CN114989299A - Composition of monoclonal antibody, application of composition, reagent, kit and method for detecting human interleukin 1 beta - Google Patents

Abstract

The application relates to the technical field of detection of human interleukin 1 beta, and particularly discloses a composition of a monoclonal antibody, application thereof, a reagent, a kit and a method for detecting human interleukin 1 beta. The composition comprises monoclonal antibody IL1 β -3F5 and/or monoclonal antibody IL1 β -5C 9; the monoclonal antibody IL1 beta-3F 5 comprises a heavy chain with a variable region amino acid sequence shown as SEQ ID NO.1 and a light chain with a variable region amino acid sequence shown as SEQ ID NO. 2; the monoclonal antibody IL1 beta-5C 9 comprises a heavy chain with a variable region amino acid sequence shown as SEQ ID NO.3 and a light chain with a variable region amino acid sequence shown as SEQ ID NO. 4. The reagent comprises the composition. The kit comprises the reagent. The method has the advantages of strong specificity, high sensitivity and good stability.

Description

Composition of monoclonal antibody, application of composition, reagent, kit and method for detecting human interleukin 1 beta

Technical Field

The application relates to the technical field of detection of human interleukin 1 beta, and particularly discloses a composition of a monoclonal antibody, application thereof, a reagent for detecting human interleukin 1 beta, a kit and a method.

Background

Interleukins (ILs) are a class of cytokines that are produced by and act on a variety of cells. The first is the cytokine produced by leukocytes and acting as a regulator among leukocytes, and the second is a cytokine whose molecular structure and biological function are basically clear, important regulation and uniform designation. Interleukins play an important role in transmitting information, activating and regulating immune cells, mediating T, B cell activation, proliferation and differentiation, and in inflammatory responses.

Interleukin 1(IL-1), also known as a lymphocyte stimulating factor, is a monomeric glycoprotein. Almost all nucleated cells secrete this factor, including monocytes, endothelial cells, fibroblasts, keratinocytes, dendritic cells and other cell types, produced primarily by activated monocyte-macrophages. Normally only skin, sweat and urine contain a certain amount of IL-1, and most cells synthesize and secrete IL-1 after stimulation by foreign antigens or mitogens. IL-1 is mainly involved in the activation of T cells, NK cells and macrophages, can stimulate the production of cytokines such as colony stimulating factors, platelet growth factors and the like, and enables T cells to produce interleukin-2, thereby playing a role in immune response and tissue repair.

IL-1 exists in two forms, IL-1 α and IL-1 β. In humans, IL-1 α and IL-1 β are expressed from different genes, and the amino acid sequence homology between the two proteins is 21%, but they are capable of binding to the same cell surface receptor and performing the same biological function. Abnormal expression of the IL-1 protein is associated with a variety of pathological conditions, including sepsis, rheumatoid arthritis, inflammatory bowel disease, acute and chronic myelogenous leukemia, insulin-dependent diabetes, atherosclerosis, neuronal damage and diseases associated with aging.

IL-1 β plays an important role in immune responses, inflammatory responses, bone remodeling, fever, carbohydrate metabolism and GH/IGF-I physiology. The IL-1. beta. gene is located on chromosome 2, long arm 2q 14. The precursor protein contains 269 amino acid molecules, has a molecular weight of about 31KD, and hardly has any biological activity. Then the mature IL-1 beta is obtained by shearing Caspase1(CASP1/ICE), the mature IL-1 beta contains 153 amino acid molecules with the molecular weight of 17.5KD and is secreted out of the cell to play a biological function. Neither IL-1. alpha. nor IL-1. beta. contain the typical hydrophobic signal peptide domain, but they can be secreted by non-classical pathways. Therefore, understanding the content changes of human interleukin 1 β is of great importance for understanding the immune regulation of various diseases. However, there is a lack in the art of a means to detect changes in the amount of human interleukin 1 β with high specificity, sensitivity and stability.

Disclosure of Invention

In order to improve the specificity, sensitivity and stability of detecting human interleukin 1 beta, the application provides a composition of a monoclonal antibody, application thereof, a reagent for detecting human interleukin 1 beta, a kit and a method.

In a first aspect, the present application provides a composition of monoclonal antibodies, which employs the following technical scheme:

a composition of monoclonal antibodies, the composition comprising monoclonal antibody IL1 β -3F5 and/or monoclonal antibody IL1 β -5C 9; the monoclonal antibody IL1 beta-3F 5 comprises a heavy chain with a variable region amino acid sequence shown as SEQ ID NO.1 and a light chain with a variable region amino acid sequence shown as SEQ ID NO. 2;

the monoclonal antibody IL1 beta-5C 9 comprises a heavy chain with a variable region amino acid sequence shown as SEQ ID NO.3 and a light chain with a variable region amino acid sequence shown as SEQ ID NO. 4.

