Interleukin 10 antibody, application thereof and interleukin 10 detection kit

Abstract

The invention provides a novel interleukin 10 antibody which is selected from an antibody a or an antibody b. The invention also provides application of the interleukin 10 antibody and an interleukin 10 detection kit. The interleukin 10 antibody provided by the invention has good specificity and high affinity with interleukin 10, avoids crossing with EB virus BCRF1 protein, and can be used for preparing a magnetic particle chemiluminescence detection product for detecting interleukin 10. The interleukin 10 detection kit provided by the invention has the advantages of automatic detection, high sensitivity, strong specificity, large flux and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to interleukin-10 antibodies and their applications, as well as interleukin-10 detection kits. Background Art

[0002] Interleukin-10 (IL-10), also known as cytokine synthesis inhibitory factor (CSIF), is a pleiotropic cytokine that regulates cell growth and differentiation, participates in inflammatory and immune responses, and is a recognized inflammation and immunosuppressive factor. IL-10 can exert immunosuppressive or immunostimulatory effects in various types of cells, plays an important role in infections, organ transplantation, the hematopoietic system, and the cardiovascular system, and is closely related to diseases of the blood, digestive system, especially the cardiovascular system.

[0003] In some studies, IL-10 promotes the growth and survival of cancer cells, including non-Hodgkin lymphoma, Burkitt lymphoma, and non-small cell lung cancer. Overexpression of IL-10 is associated with SLE and tuberculosis, while diseases such as inflammatory bowel disease, psoriasis, asthma, and rheumatoid arthritis are associated with IL-10 deficiency. Currently, commonly used clinical methods for detecting interleukin-10 include enzyme-linked immunosorbent assay, immunofluorescence assay, chemiluminescence assay, etc.

[0004] Studies have shown that the reference range of IL-10 in the serum or plasma of healthy individuals is less than 9 pg / mL, which requires high sensitivity of the detection kit and high affinity of the antibody raw material.

[0005] The IL-10 genes of humans and mice both encode polypeptides 178 amino acids in length, including a cleavable signal peptide sequence. The IL-10 genes of humans and mice both have a high degree of homology with an open reading frame BCRF1 (BamHI C right frame 1 protein) of Epstein-Barr virus. After sequence alignment analysis, the amino acid sequences of the two have about 82% homology, and there may be a certain degree of cross-reactivity during detection, which requires high specificity of the detection kit and antibody raw material. Summary of the Invention

[0006] The object of the present invention is to provide an interleukin-10 antibody that specifically recognizes interleukin-10 and avoids cross-reactivity with Epstein-Barr virus BCRF1 protein. The present invention also provides the application of the interleukin-10 antibody in the preparation of an interleukin-10 chemiluminescence immunoassay kit using magnetic microparticles. The present invention also provides an interleukin-10 chemiluminescence immunoassay kit using magnetic microparticles, including streptavidin magnetic microparticle solution, acridinyl sulfonamide-labeled antibody solution, biotin-labeled antibody solution, calibrator, and quality control product, to solve the problems in the prior art.

[0007] To achieve the above object, the present invention provides an interleukin-10 antibody, selected from antibody a or antibody b; Antibody a includes heavy chain complementarity determining regions CDR1-VH, CDR2-VH, CDR3-VH with amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.3 in sequence, and light chain complementarity determining region CDR1-VL with amino acid sequence shown in SEQ ID NO.4, light chain complementarity determining region CDR2-VL with amino acid sequence TAS, and light chain complementarity determining region CDR3-VL with amino acid sequence shown in SEQ ID NO.5; Antibody b includes heavy chain complementarity determining regions CDR1-VH, CDR2-VH, CDR3-VH with amino acid sequences shown in SEQ ID NO.6 to SEQ ID NO.8 in sequence, and light chain complementarity determining region CDR1-VL with amino acid sequence shown in SEQ ID NO.9, light chain complementarity determining region CDR2-VL with amino acid sequence TAS, and light chain complementarity determining region CDR3-VL with amino acid sequence shown in SEQ ID NO.10.

[0008] Preferably, the amino acid sequence of the heavy chain variable region of antibody a is shown in SEQ ID NO.11.

[0009] Preferably in any of the above, the amino acid sequence of the light chain variable region of antibody a is shown in SEQ ID NO.12.

[0010] Preferably in any of the above, the amino acid sequence of the heavy chain variable region of antibody b is shown in SEQ ID NO.13.

[0011] Preferably in any of the above, the amino acid sequence of the light chain variable region of antibody b is shown in SEQ ID NO.14.

[0012] Preferably in any of the above, the amino acid sequence of the light chain constant region of antibody a or antibody b is shown in SEQ ID NO.15.

[0013] Preferably in any of the above, the amino acid sequence of the heavy chain constant region of antibody a or antibody b is shown in SEQ ID NO.16.

[0014] Preferably in any of the above, antibody a is interleukin-10 monoclonal antibody 1, and antibody b is interleukin-10 monoclonal antibody 2; further, antibody a or antibody b is murine.

[0015] The present invention also provides the coding sequence of the interleukin-10 antibody described in any of the above.

[0016] Preferably, in any one of the above, the coding sequence of the heavy chain variable region of antibody a is the nucleotide sequence from nucleotide 1 to nucleotide 369 of the nucleotide sequence shown in SEQ ID NO. 17.

[0017] Preferably, in any one of the above, the coding sequence of the light chain variable region of antibody a is the nucleotide sequence from nucleotide 1 to nucleotide 327 of the nucleotide sequence shown in SEQ ID NO. 18.

[0018] Preferably, in any one of the above, the coding sequence of the heavy chain variable region of antibody b is the nucleotide sequence from nucleotide 1 to nucleotide 369 of the nucleotide sequence shown in SEQ ID NO. 19.

