CN116925218B - Antibody of small heat shock protein HSPB1, antibody composition, hybridoma cell strain and application thereof - Google Patents

Antibody of small heat shock protein HSPB1, antibody composition, hybridoma cell strain and application thereof Download PDF

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CN116925218B
CN116925218B CN202311207275.8A CN202311207275A CN116925218B CN 116925218 B CN116925218 B CN 116925218B CN 202311207275 A CN202311207275 A CN 202311207275A CN 116925218 B CN116925218 B CN 116925218B
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antibody
heat shock
hspb1
shock protein
small heat
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CN116925218A (en
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陈新新
潘悦
苑晓松
刘星
赵海龙
路轲
王芳
魏彦辉
马玉岭
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Beijing Solarbio Technology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Abstract

The invention relates to the technical field of antibodies, in particular to an antibody of a small heat shock protein HSPB1, an antibody composition, a hybridoma cell strain and application thereof. The antibody or the antigen binding fragment thereof of the small heat shock protein HSPB1 provided by the invention can specifically bind with the human small heat shock protein HSPB1 and has higher affinity; the method has higher sensitivity and specificity when detecting the human small heat shock protein HSPB1, can realize the detection of the small heat shock protein HSPB1 with low content, can meet the requirements of detection practice, and has important application value in the field of detection of the small heat shock protein HSPB1.

Description

Antibody of small heat shock protein HSPB1, antibody composition, hybridoma cell strain and application thereof
Technical Field
The invention relates to the technical field of antibodies, in particular to an antibody of a small heat shock protein HSPB1, an antibody composition, a hybridoma cell strain and application thereof.
Background
Heat shock protein is a substance with molecular chaperone function produced by cells under stress environment (such as high heat, hypoxia, wound, disease, etc.), and participates in the processes of folding, modifying, etc. of cell protein, and can improve the resistance of cells to adverse conditions. Heat shock proteins are classified into large, medium and small types according to their molecular weights. HSPB1 is one of the small heat shock proteins, 205 amino acids in full length, with a molecular weight of about 27kD. HSPB1 has repair or decomposition functions on misfolded proteins, and when a cell is stressed, the HSPB1 expression level increases, and the cell can cope with adverse environments by various ways, increase cell survival rate, and delay or inhibit apoptosis. High expression of HSPB1 occurs during the development of various diseases, such as diabetes, cardiovascular disease, neurological disease, tumor, etc. Detecting the expression level of HSPB1 is useful for assessing disease progression or prognosis.
At present, an immunohistochemical method is mainly adopted for detecting the HSPB1, and the method can evaluate the high expression or the low expression of the HSPB1, but cannot accurately quantify. The ELISA detection technology based on the double-antibody sandwich method can accurately and rapidly quantitatively detect the HSPB1. HSPB1 is low in normal human serum, and therefore, development of antibodies with high affinity is required to ensure high sensitivity of detection.
Disclosure of Invention
The invention provides an antibody of a small heat shock protein HSPB1, an antibody composition, a hybridoma cell strain and application thereof.
According to the invention, the human HSPB1 antigen is prepared, and after mice are immunized by the human HSPB1 antigen for fusion screening, two monoclonal antibodies with good affinity, specificity and stability are obtained, and an ELISA detection kit based on a double antibody sandwich method is developed by utilizing the two monoclonal antibodies, so that the content of the human HSPB1 can be accurately detected by the kit, the sensitivity and the specificity are higher, and samples with lower content of the human HSPB1 can be detected.
Specifically, the invention provides the following technical scheme:
the invention provides an antibody or antigen binding fragment thereof of a small heat shock protein HSPB1, wherein the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of a heavy chain variable region of the antibody or antigen binding fragment thereof are respectively shown as SEQ ID NO.6, 7 and 8, the amino acid sequence of complementarity determining region CDR1 of a light chain variable region is shown as SEQ ID NO.9, the amino acid sequence of CDR2 is YAS, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 10.