In a second aspect, the present application provides a composition of monoclonal antibodies for use in detecting human interleukin-1 β.

In a third aspect, the application provides an application of a monoclonal antibody composition in preparing a detection reagent or a detection kit for detecting human interleukin 1 beta.

In a fourth aspect, the present application provides a reagent for detecting human interleukin 1 β, which adopts the following technical scheme:

a reagent for detecting human interleukin 1 beta, the reagent comprising the composition of the monoclonal antibody.

Optionally, the reagent comprises an enzyme label plate coated by a monoclonal antibody IL1 beta-3F 5, a biotin-labeled monoclonal antibody IL1 beta-5C 9, a polymerase-labeled streptavidin and a color developing agent.

Optionally, the preparation method of the elisa plate coated by the monoclonal antibody IL1 β -3F5 comprises the following steps:

coating: diluting a monoclonal antibody IL1 beta-3F 5 by using a buffer solution to obtain a monoclonal antibody IL1 beta-3F 5 diluent with the concentration of 1-4 mu g/mL; adding the monoclonal antibody IL1 beta-3F 5 diluent into a reaction hole of an enzyme label plate for incubation reaction and washing the plate; and (3) sealing: the bottom of the wells of the microplate was blocked at a position not coated with monoclonal antibody IL1 β -3F 5.

Alternatively, the buffer in the coating step may be selected from a carbonate coating buffer and a PBS solution.

Optionally, the addition amount of the monoclonal antibody IL1 beta-3F 5 diluent in the coating step is 80-120 uL/hole, for example: 100 uL/well.

Optionally, the temperature of the incubation reaction in the coating step is 4 ℃.

Optionally, the sealing step specifically comprises: adding a sealing solution into the reaction hole of the ELISA plate for incubation reaction, and drying after the liquid in the hole is thrown away.

Alternatively, the blocking solution in the blocking step may be selected from the group consisting of 5 wt% skim milk powder and 1 wt% BSA.

Optionally, the addition amount of the blocking liquid in the blocking step is 200-300 uL/hole, for example 250 uL/hole.

Optionally, the temperature of the incubation reaction in the blocking step is room temperature.

Alternatively, the preparation method of the biotin-labeled monoclonal antibody IL1 beta-5C 9 comprises the following steps:

monoclonal antibody IL1 β -5C9 was diluted to 1mg/mL with 0.1mol/L sodium bicarbonate buffer (pH 8.0); the monoclonal antibody IL1 β -5C9 was extensively dialyzed against 0.1mol/L sodium bicarbonate buffer at pH 8.0 for the interaction;

dissolving N-hydroxysuccinimide biotin (NHSB,1mg) in dimethyl sulfoxide (DMSO, 1mL), adding 10uL of DMSO solution of NHSB to 1mL of monoclonal antibody IL1 beta-5C 9 solution, stirring at room temperature for 2-4 hr, and adding 1uL of 0.1mol/L NH ₄ Aqueous Cl was stirred at room temperature for 10 min. After the product was dialyzed sufficiently at 4 ℃, the sample was applied to Sephadex G-25, and slowly eluted with PBS, and the elution peak was collected to obtain the biotin-labeled monoclonal antibody IL1 β -5C 9.

Optionally, the preparation method of the streptavidin labeled with polymerase comprises the following steps:

6mg of horseradish peroxidase was dissolved in 1.5mL of double distilled water, and 0.025mL of a freshly prepared 0.1mol/L aqueous solution of sodium periodate was added, followed by stirring at room temperature for 20 min. Adding ethylene glycol into the reaction solution, and reacting for 5min to terminate the reaction. The mixture was dialyzed against 2mmol/L sodium acetate buffer (pH 4.5) at 4 ℃ for 4 hours at least 8 times, and then adjusted to pH 9.0 with 0.2mol/L carbonate buffer (pH 9.0). To the mixture was added 1.5mL of 6mg of streptavidin prepared in 0.2mol/L carbonate buffer (pH 9.0), and the mixture was stirred at room temperature for 2 hours in the dark. 0.01mL of a freshly prepared 4mg/mL aqueous solution of sodium borohydride was added dropwise thereto, and the reaction was carried out at 4 ℃ for 2 hours. Dialyzed with 0.01mol/L PBS buffer at pH7.2 overnight at 4 ℃ and centrifuged to remove the precipitate. The obtained supernatant is the streptavidin marked by the polymerase.

Optionally, the optimal coating concentration range (1-4) ug/mL of the monoclonal antibody IL1 beta-3F 5.

