Interleukin-1β antibody, its application and an interleukin-1β detection kit

Abstract

The present invention provides interleukin-1β antibodies, including antibody A or antibody B. The interleukin-1β antibodies provided herein have good specificity, high affinity for interleukin-1β, and avoid cross-reactivity with interleukin-1α. They can be used to prepare magnetic microparticle chemiluminescence assays for detecting interleukin-1β. The interleukin-1β detection kit provided herein 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 1 beta antibody and an application thereof, and an interleukin 1 beta detection kit. Background Art

[0002] Inflammasome activation activates caspase-1 through autoproteolysis, which in turn cleaves it into pro-IL-1β and pro-IL-1β51, releasing IL-1β. The rate-limiting step in IL-1β processing and secretion occurs during inflammasome activation. Atypical inflammasome activation is mediated by caspase-4 / 11, and K+ is closely associated with both the canonical and noncanonical activation pathways of the inflammasome. In the canonical pathway, a decrease in intracellular K+ concentration is a necessary and sufficient step for caspase-1 activation; in the noncanonical pathway, activated caspase-11 triggers K+ efflux, which is required for NLRP3 activation. IL-1β is expressed under the guidance of both activated signals and the inflammasome, thereby exerting its biological role in promoting inflammation.

[0003] IL-1β is one of the most important mediators of inflammation and the host response to infection. IL-1β levels increase in response to viral, bacterial, fungal, or parasitic infections. Commonly used clinical methods for detecting IL-1β include enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, and chemiluminescence assay.

[0004] Studies have shown that the reference range of IL-1β in healthy human plasma is less than 12 pg / mL, which places high demands on the sensitivity of the detection kit and the affinity of the antibody raw material.

[0005] IL-1 has two different molecular forms, one is IL-1α and the other is called IL-1β. After sequence comparison analysis, there is about 26% homology in the amino acid sequences of the two. There may be some overlap during detection, which places high requirements on the specificity of the detection kit and antibody raw materials. Summary of the Invention

[0006] The object of the present invention is to provide an interleukin-1β antibody, which has a high affinity for interleukin-1β and can improve the sensitivity of the detection product; the interleukin-1β antibody specifically recognizes interleukin-1β and avoids cross-reaction with interleukin-1α.

[0007] The present invention also provides the use of the interleukin-1β antibody in preparing an interleukin-1β magnetic particle chemiluminescence detection product.

[0008] The present invention also provides an interleukin-1β 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.

[0009] In order to achieve the above object, the present invention provides an interleukin-1β antibody, including antibody a or antibody b,

[0010] The antibody a comprises the following heavy chain complementary determining regions: CDR1-VH with an amino acid sequence as shown in SEQ ID NO.1, CDR2-VH with an amino acid sequence as shown in SEQ ID NO.2, and CDR3-VH with an amino acid sequence as shown in SEQ ID NO.3;

[0011] The antibody a comprises the following light chain complementary determining regions: CDR1-VL with an amino acid sequence as shown in SEQ ID NO.4, CDR2-VL with an amino acid sequence as TAS, and CDR3-VL with an amino acid sequence as shown in SEQ ID NO.5;

[0012] The antibody b comprises the following heavy chain complementary determining regions: CDR1-VH with an amino acid sequence as shown in SEQ ID NO.6, CDR2-VH with an amino acid sequence as shown in SEQ ID NO.7, and CDR3-VH with an amino acid sequence as shown in SEQ ID NO.8;

[0013] The antibody b comprises the following light chain complementary determining regions: CDR1-VL with an amino acid sequence as shown in SEQ ID NO.9, CDR2-VL with an amino acid sequence as DAS, and CDR3-VL with an amino acid sequence as shown in SEQ ID NO.10.

[0014] Preferably, the amino acid sequence of the heavy chain variable region of antibody a is shown in SEQ ID NO.11.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] Preferably, any of the above items is that the amino acid sequence of the light chain constant region of the antibody a or antibody b is shown as SEQ ID NO.15, and the amino acid sequence of the heavy chain constant region of the antibody a or antibody b is shown as SEQ ID NO.16.

[0019] Preferably, any of the above items is that the antibody a is interleukin-1β monoclonal antibody 1, and the antibody b is interleukin-1β monoclonal antibody 2; further, the antibody a or antibody b is of mouse origin.

