-mentioned eosinophil cationic protein antigen comprises:

[0013] Using a bifunctional cross-linking agent to couple an eosinophil cationic protein antigen epitope peptide with a carrier protein to obtain an eosinophil cationic protein antigen;

[0014] The bifunctional cross-linking agent includes sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (NHS-ester-maleimide), and the carrier protein includes human serum albumin (HSA), keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or chicken ovalbumin (OVA ovalbumin).

[0015] Preferably, the carrier protein is keyhole limpet hemocyanin.

[0016] In a third aspect, the present invention provides an eosinophil cationic protein antibody, wherein the eosinophil cationic protein antibody is a monoclonal antibody or a polyclonal antibody prepared from the above antigen.

[0017] In a fourth aspect, the present invention provides use of the above-mentioned eosinophil cationic protein antibody in the preparation of an eosinophil cationic protein quantitative detection kit.

[0018] In a fifth aspect, the present invention provides a kit for quantitatively detecting eosinophil cationic protein, comprising the above-mentioned eosinophil cationic protein antibody.

[0019] The methodologies available for the above-mentioned kit include magnetic microparticle tube chemiluminescence method, latex agglutination method and flow fluorescence luminescence method.

[0020] Chemiluminescence technology is a method of determination that has been developed in the past two or three decades. The principle of this technology is to use the free energy released by chemical reactions to excite intermediates (commonly used alkaline phosphatase-adamantanamine, horseradish peroxidase-luminol derivatives, horseradish peroxidase-luminol derivatives, etc.), so that they return from the excited state to the ground state. When the intermediate returns from the excited state to the ground state, it will release photons of equal energy level, and the photons are measured for analysis. Chemiluminescence has the specificity of fluorescence, and does not require excitation light, which avoids the influence of stray light of excitation light in fluorescence analysis, thereby improving sensitivity, and avoids environmental pollution and health hazards caused by radioactive analysis. It is a very excellent quantitative analysis method. Magnetic particle tubular luminescence uses nanomagnetic particles with extremely small particle size as solid phase carriers, which has faster reaction speed and sensitivity, and is much stronger than the binding capacity of ELISA plates or cellulose membranes, so that the solid phase can load more coatings. Superparamagnetic magnetic particles can quickly aggregate and separate under the action of a magnetic field, and washing is more thorough and fast. In comparison, plate-type luminescent solid phase carriers are mostly ELISA plates, which have a small solid / liquid phase reaction contact area, incomplete reaction, large variation in measurement results, and low sensitivity. Therefore, with the current increasingly high requirements for quantitative detection, their use is gradually restricted.

[0021] Latex enhanced immunoturbidimetry uses the rapid formation of a network of antigen-antibody molecular complexes in a buffer by antigens and antibodies, which causes turbidity in the reaction solution and a decrease in transmittance. Within a certain range of proportions, the turbidity of the reaction solution is linearly correlated with the amount of antigen and antibody. It is based on the principle of antigen-antibody specific reaction and latex enhanced amplification, and has the advantages of high sensitivity, good specificity, and strong anti-interference ability. Small-sized latex particles have a large surface area, which can increase the binding efficiency of antibodies and accelerate the reaction speed; the homogeneous reaction conditions make it easy to realize automated detection, and the detection has high accuracy and consistency.

[0022] Flow cytometry, also known as suspension array, liquid phase chip, etc., is a multi-indicator joint diagnostic technology. This technology is based on fluorescently encoded microspheres, combined with multiple technologies such as flow principle, laser analysis and high-speed digital signal processing, and can realize multi-indicator parallel analysis. Its working principle is to allow the fluorescently labeled single cell (or particle) suspension to enter the sample suction tube and enter the flow chamber with the sheath fluid. In the detection area, the liquid flow, laser and detector are perpendicular to each other and focused on one point to achieve fluid dynamic focusing. Fluorescently labeled cells or particles emit scattered light and fluorescence emission waves under laser excitation. These signals are acquired by the detector and processed and input into the computer, and analyzed and processed by the software. Flow cytometry has the characteristics of high throughput, high sensitivity and parallel detection. This technology can accurately and quantitatively detect multiple different biological molecules in one tube at most, greatly improving the detection efficiency.