[0019] Preferably, in any one of the above, the coding sequence of the light chain variable region of antibody b is the nucleotide sequence from nucleotide 1 to nucleotide 330 of the nucleotide sequence shown in SEQ ID NO. 20.

[0020] Preferably, in any one of the above, the coding sequence of the heavy chain of antibody a is the nucleotide sequence shown in SEQ ID NO. 17.

[0021] Preferably, in any one of the above, the coding sequence of the light chain of antibody a is the nucleotide sequence shown in SEQ ID NO. 18.

[0022] Preferably, in any one of the above, the coding sequence of the heavy chain of antibody b is the nucleotide sequence shown in SEQ ID NO. 19.

[0023] Preferably, in any one of the above, the coding sequence of the light chain of antibody b is the nucleotide sequence shown in SEQ ID NO. 20.

[0024] The present invention also provides a recombinant expression vector containing the coding sequence described in any one of the above.

[0025] The recombinant expression vector contains the coding sequence of antibody a or / and the coding sequence of antibody b.

[0026] Preferably, in any one of the above, the recombinant expression vector contains the coding sequence of the light chain variable region of antibody a.

[0027] Preferably, in any one of the above, the recombinant expression vector contains the coding sequence of the heavy chain variable region of antibody a.

[0028] Preferably, in any one of the above, the recombinant expression vector contains the coding sequence of the light chain variable region of antibody b.

[0029] Preferably, in any one of the above, the recombinant expression vector contains the coding sequence of the heavy chain variable region of antibody b.

[0030] Preferably, in any of the above, the recombinant expression vector contains the coding sequence of the light chain of antibody a.

[0031] Preferably, in any of the above, the recombinant expression vector contains the coding sequence of the heavy chain of antibody a.

[0032] Preferably, in any of the above, the recombinant expression vector contains the coding sequence of the light chain of antibody b.

[0033] Preferably, in any of the above, the recombinant expression vector contains the coding sequence of the heavy chain of antibody b.

[0034] Preferably, in any of the above, the recombinant expression vector is constructed by ligating the coding sequence provided by the present invention to various expression vectors by conventional methods in the art. The expression vectors are various conventional vectors in the art, and any vector that can accommodate the nucleic acid molecule of the coding sequence is applicable to the present invention. The expression vectors preferably include: various plasmids, cosmids, phage or viral vectors, etc., such as pCDNA3.4, pCDNA3.1 series vectors, etc. The expression vector is preferably a eukaryotic expression vector, and the expression vector is preferably an expression vector suitable for mammalian cells.

[0035] The present invention also provides a host cell, which contains the recombinant expression vector described in any of the above or the coding sequence described in any of the above is integrated into the genome of the host cell.

[0036] Preferably, in any of the above, the host cell is a mammalian cell.

[0037] In a preferred embodiment of the present invention, the recombinant expression vector is preferably a eukaryotic expression vector, and the host cell is preferably a mammalian cell. Further, the recombinant expression vector is preferably pCDNA3.4, and the host cell is preferably 293T cells.

[0038] The interleukin-10 antibody of the present invention can be either a monoclonal antibody prepared by hybridoma or an antibody obtained by the recombinant expression vector described in any one of the above or the host cell described in any one of the above. The recombinant expression vector containing the antibody a heavy chain coding sequence and the recombinant expression vector containing the antibody a light chain coding sequence are co-transfected into a host cell (the host cell is preferably a mammalian cell) in equal proportion. The recombinant expression vector containing the antibody a heavy chain coding sequence and the recombinant expression vector containing the antibody a light chain coding sequence will be transcribed, translated and variously modified in the host cell to form an antibody, and the antibody a is obtained after purification; the recombinant expression vector containing the antibody b heavy chain coding sequence and the recombinant expression vector containing the antibody b light chain coding sequence are co-transfected into a host cell (the host cell is preferably a mammalian cell) in equal proportion. The recombinant expression vector containing the antibody b heavy chain coding sequence and the recombinant expression vector containing the antibody b light chain coding sequence will be transcribed, translated and variously modified in the cell to form an antibody, and the antibody b is obtained after purification. In the present invention, whether it is a monoclonal antibody obtained by hybridoma or an antibody obtained by recombinant expression, it can specifically recognize interleukin-10 and avoid cross-reaction with the EBV BCRF1 protein at the same time.

[0039] The present invention also provides the application of the interleukin-10 antibody described in any one of the above, or the coding sequence of the interleukin-10 antibody described in any one of the above, or the recombinant expression vector described in any one of the above, or the host cell described in any one of the above, in the preparation of an interleukin-10 chemiluminescent immunoassay kit with magnetic microparticles.

[0040] The present invention also provides an interleukin-10 detection kit, which includes a streptavidin magnetic microparticle solution, an acridinium sulfonamide-labeled antibody solution, a biotin-labeled antibody solution, a calibrator and a quality control product. The acridinium sulfonamide-labeled antibody solution contains the interleukin-10 antibody described in any one of the above, and the interleukin-10 antibody is labeled with acridinium sulfonamide; the biotin-labeled antibody solution contains the interleukin-10 antibody described in any one of the above, and the interleukin-10 antibody is labeled with biotin; the interleukin-10 antibody in the acridinium sulfonamide-labeled antibody solution is different from the interleukin-10 antibody in the biotin-labeled antibody solution. The kit provided by the present invention can effectively detect interleukin-10 in the range of 2.5 - 2500 pg / mL.

[0041] Preferably, in any one of the above, the concentration of the streptavidin magnetic microparticle solution is 0.1 - 1.0 mg / mL, and further preferably 0.1, 0.5, 1.0 mg / mL and the ranges therebetween.