Preferably, the amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment thereof is as shown in SEQ ID NO.13 or has at least 80% similarity to the sequence as shown in SEQ ID NO.13, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.14 or has at least 80% similarity to the sequence as shown in SEQ ID NO. 14.
Preferably, the antibody or antigen binding fragment thereof is a monoclonal antibody, fab ', F (ab') 2, fd, fv, dAb, single chain antibody, bispecific antibody or multispecific antibody.
In some embodiments of the invention, antibodies to small heat shock protein HSPB1 are provided, the heavy chain variable region of which has the amino acid sequence shown in SEQ ID No.13 and the light chain variable region has the amino acid sequence shown in SEQ ID No. 14.
In addition to the antibodies or antigen-binding fragments thereof described above, the present invention provides nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof.
Based on the amino acid sequences of the above antibodies or antigen binding fragments thereof, the skilled artisan can obtain nucleotide sequences of nucleic acid molecules encoding the above antibodies or antigen binding fragments thereof. Because of the degeneracy of the codons, the nucleotide sequences of the nucleic acid molecules encoding the antibodies or antigen binding fragments thereof are not unique, and all nucleic acid molecules capable of encoding the antibodies or antigen binding fragments thereof are within the scope of the invention.
In some embodiments of the invention, the nucleotide sequence of a nucleic acid molecule encoding the heavy chain variable region of the antibody or antigen binding fragment thereof is shown in SEQ ID NO.17 and the nucleotide sequence of a nucleic acid molecule encoding the light chain variable region of the antibody or antigen binding fragment thereof is shown in SEQ ID NO. 18.
Further, the present invention provides a biological material comprising the nucleic acid molecule; the biological material is an expression cassette, a vector or a host cell.
The above-mentioned expression cassette can be obtained by ligating a transcription or translation regulatory element such as a promoter upstream of the nucleic acid molecule and/or ligating a transcription or translation regulatory element such as a terminator downstream thereof.
Such vectors include, but are not limited to, plasmid vectors, phage vectors, viral vectors, artificial chromosome vectors, and the like.
The host cells include microbial cells, insect cells, or other animal cells.
On the basis of the antibody or the antigen binding fragment thereof, the invention provides an antibody conjugate which is obtained by coupling the antibody or the antigen binding fragment thereof of the small heat shock protein HSPB1 with a marker, wherein the marker is one or more selected from enzyme markers, biotin markers, fluorescent dye markers, chemiluminescent dye markers and radioactive markers.
The present invention provides an antibody composition for small heat shock protein HSPB1 comprising the antibodies in (1) and (2) below:
(1) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown as SEQ ID NO.1, 2 and 3 respectively; the amino acid sequence of a complementarity determining region CDR1 of the light chain variable region is shown as SEQ ID NO.4, the amino acid sequence of a CDR2 is WAS, and the amino acid sequence of a CDR3 is shown as SEQ ID NO. 5;
(2) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO.6, 7 and 8 respectively; the amino acid sequence of CDR1 of the complementarity determining region of the light chain variable region is shown as SEQ ID NO.9, the amino acid sequence of CDR2 is YAS, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 10.
The antibody composition can be used as a pairing antibody for detecting the small heat shock protein HSPB1 by a double-antibody sandwich method, and two antibodies in the antibody composition are respectively used as a coating antibody and a labeling antibody in the double-antibody sandwich method.
Preferably, the amino acid sequence of the heavy chain variable region of the antibody described in (1) above is shown as SEQ ID NO.11 or has at least 80% similarity to the sequence shown as SEQ ID NO.11, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.12 or has at least 80% similarity to the sequence shown as SEQ ID NO. 12.
The heavy chain variable region of the antibody described in (2) above has an amino acid sequence shown in SEQ ID NO.13 or at least 80% similarity to the sequence shown in SEQ ID NO.13, and the light chain variable region has an amino acid sequence shown in SEQ ID NO.14 or at least 80% similarity to the sequence shown in SEQ ID NO. 14.