Optionally, the optimal detection antibody working concentration 1 of the monoclonal antibody IL1 beta-5C 9 is (1000-2000), and the lowest detection concentration can reach the level of pg grade.

Optionally, the biotin is N-hydroxysuccinimide biotin.

Optionally, the polymerase is horseradish peroxidase.

Optionally, the color-developing agent is a 3,3',5,5' -tetramethylbenzidine color-developing substrate.

Optionally, the reagent further comprises a human interleukin 1 beta standard substance.

Optionally, the reagent further comprises a stop solution. The stop solution can be an acidic stop solution or an alkaline stop solution. Wherein the acid termination solution can be 2mol/L HCl aqueous solution and 2mol/L H ₂ SO ₄ Aqueous solutions, and the like. The alkaline stop solution may be a 30 wt% to 40 wt% aqueous solution of sodium hydroxide or the like.

In a fifth aspect, the present application provides a kit for detecting human interleukin 1 β, which adopts the following technical scheme: a kit for detecting human interleukin 1 beta, which comprises the reagent.

In a sixth aspect, the present application provides a method for detecting human interleukin 1 β, which adopts the following technical scheme:

a method of detecting human interleukin 1 β, the method comprising the steps of:

(1) obtaining a gradient standard solution:

preparing a gradient standard solution with the concentration of (0-20) pg/mL by using human interleukin 1 beta as a standard substance;

(2) gradient standard solution and detection sample treatment:

firstly, adding the gradient standard solution and the detection sample into reaction holes of an enzyme label plate coated by a monoclonal antibody IL1 beta-3F 5 respectively for incubation reaction and washing the plate; then adding a biotin-labeled monoclonal antibody IL1 beta-5C 9 for incubation reaction and washing the plate; then adding streptavidin marked by polymerase to carry out incubation reaction and washing the plate; finally adding a color developing agent for incubation reaction and washing the plate; then adding a stop solution to terminate the reaction;

(3) obtaining the concentration of the sample to be detected:

determining absorbance values of the gradient standard solution and the test sample at a wavelength of 450 nm;

drawing a standard curve by taking the concentration of the gradient standard solution as an abscissa and the absorbance value of the gradient standard solution as an ordinate, and calculating to obtain a curve equation of the standard curve;

and calculating the concentration of the abscissa corresponding to the absorbance value of the sample to be detected on the standard curve according to the curve equation.

Alternatively, the concentration of the gradient standard solution may be 0pg/mL, 0.3125pg/mL, 0.625pg/mL, 1.25pg/mL, 2.5pg/mL, 5.0pg/mL, 10.0pg/mL, 20.0 pg/mL.

In summary, the present application has the following beneficial effects:

firstly, the specificity, the sensitivity and the stability of detecting the human interleukin 1 beta are improved through a monoclonal antibody IL1 beta-3F 5 and a monoclonal antibody IL1 beta-5C 9. The kit can be well applied to the detection of the content of human interleukin 1 beta in human serum, blood plasma and cell supernatant.

Secondly, the polymerase labeled streptavidin used by the reagent kit in the application can improve the sensitivity of the detection result of the human interleukin 1 beta, greatly reduce the detection lower limit and improve the detection rate of trace human interleukin 1 beta in the detection sample.

Drawings

FIG. 1 shows the result of dicer assay after ligation of IL 1. beta. gene with PET-28A expression vector;

FIG. 2 is an SDS-PAGE electrophoresis chart showing the expression of IL 1. beta. protein by SDS-PAGE electrophoresis;

FIG. 3 is a light chain and heavy chain variable region amino acid sequence;

FIG. 4 is a standard curve established based on the results of detection of a gradient standard solution.

Detailed Description

The present application will be described in further detail with reference to examples.

Reagents used for the experiment: HAT medium (purchased from Sigma), HT medium (purchased from Sigma), RPMI1640 medium (purchased from GIBCO), Pen Strep (purchased from GIBCO), HEPES (purchased from GIBCO), L-Glutamine (purchased from GIBCO), goat anti-mouse IgG-HRP antibody (purchased from Sigma), fetal bovine serum (purchased from GIBCO), DMSO (purchased from Sigma), 50% PEG (purchased from Sigma, molecular weight 1450), total RNA extraction kit (purchased from Beijing Sorboard technology Co., Ltd.), DNA polymerase (purchased from Beijing Sorboard technology Co., Ltd.), Eco RI (purchased from Beijing Sorboard technology Co., Ltd.), Xho I (purchased from Beijing Sorboard Co., Ltd.), T4 ligase (purchased from Bao Bio), DH5 alpha (purchased from Beijing Sorboard plasmid, Kao Co., Ltd.), small extract (purchased from Beijing Sorboard Co., Ltd.), Xho I (purchased from Beijing Sorboard technology Co., Ltd.), and T4 ligase, Expression strain BL21 (purchased from Beijing Solebao Tech Co., Ltd.), and DNA product purification kit (purchased from Beijing Solebao Tech Co., Ltd.).

