CN117126278B

CN117126278B - Monoclonal antibody and application thereof

Info

Publication number: CN117126278B
Application number: CN202311396908.4A
Authority: CN
Inventors: 顾雅韵; 濮科锋; 李炯; 秦静怡; 施颖
Original assignee: Suzhou Institute of Nano Tech and Nano Bionics of CAS
Current assignee: Suzhou Institute of Nano Tech and Nano Bionics of CAS
Priority date: 2023-10-26
Filing date: 2023-10-26
Publication date: 2024-01-05
Anticipated expiration: 2043-10-26
Also published as: CN117126278A

Abstract

The invention relates to a monoclonal antibody and application thereof. In particular, the invention provides antibodies, or antigen binding fragments thereof, that specifically bind to IL-1 β proteins. The antibodies of the invention are capable of specifically binding IL-1 beta protein with high affinity.

Description

Monoclonal antibody and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a monoclonal antibody and application thereof, in particular to an anti-IL-1 beta antibody and application thereof.

Background

The basic structure of IL-1β is a mature peptide chain, consisting of 153 amino acid residues. It is formed by cutting and shearing IL-1 beta precursor in cells through enzyme. Mature IL-1. Beta. Is a globular protein molecule consisting of 12 folding regions, including alpha helices, beta sheet fragments, turns, and the like. The domain of IL-1β consists of an IL-1β N-terminal domain and an IL-1β C-terminal domain, which are connected by a short linking region. The critical residues of the IL-1 β domain are located on its surface and these residues play an important role in binding to its receptor and signaling.

At present, research on IL-1 beta antibodies has made important progress and has demonstrated potential therapeutic effects in a number of disease areas. Various IL-1. Beta. Antibody drugs have been approved and used in clinical therapies. Including canavanab, anakinumab (Anakinra), li Naxi pride (Rilonacept), and the like. The antibody medicines have obvious curative effects in treating diseases such as rheumatoid arthritis, adult stele disease, periodic fever syndrome, inflammatory bowel disease and the like. These antibodies can reduce inflammatory responses, improve joint function, reduce disease activity, and improve the quality of life of the patient.

There remains a need for better-efficacy anti-IL-1 beta antibodies.

Disclosure of Invention

The invention obtains 4 antibodies, and the CDR region sequences are as follows:

TABLE 1 heavy chain CDR sequences

TABLE 2 light chain CDR sequences

The heavy and light chain sequences of these 4 antibodies are as follows:

TABLE 3 heavy chain sequences

TABLE 4 light chain sequences

In view of the instability of some hybridoma clones, the present invention provides for the preservation of the desired hybridoma variable region sequences by PCR acquisition of the antibody variable region sequences and antibody production by other cells that highly express the antibodies.

The specific operation of the invention for obtaining the variable region sequence of the light and heavy chain of the antibody is as follows: and (3) using Trizol to lyse candidate hybridoma clone strains and extract total RNA, synthesizing first-strand cDNA by using the Trizol as a template, performing subsequent PCR amplification by using the first-strand cDNA as the template to obtain nucleic acid of an antibody light-heavy chain variable region corresponding to hybridoma cells, performing agarose gel electrophoresis, cutting gel to recover TA clone, and performing Sanger sequencing to obtain an antibody variable region sequence.

Specifically, the invention provides the following technical scheme:

in one aspect, the invention provides an antibody or antigen-binding fragment thereof that specifically binds to IL-1 β protein, characterized in that the antibody comprises:

HCDR1 comprising or consisting of a sequence of SEQ ID No. 1 or SEQ ID No. 4 or SEQ ID No. 7 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to said sequence or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

HCDR2 comprising or consisting of a sequence of SEQ ID No. 2 or 5 or 8 or 10 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to said sequence or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

HCDR3 comprising or consisting of a sequence of SEQ ID No. 3 or 6 or 9 or 11 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to said sequence or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

LCDR1 comprising or consisting of a sequence of SEQ ID No. 12 or 15 or 18 or 21 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to said sequence or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

LCDR2 comprising or consisting of a sequence of SEQ ID No. 13 or SEQ ID No. 16 or SEQ ID No. 19 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to said sequence or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence; and

LCDR3 comprising or consisting of a sequence of SEQ ID No. 14 or 17 or 20 or 22 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to said sequence or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence.