The invention provides a hybridoma cell strain 9F8 which is preserved in China general microbiological culture collection center (CGMCC) for 8 months and 2 days of 2023, and has the classification name of mouse anti-human HSPB1 monoclonal antibody hybridoma cell strain and the preservation number of CGMCC No. 45646, wherein the address is 1, 3, the institute of microbiological study, the university of China, the university of Kogyo, beijing, and the North Chen West road.
The invention also provides a hybridoma cell strain 20G5 which is preserved in China general microbiological culture collection center (CGMCC) of China general microbiological culture Collection center (address: north Xila No.1, 3 of Beijing, chaoyang area, and Biotechnology institute of China academy of sciences, mail code 100101) at the 8 th month of 2023, and classified and named as a mouse anti-human HSPB1 monoclonal antibody hybridoma cell strain, wherein the preservation number is CGMCC No. 45647.
The invention also provides a monoclonal antibody produced by the hybridoma cell strain, which can specifically bind to a small heat shock protein HSPB1.
The present invention provides the use of the antibody or antigen binding fragment thereof of small heat shock protein HSPB1 or the nucleic acid molecule or the biological material or the antibody conjugate or the antibody composition of small heat shock protein HSPB1 or the hybridoma cell line or the monoclonal antibody as described above:
(1) Use of small heat shock protein HSPB1 in the detection of the presence or level of HSPB1 in a sample for non-disease diagnostic purposes;
(2) Use in the preparation of a product for detecting the presence or level of small heat shock protein HSPB1 in a sample;
(3) The application of the polypeptide in preparing a medicament for treating diseases caused by overexpression of small heat shock protein HSPB1.
In the above (1), the use comprises detecting the presence or level of small heat shock protein HSPB1 in the sample using the antibody or antigen binding fragment thereof or the antibody conjugate or the antibody composition or the monoclonal antibody. Wherein the presence of HSPB1 in the sample refers to whether HSPB1 is contained in the sample, and the level of HSPB1 in the sample refers to the level of HSPB1 in the sample. The sample comprises a sample containing small heat shock protein HSPB1 prepared in vitro and the like.
In the above (2), the product is a product for detecting small heat shock protein HSPB1, comprising a reagent or a kit. The sample includes body fluid, cells or tissues from a human body such as plasma, serum, blood, etc.
In the above (1) and (2), the method for detecting the small heat shock protein HSPB1 by using the antibody or the antigen-binding fragment thereof or the antibody conjugate or the antibody composition or the monoclonal antibody produced by the hybridoma cell line may use detection methods such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography and the like.
In some embodiments of the invention, the small heat shock protein HSPB1 is detected in a double antibody sandwich method using the antibody or antigen binding fragment thereof or the antibody conjugate or the antibody composition.
In the invention, the small heat shock protein HSPB1 is human small heat shock protein HSPB1.
In the above (3), the diseases include, but are not limited to, breast cancer and the like.
The present invention provides a kit comprising an antibody or antigen binding fragment thereof of the small heat shock protein HSPB1, or the antibody conjugate, or an antibody composition comprising the small heat shock protein HSPB1, or the monoclonal antibody.
The kit is a detection kit for the small heat shock protein HSPB1.
In the above kit, the antibody or antigen-binding fragment thereof may further comprise a detectable label; the kit may further comprise a second antibody carrying a detectable label to detect the antibody or antigen binding fragment thereof.
In some embodiments of the present invention, a double-antibody sandwich ELISA detection kit of small heat shock protein HSPB1 is provided, comprising a coated antibody and a labeled antibody, wherein the amino acid sequences of complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region of the coated antibody are as set forth in SEQ ID NO: 1. 2, 3, the amino acid sequence of the complementarity determining region CDR1 of the light chain variable region is shown as SEQ ID NO.4, the amino acid sequence of the CDR2 is WAS, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 5; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region of the labeled antibody are shown in SEQ ID NO: 6. 7 and 8; the amino acid sequence of CDR1 of the complementarity determining region of the light chain variable region is shown as SEQ ID NO.9, the amino acid sequence of CDR2 is YAS, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 10.