Example 1: preparation of monoclonal antibody against IL1 beta antigen

S1. cloning of IL1. beta. Gene

S1.1 Synthesis of IL1 beta Gene sequence

THP-1 cells are cultured for 24h, then an irritant (5 wt% sodium dodecyl sulfate SLS aqueous solution) is added for stimulation, cell samples after stimulation are collected, total RNA is extracted by a total RNA extraction kit, reverse transcription is carried out by a reverse transcription kit to obtain cDNA, the reaction is stopped after reaction at 42 ℃ for 1h and at 95 ℃ for 5min, and the human IL1 beta gene is amplified by PCR by taking the reverse transcription product cDNA as a template.

The sequences of the primers used for PCR amplification were as follows:

an upstream primer: 5'-TTTGAATTCGCACCTGTACGATCACTGAACTGC-3', respectively;

a downstream primer: 5'-TTTCTCGAGTTAGGAAGACACAAATTGCATGGT-3' are provided.

PCR instrument (ABI, 2720), PCR program: amplification was carried out for 32 cycles at 94 ℃ for 30s, 56 ℃ for 50s, 72 ℃ for 1min, and finally extension was carried out for 10min at 72 ℃. And then carrying out double enzyme digestion on the PCR product by using the introduced double enzyme digestion site Eco RI/Xho I, and carrying out gene recombination after the double enzyme digestion product is purified by a DNA product purification kit.

S1.2 gene recombination: mixing 1 mu L of an expression vector 3 mu L, PET-28A of an enzyme-digested and purified IL1 beta gene, 5 mu L of 2 Xligase buffer and 1 mu L of T4 ligase, placing the mixture at 16 ℃ for connection for 12h, then transforming a ligation product into Escherichia coli DH5 alpha competent bacteria, coating the competent bacteria on an LB plate containing 50 mu g/mL kanamycin, picking out a single colony after forming a recombinant colony, inoculating the single colony into an LB liquid culture medium for culture, centrifuging and collecting thalli after culture, and extracting a recombinant plasmid; the introduced dual-restriction enzyme sites Eco RI/Xho I are used for dual-restriction enzyme identification, and the identification result is shown in FIG. 1. And transforming the positive plasmid into an expression strain BL21, selecting a positive clone to culture and sequence, and performing subsequent induced expression on the IL1 beta protein by using the expression strain with the correct sequencing result.

S2. expression and purification of IL1. beta. protein

And (3) carrying out IPTG induced expression at 37 ℃ for 1mmol/L on the constructed recombinant expression strain with the correct sequencing result, collecting induced expression samples for inducing for 3h, 4h and 5h respectively, and simultaneously carrying out empty vector strain comparison. Protein expression was detected by SDS-PAGE, and the results are shown in FIG. 2. After the expression of the protein was confirmed, the cells were sonicated, the supernatant and the pellet were separated, and the specific expression pattern of the protein was examined by SDS-PAGE. The detection result shows that the protein is expressed in the form of insoluble inclusion bodies. Renaturation treatment and purification are carried out on the inclusion body, and the target protein is obtained.

S3, immunizing animals

The IL1 beta protein prepared above is used as antigen to carry out primary immunization on female BALB/c mice: emulsifying an IL1 beta protein solution by adopting equivalent volume of Freund complete adjuvant, and injecting the emulsified solution into the back of the mouse at four points subcutaneously; 4 weeks later, carrying out second immunization, changing to Freund incomplete adjuvant to emulsify the antigen, and injecting four points of the emulsified antigen subcutaneously to the back of the mouse; after 3 weeks, carrying out third immunization, continuously using Freund incomplete adjuvant to emulsify the antigen, and injecting four points of emulsified antigen subcutaneously to the back of the mouse; collecting the mouse tail blood after 1 week, measuring the antibody titer in the serum, and continuing to carry out immunization when the antibody titer does not meet the fusion requirement until the serum titer is increased to meet the fusion requirement. Three days prior to fusion, boosts were performed by intraperitoneal injection of antigen.

S4. cell fusion

S4.1 myeloma (SP2/0) cell activation

Thawing and resuscitating commercially available SP2/0 cells, then resuspending in cell culture medium (RPMI-1640, supplemented with fetal calf serum), placing at 37 deg.C and 5% CO ₂ Culturing in an incubator under the condition; cellsAnd subculture is carried out when the culture dish grows to about 80% of the bottom area of the culture dish.