[0042] Preferably, in any of the above items, the acridine sulfonamide-labeled antibody solution contains 0.2-2 μg / mL of the interleukin 10 antibody; more preferably, 0.2, 0.5, 1.0, 1.5, 2.0 μg / mL and ranges therebetween.

[0043] Preferably, in any of the above items, the biotin-labeled antibody solution contains 0.2-2 μg / mL of the interleukin 10 antibody; more preferably, 0.2, 0.5, 1.0, 1.5, 2.0 μg / mL and ranges therebetween.

[0044] Any of the above is preferably that the interleukin 10 antibody diluent is a 10-50 mM Tris-HCl buffer containing BSA, and each 100 mL of the diluent contains 0.1-1.0 g of BSA; further preferably, in the Tris-HCl buffer, the concentration of Tris-HCl is 10, 20, 30, 40, 50 mM and the range therebetween; further preferably, each 100 mL of the diluent contains 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 g and the range therebetween of BSA. The interleukin 10 antibody diluent is a diluent of the acridine sulfonamide labeled antibody solution or the biotin labeled antibody solution.

[0045] Preferably, any of the above items is that the calibrator is a lyophilized product of interleukin 10 protein, and preferably the calibrator also includes lyophilized excipients, and preferably, pure water is used for reconstitution during use.

[0046] Preferably, any of the above items is that the quality control product is a freeze-dried product of interleukin 10 protein, and preferably the quality control product also includes freeze-dried auxiliary materials. Preferably, pure water is used for reconstitution during use.

[0047] In any of the above, the interleukin 10 protein freeze-dried product can be interleukin 10 prepared by a prior art method, or a commercial interleukin 10 protein product. The present invention preferably uses interleukin 10 from Beijing Baixinyi Biotechnology Co., Ltd., product number A10C08, but it should be noted that the present invention is not limited to the source and preparation method of interleukin 10.

[0048] Preferably, any of the above-mentioned lyophilized excipients is a conventional lyophilized excipient in the prior art. Preferably, it includes saccharides such as trehalose, sucrose, lactose, etc.; preferably, it includes polyols such as mannitol, sorbitol, etc.; preferably, it includes amino acids such as glycine, histidine, etc.; preferably, it includes proteins such as bovine serum albumin (BSA), etc.; preferably, it includes gelatin; preferably, it includes buffer systems such as phosphate buffer solution (PBS), Tris-HCl, etc.; preferably, it includes surfactants such as Tween-20, polyvinylpyrrolidone (PVP), etc.; preferably, it includes preservatives such as Proclin 300, sodium azide, etc.

[0049] In the present invention, the interleukin-10 antibody on the acridinesulfonamide-labeled antibody solution is different from the interleukin-10 antibody on the biotin-labeled antibody solution. In a preferred embodiment of the present invention, the interleukin-10 antibody on the acridinesulfonamide-labeled antibody solution is antibody a, and the interleukin-10 antibody on the biotin-labeled antibody solution is antibody b; in a preferred embodiment of the present invention, the interleukin-10 antibody on the acridinesulfonamide-labeled antibody solution is antibody b, and the interleukin-10 antibody on the biotin-labeled antibody solution is antibody a.

[0050] Compared with the prior art, the present invention has the following beneficial effects: The interleukin-10 antibody provided by the present invention has good specificity and high affinity for interleukin-10, avoids cross-reactivity with the EB virus BCRF1 protein, and can be used to prepare a magnetic particle chemiluminescence detection product for detecting interleukin-10.

[0051] The interleukin-10 detection kit provided by the present invention has the advantages of automated detection, high sensitivity, strong specificity, large throughput, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 It is the electrophoresis diagram of the interleukin-10 monoclonal antibody in the preferred embodiment 1 of the present invention.

[0053] Figure 2 It is the interleukin-10 detection kit in the preferred embodiments 2 and 3 of the present invention.

[0054] Figure 3 It is the interleukin-10 detection reagent in the preferred embodiments 2 and 3 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0055] The present invention will be further described in detail below through specific embodiments.

[0056] It should be noted that the "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain can be referred to as "VH". The variable domain of the light chain can be referred to as "VL". These domains are generally the most variable parts of the antibody and contain the antigen-binding sites. The variable region of the light or heavy chain is composed of framework regions interrupted by three hypervariable regions called "complementary determining regions" or "CDRs". The framework regions of the antibody, that is, the framework regions constituting the combination of the light and heavy chains of the constituent elements, play a role in positioning and aligning the CDRs, and the CDRs are mainly responsible for binding to the antigen.

[0057] The "framework" or "FR" region means the region outside those regions of the antibody variable domain that are defined as CDRs. Each antibody variable domain framework can be further subdivided into adjacent regions (FR1, FR2, FR3, and FR4) separated by CDRs.

[0058] Generally, the variable regions VL / VH of the heavy and light chains can be obtained by connecting the following numbered CDRs and FRs in the following combined arrangement: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0059] In the present invention, CDR1-VH, CDR2-VH, and CDR3-VH respectively refer to the three hypervariable regions of the heavy chain variable region. Correspondingly, CDR1-VL, CDR2-VL, and CDR3-VL respectively refer to the three hypervariable regions of the light chain variable region.

[0060] In a first aspect, the present invention provides an interleukin-10 antibody selected from antibody a or antibody b; Antibody a includes heavy chain complementary determining regions CDR1-VH, CDR2-VH, CDR3-VH with amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.3 in sequence, and light chain complementary determining regions CDR1-VL with amino acid sequence shown in SEQ ID NO.4, light chain complementary determining region CDR2-VL with amino acid sequence TAS, and light chain complementary determining region CDR3-VL with amino acid sequence shown in SEQ ID NO.5; Antibody b includes heavy chain complementary determining regions CDR1-VH, CDR2-VH, CDR3-VH with amino acid sequences shown in SEQ ID NO.6 to SEQ ID NO.8 in sequence, and light chain complementary determining regions CDR1-VL with amino acid sequence shown in SEQ ID NO.9, light chain complementary determining region CDR2-VL with amino acid sequence TAS, and light chain complementary determining region CDR3-VL with amino acid sequence shown in SEQ ID NO.10.