[0020] The present invention also provides a coding sequence of any of the above-mentioned interleukin-1β antibodies.

[0021] Preferably, in any of the above items, the coding sequence of the heavy chain variable region of antibody a is nucleotides 1 to 363 of the nucleotide sequence shown in SEQ ID NO.17.

[0022] Preferably, any of the above items is that the coding sequence of the antibody a light chain variable region is nucleotides 1 to 321 of the nucleotide sequence shown in SEQ ID NO.18.

[0023] Preferably, in any of the above items, the coding sequence of the heavy chain variable region of antibody b is nucleotides 1 to 375 of the nucleotide sequence shown in SEQ ID NO.19.

[0024] Preferably, any of the above items is that the coding sequence of the light chain variable region of antibody b is nucleotides 1 to 318 of the nucleotide sequence shown in SEQ ID NO.20.

[0025] 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.

[0026] Preferably, any of the above items is that the coding sequence of the antibody a light chain is the nucleotide sequence shown in SEQ ID NO.18.

[0027] 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.

[0028] Preferably, any of the above items is that the coding sequence of the antibody b light chain is the nucleotide sequence shown in SEQ ID NO.20.

[0029] The present invention also provides a recombinant expression vector comprising any of the above-mentioned coding sequences, wherein the recombinant expression vector comprises the coding sequence of the antibody a and / or the coding sequence of the antibody b.

[0030] Preferably, any of the above items is that the recombinant expression vector comprises the coding sequence of the antibody a 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 a.

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

[0033] 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.

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

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

[0036] Preferably, any of the above items is that the recombinant expression vector comprises the coding sequence of the antibody b light chain.

[0037] Preferably, any of the above items is that the recombinant expression vector comprises an antibody b heavy chain coding sequence.

[0038] 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 by conventional methods in the art. The expression vector is any conventional vector in the art, and any vector that can carry the nucleic acid molecule of the coding sequence is suitable for use in the present invention. The expression vector preferably includes various plasmids, cosmids, phage or viral vectors, such as pCDNA3.4 and pCDNA3.1 series vectors. The expression vector is preferably a eukaryotic expression vector, and the expression vector is preferably an expression vector suitable for use in mammalian cells.

[0039] The present invention also provides a host cell, the host cell comprising any of the above-mentioned recombinant expression vectors or the host cell genome having any of the above-mentioned coding sequences integrated therein. The host cell is preferably a mammalian cell.

[0040] 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. Furthermore, the recombinant expression vector is preferably pCDNA3.4, and the host cell is preferably a 293T cell.

[0041] The interleukin-1β antibody described herein can be either a monoclonal antibody produced by a hybridoma or an antibody 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 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 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 by hybridoma and the recombinantly expressed antibody can specifically recognize interleukin-1β while avoiding cross-reactivity with interleukin-1α.

[0042] The present invention provides use of any of the above-mentioned interleukin-1β antibodies, or the coding sequence of any of the above-mentioned interleukin-1β antibodies, or the recombinant expression vector, or the host cell in the preparation of an interleukin-1β magnetic particle chemiluminescence detection product.

[0043] The present invention provides an interleukin-1β detection kit, which comprises a streptavidin magnetic particle solution, an acridine sulfonamide labeled antibody solution, a biotin labeled antibody solution, a calibrator and a quality control product.

[0044] The kit provided by the present invention can effectively detect 2.5-2500 pg / mL of interleukin-1β. Furthermore, the detection range is preferably 8-1000 pg / mL.

[0045] Preferably, in any of the above items, the acridine sulfonamide-labeled antibody solution contains the interleukin-1β antibody described in any of the above items, and the interleukin-1β antibody is labeled with acridine sulfonamide.

[0046] Preferably, any of the above items is that the biotin-labeled antibody solution contains the interleukin-1β antibody described in any of the above items, and the interleukin-1β antibody is labeled with biotin.

[0047] Preferably, in any of the above items, the acridine sulfonamide-labeled antibody solution and the biotin-labeled antibody solution contain different interleukin-1β antibodies.

[0048] In a preferred embodiment of the present invention, the interleukin 1β antibody on the acridine sulfonamide-labeled antibody solution is antibody a, and the interleukin 1β antibody on the biotin-labeled antibody solution is antibody b; in a preferred embodiment of the present invention, the interleukin 1β antibody on the acridine sulfonamide-labeled antibody solution is antibody b, and the interleukin 1β antibody on the biotin-labeled antibody solution is antibody a.