Collecting cells, suspending the cells in 1640 basic solution, counting, and taking (0.5-1) × 10 ⁶ Injecting the cells into the subcutaneous back of a BALB/c mouse, continuously culturing for 9-10 days, pulling the neck of the mouse to kill the mouse after the tumor volume on the back is increased to about 0.8cm, soaking the mouse in 75% alcohol for 5min, and taking out the tumor by aseptic operation.

Shearing off tumor blocks, placing the tumor blocks in a sterilized homogenizer, adding 1640 basic liquid, fully grinding, adding 10mL of 1640 basic liquid, standing for 2min, sucking the upper-layer cell suspension, placing the upper-layer cell suspension in another centrifugal tube, adding 10mL of 1640 basic liquid, and repeatedly grinding twice; the cell suspension obtained above was centrifuged at 1000r/min for 10min to remove the supernatant, and then resuspended in 30mL 1640 base solution.

Adding 15mL of lymphocyte separation solution into another centrifuge tube, and carefully placing the cell suspension on the separation solution; and then centrifuging at 1200r/min for 15min, sucking the white cell layer with compact interface by a pipette, washing the cells for 2 times by using 1640 basic solution, then suspending the cells in 10mL 1640 basic solution, and counting for later use.

S4.2 preparation of immune splenocytes

Taking one BALB/c mouse for strengthening immunity, draining blood from an orbit, killing the mouse (collecting serum, namely positive serum), soaking the mouse in 75% alcohol for 5-10 min for disinfection, fixing the mouse on a dissection plate for dissection, taking out a spleen, cutting the spleen, and placing the spleen in a sterilized homogenizer; the grinding and cell suspension preparation were as described in SP2/0 and counted for further use.

S4.3 preparation of feeder cells

One uninmmunized BALB/c mouse was bled from the orbit and the serum collected as negative serum. 2-3 mL of 1640 basic liquid is injected into the abdominal cavity of the mouse, the liquid is sucked out after being blown and is placed into another centrifugal tube for later use, and the liquid contains abdominal cavity macrophages. Splenocytes suspensions were prepared as above and placed in peritoneal macrophage tubes. Centrifuging at 1000r/min for 10min to remove supernatant, suspending cells in HAT medium, standing at 37 deg.C and 5% CO ₂ And (5) the incubator is used for later use.

S4.4 cell fusion and Selective culture

Will be (1-2) × 10 ⁷ SP2/0 and 10 ⁸ The immune cells are mixed evenly in a 50mL centrifuge tube, and centrifuged for 8min at 1000 r/min. After discarding the supernatant, the centrifuge tube containing the cell mixture was placed in a 37 ℃ water bath, and then 50 wt% PEG0.8mL pre-warmed to 37 ℃ was added, stirred, and then allowed to stand for 30 seconds. After standing, 30mL of 1640 basic solution pre-warmed at 37 ℃ was added. And (4) centrifuging for 5min at 1000r/min after uniformly mixing, removing supernatant, and standing at 37 ℃ for 5-8 min. Then mixed with the feeder cell suspension, seeded in 96-well plates at 150 uL/well in a medium at 37 ℃ with 5% CO ₂ Culturing in an incubator. And 4 days after the fusion, the culture is continued by changing the HT medium. Antibody detection was performed when the fused cell colonies grew to culture well 1/4 and the medium turned slightly yellow. The day before detection, the liquid changing treatment is carried out to reduce false positive results.

S5, screening of hybridoma positive clones and cloning of cells

S5.1 screening of Positive hybridoma cells by Indirect ELISA

Coating of known antigens: the purified coating antigen was diluted to 1-5 ug/mL with coating buffer (0.05 mol/L carbonate buffer pH 9.6), added to the elisa plate, incubated at 100 uL/well overnight at 4 ℃, the liquid in the wells was spun off and the plate was washed 1 time.

Sealing the position of the bottom of the enzyme label plate hole which is not coated by the antigen: adding a blocking solution (5 wt% of skimmed milk powder or 1 wt% of BSA) into the plate hole, and incubating for 2h at room temperature at 250 uL/hole; spin off the liquid in the wells and wash the plate 3 times.

Sample adding: and (3) taking 50uL of supernatant from each hole of the hybridoma to be detected, sequentially adding the supernatant into an enzyme label plate, incubating for 1h at 37 ℃, throwing off liquid in the hole, washing the plate for 4 times, and patting dry.

Secondary antibody: adding HRP labeled goat anti-mouse IgG, diluting the enzyme-labeled secondary antibody with a diluent to a working concentration according to an instruction, incubating for 30min at 37 ℃, throwing off liquid in the hole, washing the plate for 4 times, and patting dry.

Color development: adding TMB developing solution, 100 uL/hole, and incubating at 37 deg.C for 10 min.

And (4) terminating: stop solution, 50 uL/well, was added.

