An interleukin-10 antibody, its application, and an interleukin-10 detection kit

Abstract

The present invention provides novel interleukin-10 antibodies selected from antibody a or antibody b. Also provided are uses of the interleukin-10 antibodies and an interleukin-10 detection kit. The interleukin-10 antibodies provided by the present invention have good specificity and high affinity for interleukin-10, avoid cross-reaction with the EB virus BCRF1 protein, and can be used to prepare magnetic microparticle chemiluminescence detection products for detecting interleukin-10. The interleukin-10 detection kit provided by the present invention has the advantages of automated detection, high sensitivity, strong specificity, and high throughput.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to an interleukin 10 antibody and application thereof, and an interleukin 10 detection kit. 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 inflammatory and immunosuppressive factor. IL-10 can exert both immunosuppressive and immunostimulatory effects on various cell types, playing a crucial role in infection, organ transplantation, the hematopoietic system, and the cardiovascular system. It is closely associated with diseases of the blood, digestive system, and, in particular, the cardiovascular system.

[0003] In some studies, IL-10 promotes the growth and survival of cancer cells, including non-Hodgkin's lymphoma, Burkitt's lymphoma, and non-small cell lung cancer. IL-10 overexpression is associated with systemic lupus erythematosus (SLE) and tuberculosis, while IL-10 deficiency is associated with inflammatory bowel disease, psoriasis, asthma, and rheumatoid arthritis. Currently, commonly used clinical methods for detecting interleukin-10 include enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, and chemiluminescence assay.

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

[0005] Both human and mouse IL-10 genes encode a 178-amino acid polypeptide, including a cleavable signal peptide sequence. Both human and mouse IL-10 genes share a high degree of homology with BCRF1 (BamHI C right frame 1 protein), an open reading frame of the Epstein-Barr virus. Sequence alignment analysis revealed approximately 82% amino acid sequence identity between the two, potentially leading to some overlap during detection. This necessitates high specificity requirements for both detection kits and antibody raw materials. Summary of the Invention

[0006] The object of the present invention is to provide an interleukin 10 antibody, which specifically recognizes interleukin 10 and avoids cross-reaction with the EB virus BCRF1 protein; the present invention also provides the use of the interleukin 10 antibody in the preparation of an interleukin 10 magnetic particle chemiluminescence detection product; the present invention also provides an interleukin 10 magnetic particle chemiluminescence detection product, comprising a streptavidin magnetic particle solution, an acridine sulfonamide-labeled antibody solution, a biotin-labeled antibody solution, a calibrator and a quality control product, to solve the problems in the prior art.

[0007] In order to achieve the above object, the present invention provides an interleukin-10 antibody selected from antibody a or antibody b;

[0008] The antibody a comprises heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH, whose amino acid sequences are shown in SEQ ID NO.1 to SEQ ID NO.3, and a light chain complementary determining region CDR1-VL, whose amino acid sequence is shown in SEQ ID NO.4, 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.5;

[0009] The antibody b includes heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH with amino acid sequences as shown in SEQ ID NO.6 to SEQ ID NO.8, and a light chain complementary determining region CDR1-VL with an amino acid sequence as shown in SEQ ID NO.9, a light chain complementary determining region CDR2-VL with an amino acid sequence of TAS, and a light chain complementary determining region CDR3-VL with an amino acid sequence as shown in SEQ ID NO.10.

[0010] Preferably, the amino acid sequence of the heavy chain variable region of antibody a is shown in SEQ ID NO.11.

[0011] Preferably, any of the above items is that the amino acid sequence of the light chain variable region of the antibody a is as shown in SEQ ID NO.12.

[0012] Preferably, in any of the above items, the amino acid sequence of the heavy chain variable region of the antibody b is as shown in SEQ ID NO.13.

[0013] Preferably, in any of the above items, the amino acid sequence of the light chain variable region of the antibody b is as shown in SEQ ID NO.14.

[0014] Preferably, in any of the above items, the amino acid sequence of the light chain constant region of the antibody a or antibody b is as shown in SEQ ID NO.15.

[0015] Preferably, any of the above items is that the amino acid sequence of the heavy chain constant region of the anti-a or anti-b antibody is as shown in SEQ ID NO.16.

[0016] Preferably, any of the above items is that the antibody a is interleukin-10 monoclonal antibody 1, and the antibody b is interleukin-10 monoclonal antibody 2; further, the antibody a or antibody b is of mouse origin.

[0017] The present invention also provides a coding sequence of any of the above-mentioned interleukin-10 antibodies.