[0049] Preferably, in any of the above items, the biotin-labeled antibody solution contains 0.2-2 μg / mL of the interleukin-1β antibody; more preferably, 0.2, 0.5, 1.0, 1.5, 2.0 μg / mL and ranges therebetween.

[0050] In any of the above, preferably, the acridine sulfonamide-labeled antibody solution contains 0.2-2 μg / mL of the interleukin-1β antibody, and more preferably, 0.2, 0.5, 1.0, 1.5, 2.0 μg / mL, and ranges therebetween.

[0051] Preferably, any of the above items is that the interleukin-1β 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, the concentration of the Tris-HCl buffer in the Tris-HCl buffer is 10, 20, 30, 40, 50 mM, and ranges 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 ranges therebetween of BSA. The interleukin-1β antibody diluent is a diluent of the acridine sulfonamide-labeled antibody solution or a diluent of the biotin-labeled antibody solution.

[0052] 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.

[0053] Preferably, any of the above items is that the calibrator is a freeze-dried product of interleukin-1β, preferably further comprising freeze-dried excipients. Preferably, pure water is used for reconstitution during use.

[0054] Preferably, any of the above items is that the quality control product is a freeze-dried product of interleukin-1β, and preferably also includes freeze-dried excipients. Preferably, pure water is used for reconstitution during use.

[0055] In any of the above, preferably, the lyophilized interleukin-1β product is interleukin-1β prepared using existing technology, or a commercial interleukin-1β protein product. The present invention uses interleukin-1β produced by Beijing Baixinyi Biotechnology Co., Ltd., but it should be noted that the present invention is not limited to the source and preparation method of interleukin-1β.

[0056] 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.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] The interleukin 1β antibody provided by the present invention has good specificity, high affinity with interleukin 1β, avoids crosstalk with interleukin 1α, and can be used to prepare a magnetic particle chemiluminescence detection product for detecting interleukin 1β.

[0059] The interleukin-1β 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

[0060] Figure 1 This is the electrophoresis diagram of the interleukin-1β monoclonal antibody in the preferred embodiment 1 of the present invention.

[0061] Figure 2 This is the interleukin-1β detection kit in preferred embodiments 2 and 3 of the present invention.

[0062] Figure 3 This is the interleukin-1β detection reagent in preferred embodiments 2 and 3 of the present invention. DETAILED DESCRIPTION

[0063] The present invention is further described in detail below through specific embodiments.

[0064] 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.

[0065] "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.

[0066] 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.

[0067] 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.

[0068] In a first aspect, the present invention provides an interleukin-1β antibody selected from antibody a and / or antibody b;

[0069] 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 a light chain complementary determining region CDR1-VL, whose amino acid sequence is sequentially 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;

[0070] 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 DAS, and a light chain complementary determining region CDR3-VL whose amino acid sequence is shown in SEQ ID NO.10.

[0071] The amino acid sequences shown in SEQ ID NO. 1 to SEQ ID NO. 10 are shown in Table 1.

[0072] Table 1

[0073]

[0074] The interleukin 1β antibody provided by the present invention has good specificity, high biological activity, and high affinity for interleukin 1β, and can be used to prepare products for detecting interleukin 1β.

[0075] 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;

[0076] 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;

[0077] Preferably, the interleukin-1β antibody is of murine origin.

[0078] The amino acid sequences represented by SEQ ID NO.11 to SEQ ID NO.14 are shown in Table 2.

[0079] Table 2

[0080]

[0081] The heavy chain of antibody a, i.e., interleukin-1β monoclonal antibody 1, consists of the heavy chain variable region and constant region of interleukin-1β monoclonal antibody 1, and the light chain of interleukin-1β monoclonal antibody 1 consists of the light chain variable region and constant region of interleukin-1β monoclonal antibody 1; the heavy chain of antibody b, i.e., interleukin-1β monoclonal antibody 2, consists of the heavy chain variable region and constant region of interleukin-1β monoclonal antibody 2, and the light chain of interleukin-1β monoclonal antibody 2 consists of the light chain variable region and constant region of interleukin-1β monoclonal antibody 2. 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, as shown in Table 3. Accordingly, the amino acid sequence of the heavy chain of antibody a is composed, in sequence, of the amino acid sequences shown in SEQ ID NO.11 and SEQ ID NO.16, and the amino acid sequence of the light chain of antibody a is composed, in sequence, of the amino acid sequences shown in SEQ ID NO.12 and SEQ ID NO.15; the amino acid sequence of the heavy chain of antibody b is composed, in sequence, of the amino acid sequences shown in SEQ ID NO.13 and SEQ ID NO.16, and the amino acid sequence of the light chain of antibody b is composed, in sequence, of the amino acid sequences shown in SEQ ID NO.14 and SEQ ID NO.15.