As a result: and (3) reading the OD450 light absorption value by using a microplate reader, wherein the higher the reading value is, the stronger the positive result is.

S5.2 cloning of hybridoma cells (limiting dilution method)

Preparing a mouse feeder cell layer before cloning; selecting a positive hole hybridoma cell for subcloning, slightly blowing down the hybridoma cell to be cloned from a culture hole, and counting the cell by using a cell counting plate; adding 50-70 cells into 5mL of complete culture medium, transferring the 5mL of cell suspension into a 96-well plate, wherein each positive cell is transferred into a half 96-well plate at 100 uL/well. And (5) culturing until one drop of fluid is replenished at the 4 th day, and carefully observing and recording the growth condition of cells in each hole at the 5 th-6 th day.

Detection of specific antibodies: and (3) detecting when the cell clone grows to the bottom area of the hole of 1/3-1/2 7-9 days after cloning, and performing liquid change treatment one day before detection, wherein the detection process is the same as the screening process of the positive hybridoma cells.

Hybridoma cell line determination: the hybridoma cells detected as the positive holes can be continuously subcloned by a limiting dilution method, and the subcloning is repeated for 3 times to obtain a monoclonal positive cell strain, namely a definite strain cell.

S6, large-scale preparation of monoclonal antibody

And (3) carrying out amplification culture on the cell strain after the selected strain is screened, inoculating the abdominal cavity of the mouse to prepare ascites when the cell grows well, collecting the ascites, measuring the titer by using indirect ELISA, and purifying the ascites to obtain the monoclonal antibody.

S7, sequence determination of variable regions of monoclonal antibodies IL1 beta-3F 5 and IL1 beta-5C 9

Extracting total RNA of hybridoma cells: respectively extracting total RNA of two cells by adopting a total RNA extraction kit according to the operation of an instruction;

synthesis of cDNA: synthesizing a first cDNA strand by using a reverse transcription kit;

PCR cloning of variable region sequences: primers are designed according to conserved sites of a mouse antibody sequence in the national center for Biotechnology information (GenBank), and cDNA is used as a template to amplify the light chain variable region gene and the heavy chain variable region gene of the antibody. The PCR program is 94 ℃ for 30s, 56 ℃ for 50s, 72 ℃ for 1min, amplification is carried out for 35 cycles, and finally extension is carried out for 10min at 72 ℃. The PCR product is recovered and connected to the T vector through agarose gel, and positive transformants are picked and sent to the company Limited Biotechnology (Shanghai) for sequencing. Wherein the primer sequences are respectively as follows: heavy chain variable region sequences 5 '-AGGTSMARCTGCAGSAGTCWG-3' and 5'-TGAGGAGACGGTGACCGTGGTCCCTTGGCCC-3' wherein S, M, R, W is a degenerate base, M ═ a/C, R ═ a/G, S ═ C/G, W ═ a/T, light chain variable region primers 5'-GACATTGAGCTCACCCAGTCTCCA-3' and 5'-CCGTTTTATTTCCAGCTTGGTCCC-3'.

Obtaining a gene sequencing result: the heavy chain variable region sequence of the 3F5 cell strain is 339bp in length, 113 amino acids are coded, and the sequence is shown as SEQ ID NO. 1; the light chain variable region sequence is 321bp long, 107 amino acids are coded, and the sequence is shown as SEQ ID NO. 2. The heavy chain variable region sequence of the 5C9 cell strain is 345bp in length, 115 amino acids are coded, and the sequence is shown as SEQ ID NO. 3; the light chain variable region has a sequence length of 309bp, encodes 103 amino acids, and has a sequence shown in SEQ ID NO. 4.

Example 2: reagent for detecting human interleukin 1 beta

ELISA plate coated by monoclonal antibody IL1 beta-3F 5Coating: diluting IL1 beta-3F 5 to 1-4 mu g/mL by using carbonate coating buffer solution or PBS solution, adding the diluted IL1 beta-3F 5 into an enzyme label plate, incubating the mixture at the temperature of 4 ℃ overnight at the concentration of 100 uL/hole, throwing off liquid in the hole, and washing the plate for 1 time.

And (3) sealing: blocking the position of the bottom of the plate hole of the enzyme label plate which is not coated by the antibody, adding blocking liquid (5 wt% of skimmed milk powder or 1 wt% of BSA) into the plate hole, incubating for 2h at room temperature for 250 uL/hole, drying the plate after the liquid in the hole is thrown off, drying overnight in a dry room, then vacuumizing, and storing at 4 ℃.