[0018] Preferably, in any of the above items, the coding sequence of the heavy chain variable region of antibody a is nucleotides 1 to 369 of the nucleotide sequence shown in SEQ ID NO.17.

[0019] Preferably, in any of the above items, the coding sequence of the antibody a light chain variable region is nucleotides 1 to 327 of the nucleotide sequence shown in SEQ ID NO.18.

[0020] Preferably, in any of the above items, the coding sequence of the heavy chain variable region of antibody b is nucleotides 1 to 369 of the nucleotide sequence shown in SEQ ID NO.19.

[0021] Preferably, in any of the above items, the coding sequence of the light chain variable region of antibody b is nucleotides 1 to 330 of the nucleotide sequence shown in SEQ ID NO.20.

[0022] Preferably, any of the above items is that the coding sequence of the antibody a heavy chain is the nucleotide sequence shown in SEQ ID NO.17.

[0023] Preferably, in any of the above items, the coding sequence of the antibody a light chain is the nucleotide sequence shown in SEQ ID NO.18.

[0024] Preferably, in any of the above items, the coding sequence of the heavy chain of antibody b is the nucleotide sequence shown in SEQ ID NO.19.

[0025] Preferably, in any of the above items, the coding sequence of the antibody b light chain is the nucleotide sequence shown in SEQ ID NO.20.

[0026] The present invention also provides a recombinant expression vector comprising any one of the above coding sequences.

[0027] The recombinant expression vector comprises the coding sequence of the antibody a and / or the coding sequence of the antibody b.

[0028] Preferably, any of the above items is that the recombinant expression vector comprises the coding sequence of the antibody a light chain variable region.

[0029] Preferably, any of the above items is that the recombinant expression vector comprises the coding sequence of the heavy chain variable region of antibody a.

[0030] Preferably, any of the above items is that the recombinant expression vector comprises the coding sequence of the antibody b light chain variable region.

[0031] Preferably, any of the above items is that the recombinant expression vector comprises the coding sequence of the heavy chain variable region of antibody b.

[0032] Preferably, any of the above items is that the recombinant expression vector comprises the coding sequence of the antibody a light chain.

[0033] Preferably, any of the above items is that the recombinant expression vector comprises the coding sequence of antibody a heavy chain.

[0034] Preferably, any of the above items is that the recombinant expression vector comprises the coding sequence of the antibody b light chain.

[0035] Preferably, any of the above items is that the recombinant expression vector comprises the coding sequence of antibody b heavy chain.

[0036] In any of the above, preferably, the recombinant expression vector is constructed by connecting the coding sequence provided by the present invention to various expression vectors using conventional methods in the art. The expression vectors are any conventional vectors in the art, and any vectors capable of carrying the nucleic acid molecule containing the coding sequence are suitable for use in the present invention. The expression vectors preferably include various plasmids, cosmids, phages, or viral vectors, such as the pCDNA3.4 and pCDNA3.1 series vectors. The expression vectors are preferably eukaryotic expression vectors, and are preferably expression vectors suitable for use in mammalian cells.

[0037] The present invention also provides a host cell, which comprises any of the above-mentioned recombinant expression vectors or any of the above-mentioned coding sequences are integrated into the genome of the host cell.

[0038] Preferably, any of the above items is that the host cell is a mammalian cell.

[0039] 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 a 293T cell.

[0040] The interleukin-10 antibody described herein can be a monoclonal antibody produced by a hybridoma or obtained using any of the recombinant expression vectors or host cells described above. A recombinant expression vector containing the heavy chain coding sequence of Antibody A and a recombinant expression vector containing the light chain coding sequence of Antibody A are co-transfected into a host cell (preferably a mammalian cell) in equal proportions. The recombinant expression vectors containing the heavy chain coding sequence of Antibody A and the light chain coding sequence of Antibody A undergo transcription, translation, and various modifications within the host cell to form the antibody, which is then purified to obtain Antibody A. A recombinant expression vector containing the heavy chain coding sequence of Antibody B and a recombinant expression vector containing the light chain coding sequence of Antibody B are co-transfected into a host cell (preferably a mammalian cell) in equal proportions. The recombinant expression vectors containing the heavy chain coding sequence of Antibody B and the light chain coding sequence of Antibody B undergo transcription, translation, and various modifications within the cell to form the antibody, which is then purified to obtain Antibody B. In the present invention, both the monoclonal antibody obtained from hybridoma and the antibody obtained by recombinant expression can specifically recognize interleukin 10 while avoiding cross-reaction with EB virus BCRF1 protein.