[0082] Table 3

[0083]

[0084] In a second aspect, the present invention provides the use of the interleukin-1β antibody in the preparation of an interleukin-1β magnetic microparticle chemiluminescence detection product.

[0085] In a third aspect, the present invention provides an interleukin-1β 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.

[0086] Preferably, the concentration of the streptavidin magnetic particle solution is 0.1-1.0 mg / mL.

[0087] Preferably, the acridine sulfonamide-labeled antibody solution contains any one of the interleukin-1β antibodies described above, wherein the interleukin-1β antibody is labeled with acridine sulfonamide, and the concentration of acridine sulfonamide is identified by a detection instrument.

[0088] Preferably, the acridine sulfonamide-labeled antibody solution contains 0.2-2 μg / mL of the interleukin-1β antibody.

[0089] Preferably, the biotin-labeled antibody solution contains 0.2-2 μg / mL of the interleukin-1β antibody.

[0090] 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.

[0091] In a preferred embodiment of the present invention, the interleukin-1β antibody in the acridine sulfonamide-labeled antibody solution is different from the interleukin-1β antibody in the biotin-labeled antibody solution.

[0092] In a preferred embodiment of the present invention, the antibody in the acridine sulfonamide-labeled antibody solution is antibody a, which is interleukin-1β monoclonal antibody 1; the antibody in the biotin-labeled antibody solution is antibody b, which is interleukin-1β monoclonal antibody 2.

[0093] In a preferred embodiment of the present invention, the antibody in the acridine sulfonamide-labeled antibody solution is antibody b, and the antibody in the biotin-labeled antibody solution is antibody a.

[0094] Preferably, the calibrator is a freeze-dried product of interleukin-1β, and preferably also includes freeze-dried excipients. Preferably, pure water is used for reconstitution during use.

[0095] Preferably, the quality control product is a freeze-dried product of interleukin-1β, and preferably also includes freeze-dried excipients. Preferably, pure water is used for reconstitution during use.

[0096] Preferably, the interleukin-1β lyophilized product is interleukin-1β prepared using existing technology, or a commercial interleukin-1β protein product. The present invention uses interleukin-1β produced by Beijing Baixinyi Biotechnology Co., Ltd., but the present invention is not limited to the source and preparation method of interleukin-1β.

[0097] 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.

[0098] The interleukin-1β detection kit provided by the present invention has the advantages of automated operation, high sensitivity, strong specificity, and high throughput.

[0099] The reagent provided by the present invention is based on the principle of antigen-antibody reaction. After labeling the interleukin-1β antibody with acridine sulfonamide, a diluent is used to prepare a biotin-labeled antibody solution. The biotin-labeled antibody solution contains another antibody labeled with biotin for interleukin-1β. Based on the principle of antigen-antibody reaction, the reagent can be detected by a supporting instrument after the reaction is completed. If interleukin-1β is present in the sample, a double-antibody sandwich structure is formed, and the instrument will detect a strong light signal. If interleukin-1β is absent in the sample, the instrument will detect a weak light signal. The intensity of the light signal is used to predict the amount of the analyte.

[0100] 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.

[0101] It should be noted that the diluent for the interleukin-1β antibody of the present invention 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; unless otherwise specified in the following embodiments, preferably, the diluent for the interleukin-1β antibody is a 20 mM Tris-HCl buffer containing BSA, and each 100 mL of the diluent contains 0.5 g of BSA.