[0023] The above three methodologies can also achieve a high degree of automation, and are good methods for achieving the repeatability, linearity, accuracy and other indicators required for quantitative detection and analysis. In comparison, immunochromatography (colloidal gold, fluorescence chromatography), plate luminescence or enzyme-linked immunosorbent assay (ELISA) and other methodologies have the disadvantages of low automation and heterogeneous reactions, resulting in poor repeatability and accuracy, and do not meet the requirements of the quantitative analysis of the present invention.

[0024] As a possible design, the above includes a magnetic particle chemiluminescence reaction system, which includes a buffer, a monoclonal antibody and a calibrator, and the monoclonal antibody is the above monoclonal antibody.

[0025] As a possible design, the above includes a latex agglutination reaction system, which includes R1 reagent and R2 reagent, the R1 reagent includes a buffer, a surfactant and a preservative, and the R2 reagent includes eosinophil cationic protein latex particles, an antibacterial agent and a surfactant.

[0026] As a possible design, the above-mentioned eosinophil cationic protein latex particles are prepared by the following method:

[0027] Two hydroxyl microspheres of different particle sizes are mixed and then a buffer solution is added to react and activate to obtain a microsphere mixture;

[0028] After the microsphere mixture is reacted with the double cross-linking agent, it is washed, centrifuged, and ultrasonicated to obtain a resuspended microsphere solution;

[0029] The above-mentioned polyclonal antibody is diluted with a buffer solution, mixed with the resuspended microsphere solution, incubated, washed, centrifuged, and ultrasonicated to obtain eosinophil cationic protein latex particles.

[0030] Microspheres of different particle sizes can produce the best immune response with target analytes in different concentration ranges. Small-sized microspheres have better binding effects with low-concentration analytes, while large-sized microspheres have better binding effects with high-concentration analytes. The combination of microspheres of two particle sizes can expand the linear range of the method and improve sensitivity.

[0031] Preferably, the above-mentioned hydroxy microspheres are produced by JSR Life Sciences.

[0032] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0033] The present invention provides an ECP antigen epitope peptide, and the antibody prepared based on the epitope peptide has high specificity and strong binding force, does not cross-react with other proteins of EDN or RNase A superfamily, and can detect glycosylated and non-glycosylated ECP. The kit containing these two antibodies is used for the quantitative detection of ECP and has good clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without creative work. In the drawings:

[0035] Figure 1 The sequence similarity analysis diagram of RNAse 1-8, members of the RNAse A protein superfamily, in Example 1;

[0036] Figure 2 is the gel electrophoresis diagram of Example 2;

[0037] Figure 3 This is the standard curve diagram of Example 3. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with examples and drawings. The illustrative embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0039] Unless otherwise specified, the reagents, materials, and equipment used in this example are all commercially available; the experimental methods are all conventional experimental methods in the art unless otherwise specified.

[0040] Example 1

[0041] Preparation of ECP antigen epitope peptides.

[0042] Sequence similarity analysis of RNAse 1-8 members of the RNAse A protein superfamily Figure 1 As shown; Asn57, Asn65 and Asn92 of the ECP sequence are glycosylation sites.

[0043] The sequences of ECP (RNAse 3) and EDN (RNAse 3) had the highest similarity, reaching 71.64%.

[0044] The ECP protein sequence was predicted using algorithms such as Chou & Fasman Beta-Turn Prediction, Emini Surface Accessibility Prediction, Karplus & Schulz Flexibility Prediction, Kolaskar & Tongaonkar Antigenicity, and Parker Hydrophilicity Prediction. The results are shown in Table 1:

[0045] Table 1

[0046] Prediction Methods Predict epitope location Kolaskar&Tongaonkar Antigenicity 11-24，49-57，77-86，105-112，121-130 Parker Hydrophilicity Prediction 17-24，36-42，54-75，88-104，110-122 Chou&Fasman Beta-Turn Prediction 17-23，31-41，53-73，85-105，112-123 Karplus&Schulz Flexibility Prediction 18-23，37-48，55-76，87-96，99-105，111-123 Emini Surface Accessibility Prediction 16-22，30-40，58-77，88-94，97-105，113-122

[0047] The overlapping regions of the prediction results of several algorithms were integrated as the predicted linear epitopes of ECP, and the following 5 sequences were obtained; similarity analysis was performed with the corresponding sequences of EDN (RNAse 2), and the results are shown in Table 2:

[0048] Table 2

[0049] Serial number Location sequence length The same number of amino acid residues as END Number of amino acid residues similar to END Similarity 1 17-23 SLNPPRC 7 1 0 14.29% 2 31-41 NNYRWRCKNQN 11 9 0 81.82% 3 54-75 VCGNQSIRCPHNRTLNNCHRSR 22 12 2 63.64% 4 87-105 NPGAQNISNCTYADRPGRR 19 10 2 63.16% 5 112-123 DNRDPRDSPRYP 12 8 1 69.23%

[0050] Comprehensive structural analysis and sequence similarity analysis screened out two sequences:

[0051] Sequence ①Ser17-Cys23: It is a protruding structure of the β-turn structure, which appears on the surface of the protein antigen, is conducive to chimerism with antibodies, and has low similarity with the EDN sequence;

[0052] Sequence ② Asp112-Pro123: This sequence has a certain flexibility and is easy to chimerize with antibodies to form epitopes. Although the sequence has a similarity of 69.23% with EDN, the superposition analysis and structural prediction of the main chain backbone of the primary sequence of human ECP on the three-dimensional structure of EDN show that the loop region corresponding to D115-Y122 is the region with the greatest difference in the main chain backbone of the two proteins (Mosimann SC, Newton DL, Youle RJ, et al. X-ray Crystallographic Structure of Recombinant Eosinophil-derived Neurotoxin at 1.83 Å Resolution[J]. Journal of Molecular Biology, 1996, 260(4):540-552.). And this sequence corresponds to the exposed loop in the three-dimensional structure of ECP.

[0053] The three peptides 31-41, 54-75, and 87-105 are too similar to EDN and have no major structural differences. Moreover, 54-75 and 87-105 cover the glycosylation sites of three ECPs, Asn57, Asn65, and Asn92, which may affect the detection of different glycosylated ECPs in the natural state. Therefore, these three peptides were not selected as ECP antigen epitope peptides.

[0054] After sequence confirmation, two amino acids K and S are added to the N-terminus to form the ECP antigen epitope peptide:

[0055] ① SEQ ID No.1:KSISLNPPRCTI

[0056] ② SEQ ID No.2: KSDNRDPRDSPRYP

[0057] Sangon Biotech (Shanghai) Co., Ltd. was commissioned to carry out peptide synthesis.

[0058] Example 2

[0059] Preparation of ECP epitope peptide antibodies.

[0060] (1) ECP antigen epitope peptide ① and ECP antigen epitope peptide ② were coupled to the carrier protein KLH respectively through sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC, Thermo Scientific, 22322). The specific steps include:

[0061] a. Activated KLH: After dialyzing KLH (hemocyanin) against 20 mM phosphate buffer solution (PBS, pH 7.4) overnight, react with Sulfo-SMCC at room temperature for 0.5 h, with a molar ratio of 1:10; after the reaction is completed, dialyze against 20 mM PBS (pH 7.4) overnight again;

[0062] b. Activation of peptides: Dissolve the peptides to 2 mg / mL with 0.1 M sodium borate (pH 8.0), add 2-iminothiolane hydrochloride and react at room temperature for 0.5 h, with a mol ratio of 1:50; purify the activated peptides by ultrafiltration, and replace the buffer with 20 mM PBS (pH 7.4);

[0063] c. KLH-peptide coupling: The activated KLH and peptide were reacted at room temperature for 1.5-2h, with a mol ratio of 1:20; the conjugate was purified by dialysis, and the dialysate was treated with 20mM PBS (pH7.4). ECP antigen epitope peptide ① and ECP antigen epitope peptide ② were coupled separately to obtain KLH-ECP antigen epitope peptide ① and KLH-ECP antigen epitope peptide ②.

[0064] (2) Based on hybridoma technology, mice were immunized with KLH-ECP antigen epitope peptide ① and KLH-ECP antigen epitope peptide ② to prepare monoclonal antibodies, and two monoclonal antibodies were screened: ECP monoclonal antibody ① and ECP monoclonal antibody ②; rabbits were immunized with KLH-ECP antigen epitope peptide ② to screen and obtain an ECP polyclonal antibody;

[0065] The preparation of monoclonal antibodies and polyclonal antibodies was entrusted to Beijing Sino Biological Technology Co., Ltd.