[0041] The present invention also provides the use of any of the above-mentioned interleukin-10 antibodies, or the coding sequence of any of the above-mentioned interleukin-10 antibodies, or the recombinant expression vector, or the host cell in the preparation of an interleukin-10 magnetic particle chemiluminescence detection product.

[0042] The present invention also provides 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. The acridine sulfonamide-labeled antibody solution contains any of the interleukin-10 antibodies described above, labeled with acridine sulfonamide; the biotin-labeled antibody solution contains any of the interleukin-10 antibodies described above, labeled with biotin; and the interleukin-10 antibody in the acridine 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 at concentrations of 2.5 to 2500 pg / mL.

[0043] Preferably, in any of the above items, the concentration of the streptavidin magnetic particle solution is 0.1-1.0 mg / mL, and more preferably 0.1, 0.5, 1.0 mg / mL and ranges therebetween.

[0044] 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.

[0045] 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.

[0046] In any of the above, preferably, the interleukin-10 antibody diluent is a 10-50 mM Tris-HCl buffer containing BSA, with each 100 mL of diluent containing 0.1-1.0 g of BSA; further preferably, the concentration of Tris-HCl in the Tris-HCl buffer is 10, 20, 30, 40, 50 mM, and ranges therebetween; further preferably, each 100 mL of diluent contains 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 g, and ranges therebetween of BSA. The interleukin-10 antibody diluent is a diluent of the acridine sulfonamide-labeled antibody solution or the biotin-labeled antibody solution.

[0047] Preferably, any of the above items is that the calibrator is a freeze-dried product of interleukin-10 protein, and preferably the calibrator also includes freeze-dried excipients. Preferably, pure water is used for reconstitution during use.

[0048] 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 excipients. Preferably, pure water is used for reconstitution during use.

[0049] In any of the above, the lyophilized interleukin 10 protein may be interleukin 10 prepared using existing methods or a commercial interleukin 10 protein product. The present invention preferably uses interleukin 10 produced by 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.

[0050] Preferably, any of the above items is that the lyophilization excipient is a conventional lyophilization excipient in the prior art, preferably including saccharides such as trehalose, sucrose, and lactose; preferably including polyols such as mannitol and sorbitol; preferably including amino acids such as glycine and histidine; preferably including proteins such as bovine serum albumin (BSA); preferably including gelatin; preferably including buffer systems such as phosphate buffered saline (PBS) and Tris-HCl; preferably including surfactants such as Tween-20 and polyvinylpyrrolidone (PVP); preferably including preservatives such as Proclin 300 and sodium azide.

[0051] In the present invention, the interleukin-10 antibody on the acridine sulfonamide-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 acridine sulfonamide-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 acridine sulfonamide-labeled antibody solution is antibody b, and the interleukin-10 antibody on the biotin-labeled antibody solution is antibody a.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] The interleukin 10 antibody provided by the present invention has good specificity and high affinity for interleukin 10, avoids cross-reaction with EB virus BCRF1 protein, and can be used to prepare a magnetic particle chemiluminescence detection product for detecting interleukin 10.

[0054] The interleukin-10 detection kit provided by the present invention has the advantages of automated detection, high sensitivity, strong specificity, and high throughput. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is the electrophoresis diagram of the interleukin-10 monoclonal antibody in the preferred embodiment 1 of the present invention.

[0056] Figure 2 This is the interleukin-10 detection kit in preferred embodiments 2 and 3 of the present invention.

[0057] Figure 3 This is the interleukin-10 detection reagent in preferred embodiments 2 and 3 of the present invention. DETAILED DESCRIPTION

[0058] The present invention is further described in detail below through specific embodiments.

[0059] 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 an 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 usually the most variable parts of the antibody and contain the antigen binding site. The light or heavy chain variable region is composed of a framework region interrupted by three hypervariable regions called "complementarity determining regions" or "CDRs". The framework region of an antibody, that is, the combined framework region of the constituent light and heavy chains, plays a role in positioning and aligning the CDRs, which are primarily responsible for binding to the antigen.

[0060] "Framework" or "FR" regions refer to the regions of an antibody variable domain excluding those defined as CDRs. Each antibody variable domain framework can be further subdivided into contiguous regions (FR1, FR2, FR3, and FR4) separated by CDRs.

[0061] Typically, the variable regions VL / VH of the heavy and light chains can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0062] In the present invention, CDR1-VH, CDR2-VH and CDR3-VH refer to the three hypervariable regions of the heavy chain variable region, respectively. Correspondingly, CDR1-VL, CDR2-VL and CDR3-VL refer to the three hypervariable regions of the light chain variable region, respectively.