[0061] The amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.10 are shown in Table 1 as follows.

[0062] Table 1

[0063] In a preferred embodiment of the present invention, the antibody a comprises a heavy chain variable region and a light chain variable region with amino acid sequences as shown in SEQ ID NO.11 and SEQ ID NO.12 in sequence; The antibody b comprises a heavy chain variable region and a light chain variable region with amino acid sequences as shown in SEQ ID NO.13 and SEQ ID NO.14 in sequence; Preferably, the interleukin-10 antibody is murine-derived.

[0064] The amino acid sequences shown in SEQ ID NO.11 to SEQ ID NO.14 are shown in Table 2 as follows.

[0065] Table 2

[0066] The light chain constant region of the antibody a or antibody b is as shown in SEQ ID NO.15, and the heavy chain constant region of the antibody a or antibody b is as shown in SEQ ID NO.16, as shown in Table 3.

[0067] The amino acid sequence of the heavy chain of antibody a consists of the amino acid sequences shown in SEQ ID NO.11 and SEQ ID NO.16 in sequence; The amino acid sequence of the light chain of antibody a consists of the amino acid sequences shown in SEQ ID NO.12 and SEQ ID NO.15 in sequence; The amino acid sequence of the heavy chain of antibody b consists of the amino acid sequences shown in SEQ ID NO.13 and SEQ ID NO.16 in sequence; The amino acid sequence of the light chain of antibody b consists of the amino acid sequences shown in SEQ ID NO.14 and SEQ ID NO.15 in sequence.

[0068] Table 3

[0069] The interleukin-10 antibody provided by the present invention has good specificity, high biological activity, and high affinity for interleukin-10, and can be used to prepare products for detecting interleukin-10.

[0070] In a second aspect, the present invention provides the use of the interleukin-10 antibody in the preparation of a chemiluminescence immunoassay product for interleukin-10 using magnetic microparticles.

[0071] In a third aspect, the present invention provides an interleukin-10 detection kit, which includes a streptavidin magnetic microparticle solution, an acridinium sulfonamide-labeled antibody solution, a biotin-labeled antibody solution, a calibrator, and a quality control product.

[0072] Preferably, the concentration of the streptavidin magnetic microparticle solution is 0.1 - 1.0 mg / mL.

[0073] Preferably, the acridinium sulfonamide-labeled antibody solution contains the interleukin-10 antibody, and the interleukin-10 antibody is labeled with acridinium sulfonamide, and the concentration of acridinium sulfonamide is recognized by a detection instrument.

[0074] Preferably, the biotin-labeled antibody solution contains 0.2 - 2 μg / mL of the interleukin-10 antibody.

[0075] Preferably, the acridinium sulfonamide-labeled antibody solution contains 0.2 - 2 μg / mL of the interleukin-10 antibody.

[0076] Preferably, the diluents of the biotin-labeled antibody solution and the acridinium sulfonamide-labeled antibody solution are 10 - 50 mM Tris-HCl buffer containing BSA, and each 100 mL of the diluent contains 0.1 - 1.0 g of BSA.

[0077] Preferably, the interleukin-10 antibody on the acridinium sulfonamide-labeled antibody solution is different from the interleukin-10 antibody on the biotin-labeled antibody solution.

[0078] In a preferred embodiment of the present invention, the antibody in the acridinium sulfonamide-labeled antibody solution is interleukin-10 antibody a, where antibody a is interleukin-10 monoclonal antibody 1, and the antibody in the biotin-labeled antibody solution is interleukin-10 antibody b, where antibody b is interleukin-10 monoclonal antibody 2.

[0079] In a preferred embodiment of the present invention, the antibody in the acridinium sulfonamide-labeled antibody solution is interleukin-10 antibody b, and the antibody in the biotin-labeled antibody solution is interleukin-10 antibody a.

[0080] Preferably, the calibrator is a freeze-dried product of interleukin-10 protein. Preferably, the calibrator further includes freeze-dried excipients. Preferably, it is reconstituted with pure water before use.

[0081] Preferably, the quality control product is a freeze-dried product of interleukin-10 protein. Preferably, the quality control product further comprises freeze-drying excipients. Preferably, it is reconstituted with pure water during use.

[0082] Preferably, the freeze-dried product of interleukin-10 protein can be interleukin-10 prepared by using the existing technology, or a commercial interleukin-10 protein product. However, the present invention is not limited to the source and preparation method of interleukin-10.

[0083] Preferably, the freeze-drying excipients are conventional freeze-drying excipients in the existing technology. Preferably, they include saccharides such as trehalose, sucrose, lactose, etc.; preferably, they include polyols such as mannitol, sorbitol, etc.; preferably, they include amino acids such as glycine, histidine, etc.; preferably, they include proteins such as bovine serum albumin (BSA), etc.; preferably, they include gelatin; preferably, they include buffer systems such as phosphate buffer solution (PBS), Tris-HCl, etc.; preferably, they include surfactants such as Tween-20, polyvinylpyrrolidone (PVP), etc.; preferably, they include preservatives such as Proclin 300, sodium azide, etc.

[0084] The interleukin-10 detection kit provided by the present invention has the advantages of automated operation, high sensitivity, strong specificity, large throughput, etc.