The kit has higher sensitivity when being used for detecting the small heat shock protein HSPB1, and the detection range can reach 0.078-5ng/mL; the kit has good stability, and the kit has no obvious change in 13 days when an acceleration stability experiment is carried out at 37 ℃; and the protein has higher specificity, and non-target proteins such as human IgG, human albumin, mmp2, human type 1 collagen alpha, human Golgi membrane protein 1, human IL-10, human IL-2, human IL-6, human IL-12, human IL-1β, human TNF-alpha, human IFN-alpha, mouse IgG, rat IgG and the like are not identified in the protein crossing experiment.
For ease of detection, the kit may also contain other reagents for ELISA detection including, but not limited to, an ELISA plate, a small heat shock protein HSPB1 standard, a PBST wash, a blocking solution, a chromogenic solution, a stop solution, and the like.
The present invention provides a medicament comprising an antibody or antigen binding fragment thereof to the small heat shock protein HSPB1 or comprising the monoclonal antibody.
The invention provides a detection method of a small heat shock protein HSPB1, which comprises the following steps: detecting the content of the small heat shock protein HSPB1 in the sample to be detected by using the antibody or the antigen binding fragment thereof or the antibody conjugate or the antibody composition or the kit.
The detection method can be selected from enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay or immunochromatography.
The beneficial effects of the invention at least comprise: the antibody or the antigen binding fragment thereof of the small heat shock protein HSPB1 provided by the invention can specifically bind to human small heat shock protein HSPB1 and has higher affinity; the method has higher sensitivity (the detection range can reach 0.078-5 ng/mL) and specificity when the detection of the human small heat shock protein HSPB1 is carried out, can realize the detection of the small heat shock protein HSPB1 with low content, can meet the requirements of detection practice, and has important application value in the field of detection of the small heat shock protein HSPB1.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing the result of SDS-PAGE electrophoresis of purified HSPB1 protein in example 1 of the present invention, wherein M is a protein molecular weight marker.
FIG. 2 is a diagram showing the result of SDS-PAGE electrophoresis of the purified monoclonal antibodies 9F8 and 20G5 of example 1 of the present invention, wherein M is a molecular weight marker of protein.
FIG. 3 is a standard curve of the detection of HSPB1 protein by the double antibody sandwich ELISA method of example 3 of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 preparation of monoclonal antibodies against human HSPB1 protein
1. Purification of HSPB1 proteins
The HSPB1 gene is synthesized on a pET-28A vector to obtain a recombinant plasmid, which is named as HSPB1-28A. And (3) transforming the recombinant plasmid HSPB1-28A into an expression strain escherichia coli Rosseta, picking up a positive clone, culturing and sequencing (the nucleotide sequence of the HSPB1 gene is shown as SEQ ID NO. 19) to obtain the recombinant strain. The recombinant strain was induced to express while the uninduced strain and empty vector strain controls were set. HSPB1 protein expression was detected by SDS-PAGE. After determining the expression of the HSPB1 protein, the cells were sonicated, the supernatant and the pellet were separated, and the form in which the HSPB1 protein was expressed was examined by SDS-PAGE. The detection result shows that the HSPB1 protein is expressed in an insoluble inclusion body form, and the inclusion body is subjected to renaturation treatment and purification to obtain the target protein. SDS-PAGE detection results are shown in FIG. 1.
2. Immunization of animals
And (3) selecting a female Balb/c mouse with the age of 6-8 weeks, emulsifying the purified HSPB1 antigen prepared in the step (1) with an equal volume of Freund's adjuvant, performing immunization, wherein the immunization period is two weeks, taking blood after 3 times of immunization, measuring the titer, and boosting again three days before fusion.