[0066] (3) Monoclonal antibody and polyclonal antibody identification:

[0067] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (SDS-PAGE analysis) was performed using ECP protein from human neutrophils (AROTECH Diagnostic, ATE03), which contains three naturally glycosylated ECPs. The primary antibodies were ECP monoclonal antibody ①, ECP monoclonal antibody ②, and ECP polyclonal antibody at a concentration of 0.5 mg / mL, and the secondary antibodies were enzyme-labeled goat anti-mouse and enzyme-labeled goat anti-rabbit. The bands were as follows Figure 2 shown.

[0068] observe Figure 2 , Figure 2 The first band is ECP polyclonal antibody, the second band is ECP monoclonal antibody ①, and the third band is ECP monoclonal antibody ②. It can be seen from the bands that ECP monoclonal antibody ①, ECP monoclonal antibody ② and ECP polyclonal antibody in this example can recognize three naturally glycosylated ECP proteins.

[0069] Example 3

[0070] Study on the performance of ECP magnetic microparticle chemiluminescence quantitative detection kit.

[0071] 1. Preparation of magnetic particle chemiluminescence kit (M, R1, R2, calibrator)

[0072] (1) 0.4 mg / mL streptavidin magnetic particles were dispersed in 0.02 M PBS (pH 7.4) buffer containing 1% bovine serum albumin (BSA), 0.01% TritonX-100, and 0.1% Proclin-300 preservative as reagent M;

[0073] (2) ECP monoclonal antibody ① was diluted to 1 mg / mL, and biotin was labeled with biotin N-hydroxysulfosuccinimide (NHS) ester (NHS-Biotin, Thermo Scientific, 20217). The molar ratio of ECP monoclonal antibody ① to Biotin was 1:20, and the operation was carried out according to the instructions of the coupling agent. After labeling, it was diluted to 0.1 μg / mL with 0.01 M tris (Tris buffer, pH 7.4) containing 1% BSA, 0.1% Tween-20 and 0.1% Proclin-300 preservatives as R1 reagent;

[0074] (3) ECP monoclonal antibody ② was coupled to an equimolar amount of alkaline phosphatase using sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC, Thermo Scientific, 22322) crosslinker according to the instructions of the coupling agent. After labeling, it was diluted to 0.2 μg / mL with 0.02 M M ES (pH 6.0) buffer containing 1% BSA, 0.1% Tween-20 and 0.1% Proclin-300 preservatives as R2 reagent;

[0075] (4) The recombinant ECP antigen was diluted to 0, 2, 5, 15, 100, and 200 μg / L in a certain ratio. The quantitative product traceability method was based on the national standard GBT 21415-2008 Metrological traceability of the value of calibration materials and control materials for the measurement of quantities in biological samples of in vitro diagnostic medical devices: There is a measurement procedure selected by the manufacturer, but there is neither an internationally agreed reference measurement procedure nor an internationally agreed calibration material, and it cannot be traced back to the SI in metrology. The antigen was determined using the manufacturer's selected measurement procedure, that is, the industry's "gold standard" ImmunoCAP™ ECP detection system (Thermo Scientific, PhadiaAB), as the product calibrator.

[0076] (5) Equipped with a fully automatic chemiluminescence analyzer (Sichuan Xieguang Biology), R1, the sample to be tested, and M magnetic particles were mixed and incubated for 10 minutes; after washing, R2 was added and mixed for 10 minutes to form a solid-phase antibody-antigen-antibody complex. The unbound enzyme-labeled antibody and other substances were removed by washing. The chemiluminescent substrate AMPPD was added. The luminescent substrate emitted photons under the catalysis of alkaline phosphatase. The number of photons generated was proportional to the concentration of ECP in the sample. The sample luminescence value (RLU) was substituted into the curve of the calibration sample concentration and luminescence value at each point, and the concentration of ECP in the sample was calculated back.

[0077] 2. Testing and verification:

[0078] The above designed kits were investigated for precision, linearity and clinical comparison analysis.