[0063] In a first aspect, the present invention provides an interleukin-10 antibody selected from antibody a or antibody b;

[0064] The antibody a comprises heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH, whose amino acid sequences are shown in SEQ ID NO.1 to SEQ ID NO.3, and a light chain complementary determining region CDR1-VL, whose amino acid sequence is shown in SEQ ID NO.4, 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.5;

[0065] The antibody b includes heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH with amino acid sequences as shown in SEQ ID NO.6 to SEQ ID NO.8, and a light chain complementary determining region CDR1-VL with an amino acid sequence as shown in SEQ ID NO.9, a light chain complementary determining region CDR2-VL with an amino acid sequence of TAS, and a light chain complementary determining region CDR3-VL with an amino acid sequence as shown in SEQ ID NO.10.

[0066] The amino acid sequences shown in SEQ ID NO. 1 to SEQ ID NO. 10 are shown in Table 1.

[0067] Table 1

[0068]

[0069] In a preferred embodiment of the present invention, the antibody a comprises a heavy chain variable region and a light chain variable region whose amino acid sequences are shown in SEQ ID NO.11 and SEQ ID NO.12, respectively;

[0070] The antibody b comprises a heavy chain variable region and a light chain variable region whose amino acid sequences are shown in SEQ ID NO.13 and SEQ ID NO.14, respectively;

[0071] Preferably, the interleukin-10 antibody is of murine origin.

[0072] The amino acid sequences represented by SEQ ID NO.11 to SEQ ID NO.14 are shown in Table 2.

[0073] Table 2

[0074]

[0075] The light chain constant region of the antibody a or antibody b is shown in SEQ ID NO.15, and the heavy chain constant region of the antibody a or antibody b is shown in SEQ ID NO.16, as shown in Table 3.

[0076] 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;

[0077] The amino acid sequence of the antibody a light chain consists of the amino acid sequences shown in SEQ ID NO. 12 and SEQ ID NO. 15, in sequence;

[0078] 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;

[0079] The amino acid sequence of the antibody b light chain consists of the amino acid sequences shown in SEQ ID NO. 14 and SEQ ID NO. 15 in sequence.

[0080] Table 3

[0081]

[0082] 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.

[0083] In a second aspect, the present invention provides the use of the interleukin-10 antibody in the preparation of an interleukin-10 magnetic particle chemiluminescence detection product.

[0084] In a third aspect, the present invention provides an interleukin 10 detection kit, which includes a streptavidin magnetic particle solution, an acridine sulfonamide-labeled antibody solution, a biotin-labeled antibody solution, a calibrator, and a quality control product.

[0085] Preferably, the concentration of the streptavidin magnetic particle solution is 0.1-1.0 mg / mL.

[0086] Preferably, the acridine sulfonamide-labeled antibody solution contains the interleukin-10 antibody, the interleukin-10 antibody is labeled with acridine sulfonamide, and the concentration of acridine sulfonamide is identified by a detection instrument.

[0087] Preferably, the biotin-labeled antibody solution contains 0.2-2 μg / mL of the interleukin-10 antibody.

[0088] Preferably, the acridine sulfonamide-labeled antibody solution contains 0.2-2 μg / mL of the interleukin-10 antibody.

[0089] Preferably, the diluent for the biotin-labeled antibody solution and the acridine sulfonamide-labeled antibody solution 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.

[0090] Preferably, the interleukin-10 antibody on the acridine sulfonamide-labeled antibody solution is different from the interleukin-10 antibody on the biotin-labeled antibody solution.

[0091] In a preferred embodiment of the present invention, the antibody in the acridine sulfonamide-labeled antibody solution is interleukin 10 antibody a, wherein antibody a is interleukin 10 monoclonal antibody 1, and the antibody in the biotin-labeled antibody solution is interleukin 10 antibody b, wherein antibody b is interleukin 10 monoclonal antibody 2.

[0092] In a preferred embodiment of the present invention, the antibody in the acridine sulfonamide-labeled antibody solution is interleukin-10 antibody b, and the antibody in the biotin-labeled antibody solution is interleukin-10 antibody a.

[0093] Preferably, the calibrator is a freeze-dried product of interleukin-10 protein, and preferably the calibrator also includes freeze-dried excipients. Preferably, pure water is used for reconstitution during use.

[0094] Preferably, the quality control product is a freeze-dried product of interleukin-10 protein, and preferably the quality control product also includes freeze-dried excipients. Preferably, pure water is used for reconstitution during use.