In some embodiments, the antibody comprises a sequence selected from the group consisting of seq id no:

(a) HCDR1 shown as SEQ ID NO. 1, HCDR2 shown as SEQ ID NO. 2, HCDR3 shown as SEQ ID NO. 3, LCDR1 shown as SEQ ID NO. 12, LCDR2 shown as SEQ ID NO. 13, LCDR3 shown as SEQ ID NO. 14;

(b) HCDR1 shown as SEQ ID NO. 4, HCDR2 shown as SEQ ID NO. 5, HCDR3 shown as SEQ ID NO. 6, LCDR1 shown as SEQ ID NO. 15, LCDR2 shown as SEQ ID NO. 16, LCDR3 shown as SEQ ID NO. 17;

(c) HCDR1 shown as SEQ ID NO. 7, HCDR2 shown as SEQ ID NO. 8, HCDR3 shown as SEQ ID NO. 9, LCDR1 shown as SEQ ID NO. 18, LCDR2 shown as SEQ ID NO. 19, LCDR3 shown as SEQ ID NO. 20; and

(d) HCDR1 shown as SEQ ID NO. 4, HCDR2 shown as SEQ ID NO. 10, HCDR3 shown as SEQ ID NO. 11, LCDR1 shown as SEQ ID NO. 21, LCDR2 shown as SEQ ID NO. 16, LCDR3 shown as SEQ ID NO. 22.

In some embodiments, wherein the antibody is selected from the group consisting of:

(a) An antibody comprising the amino acid sequence set forth in SEQ ID NO:23 and a heavy chain amino acid sequence as set forth in SEQ ID NO:27 or consists of the light chain amino acid sequence shown in seq id no;

(b) An antibody comprising a sequence identical to the sequence set forth in SEQ ID NO:23, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence and having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to said sequence and having the amino acid sequence set forth in SEQ ID NO:27, or an amino acid sequence having or consisting of one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

(c) An antibody comprising the amino acid sequence set forth in SEQ ID NO:24 and a heavy chain amino acid sequence as set forth in SEQ ID NO:28 or consists of the light chain amino acid sequence shown in seq id no;

(d) An antibody comprising a sequence identical to the sequence set forth in SEQ ID NO:24, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence and having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to said sequence and having the amino acid sequence set forth in SEQ ID NO:28, or an amino acid sequence having or consisting of one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

(e) An antibody comprising the amino acid sequence set forth in SEQ ID NO:25 and a heavy chain amino acid sequence as set forth in SEQ ID NO:29 or consists of the light chain amino acid sequence shown in seq id no;

(f) An antibody comprising a sequence identical to the sequence set forth in SEQ ID NO:25, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence and having a sequence as set forth in SEQ ID NO:29, or an amino acid sequence having or consisting of one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

(g) An antibody comprising the amino acid sequence set forth in SEQ ID NO:26 and a heavy chain amino acid sequence as set forth in SEQ ID NO:30 or consists of the light chain amino acid sequence shown in seq id no; and

(h) An antibody comprising a sequence identical to the sequence set forth in SEQ ID NO:26, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence and having a sequence as set forth in SEQ ID NO:30, or an amino acid sequence having or consisting of one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence.

In some embodiments, the antigen binding fragment is selected from the group consisting of: fab, fab ', F (ab') 2, fd, fv, dAb, fab/c, complementarity Determining Region (CDR) fragments, single chain antibodies, diabodies, domain antibodies.

In another aspect, the invention provides a polynucleotide encoding an antibody or antigen-binding fragment thereof as described above.

In another aspect, the invention provides an expression vector comprising a polynucleotide as described above.

In another aspect, the invention provides a host cell comprising an expression vector as described above, said host cell being a host cell for expressing a foreign protein, e.g. a bacterial, yeast, insect cell or mammalian cell.

In another aspect, the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described above, and a pharmaceutically acceptable carrier.

In another aspect, the invention provides a fusion protein comprising an antibody or antigen-binding fragment thereof as described above.

In another aspect, the invention provides a method for detecting the amount of IL-1 β in a sample, characterized in that the method comprises the step of binding the sample to an antibody or antigen binding fragment thereof as described above.

In another aspect, the invention provides the use of an antibody or antigen binding fragment thereof as described above in the preparation of a kit for detecting the amount of IL-1β in a sample.

Drawings

Fig. 1 shows the results of evaluation of serum titers after immunization of mice. The binding capacity of the anti-IL-1 beta antibody and IL-1 beta protein in the serum of the mice is detected by a protein ELISA method, and the binding capacity of the antibody under different serum dilution factors is shown.