[0102] The methods of the present invention for preparing interleukin-1β monoclonal antibody 1 represented by antibody a and interleukin-1β 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-1β purchased from Beijing Baixinyi Biotechnology Co., Ltd. was mixed with Freund's adjuvant in equal volumes 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 ELISA indirect method was used to coat interleukin-1β and test the titer of the antibody in the serum. 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 363 are the antibody a heavy chain variable region encoding sequence, and nucleotides 364 to 1335 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 321 are the antibody a light chain variable region encoding sequence, and nucleotides 322 to 642 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 375 are the antibody b heavy chain variable region encoding sequence, and nucleotides 376 to 1347 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 318 are the antibody b light chain variable region encoding sequence, and nucleotides 319 to 639 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 validated by SDS-PAGE and ELISA, respectively, using the methods described in Example 1. Both Antibodies a and b exhibited two characteristic bands at approximately 25 kD and 50 kD, representing the light and heavy chains of IgG, respectively. Indirect ELISA titers demonstrated that both Antibodies a and b specifically recognized interleukin-1β and avoided cross-reactivity with interleukin-1α.

[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. Example 1

[0127] Interleukin-1β antibodies include: interleukin-1β monoclonal antibody 1 (antibody a) and interleukin-1β 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.

[0128] 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-1β 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%.

[0129] Figure 1 In the figure, lane 1 is a marker; lane 2 is interleukin-1β monoclonal antibody 1; and lane 3 is interleukin-1β monoclonal antibody 2.

[0130] The antibodies used in the following examples are the same as those in Example 1.

[0131] Verification of the specificity of interleukin-1β monoclonal antibody 1 (antibody a): The titer was determined by the indirect ELISA method as follows: Interleukin-1α (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number rp174289) and interleukin-1β (purchased from Beijing Baixinyi Biotechnology Co., Ltd., catalog number A10C02) were coated on the ELISA plate, and antibody a was diluted to 100, 10, 1, and 0.1 ng / mL, respectively. The affinity of antibody a for different antigens was measured. The results are shown in Table 4.

[0132] Table 4

[0133]

[0134] Verification of the specificity of interleukin-1β monoclonal antibody 2 (antibody b): The ELISA indirect method was used as follows: ELISA plates were coated with interleukin-1α and interleukin-1β, respectively, and antibody b was diluted to 100, 10, 1, and 0.1 ng / mL, respectively. The affinity of antibody b for different antigens was measured. The results are shown in Table 5.

[0135] Table 5

[0136]

[0137] Both antibody a and antibody b have a strong affinity for interleukin-1β and no recognition for interleukin-1α.

[0138] Example 2

[0139] An interleukin 1β 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 antibody a and a diluent. The acridine sulfonamide-labeled antibody solution includes acridine sulfonamide-labeled antibody b and a diluent. The calibrator and quality control are lyophilized products, including interleukin-1β and lyophilized excipients.

[0140] The diluent for the antibody a or antibody b 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.

[0141] Example 3

[0142] Example 3 provides an interleukin-1β detection kit.

[0143] 1 Main Materials

[0144] 1.1 Antibodies: The mouse anti-monoclonal antibodies are both interleukin-1β antibodies (antibody a and antibody b) described in the present invention, which are mouse monoclonal antibodies and are used to label with 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-1β: purchased from Beijing Baixinyi Biotechnology Co., Ltd., product number A10C02.

[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-mentioned PBS buffer containing the antibody, mix well, and place it at room temperature in the dark for 1-2 hours; dialyze it 5-10 times (each time with an interval of 2 hours) with 0.01 M PBS (pH 7.2) buffer at 2-8°C in the dark;

[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, and each 100 mL of the diluent contains 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 a diluent containing 10-50 mM Tris-HCl buffer containing BSA, with 0.1-1.0 g of BSA per 100 mL of diluent. Aliquot 6 mL into a vial.

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

[0166] Dilute interleukin-1β to 2.5-2500 pg / mL with lyophilized solution (further, 2000 pg / mL interleukin-1β is preferred for the preparation of calibrators and quality controls), and dispense into 0.6 mL / vial for lyophilization.

[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; preferably, the sample of the present invention is serum.

[0171] Step 2: Take out the refrigerated test kit and Figure 3 The 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 minutes.

[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 according to the detection luminescence value of the calibrator to obtain a calibration curve; the concentration values of interleukin-1β in the quality control product and the sample to be tested are calculated through the calibration curve.

[0179] In Example 3, 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; the lyophilized 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.