[0095] Preferably, the lyophilized interleukin 10 protein can be interleukin 10 prepared using 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.

[0096] Preferably, the lyophilization excipients are conventional lyophilization excipients in the prior art, preferably including sugars such as trehalose, sucrose, and lactose; preferably including polyols such as mannitol and sorbitol; preferably including amino acids such as glycine and histidine; preferably including proteins such as bovine serum albumin (BSA); preferably including gelatin; preferably including buffer systems such as phosphate buffered saline (PBS) and Tris-HCl; preferably including surfactants such as Tween-20 and polyvinylpyrrolidone (PVP); preferably including preservatives such as Proclin 300 and sodium azide.

[0097] The interleukin-10 detection kit provided by the present invention has the advantages of automated operation, high sensitivity, strong specificity, and high throughput.

[0098] The reagent provided by the present invention is based on the principle of antigen-antibody reaction. After labeling the interleukin-10 antibody with acridine sulfonamide, a diluent is used to prepare a biotin-labeled antibody solution. The biotin-labeled antibody solution contains another biotin-labeled interleukin-10 antibody. Based on the principle of antigen-antibody reaction, the reaction can be detected by a supporting instrument after completion. If interleukin-10 is present in the sample, a double antibody sandwich structure is formed, and the instrument will detect a strong light signal. If interleukin-10 is absent, the instrument will detect a weak light signal. The intensity of the light signal is used to predict the amount of the analyte.

[0099] The test kit provided by the present invention is used for testing, and the test results can be obtained within 30 minutes of the entire process. This is fast and efficient, which helps medical personnel to obtain test results in a timely manner, make comprehensive judgments based on the results, and take timely measures.

[0100] Add test sample to the kit:

[0101] It should be noted that the diluent for the interleukin-10 antibody described in the present invention is a 10-50 mM Tris-HCl buffer containing BSA, with each 100 mL of the diluent containing 0.1-1.0 g of BSA. Unless otherwise specified in the following examples, the diluent for the interleukin-10 antibody is preferably a 20 mM Tris-HCl buffer containing BSA, with each 100 mL of the diluent containing 0.5 g of BSA.

[0102] The methods of the present invention for preparing interleukin-10 monoclonal antibody 1 represented by antibody a and interleukin-10 monoclonal antibody 2 represented by antibody b are conventional monoclonal antibody preparation methods in the prior art, and are briefly described as follows:

[0103] (1) Animal immunization:

[0104] A: Interleukin-10 (catalog number A10C08) purchased from Beijing Baixinyi Biotechnology Co., Ltd. was mixed with equal volumes of Freund's adjuvant to an appropriate volume and emulsified completely. Mice were immunized by intraperitoneal injection, with 50 μg of immunogen injected into each mouse in a volume of 100 μL, once a week.

[0105] B: After immunization for 4 times, the titer of the antibody in the serum was tested by indirect ELISA method with interleukin-10 coated. Mice with an OD value greater than 1.0 detected by 16,000-fold serum dilution were screened.

[0106] (2) Preparation of monoclonal antibodies:

[0107] A: Splenocytes from screened mice were fused with myeloma cells, and the fused cells were plated and cultured using the limiting dilution method.

[0108] B: Screen the monoclonal cell wells and culture and expand them. The monoclonal cell well with the highest OD value detected in the cell culture supernatant is used as the target hybridoma cell, culture and expand it to obtain the hybridoma cell line and freeze it;

[0109] C: Isolation of antibody variable region genes from hybridoma cells using RT-PCR: After homogenization of the hybridoma cells, add cell lysis buffer for RNA extraction. Precipitate RNA from the aqueous phase with isopropanol. Wash the precipitated RNA after centrifugation to remove impurities, resuspend it, and perform reverse transcription to obtain cDNA.

[0110] D: PCR was performed using mouse-specific primers known in the prior art, using hybridoma cell cDNA as a template to amplify the heavy and light chain variable region genes of the antibody. A 50 μL system contained 5 μL of cDNA, HotStarTaq Plus enzyme, dNTPs, and 0.5 μM of specific primers. PCR amplification was performed under the following conditions: pre-denaturation at 94°C for 5 min; 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 50 s; and 72°C for 7 min. The obtained PCR products were identified by 1% agarose gel electrophoresis, and the target fragments were recovered and sent for sequencing. Based on the antibody gene sequences obtained by sequencing, the antibody variable region amino acid sequences shown in SEQ ID NOs: 11 to 14 were further obtained; wherein the light chain variable region amino acid sequence of antibody a is shown in SEQ ID NO.12, the heavy chain variable region amino acid sequence of antibody a is shown in SEQ ID NO.11, the light chain variable region amino acid sequence of antibody b is shown in SEQ ID NO.14, and the heavy chain variable region amino acid sequence 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 SEQ ID 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.