FIGS. 2A, 2B and 2C show the results of detection sensitivity of the antibody pair combinations of the present invention to IL-1β protein. Wherein FIG. 2A shows the detection results of the B1-B2 combination; FIG. 2B shows the detection results of the combination of B1-B3; FIG. 2C shows the detection results of the B1-B4 combination.

FIGS. 3A, 3B and 3C show the results of detection specificity of the combination of antibody pairs of the invention for IL-1β protein. Wherein FIG. 3A shows the detection results of the B1-B2 combination; FIG. 3B shows the detection results of the combination B1-B3; FIG. 3C shows the detection results of the B1-B4 combination.

Fig. 4A, 4B, and 4C show the results of an experiment for serum incorporation of an antibody of the present invention. Wherein FIG. 4A shows the detection results of the B1-B2 combination; FIG. 4B shows the detection results of the combination of B1-B3; FIG. 4C shows the detection results of the B1-B4 combination.

FIG. 5 shows ELISA detection results of the antibody pair B1-B2 combinations of the present invention.

FIG. 6 shows the chemiluminescent detection results of the antibody pair B1-B2 combinations of the invention.

Detailed Description

The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.

Example 1 screening of antibodies

IL-1 beta immune mice are screened to obtain specific target antibodies

The hybridoma antibody screening technology provides a platform for screening monoclonal antibodies with target specificity and high affinity. The technology mainly fuses plasma B cells which can secrete antibodies but cannot be immortalized with myeloma cells which can be proliferated in vitro and cultured for a long time, and the hybridoma cells which can secrete specific antibodies and can be cultured for a long time are obtained through screening. The production and preparation of the monoclonal antibody are realized through the enlarged culture of the hybridoma cells, and the hybridoma cells can be stored by freezing with liquid nitrogen, so that convenience is provided for the production of the later-stage antibody.

1. Immunized mice and serologic potency assays

Target immune-immune mice and serologic potency assays

To obtain higher mouse titers, C57/BL6 mice were used for 6-8 weeks, 4 days prior to immunization, the orbit was bled, 3000g,5min, centrifuged to obtain about 20ul serum, and frozen at-20℃as a negative control. Freund's adjuvant (Sigma) was used as an immunological adjuvant, and the antigen was IL-1. Beta. Protein (MAEVPELASEMMAYYSGNEDDLFFEADGPKQMKCSFQDLDLCPLDGGIQLRISDHHYSKGFRQAASVVVAMDKLRKMLVPCPQTFQENDLSTFFPFIFEEEPIFFDTWDNEAYVHDAPVRSLNCTLRDSQQKSLVMSGPYELKALHLQGQDMEQQVVFSMSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKGGQDITDFTMQFVSS, SEQ ID NO: 31). The method adopts a multipoint subcutaneous injection mode, after primary immunization, boosting is carried out every two weeks, 100ug of the mice are subjected to three times of immunization, and the antibody titer in the serum of the mice is detected by protein ELISA. The immune mode is simple to operate, and the antibody titer in serum is detected, so that the mice have better titers.

Protein ELISA detection: the 96-well ELISA plate was coated with IL-1. Beta. Protein (1 ug/ml) and incubated at 37℃for 2 hours. The fluid in the wells was then discarded, washed three times with PBST wash buffer (Solarbio), blocked overnight at 4℃with the addition of PBS (Solarbio) solution containing 2% BSA (Solarbio). The following day after the blocking plate was washed with PBST wash buffer, mouse serum samples of different dilution were added, incubated for 1 hour at 37 ℃ and after washing with PBST wash buffer, 2% BSA-PBST was added to 1: 10000-fold dilution of HPR-labeled goat anti-mouse IgG (H+L) (Sigma), incubated at 37℃for 1 hour. After washing with PBST wash buffer, TMB substrate solution (Beyotidme) was added for color development, and after 15 minutes of reaction at room temperature in the dark, the reaction was terminated with 2M hydrochloric acid solution and absorbance was read at 450nm, and the results are shown in FIG. 1.

Cell fusion

3 days after the last boost, one mouse with the highest antibody titer in the serum of the mouse was selected, the spleen was removed and ground in RPMI-1640 basal medium to obtain a lymphocyte-enriched suspension. To obtain hybridomas for selection, we fused mouse spleen cells with mouse myeloma cells (SP 2/0) in a 10:1 ratio using efficient electrotransformation. After 7 days of fusion, the supernatant was assayed by protein ELISA to determine whether the fused cells produced antibodies.