Biotin-labeled monoclonal antibody IL1 beta-5C 9

The purified monoclonal antibody IL1 β -5C9 was diluted to 1mg/mL with 0.1mol/L sodium bicarbonate buffer (pH 8.0); the monoclonal antibody IL1 β -5C9 was extensively dialyzed against 0.1mol/L sodium bicarbonate buffer at pH 8.0 for the interaction;

n-hydroxysuccinimide biotin (NHSB,1mg) was dissolved in dimethyl sulfoxide (DMSO, 1mL), 10uL of NHSB in DMSO was added to 1mL of the monoclonal antibody IL1 beta-5C 9 solution, stirring was continued at room temperature for 2-4 hours, followed by 1uL of 0.1mol/L NH ₄ Aqueous Cl solution at room temperatureStirred for 10 minutes. After the above products were dialyzed sufficiently at 4 ℃ the sample was applied to Sephadex G-25, slowly eluted with PBS, the peak was collected, and the biotin-labeled monoclonal antibody IL 1. beta. -5C9 was stored at-20 ℃.

Polymerase labeled streptavidin

6mg of horseradish peroxidase was dissolved in 1.5mL of double distilled water, and 0.025mL of a freshly prepared 0.1mol/L aqueous solution of sodium periodate was added thereto, followed by stirring at room temperature for 20 min. Adding ethylene glycol into the reaction solution, and reacting for 5min to terminate the reaction. Dialyzed against 2mmol/L sodium acetate buffer (pH 4.5) at 4 ℃ for 4 hours, exchanged at least 8 times, and then adjusted to pH 9.0 with 0.2mol/L carbonate buffer (pH 9.0). To the mixture was added 1.5mL of 6mg of streptavidin prepared in 0.2mol/L carbonate buffer (pH 9.0), and the mixture was stirred at room temperature for 2 hours in the dark. 0.01mL of a freshly prepared 4mg/mL aqueous solution of sodium borohydride was added dropwise and the reaction was carried out at 4 ℃ for 2 hours. Dialyzed overnight at 4 ℃ against 0.01mol/L PBS buffer at pH7.2, and centrifuged to remove the precipitate. The obtained supernatant is polymerase-labeled streptavidin, and is stored at-20 ℃.

Color developing agent

The color developing agent is a 3,3',5,5' -tetramethyl benzidine color developing substrate.

Example 3: method for detecting human interleukin 1 beta

This example uses a double-anti-sandwich enzyme-linked immunoassay to determine the level of interleukin 1 β in a human serum sample.

A method for detecting human interleukin 1 β, comprising the steps of:

(1) obtaining a gradient standard solution:

the concentrations of the gradient standard solutions prepared from the human interleukin 1 beta as the standard substance are respectively 20pg/mL, 10pg/mL, 5pg/mL, 2.5pg/mL, 1.25pg/mL, 0.625pg/mL, 0.312pg/mL and 0 pg/mL.

(2) Gradient standard solution and detection sample treatment:

washing the ELISA plate for 3 times before use, respectively adding the gradient standard solution into the reaction holes of the ELISA plate at 100 uL/hole, and simultaneously adding the sample to be detected into the reaction holes of the ELISA plate at 100 uL/hole; after this time, incubation was carried out for 90min at 37 ℃ and the wells were spin-dried and the plates were washed 4 times. Blank wells and negative control wells were also made.

Respectively adding a working solution (dilution after titration) of a biotin-labeled monoclonal antibody IL1 beta-5C 9 into reaction holes of an enzyme label plate coated by a monoclonal antibody IL1 beta-3F 5, wherein the dilution is 100 uL/hole; incubating at 37 ℃ for 60min, spin-drying the liquid in the pores and washing the plate for 4 times;

respectively adding 100 uL/hole of a polymerase labeled streptavidin working solution (dilution after titration) into reaction holes of an enzyme label plate coated by a monoclonal antibody IL1 beta-3F 5; incubating at 37 ℃ for 60min, spin-drying the liquid in the pores and washing the plate for 5 times;

respectively adding 3,3',5,5' -tetramethyl benzidine chromogenic substrate into reaction holes of an enzyme label plate coated by a monoclonal antibody IL1 beta-3F 5, wherein the amount of the reaction holes is 100 uL/hole; incubating for 10-30 min at 37 ℃, spin-drying liquid in the holes and washing the plate for 1 time;

stop solution, 50 uL/well, was added to an ELISA plate coated with monoclonal antibody IL1 β -3F 5.

(3) Obtaining the concentration of the sample to be detected:

determining absorbance values of the gradient standard solution and the test sample at a wavelength of 450 nm; the detection data of the gradient standard solution are shown in table 1.

TABLE 1 detection data for gradient standard solutions

Concentration of standards (pg/mL)	OD450 value
		0	0.081
0.312	0.146
		0.625	0.184
1.25	0.280
		2.5	0.458
5.0	0.839
		10.0	1.474
20.0	2.286

And drawing a standard curve by taking the concentration of the gradient standard solution as an abscissa and the absorbance value of the gradient standard solution as an ordinate, wherein the standard curve is shown in figure 4.