[0085] The reagent provided by the present invention is based on the principle of antigen-antibody reaction. After labeling interleukin-10 antibody with acridinesulfonamide, it is formulated into an acridinesulfonamide-labeled antibody solution with a diluent; another antibody of biotin-labeled interleukin-10 is included in the biotin-labeled antibody solution; based on the principle of antigen-antibody reaction, it can be detected by a supporting instrument after the reaction is completed. If interleukin-10 exists in the sample, a sandwich structure of double antibodies will be formed, and the instrument will detect a strong light signal. If there is no interleukin-10 in the sample, the instrument will detect a weak light signal. The load of the analyte is predicted according to the intensity of the light signal.

[0086] Using the kit provided by the present invention for detection, the detection result can be obtained within 30 minutes for the whole process, which is fast and efficient, helps medical staff to obtain the detection result in time, and make a comprehensive judgment and timely treatment according to the result.

[0087] Sample loading of the kit for testing: It should be noted that the diluent of the interleukin-10 antibody described in the present invention is a 10-50 mM Tris-HCl buffer solution containing BSA, and each 100 mL of the diluent contains 0.1-1.0 g of BSA. In the following examples without special instructions, preferably, the diluent of the interleukin-10 antibody is a 20 mM Tris-HCl buffer solution containing BSA, and each 100 mL of the diluent contains 0.5 g of BSA.

[0088] The method for preparing the interleukin 10 monoclonal antibody 1 shown in antibody a and the interleukin 10 monoclonal antibody 2 shown in antibody b of the present invention is a conventional monoclonal antibody preparation method in the prior art, which is briefly described as follows: (1) Animal immunization: A: Interleukin 10 purchased from Beijing Baixinyi Biotechnology Co., Ltd., product number A10C08, was mixed with equal volumes of Freund's adjuvant to a suitable volume and emulsified completely. Mice were immunized by intraperitoneal injection. Each mouse was injected with 50 μg of immunogen, the injection volume was 100 μL / mouse, and the injection was performed once a week. B: After immunization for 4 times, the ELISA indirect method was used to coat interleukin 10 and test the titer of the antibody in the serum, and mice with an OD value greater than 1.0 detected by 16,000-fold serum dilution were screened.

[0089] (2) Preparation of monoclonal antibodies: A: The spleen cells of the screened mice were fused with myeloma cells, and the fused cells were plated and cultured using the limiting dilution method; B: Screen the monoclonal cell wells and culture and expand them. The monoclonal cell wells with the highest OD value detected in the cell culture supernatant are used as target hybridoma cells, culture and expand them, obtain hybridoma cell lines and freeze them; C: Isolation of antibody variable region genes from hybridoma cells using RT-PCR: After the hybridoma cells are homogenized, cell lysis buffer is added to extract RNA, RNA is precipitated from the aqueous phase with isopropanol, the precipitated RNA is washed after centrifugation, impurities are removed, and reverse transcription is performed after resuspension to obtain cDNA; D: PCR was performed using specific primers for mice known in the prior art, with the cDNA of hybridoma cells as the template to amplify the variable region genes of the heavy and light chains of the antibody. The 50 μL system contained 5 μL cDNA, HotStarTaq Plus enzyme, dNTPs, and 0.5 μM specific primers. PCR amplification was carried out under the following conditions: pre-denaturation at 94 °C for 5 min; 94 °C for 30 s, 55 °C for 30 s, 72 °C for 50 s, for 35 cycles; 72 °C for 7 min; The obtained PCR products were identified by 1% agarose gel electrophoresis, the target fragments were recovered, sent for sequencing, and according to the antibody gene sequences obtained by sequencing, the amino acid sequences of the antibody variable regions shown in SEQ ID NO: 11-14 were further obtained; among them, the amino acid sequence of the light chain variable region of antibody a is shown in SEQ ID NO.12, the amino acid sequence of the heavy chain variable region of antibody a is shown in SEQ ID NO.11, the amino acid sequence of the light chain variable region of antibody b is shown in SEQ ID NO.14, and the amino acid sequence of the heavy chain variable region of antibody b is shown in SEQ ID NO.13. The amino acid sequence of the light chain constant region of antibody a or antibody b is shown in SEQID NO.15, and the amino acid sequence of the heavy chain constant region of antibody a or antibody b is shown in SEQ ID NO.16.

[0090] E: Construction of the expression vector for monoclonal antibodies: According to the antibody gene sequences obtained by sequencing in step D, the expression vector for monoclonal antibodies was constructed by conventional methods in the art. Homologous recombination arms were added to both ends of the heavy chain variable region gene and the light chain variable region gene of the antibody using homologous recombination primers, and the expression plasmid containing the murine heavy and light chain IgG1 constant regions was linearized using double enzymes to generate homologous recombination arms; the variable region gene fragments with added homologous recombination arms and the linearized plasmid were ligated by homologous recombination to form a complete expression vector. The expression vector was pCDNA3.4, and the recombinant product was transformed into TOP10 Escherichia coli competent cells to amplify the plasmid; pCDNA3.4-antibody a heavy chain plasmid, pCDNA3.4-antibody a light chain plasmid, pCDNA3.4-antibody b heavy chain plasmid, and pCDNA3.4-antibody b light chain plasmid were obtained respectively.

[0091] The nucleotide sequence of the gene fragment encoding the heavy chain of antibody a inserted into the pCDNA3.4 vector is the nucleotide sequence shown in SEQ ID NO.17, where nucleotides 1-369 are the coding sequence of the heavy chain variable region of antibody a, and nucleotides 370-1341 are the coding sequence of the heavy chain constant region of antibody a.