Protein ELISA detection: the 96-well ELISA plate was coated with IL-1. Beta. Protein (1 ug/ml) and incubated at 37℃for 2 hours. The well fluid was then discarded, washed three times with PBST wash buffer, blocked overnight at 4℃with the addition of PBS containing 2% BSA. The following day after the blocking plate was washed with PBST wash buffer, mouse serum samples of different dilution were added, incubated for 1 hour at 37 ℃ and after washing with PBST wash buffer, 2% BSA-PBST was added to 1: 10000-fold dilution of HPR-labeled goat anti-mouse IgG (H+L), incubation at 37℃for 1 hour. After washing with PBST wash buffer, the color development was performed by adding TMB substrate solution, and after 15 minutes of reaction at room temperature in the dark, the reaction was terminated with 2M hydrochloric acid solution and the absorbance was read at 450 nm. Wells with high OD were selected for subcloning.

In the project verification, a plurality of antibody expression clone strains capable of combining IL-1 beta protein are screened out from 53 screening plates by the method, and a plurality of antibody expression clone strains have the capability of stably expressing high-affinity antibodies aiming at the IL-1 beta protein. Negative value 0.15.

TABLE 5 Elisa values of subcloned hybridomas

Example 2 characterization of antibodies

1. Affinity assay for antibodies

Affinity assays for antibodies using biocore T200, 4 antibodies affinity activities were as follows:

TABLE 6 antibody affinity

2. Detection of IL-1 beta factor

Magnetic bead coated capture antibody (Experimental procedures refer to Solarbio's magnetic bead coupling instructions)

1. Preparation of Capture antibody solution

100ug of capture antibody is dissolved by coupling buffer solution, and the capture antibody is prepared into solution with the concentration of more than or equal to 3.0 mg/mL. And (5) storing the prepared capture antibody solution at the temperature of 4 ℃ for standby.

2. Magnetic bead cleaning

After thoroughly mixing the beads using a vortex shaker, 500 μl of 20% bead suspension was mixed well in a 1.5 mL EP tube.

Place the EP tube in a magnetic separation rack, enrich the beads, remove supernatant.

Adding 1 mL of washing buffer solution (1 mM hydrochloric acid) at 2-8 ℃ into a centrifuge tube, and swirling 15 and s to uniformly mix the magnetic beads.

3. Protein immobilization

Add 200 μl of capture antibody solution to the EP tube, vortex 30 s, mix well.

EP tube vortex 15 s is placed on a vertical mixer and mixed for 2-4 hours at room temperature.

-enriching the beads with a magnetic separation rack, preserving the flow-through.

4. Magnetic bead closure

Add 1 mL blocking buffer (100 mM Tris-HCl,150mM NaCl,pH 8.0) to EP tube, vortex 30 s, place EP tube in magnetic separation rack, enrich magnetic beads, discard supernatant. Four replicates were performed.

Add 1 mL blocking buffer to the EP tube, vortex 30 s, place the EP tube in a vertical mixer for 2h at room temperature.

Place EP tube in magnetic separation rack, enrich magnetic beads, discard supernatant.

Add 1 mL ultra pure water to the EP tube, mix well, enrich the beads with magnetic rack, discard supernatant.

Add 1 mLPBS buffer to the EP tube, mix well, enrich the beads with magnetic rack, discard supernatant. This operation was repeated 2 times.

Adding the mixture into a PBS buffer EP tube, fully mixing, and storing at 4 ℃ for standby.

Antibody biotin labelling (for experimental procedures reference to the Frdbbio biotin labelling instructions)

1. Four antibodies obtained in the present invention were taken 1mg in ultrafiltration tubes, respectively, and 500ul of a labeling buffer (Frdbbio) was added thereto so that the final concentration of the antibodies was 2mg/mL, and centrifuged at 12,000g for 10min.

2. 13.3. Mu.L of biotin solution and an appropriate amount of labeling buffer were added to the above-mentioned ultrafilter tube to a final volume of 0.5ml, and gently stirred and mixed. Placing in a 37 ℃ incubator to incubate for 30min in the dark.

Centrifuge at 3.12,000 g for 10min.

4. Adding a proper amount of marking buffer solution into the ultrafiltration tube, gently blowing and uniformly mixing, centrifuging at 12,000g for 10min, and repeating for 3 times.