[0079] (1) Precision: Select two samples with low and high values, repeat the test 10 times, calculate the mean value and coefficient of variation CV, and the results are shown in Table 3:

[0080] Table 3

[0081]

[0082] (2) Linearity: The high-value sample was diluted with saline in multiple ratios, and the correlation between the theoretical concentration and the measured concentration was calculated. The results are shown in Table 4:

[0083] Table 4

[0084]

[0085] (3) Clinical comparative analysis: Clinical serum samples were collected and tested using the industry's "gold standard" ImmunoCAP™ ECP detection system (allergen detection system) and the above-mentioned chemiluminescent ECP kit to analyze the consistency of the detection. The results are as follows: Figure 3 shown.

[0086] observe Figure 3 , the vertical axis is the ECP concentration detected by the ImmunoCAP™ ECP detection system, and the horizontal axis is the ECP concentration detected by the chemiluminescent ECP quantitative detection kit. The precision CV of the chemiluminescent ECP quantitative detection kit is <2%, and the dilution linear correlation R=0.9997 in the range of about 200μg / L, and the slope k=0.9918 and correlation R=0.9961 compared with the "gold standard" clinical comparison. In summary, the chemiluminescent ECP quantitative detection kit prepared with ECP monoclonal antibody ① and ECP monoclonal antibody ② has good quantitative analysis performance and good consistency with the ImmunoCAP™ ECP detection system.

[0087] Example 4

[0088] Preparation of quantitative detection kit for ECP latex agglutination method.

[0089] The ECP latex agglutination quantitative detection kit consists of reagent R1 and reagent R2:

[0090] (1) Reagent R1 is 0.02 M, pH 5.00 glycine-tris buffer, the surfactant is polyoxyethylene alkyl alcohol amide, and the preservative is ProClin 300;

[0091] (2) Reagent R2, mainly comprising ECP latex particles, ProClin 300 and surfactants: polyoxyethylene alkyl alcohol amide, Triton X-100.

[0092] ECP latex particles were prepared by the following method:

[0093] S1, adding carboxyl microspheres A with a particle size of 80 nm and carboxyl microspheres B with a particle size of 350 nm in a mass ratio of 1:9 into 0.05 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES buffer) with a pH of 7.00, stirring and reacting at room temperature for 30 minutes to obtain an activated microsphere mixture;

[0094] S2, adding the double cross-linking agent: 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the activated microsphere mixture obtained in step S1, the mass ratio of EDC: microsphere is 1:40, the mass ratio of NHS: microsphere is 1:20, and the reaction is stirred at room temperature for 30 minutes;

[0095] S3, washing the activated microsphere conjugate obtained in step S2 with 0.05 mol / L 2-(N-morpholino)ethanesulfonic acid (MES buffer) at pH 7.40 for 2 to 3 times, centrifuging and sonicating, and resuspending the microspheres to a final concentration of 1.0%;

[0096] S4, diluting the ECP polyclonal antibody provided in Example 2 with 0.05 mol / L, pH 7.40 MES buffer to a final concentration of 1.0 mg / mL;

[0097] S5, mixing the activated microsphere conjugate obtained in step S3 and the ECP polyclonal antibody solution obtained in step S4 in a ratio of 1:1, incubating with stirring at room temperature for 120 min, adding blocking solution, and incubating with stirring at room temperature for 60 min;

[0098] S6. Wash the ECP-sensitized latex obtained in step S5 2-3 times using 0.02 M glycine storage buffer at pH 8.00, centrifuge and sonicate, and dilute the microspheres to a final concentration of 0.35%, thereby obtaining ECP latex particles.

[0099] Experimental Example 1

[0100] The commercial ECP mouse monoclonal antibody (AA30-150) and the commercial ECP rabbit polyclonal antibody were used as comparative examples 1 and 2, respectively, to verify the antibody specificity:

[0101] (1) ELISA plate coating: dilute 50 ng / mL RNAse 1-8 with CB buffer at pH 9.6, add 100 μL RNAse 1-8 to each well of a 96-well plate, and incubate at 2-8°C overnight for coating;

[0102] (2) Wash the plate three times and spin dry. Add 200 μL of 10% BSA-sucrose blocking solution to each well. Block at room temperature for 4 h, spin dry, and place in a 37°C oven to dry for 4 h.