[0092] The gene fragment encoding the antibody a light chain inserted into the pCDNA3.4 vector is the nucleotide sequence shown in SEQ ID NO.18, wherein nucleotides 1 to 327 are the antibody a light chain variable region encoding sequence, and nucleotides 328 to 648 are the antibody a light chain constant region encoding sequence.

[0093] The gene fragment encoding the antibody b heavy chain inserted into the pCDNA3.4 vector is the nucleotide sequence shown in SEQ ID NO.19, wherein nucleotides 1 to 369 are the antibody b heavy chain variable region encoding sequence, and nucleotides 370 to 1341 are the antibody b heavy chain constant region encoding sequence.

[0094] The gene fragment encoding the antibody b light chain inserted into the pCDNA3.4 vector is the nucleotide sequence shown in SEQ ID NO.20, wherein nucleotides 1 to 330 are the antibody b light chain variable region encoding sequence, and nucleotides 331 to 651 are the antibody b light chain constant region encoding sequence.

[0095] F: Expression and purification of monoclonal antibodies: The monoclonal antibody heavy and light chain expression plasmids obtained in step E were added to the Opti-Mem transfection medium at a ratio of 1:1, and after being fully mixed, a transfection reagent PEI with a mass 4 times that of the DNA was added. After mixing, the mixture was placed in the dark at room temperature for 10 minutes, and then added to 293T cells. After incubation for 6 hours, the transfection system was removed, and FreeStyleTM293 expression medium was added. After culturing for 5 days, the cell culture supernatant was collected, and the expressed cell culture supernatant was purified by affinity purification (Protein A) to obtain the monoclonal antibody. The specific steps are as follows: (1) Centrifuge the expressed antibody supernatant at 2500 × g for 10 min at room temperature to remove the precipitate; (2) Wash the affinity purification column containing Protein A with 10 volumes of binding buffer; (3) The expression supernatant was passed through the purification column at a flow rate of 5 mL / min; (4) Use 20 times the volume of the purification column binding buffer to thoroughly wash the purification column; (5) eluting the purification column with 0.1 M pH = 3.0-3.5 citric acid buffer until the elution peak drops to equilibrium, and adjusting the pH to 7.0 with 1 M pH = 9.0 Tris-HCl buffer; (6) The purified monoclonal antibody is concentrated using a centrifugal column, PBS is used as a buffer for antibody storage, and finally, the concentration of the concentrated antibody is measured using an ultra-micro UV spectrophotometer.

[0096] In a preferred embodiment of the present invention, the pCDNA3.4-antibody a heavy chain plasmid and the pCDNA3.4-antibody a light chain plasmid are co-transfected in a ratio of 1:1, and expressed and purified according to the method of step F to obtain antibody a. The pCDNA3.4-antibody b heavy chain plasmid and the pCDNA3.4-antibody b light chain plasmid are expressed and purified according to the method of step F to obtain antibody b. The obtained antibody a and antibody b are respectively verified by SDS-PAGE and ELISA according to the method described in Example 1. Both antibody a and antibody b show two characteristic bands of about 25KD and 50KD, which are the light chain and heavy chain of IgG respectively; the titers measured by the indirect ELISA method prove that both antibody a and antibody b can specifically recognize interleukin 10 and avoid cross-reaction with the EB virus BCRF1 protein.

[0097] The methods applied in the processes of antibody preparation, antibody gene sequencing, construction and identification of monoclonal antibody expression vectors in the present invention are all conventional methods in molecular biology. The relevant sequences such as primers involved can be obtained from the relevant information recorded in existing technologies such as gene databases or existing literature, or obtained from the heavy chain target gene fragment or light chain target gene fragment of antibody a or b, such as according to the nucleotide sequences shown in SEQ ID NO.17 to SEQ ID NO.20, which will not be elaborated here.

[0098] Example 1

[0099] The interleukin 10 antibodies include: interleukin 10 monoclonal antibody 1 (antibody a), interleukin 10 monoclonal antibody 2 (antibody b). The variable region sequences of the above antibodies are shown in Table 2, and the constant region sequences are shown in Table 3.

[0100] Prepare a 12% SDS-PAGE gel by conventional method, load 5 μg of the above antibody, and perform electrophoresis using a protein molecular weight standard as a reference. The results show that the two interleukin 10 monoclonal antibodies show two characteristic bands of about 25KD and 50KD, which are the light chain and heavy chain of IgG respectively ( Figure 1 ). After scanning the bands, the antibody content is analyzed to be above 90%.

[0101] Figure 1 In the figure, 1: Lane 1 is Marker, Lane 2 is interleukin 10 monoclonal antibody 1; Lane 3 is interleukin 10 monoclonal antibody 2.

[0102] The antibodies used in the following examples are the same as those in Example 1.

[0103] Verification of the specificity of interleukin 10 monoclonal antibody 1 (antibody a): The titer was determined by the indirect ELISA method as follows: the ELISA plate was coated with BCRF1 protein (protein expressed by Nanjing GenScript Biotechnology Co., Ltd., the amino acid sequence is shown in SEQID NO.21) and interleukin 10 (purchased from Beijing Baixinyi Biotechnology Co., Ltd., catalog number A10C08), and the antibody was diluted to 100, 10, 1, and 0.1 ng / mL, respectively, and the affinity of the antibody to different antigens was determined. The results are shown in Table 4.

[0104] Table 4

[0105] Verification of the specificity of interleukin 10 monoclonal antibody 2 (antibody b): The ELISA indirect method was used for determination, and the method was as follows: the ELISA plate was coated with BCRF1 protein and interleukin 10, respectively, and the antibody was diluted to 100, 10, 1, and 0.1 ng / mL, respectively, and the affinity of the antibody to different antigens was measured. The results are shown in Table 5.