[0111] E: Construction of monoclonal antibody expression vector:

[0112] The antibody gene sequence obtained by sequencing in step D is used to construct a monoclonal antibody expression vector using conventional methods in the art. Homologous recombination primers are used to add homologous recombination arms to both ends of the antibody heavy chain variable region gene and the light chain variable region gene, respectively. The expression plasmid containing the mouse antibody heavy and light chain IgG1 constant regions is linearized using a dual enzyme to generate homologous recombination arms. The variable region gene fragments added with the homologous recombination arms and the linearized plasmid are connected by homologous recombination to form a complete expression vector, the expression vector being pCDNA3.4. The recombinant product is transformed into a TOP10 competent Escherichia coli, and the plasmid is amplified. The pCDNA3.4-antibody a heavy chain plasmid, pCDNA3.4-antibody a light chain plasmid, and the pCDNA3.4-antibody b heavy chain plasmid and pCDNA3.4-antibody b light chain plasmid are obtained, respectively.

[0113] The gene fragment encoding the antibody a heavy chain inserted into the pCDNA3.4 vector is the nucleotide sequence shown in SEQ ID NO.17, wherein nucleotides 1 to 369 are the antibody a heavy chain variable region encoding sequence, and nucleotides 370 to 1341 are the antibody a heavy chain constant region encoding sequence.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] F: Expression and purification of monoclonal antibodies:

[0118] The monoclonal antibody heavy and light chain expression plasmids obtained in step E were added to Opti-Mem transfection medium at a ratio of 1:1. After thorough mixing, PEI transfection reagent (4 times the mass of 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:

[0119] (1) Centrifuge the expressed antibody supernatant at 2500 × g for 10 min at room temperature to remove the precipitate;

[0120] (2) The affinity purification column containing Protein A was thoroughly washed with 10 volumes of binding buffer;

[0121] (3) The expression supernatant was passed through the purification column at a flow rate of 5 mL / min;

[0122] (4) Wash the purification column thoroughly with 20 times the volume of the purification column binding buffer;

[0123] (5) Elute the purification column with 0.1 M pH = 3.0-3.5 citric acid buffer until the elution peak drops to equilibrium, and adjust the pH to 7.0 with 1 M pH = 9.0 Tris-HCl buffer;

[0124] (6) The purified monoclonal antibody was concentrated using a centrifugal column, PBS was used as the buffer for antibody storage, and the concentration of the concentrated antibody was measured using an ultra-micro UV spectrophotometer.

[0125] 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 were co-transfected at a 1:1 ratio and expressed and purified according to the method in step F to obtain antibody a. The pCDNA3.4-antibody b heavy chain plasmid and the pCDNA3.4-antibody b light chain plasmid were expressed and purified according to the method in step F to obtain antibody b. The obtained antibodies a and b were verified by SDS-PAGE and ELISA, respectively, using the methods described in Example 1. Both antibodies a and b exhibited two characteristic bands of approximately 25 kD and 50 kD, representing the light and heavy chains of IgG, respectively. Indirect ELISA titer determination demonstrated that both antibodies a and b could specifically recognize interleukin 10 and avoid cross-reaction with the Epstein-Barr virus BCRF1 protein.

[0126] The methods used in the present invention for antibody preparation, antibody gene sequencing, construction and identification of monoclonal antibody expression vectors, etc., are all conventional methods in molecular biology. The primers and other related sequences involved can be obtained through existing technologies, such as relevant information recorded in gene databases or existing literature, or can be obtained from the heavy chain target gene fragment or light chain target gene fragment of antibody a or b, such as the nucleotide sequences shown in SEQ ID NO. 17 to SEQ ID NO. 20, and are not described in detail here.

[0127] Example 1

[0128] Interleukin 10 antibodies include: interleukin 10 monoclonal antibody 1 (antibody a) and 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.

[0129] A 12% SDS-PAGE gel was prepared according to conventional methods, and 5 μg of the above antibodies were loaded and electrophoresed using a protein molecular weight standard as a reference. The results showed that the two interleukin-10 monoclonal antibodies showed two characteristic bands of about 25KD and 50KD, which were the light chain and heavy chain of IgG, respectively ( Figure 1 ). After scanning and analysis, the antibody content of the bands was above 90%.