5. The solution (i.e., biotin-labeled antibody) in the ultrafilter tube was collected and stored at-20℃for further use.

Double antibody sandwich assay

Incubating the coated magnetic beads with IL-1 beta protein to be detected, wherein the protein concentration is 10ng/ul. The four antibodies obtained by the invention are respectively labeled with biotin to form immune complexes of antibody coated microsphere-cytokine-detection antibody. Finally, adding Phycoerythrin (PE) marked streptavidin (Invitrogen), combining with biotin, and detecting by a flow cytometry to obtain the fluorescence intensity of the object to be detected. The control group was an antibody without biotin labeling.

TABLE 7 double antibody sandwich assay results

The significant difference between the fluorescence intensity of the antibody and the fluorescence intensity of the control shows that the antibodies screened by the invention can be used for detecting the protein.

3. Antibody pair sensitivity detection

The 4 antibodies of the invention can capture and detect the antibodies for pairing. Now taking B1 as capture antibody and B2 as detection antibody as an example:

the magnetic beads are coated with B1 antibody and incubated with IL-1 beta protein to be tested, wherein the protein concentration is 20000pg/ml,4000pg/ml,800pg/ml,160pg/ml,32pg/ml,6.4pg/ml,1.28pg/ml and 0.256pg/ml respectively. And (3) carrying out biotin labeling on the B2 antibody obtained by the invention to form an immune complex of the antibody coated microsphere-cytokine-detection antibody. Finally, adding Phycoerythrin (PE) marked streptavidin (Invitrogen), combining with biotin, and detecting by a flow cytometry to obtain the fluorescence intensity of the IL-1 beta protein of the object to be detected.

The detection sensitivity of the antibody disclosed by the invention on the combination of B1-B2, B1-B3 and B1-B4 on IL-1 beta protein can reach 6.4pg/ml, and the results are shown in FIG. 2A, FIG. 2B and FIG. 2C. Antibody pair sensitivity detection may be performed using, but not limited to, combinations of the above, e.g., B2-B3, B2-B4 combinations, paired detection may also be performed, and capture antibody and detection antibody may be used interchangeably.

4. Antibody pair specificity detection

The 4 antibodies of the invention can capture and detect the antibodies for pairing. To test the capture capacity of an antibody pair for the specificity of the IL-1 β protein in the presence of various other factors, 11 factor mixtures are used, with B1 as capture antibody and B2 as test antibody as an example:

the beads were coated with B1 antibody, incubated with the remaining beads coated with 11 proteins (IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-17, IFN- α, IFN- γ, TNF- α) antibodies (Raiseart), and the concentration of the proteins was 10ng/ul. And (3) carrying out biotin labeling on the B2 antibody obtained by the invention to form an immune complex of the antibody coated microsphere-cytokine-detection antibody. Finally, adding Phycoerythrin (PE) marked streptavidin (Invitrogen), combining with biotin, and detecting by a flow cytometry to obtain the fluorescence intensity of the IL-1 beta protein of the object to be detected.

IL-1. Beta. Proteins were mixed into the other 11 proteins (IL-10, IL-2, IL-4, IL-5, IL-6, IL-8, IL-12, IL-17, IFN-. Alpha., IFN-. Gamma., TNF-. Alpha.) and incubated together. IL-1 beta protein can be detected from the mixed protein by the pairing combination of three kinds of B1-B2, B1-B3 and B1-B4. And have significant differences from other protein signals, the results are shown in fig. 3A, 3B and 3C. The antibody pair specificity detection may be performed using, but not limited to, combinations of the above, e.g., B2-B3, B2-B4 combinations, paired detection may also be performed, and the capture antibody and detection antibody may be used interchangeably.

5. Serum incorporation experiments

the protein IL-1β to be tested was diluted with PBS and negative serum FBS (Gibco), respectively, and the protein concentrations after dilution were 20000pg/ml,4000pg/ml,800pg/ml,160pg/ml,32pg/ml,6.4pg/ml,1.28pg/ml, and 0.256pg/ml, respectively. The magnetic beads are coated with the B1 antibody, and the coated magnetic beads are respectively incubated with the antibodies B2, B3 and B4. The PBS group and the serogroup were used. And (3) carrying out biotin labeling on the B2 antibody obtained by the invention to form an immune complex of the antibody coated microsphere-cytokine-detection antibody. Finally, adding Phycoerythrin (PE) marked streptavidin (Invitrogen), combining with biotin, and detecting by a flow cytometry to obtain the fluorescence intensity of the object to be detected.