[0106] Table 5

[0107] Both antibodies have a stronger affinity for interleukin-10 and almost no recognition for BCRF1 protein.

[0108] Example 2

[0109] An interleukin 10 detection kit, such as Figure 2 , Figure 3 As shown, M is a streptavidin magnetic particle solution, B is a biotin labeled antibody solution, A is an acridine sulfonamide labeled antibody solution, CAL is a calibrator, and QC is a quality control product. The biotin labeled antibody solution includes a biotin labeled interleukin 10 antibody a and a diluent. The acridine sulfonamide labeled antibody solution includes an acridine sulfonamide labeled interleukin 10 antibody b and a diluent. The calibrator and quality control products are lyophilized products, including interleukin 10 and lyophilized excipients.

[0110] Example 3

[0111] Interleukin 10 preparation Interleukin 10 detection kit 1 Main Materials 1.1 Antibodies: Mouse anti-monoclonal antibodies are all interleukin-10 antibodies described in the present invention, which are mouse monoclonal antibodies and are used to label biotin and acridine sulfonamide, respectively.

[0112] 1.2 Streptavidin magnetic particles: JSR.

[0113] 1.3 Acridine sulfonamide NSP-SA-NHS: Helison (Xiamen) Biotechnology Co., Ltd.

[0114] 1.4 Sulfo-NHS-LC-Biotin: Thermo.

[0115] 1.5 Other consumables: The chemiluminescence reagent rack set is provided by Clase Biotech (Chongqing) Co., Ltd.; commonly used reagents are all analytical grade reagents.

[0116] 1.6 Interleukin 10: purchased from Beijing Baixinyi Biotechnology Co., Ltd., product number A10C08.

[0117] 2 Methods 2.1 Preparation of biotin-labeled antibody solution: The steps for preparing the biotin-labeled antibody solution are as follows: (1) Place 1 mg of the antibody to be labeled in a glass bottle and dilute it to 1 mg / mL with 0.01 M PBS (pH 7.2) buffer; (2) Take 14.85 μL of 10 mg / mL Sulfo-NHS-LC-Biotin solution and add it to the above PBS buffer containing the antibody, mix well, and place it at room temperature in the dark for 1-2 hours; dialyze it with 0.01 M PBS (pH 7.2) buffer at 2-8°C in the dark for 5-10 times (each time with an interval of 2 hours); (3) Dilute the labeled product to 0.2-2.0 μg / mL with a diluent; the diluent is a 10-50 mM Tris-HCl buffer containing BSA, and each 100 mL of the diluent contains 0.1-1.0 g of BSA.

[0118] (4) Package the solution in 4 mL / bottle.

[0119] 2.2 Preparation of acridine sulfonamide labeled antibody solution: The steps for preparing the acridine sulfonamide labeled antibody solution are as follows: (1) Place 1 mg of the antibody to be labeled in a glass bottle and dilute it to 1 mg / mL with 0.1 M CBS (pH 9.0); (2) Take 45.5 μL of 2 mg / mL acridine sulfonamide NSP-SA-NHS and add it to the above CBS buffer containing the antibody, mix well, and place it at room temperature in the dark for 1-2 hours; dialyze it with 0.01 M PBS (pH 7.2) buffer at 2-8°C in the dark for 5-10 times (each time with an interval of 2 hours); (3) Dilute the labeled product with a diluent to 0.2 - 2.0 μg / mL; the diluent is a 10 - 50 mM Tris-HCl buffer containing BSA, and each 100 mL of the diluent contains 0.1 - 1.0 g of BSA; (4) Aliquot at 4 mL / vial.

[0120] 2.3 Preparation of streptavidin magnetic particle solution: Dilute streptavidin magnetic particles with a diluent to 0.1 - 1.0 mg / mL; aliquot at 6 mL / vial. The diluent is a 10 - 50 mM Tris-HCl buffer containing BSA, and each 100 mL of the diluent contains 0.1 - 1.0 g of BSA.

[0121] 2.4 Preparation of calibrators and quality control products: Dilute the antigen with a lyophilization solution to 2.5 - 2500 pg / mL (preferably, the preparation of both the calibrator and the quality control product is interleukin 10 at 2000 pg / mL), and aliquot at 0.6 mL / vial for lyophilization. The lyophilization solution is a 10 - 50 mM Tris-HCl buffer containing mannitol, BSA, and PVP, and each 100 mL of the diluent contains 1 - 10 g of mannitol, 0.1 - 1.0 g of BSA, and 0.02 - 0.5 g of PVP.

[0122] 2.5 Assembly of the kit: Refer to Figure 2 the layout, and place the biotin-labeled antibody solution, acridinium sulfonamide-labeled antibody solution, streptavidin magnetic particle solution, calibrators, and quality control products in their respective positions.

[0123] 2.6 Detection: Step 1: Take out the sample to be tested and equilibrate it to room temperature; the kit of the present invention is applicable to serum or plasma samples, preferably serum samples; Step 2: Take out the refrigerated kit and Figure 3 install the reagents shown in the reagent position of the analyzer; Step 3: Reconstitute the calibrators and quality control products with 600 μL of pure water, and then take 60 μL of the reconstituted calibrator or quality control product and dilute it 10-fold with pure water; Step 4: Detect the reconstituted and diluted calibrators, quality control products, and the sample to be tested on the machine; Step 5: The analyzer aspirates 50 - 100 μL of the calibrator, quality control product, or the sample to be tested and adds it to the reaction cup, then adds 10 - 30 μL of the biotin-labeled antibody solution and 10 - 30 μL of the acridinium sulfonamide-labeled antibody, mixes well, and incubates at 37°C for 5 - 25 min; Step 6: Add 10 - 50 μL of streptavidin magnetic particle solution, mix well, and incubate at 37°C for 2 - 10 min; Step 7: After washing, add the pre - excitation solution and the excitation solution, and the analyzer detects the luminescence value.