[0130] Figure 1 In the figure, 1: lane 1 is a marker, lane 2 is interleukin-10 monoclonal antibody 1; lane 3 is interleukin-10 monoclonal antibody 2.

[0131] The antibodies used in the following examples are the same as those in Example 1.

[0132] Verification of the specificity of interleukin-10 monoclonal antibody 1 (antibody a): The titer was determined by the indirect ELISA method as follows: BCRF1 protein (expressed by Nanjing GenScript Biotechnology Co., Ltd., with the amino acid sequence shown in SEQ ID NO. 21) and interleukin-10 (purchased from Beijing Baixinyi Biotechnology Co., Ltd., catalog number A10C08) were coated on an ELISA plate, and the antibodies were diluted to 100, 10, 1, and 0.1 ng / mL, respectively. The affinity of the antibodies for different antigens was measured. The results are shown in Table 4.

[0133] Table 4

[0134]

[0135] Verification of the specificity of interleukin-10 monoclonal antibody 2 (antibody b): The ELISA indirect method was used as follows: BCRF1 protein and interleukin-10 were coated on the enzyme-labeled plate, respectively, and the antibody was diluted to 100, 10, 1, and 0.1 ng / mL, respectively. The affinity of the antibody for different antigens was measured. The results are shown in Table 5.

[0136] Table 5

[0137]

[0138] The two antibodies have a strong affinity for interleukin-10 and almost no recognition for BCRF1 protein.

[0139] Example 2

[0140] 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. The biotin-labeled antibody solution includes biotin-labeled interleukin-10 antibody a and a diluent. The acridine sulfonamide-labeled antibody solution includes acridine sulfonamide-labeled interleukin-10 antibody b and a diluent. The calibrator and quality control are lyophilized products, including interleukin-10 and lyophilized excipients.

[0141] Example 3

[0142] Interleukin 10 preparation Interleukin 10 detection kit

[0143] 1 Main Materials

[0144] 1.1 Antibodies: The 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.

[0145] 1.2 Streptavidin magnetic particles: JSR.

[0146] 1.3 Acridinium sulfonamide NSP-SA-NHS: Helison (Xiamen) Biotechnology Co., Ltd.

[0147] 1.4 Sulfo-NHS-LC-Biotin: Thermo.

[0148] 1.5 Other consumables: The chemiluminescence reagent rack set is provided by Clase Biotechnology (Chongqing) Co., Ltd.; commonly used reagents are all analytical grade reagents.

[0149] 1.6 Interleukin-10: purchased from Beijing Baixinyi Biotechnology Co., Ltd., product number A10C08.

[0150] 2 Methods

[0151] 2.1 Preparation of biotin-labeled antibody solution:

[0152] The steps for preparing biotin-labeled antibody solution are as follows:

[0153] (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;

[0154] (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 in the dark at room temperature 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);

[0155] (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.

[0156] (4) Dispense the solution at 4 mL / bottle.

[0157] 2.2 Preparation of Acridine Sulfonamide Labeled Antibody Solution:

[0158] The steps for preparing the acridine sulfonamide labeled antibody solution are as follows:

[0159] (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);

[0160] (2) Take 45.5 μL of 2 mg / mL acridine sulfonamide NSP-SA-NHS and add it to the above-mentioned CBS buffer containing the antibody, mix well, and place it in the dark at room temperature 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);

[0161] (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, with each 100 mL of diluent containing 0.1-1.0 g of BSA;

[0162] (4) Dispense the solution at 4 mL / bottle.

[0163] 2.3 Preparation of Streptavidin Magnetic Particle Solution:

[0164] Dilute the streptavidin magnetic particles to 0.1-1.0 mg / mL with diluent. Aliquot and package at 6 mL / bottle. The diluent is 10-50 mM Tris-HCl buffer containing BSA, with 0.1-1.0 g of BSA per 100 mL.

[0165] 2.4 Preparation of calibrators and quality control products:

[0166] Dilute the antigen to 2.5-2500 pg / mL (preferably, the calibrator or quality control is prepared at 2000 pg / mL of interleukin-10) using a lyophilization solution. Aliquot and freeze-dry in 0.6 mL tubes. The lyophilization solution is a 10-50 mM Tris-HCl buffer containing mannitol, BSA, and PVP. Each 100 mL of diluent contains 1-10 g mannitol, 0.1-1.0 g BSA, and 0.02-0.5 g PVP.