Antibody to serum incorporation experiments can be used, but are not limited to, B1-B2, B1-B3, B1-B4 combinations. Other B2-B3, B2-B4 combinations may also be paired for detection, and capture antibodies and detection antibodies may be used interchangeably.

From the results of fig. 4A, 4B and 4C, it is clear that the antibody pair can well detect specific proteins in not only PBS but also other media such as FBS (fetal bovine serum), and has good coincidence.

6. ELISA detection.

Take the B1-B2 antibody pair as an example:

the 96-well ELISA plate was coated with B1 antibody (1 ug/ml) and incubated at 37℃for 2 hours. The fluid in the wells was then discarded, washed three times with PBST wash buffer (Solarbio), blocked overnight at 4℃with the addition of PBS (Solarbio) solution containing 2% BSA (Solarbio). The next day, the microtiter plates were washed twice with 200 μl PBS. The diluted IL-1. Beta. Protein samples were added to the wells at a concentration of 24. Mu.g/L, 16. Mu.g/L, 8. Mu.g/L, 4. Mu.g/L, 2. Mu.g/L, and incubated at 37℃for 1.5 hours. And after washing with PBST wash buffer, 100 μl of diluted detection antibody B1 was added to each well and incubated for 2 hours at room temperature. Washed three times with PBS. 100 μl of biotin-labeled B2 was added, incubated for 1h at room temperature, and the microtiter plates were washed three times with PBS. SA-HRP (Thermofisher) was added and incubated at room temperature for 1 h. Chemiluminescent HRP substrate (thermofiser) was added vertically in suspension, and each well was incubated at 37℃for 30min at 90. Mu.L and the ELISA plate was removed. Stop solution (thermofilter) was added vertically in suspension, 50 μl per well. ELISA plate reading OD450. Standard curves were plotted using serial diluent data, with concentration on the X-axis (logarithmic scale) and absorbance on the Y-axis (linear scale).

Combinations of B1-B2 may be used, but are not limited to. Other B1-B3, B1-B4, B2-B3, B2-B4 combinations may also be paired, and capture antibodies and detection antibodies may be used interchangeably.

As can be seen from the results of FIG. 5, the antibody pairs of the present invention also find good application in a double antibody sandwich ELISA.

7. Chemiluminescent detection

the magnetic beads are coated with B1 antibody and incubated with IL-1 beta protein to be tested, wherein the protein concentration is 7650pg/ml,2393pg/ml,499pg/ml,100pg/ml and 50pg/ml respectively. The B2 antibody obtained by the invention is mixed with acridinium ester to form an immune complex of antibody coated microsphere-cytokine-detection antibody. And finally adding hydrogen peroxide alkaline solution, and detecting by using a chemiluminescent instrument.

Chemiluminescent detection may be used, but is not limited to, a B1-B2 combination. Other combinations of B1-B3, B1-B4, B2-B3, B2-B4 may also be paired, and capture antibodies and detection antibodies may be used interchangeably.

As can be seen from the results of FIG. 6, the antibody pairs of the present invention also find good application in chemiluminescent detection.

The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to IL-1 β protein, wherein the antibody comprises the sequence: HCDR1 shown as SEQ ID NO. 1, HCDR2 shown as SEQ ID NO. 2, HCDR3 shown as SEQ ID NO. 3, LCDR1 shown as SEQ ID NO. 12, LCDR2 shown as SEQ ID NO. 13, LCDR3 shown as SEQ ID NO. 14.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody comprises the amino acid sequence set forth in SEQ ID NO:23 and a heavy chain amino acid sequence as set forth in SEQ ID NO:27 or consists of the light chain amino acid sequence shown in seq id no.

3. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-2.

4. An expression vector comprising the polynucleotide of claim 3.

5. A host cell comprising the expression vector of claim 4, said host cell being a host cell for expressing a foreign protein.

6. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-2, and a pharmaceutically acceptable carrier.

7. A fusion protein comprising the antibody or antigen-binding fragment thereof of any one of claims 1-2.

8. A method for detecting the amount of IL-1 β in a sample, characterized in that the method comprises the step of binding the sample to an antibody or antigen binding fragment thereof according to any one of claims 1-2.

9. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-2 in the preparation of a kit for detecting the amount of IL-1β in a sample.