[0124] 3 Results: The analyzer automatically calibrates the built - in curve according to the detected luminescence value of the calibrator to obtain the calibration curve; the concentration values of interleukin - 10 in the quality control product and the sample to be tested are calculated through the calibration curve. Preferably, the analyzer is the full - automatic chemiluminescence immunoassay analyzer of Kolas Biotech (Chongqing) Co., Ltd., model: Venus 100S.

[0125] Example 4

[0126] Example 4 provides the detection process of the kit.

[0127] Sample preparation to be tested: Dilute interleukin - 10 and BCRF1 protein to 2500, 500, 100, 20, 5, 2.5 pg / mL respectively with 0.01 M PBS containing 1% BSA, and mix well.

[0128] Sample detection: Use the analyzer to detect the reconstituted calibrator, quality control product and sample to be tested according to the method described in Example 3.

[0129] Use the full - automatic chemiluminescence immunoassay analyzer of Kolas Biotech (Chongqing) Co., Ltd., model: Venus100S. Use the built - in program of the instrument for calculation. Among them, the built - in curve fitting method: double - logarithmic four - parameter curve fitting (automatically fitted by the equipment software system); calibration method: two - point segmented calibration; sample concentration calculation: obtained by substituting the sample detection optical signal into the calibration curve. Specifically, import the built - in curve and calibrate to obtain the calibration curve, and calculate the concentration values of interleukin - 10 in the quality control product and the sample to be tested through the calibration curve. The test results are shown in Table 6.

[0130] Table 6 Test results of interleukin - 10 and BCRF1 protein

[0131] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An interleukin 10 antibody selected from antibody a or antibody b, characterized in that: The antibody a comprises heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH, whose amino acid sequences are sequentially shown in SEQ ID NO.1 to SEQ ID NO.3, and light chain complementary determining region CDR1-VL, whose amino acid sequence is shown in SEQ ID NO.4, light chain complementary determining region CDR2-VL, whose amino acid sequence is TAS, and light chain complementary determining region CDR3-VL, whose amino acid sequence is shown in SEQ ID NO.5; The antibody b includes heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH whose amino acid sequences are shown in SEQ ID NO.6 to SEQ ID NO.8, and a light chain complementary determining region CDR1-VL whose amino acid sequence is shown in SEQ ID NO.9, a light chain complementary determining region CDR2-VL whose amino acid sequence is TAS, and a light chain complementary determining region CDR3-VL whose amino acid sequence is shown in SEQ ID NO.

10.

2. The interleukin 10 antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the antibody a is shown in SEQ ID NO.11; the amino acid sequence of the light chain variable region of the antibody a is shown in SEQ ID NO.

12.

3. The interleukin 10 antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the antibody b is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the antibody b is shown in SEQ ID NO.

14.

4. The coding sequence of the interleukin 10 antibody according to any one of claims 1 to 3, characterized in that: The coding sequence of the heavy chain variable region of the antibody a is nucleotides 1 to 369 of the nucleotide sequence shown in SEQ ID NO.17; the coding sequence of the light chain variable region of the antibody a is nucleotides 1 to 327 of the nucleotide sequence shown in SEQ ID NO.18; the coding sequence of the heavy chain variable region of the antibody b is nucleotides 1 to 369 of the nucleotide sequence shown in SEQ ID NO.19; the coding sequence of the light chain variable region of the antibody b is nucleotides 1 to 330 of the nucleotide sequence shown in SEQ ID NO.

20.

5. The coding sequence according to claim 4, characterized in that The coding sequence of the heavy chain of antibody a is the nucleotide sequence shown in SEQ ID NO.17; the coding sequence of the light chain of antibody a is the nucleotide sequence shown in SEQ ID NO.18; the coding sequence of the heavy chain of antibody b is the nucleotide sequence shown in SEQ ID NO.19; and the coding sequence of the light chain of antibody b is the nucleotide sequence shown in SEQ ID NO.

20.

6. A recombinant expression vector comprising the coding sequence of claim 4 or 5.

7. A host cell, characterized in that The host cell comprises the recombinant expression vector of claim 6 or the coding sequence of claim 4 or 5 is integrated into the genome of the host cell.

8. Use of the interleukin 10 antibody according to any one of claims 1 to 3, or the coding sequence of the interleukin 10 antibody according to claim 4 or 5, or the recombinant expression vector according to claim 6, or the host cell according to claim 7 in the preparation of an interleukin 10 magnetic particle chemiluminescence detection product.

9. An interleukin 10 detection kit, comprising a streptavidin magnetic particle solution, an acridine sulfonamide labeled antibody solution, a biotin labeled antibody solution, a calibrator and a quality control product, characterized in that: The acridine sulfonamide-labeled antibody solution contains the interleukin 10 antibody according to any one of claims 1 to 3, and the interleukin 10 antibody is labeled with acridine sulfonamide; the biotin-labeled antibody solution contains the interleukin 10 antibody according to any one of claims 1 to 3, and the interleukin 10 antibody is labeled with biotin; the interleukin 10 antibody in the acridine sulfonamide-labeled antibody solution is different from the interleukin 10 antibody in the biotin-labeled antibody solution.

10. The kit according to claim 9, characterized in that The acridine sulfonamide labeled antibody solution contains 0.2-2 μg / mL of the interleukin 10 antibody; the biotin labeled antibody solution contains 0.2-2 μg / mL of the interleukin 10 antibody; the interleukin 10 antibody diluent is a 10-50 mM Tris-HCl buffer containing BSA, and each 100 mL of the diluent contains 0.1-1.0 g of BSA.

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