[0167] 2.5 Assembly of the kit:

[0168] refer to Figure 2 Layout, place the biotin-labeled antibody solution, acridine sulfonamide-labeled antibody solution, streptavidin magnetic particle solution, calibrator and quality control products in the corresponding positions respectively.

[0169] 2.6 Testing:

[0170] Step 1: Take out the sample to be tested and equilibrate it to room temperature; the kit of the present invention is suitable for serum or plasma samples, preferably serum samples;

[0171] Step 2: Take out the refrigerated test kit and Figure 3The reagents shown are installed in the reagent position of the analyzer;

[0172] Step 3: Reconstitute the calibrator and quality control with 600 μL of pure water, then dilute 60 μL of the reconstituted calibrator or quality control 10-fold with pure water.

[0173] Step 4: Load the reconstituted and diluted calibrators and quality control materials and the samples to be tested onto the instrument for testing;

[0174] Step 5. Pipette 50-100 μL of calibrator, quality control, or sample into the reaction cup. Then add 10-30 μL of biotin-labeled antibody solution and 10-30 μL of acridine sulfonamide-labeled antibody. Mix well and incubate at 37°C for 5-25 minutes.

[0175] Step 6: Add 10-50 μL of streptavidin magnetic particle solution, mix well, and incubate at 37°C for 2-10 min.

[0176] Step 7: After washing, add pre-excitation solution and excitation solution, and use an analyzer to detect the luminescence value.

[0177] 3 Results:

[0178] The analyzer automatically calibrates the built-in curve based on the luminescence value of the calibrator to obtain a calibration curve; the concentration of interleukin-10 in the quality control sample and the test sample is calculated using the calibration curve. Preferably, the analyzer is a fully automatic chemiluminescence immunoassay analyzer manufactured by Clus Biotech (Chongqing) Co., Ltd., model: Venus 100S.

[0179] Example 4

[0180] Example 4 provides the kit detection process.

[0181] Preparation of test samples: Dilute interleukin-10 and BCRF1 proteins to 2500, 500, 100, 20, 5, and 2.5 pg / mL, respectively, with 0.01 M PBS containing 1% BSA and mix well.

[0182] Sample detection: Use an analyzer to detect the reconstituted calibrators, quality control products and test samples according to the method described in Example 3.

[0183] A fully automated chemiluminescence immunoassay analyzer, model Venus100S, from Clarith Biotech (Chongqing) Co., Ltd. was used. Calculations were performed using the instrument's built-in program, which included a double-logarithmic four-parameter curve fit (automatically fitted by the instrument software); a two-point segmented calibration method; and sample concentrations were calculated by substituting the sample detection light signal into the calibration curve. Specifically, the built-in curve was imported and calibrated to obtain a calibration curve. Interleukin-10 concentrations in the control sample and test samples were calculated using the calibration curve. The test results are shown in Table 6.

[0184] Table 6 Interleukin-10 and BCRF1 protein test results

[0185]

[0186] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to 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 shown in SEQ ID NO.1 to SEQ ID NO.3, and a light chain complementary determining region CDR1-VL, whose amino acid sequence is shown in SEQ ID NO.4, 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.5; The antibody b includes heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH with amino acid sequences as shown in SEQ ID NO.6 to SEQ ID NO.8, and a light chain complementary determining region CDR1-VL with an amino acid sequence as shown in SEQ ID NO.9, a light chain complementary determining region CDR2-VL with an amino acid sequence of TAS, and a light chain complementary determining region CDR3-VL with an amino acid sequence as shown in SEQ ID NO.

10.

2. The interleukin-10 antibody according to claim 1, wherein 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, wherein 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 nucleic acid encoding the interleukin-10 antibody according to any one of claims 1 to 3, characterized in that The nucleic acid encoding the heavy chain variable region of antibody a is nucleotides 1 to 369 of the nucleotide sequence shown in SEQ ID NO.17; the nucleic acid encoding the light chain variable region of antibody a is nucleotides 1 to 327 of the nucleotide sequence shown in SEQ ID NO.18; the nucleic acid encoding the heavy chain variable region of antibody b is nucleotides 1 to 369 of the nucleotide sequence shown in SEQ ID NO.19; and the nucleic acid encoding the light chain variable region of antibody b is nucleotides 1 to 330 of the nucleotide sequence shown in SEQ ID NO.

20.

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

20.

6. A recombinant expression vector comprising the encoding nucleic acid according to claim 4 or 5.

7. A host cell, characterized in that The host cell comprises the recombinant expression vector of claim 6 or the encoding nucleic acid 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 nucleic acid encoding 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, wherein 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 diluent contains 0.1-1.0 g of BSA.

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