The curve equation of the standard curve obtained by analysis with four-parameter Logistic curve fitting of the ELISA Cale regression/fitting calculation program software is:

y ═ a-D/[ 1+ (X/C) ^ B ] + D, where a ═ 4.29894, B ═ -1.15465, C ═ 19.21773, D ═ 0.01612, r ^2 ^ 0.99984.

In the above-mentioned curve equation Y ═ a-D)/(1 + (X/C) ^ B ] + D, a represents the maximum value when X tends to infinity or infinity, B represents the slope, C represents the inflection point concentration, and D represents the minimum value when X tends to infinity or infinity.

And substituting the absorbance value of the detection sample into a curve equation, and calculating the concentration of the abscissa corresponding to the absorbance value of the sample to be detected on the standard curve, namely the content of the human interleukin 1 beta in the sample to be detected.

After matrix titration test debugging, the optimal coating concentration range (1-4) ug/mL of the monoclonal antibody IL1 beta-3F 5 and the optimal detection antibody working concentration (1000-2000) of the monoclonal antibody IL1 beta-5C 9 are measured, and the lowest detection concentration can reach pg level.

TABLE 2 detection data for the matrix titration test

The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.

Sequence listing

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Claims (10)

1. A composition of monoclonal antibodies, comprising monoclonal antibody IL1 β -3F5 and/or monoclonal antibody IL1 β -5C 9;

the monoclonal antibody IL1 beta-3F 5 comprises a heavy chain with a variable region amino acid sequence shown as SEQ ID NO.1 and a light chain with a variable region amino acid sequence shown as SEQ ID NO. 2;

the monoclonal antibody IL1 beta-5C 9 comprises a heavy chain with a variable region amino acid sequence shown as SEQ ID NO.3 and a light chain with a variable region amino acid sequence shown as SEQ ID NO. 4.

2. Use of a composition of monoclonal antibodies of claim 1 for detecting human interleukin-1 β.

3. Use of a composition of monoclonal antibodies according to claim 1 in the preparation of a detection reagent or a detection kit for detecting human interleukin 1 β.

4. A reagent for detecting human interleukin 1 β, comprising the composition of monoclonal antibodies of claim 1.

5. The reagent of claim 4, wherein the reagent comprises an ELISA plate coated with monoclonal antibody IL1 beta-3F 5, biotin-labeled monoclonal antibody IL1 beta-5C 9, polymerase-labeled streptavidin, and a color-developing agent.

6. A reagent according to claim 5, wherein the biotin is N-hydroxysuccinimide biotin.

7. The reagent of claim 5, wherein the polymerase is a horseradish peroxidase.

8. The reagent according to claim 5, wherein the color-developing agent is a 3,3',5,5' -tetramethylbenzidine color-developing substrate.

9. A kit for detecting human interleukin 1 β, comprising the reagent of any one of claims 4 to 8.

10. A method for detecting human interleukin 1 β, said method comprising the steps of:

(1) obtaining a gradient standard solution:

preparing a gradient standard solution with the concentration of (0-20) pg/mL by using human interleukin 1 beta as a standard substance;

(2) gradient standard solution and detection sample treatment:

firstly, adding the gradient standard solution and the detection sample into reaction holes of an enzyme label plate coated by a monoclonal antibody IL1 beta-3F 5 respectively for incubation reaction and washing the plate; then adding a biotin-labeled monoclonal antibody IL1 beta-5C 9 for incubation reaction and washing the plate; then adding streptavidin marked by polymerase to perform incubation reaction and washing the plate; finally adding a color developing agent for incubation reaction and washing the plate; then adding a stop solution to stop the reaction;

the monoclonal antibody IL1 beta-3F 5 comprises a heavy chain with a variable region amino acid sequence shown as SEQ ID NO.1 and a light chain with a variable region amino acid sequence shown as SEQ ID NO. 2;

the monoclonal antibody IL1 beta-5C 9 comprises a heavy chain with a variable region amino acid sequence shown as SEQ ID NO.3 and a light chain with a variable region amino acid sequence shown as SEQ ID NO. 4;

(3) obtaining the concentration of the sample to be detected:

determining absorbance values of the gradient standard solution and the test sample at a wavelength of 450 nm;

drawing a standard curve by taking the concentration of the gradient standard solution as an abscissa and the absorbance value of the gradient standard solution as an ordinate, and calculating to obtain a curve equation of the standard curve;

and calculating the concentration of the abscissa corresponding to the absorbance value of the sample to be detected on the standard curve according to the curve equation.

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