[0103] (3) Dilute the commercial ECP mouse monoclonal antibody (AA30-150), commercial ECP rabbit polyclonal antibody, and ECP monoclonal antibody ①, ECP monoclonal antibody ② and ECP polyclonal antibody provided in Example 2 to 0.2 μg / mL with 0.02M PBS (pH 7.4), and add 50 μL of the four antibodies to each well of a 96-well plate coated with RNAse 1-8; add enzyme-labeled goat anti-mouse secondary antibody to the mouse monoclonal antibody well at a ratio of 1:1000; add enzyme-labeled goat anti-rabbit secondary antibody to the rabbit polyclonal antibody well at a ratio of 1:1000, react at room temperature for 2 hours, wash three times, and spin dry, add ELISA substrate to react at room temperature for 10 minutes, and read the results with a microplate reader at a wavelength of 450. The results are shown in Table 5.

[0104] Table 5

[0105]

[0106] According to Table 5, the commercial ECP mouse monoclonal antibody (AA30-150) of Comparative Example 1 has a strong reactivity with EDN; the commercial ECP rabbit polyclonal antibody of Comparative Example 2 has cross-reactivity with RNAse 1, EDN, and RNAse 4-8. In Example 2, ECP monoclonal antibody ①, ECP monoclonal antibody ②, and ECP polyclonal antibody are only reactive with ECP, and have good specificity.

[0107] Experimental Example 2

[0108] Comparison of samples from commercial kits:

[0109] Taking the eosinophil cationic protein (ECP) enzyme-immunoassay kit (scientific research reagent) as comparative example 3, the chemiluminescent ECP detection kit of Example 3 and the eosinophil cationic protein (ECP) enzyme-immunoassay kit (scientific research reagent) were used to simultaneously detect samples determined by the ImmunoCAP™ ECP detection system. The results are shown in Table 6.

[0110] Table 6

[0111]

[0112] It can be seen from Table 6 that the eosinophil cationic protein (ECP) enzyme-immune assay kit (scientific research reagent) of Comparative Example 3 was not quantitatively traced according to standardized procedures, the measurement units were not unified, and the difference from the "gold standard" measurement value was large; in comparison, the chemiluminescent ECP detection kit of Example 3 had metrological traceability and the accuracy of quantitative analysis performance.

[0113] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An eosinophil cationic protein antigen epitope peptide, characterized in that: The eosinophil cationic protein antigen epitope peptide is a composition of a combination of polypeptides shown in the amino acid sequences of SEQ ID No. 1 and SEQ ID No.

2.

2. An eosinophil cationic protein antigen, characterized in that Comprising the eosinophil cationic protein antigen epitope peptide according to claim 1.

3. The method for preparing an eosinophil cationic protein antigen according to claim 2, characterized in that: include: Using a bifunctional cross-linking agent to couple an eosinophil cationic protein antigen epitope peptide with a carrier protein to obtain an eosinophil cationic protein antigen; The bifunctional cross-linking agent includes sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and the carrier protein includes human serum albumin, keyhole limpet hemocyanin, bovine serum albumin or chicken ovalbumin.

4. An eosinophil cationic protein antibody, characterized in that: The eosinophil cationic protein antibody is a polyclonal antibody prepared from the antigen according to claim 2.

5. Use of the eosinophil cationic protein antibody according to claim 4 in preparing a kit for quantitative detection of eosinophil cationic protein.

6. A kit for quantitatively detecting eosinophil cationic protein, characterized in that: The invention comprises the eosinophil cationic protein antibody according to claim 4.

7. A kit for quantitatively detecting eosinophil cationic protein according to claim 6, characterized in that: The invention comprises a latex agglutination reaction system, wherein the latex agglutination reaction system comprises an R1 reagent and an R2 reagent, wherein the R1 reagent comprises a buffer, a surfactant and a preservative, and the R2 reagent comprises eosinophil cationic protein latex particles, an antibacterial agent and a surfactant.

8. A kit for quantitatively detecting eosinophil cationic protein according to claim 7, characterized in that: The eosinophil cationic protein latex particles are prepared by the following method: Two hydroxyl microspheres of different particle sizes are mixed and then a buffer solution is added to react and activate to obtain a microsphere mixture; After the microsphere mixture is reacted with the double cross-linking agent, it is washed, centrifuged, and ultrasonicated to obtain a resuspended microsphere solution; The polyclonal antibody described in claim 4 is diluted with a buffer solution, mixed with a resuspended microsphere solution, incubated, washed, centrifuged, and ultrasonicated to obtain eosinophil cationic protein latex particles.

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