[0180] In Example 3, the fully automatic chemiluminescence immunoassay analyzer produced by Clarith Biotechnology (Chongqing) Co., Ltd., model: Venus 100S, is preferably used.

[0181] Example 4

[0182] Example 4 provides the kit detection process.

[0183] Preparation of test samples: Dilute interleukin-1β and interleukin-1α to 5000, 1000, 200, 40, 8, and 1.6 pg / mL, respectively, with 0.01 M PBS containing 1% BSA and mix well.

[0184] Sample detection: Use an analyzer to detect the reconstituted calibrators, quality control products and test samples according to the method described in Example 3.

[0185] Example 4 employed a fully automated chemiluminescence immunoassay analyzer, model Venus 100S, from Clarith Biotechnology (Chongqing) Co., Ltd. Calculations were performed using the instrument's built-in program. The built-in curve fitting method employed a double-logarithmic four-parameter curve fit (automatically fitted by the instrument software); the calibration method employed a two-point segmented calibration; 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. The interleukin-1β concentrations in the control sample and the test sample were calculated using the calibration curve. The test results are shown in Table 6.

[0186] Table 6 Interleukin-1β and interleukin-1α test results

[0187]

[0188] The above embodiments are provided for illustrative purposes only and are not intended to limit the scope of implementation. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to provide an exhaustive list of all implementations. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. An interleukin-1β antibody, which is antibody a or antibody b, characterized in that: The antibody a comprises the following heavy chain complementary determining regions: CDR1-VH with an amino acid sequence as shown in SEQ ID NO.1, CDR2-VH with an amino acid sequence as shown in SEQ ID NO.2, and CDR3-VH with an amino acid sequence as shown in SEQ ID NO.3; The antibody a comprises the following light chain complementary determining regions: CDR1-VL with an amino acid sequence as shown in SEQ ID NO.4, CDR2-VL with an amino acid sequence as TAS, and CDR3-VL with an amino acid sequence as shown in SEQ ID NO.5; The antibody b comprises the following heavy chain complementary determining regions: CDR1-VH with an amino acid sequence as shown in SEQ ID NO.6, CDR2-VH with an amino acid sequence as shown in SEQ ID NO.7, and CDR3-VH with an amino acid sequence as shown in SEQ ID NO.8; The antibody b comprises the following light chain complementary determining regions: CDR1-VL with an amino acid sequence as shown in SEQ ID NO.9, CDR2-VL with an amino acid sequence as DAS, and CDR3-VL with an amino acid sequence as shown in SEQ ID NO.

10.

2. The interleukin-1β 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-1β 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 gene encoding the interleukin-1β antibody according to any one of claims 1 to 3, characterized in that The nucleotide sequence of the heavy chain variable region of antibody a is nucleotides 1 to 363 of the nucleotide sequence shown in SEQ ID NO.17; the nucleotide sequence of the light chain variable region of antibody a is nucleotides 1 to 321 of the nucleotide sequence shown in SEQ ID NO.18; the nucleotide sequence of the heavy chain variable region of antibody b is nucleotides 1 to 375 of the nucleotide sequence shown in SEQ ID NO.19; and the nucleotide sequence of the light chain variable region of antibody b is nucleotides 1 to 318 of the nucleotide sequence shown in SEQ ID NO.

20.

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

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

8. Use of the interleukin-1β antibody according to any one of claims 1 to 3, or the gene encoding the interleukin-1β 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-1β magnetic microparticle chemiluminescence detection product.

9. An interleukin-1β 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 1β antibody according to any one of claims 1 to 3, and the interleukin 1β antibody is labeled with acridine sulfonamide; the biotin-labeled antibody solution contains the interleukin 1β antibody according to any one of claims 1 to 3, and the interleukin 1β antibody is labeled with biotin; the interleukin 1β antibodies contained in the acridine sulfonamide-labeled antibody solution and the biotin-labeled antibody solution are different, that is, when antibody a is labeled with acridine sulfonamide, antibody b is labeled with biotin; when antibody b is labeled with acridine sulfonamide, antibody a is labeled with biotin.

10. The kit according to claim 9, wherein The interleukin-1β 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; the biotin-labeled antibody solution contains 0.2-2 μg / mL of the interleukin-1β antibody; and the acridine sulfonamide-labeled antibody solution contains 0.2-2 μg / mL of the interleukin-1β antibody.

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