3. Cell fusion
Mice were sacrificed by cervical scission, spleens were removed by aseptic manipulation, and spleen cell suspensions were prepared by squeeze milling in a plate. The syngeneic myeloma cells and the spleen cells of the mice are mixed according to a certain proportion, and a fusogenic agent polyethylene glycol (PEG) is added. Under the action of polyethylene glycol, various lymphocytes can be fused with myeloma cells to form hybridoma cells. The specific operation is as follows:
(1) Myeloma (SP 2/0) cell activation
Thawing and resuscitating commercial SP2/0 cells, and then re-suspending in nutrient solution (RPMI-1640 basal medium supplemented with 20% fetal bovine serum), placing at 37deg.C and 5% CO 2 Culturing in an incubator under the condition; passaging is carried out after 3-5 d;
collecting cells and suspending in RPMI-1640 basic culture medium, counting, and collecting 0.5-1×10 6 The individual cells are injected into the back of a BALB/c mouse subcutaneously and cultured continuously for 9-10 d. After the tumor volume of the back is increased to about 0.8cm in diameter, the mice are killed by pulling the neck, and the tumor is taken out after 75% alcohol soaking for 5min.
Cutting off tumor blocks, placing the cut tumor blocks in a sterilized homogenizer, adding an RPMI-1640 basic culture medium, fully grinding, adding 10mL of the RPMI-1640 basic culture medium, standing for 2 min, sucking the upper cell suspension, placing in another centrifuge tube, adding 10mL of the RPMI-1640 basic culture medium, and repeatedly grinding twice; the cell suspension thus obtained was centrifuged at 1000 r/min for 10min to remove the supernatant, followed by resuspension in 30 mL of RPMI-1640 basal medium to obtain a cell suspension.
Adding 15mL of lymphocyte separation liquid into another centrifuge tube, and carefully placing the cell suspension on the separation liquid; and then centrifuging at 1200 r/min for 15min, sucking a white cell layer positioned at the interface compact by a suction tube, cleaning the cells by using RPMI-1640 basal medium for 2 times, then re-suspending the cells in 10mL of the RPMI-1640 basal medium, and counting for later use.
(2) Preparation of immune spleen cells
Taking one BALB/c mouse with enhanced immunity, killing the eye socket by bleeding (collecting serum, namely positive serum), soaking in 75% alcohol for 5-10 min for sterilization, then fixing the BALB/c mouse on an dissecting plate for dissection, taking out spleen, shearing the spleen, and placing the mouse in a sterilized homogenizer; grinding and preparing cell suspension, wherein the method is the same as SP2/0 in the step (1), and counting for later use.
(3) Preparation of feeder cells
1 non-immunized BALB/c mouse was taken, the orbit was exsanguinated, and serum was collected as negative serum. Injecting 2-3 mL of RPMI-1640 basic culture medium into the abdominal cavity of the mouse, sucking out the basic culture medium after blowing, and placing the basic culture medium into another centrifuge tube for standby, wherein the liquid contains abdominal macrophages. Spleen cell suspensions were prepared and placed into peritoneal macrophage tubes. 1000 Centrifuging at r/min for 10min to remove supernatant, suspending cells in HAT medium, standing at 37deg.C, and 5% CO 2 And (5) placing the mixture in an incubator for later use.
(4) Cell fusion
1 to 2X 10 7 SP2/0 and 10 8 The individual immunocytes were mixed well in a 50mL centrifuge tube, 1000 r/min and centrifuged for 8min. After discarding the supernatant, the centrifuge tube containing the cell mixture was placed in a 37℃water bath, followed by addition of 50% PEG 0.8 mL (Sigma) pre-warmed to 37℃and allowed to stand for 30s after stirring. After standing, 10mL of RPMI-1640 basal medium was added at 37℃for preheating. Centrifuging at 1000 r/min for 5min, removing supernatant, and standing at 37deg.C for 5-8min. Subsequently mixed with feeder cell suspension, seeded in 96-well plates at 250. Mu.L/well, at 37℃with 5% CO 2 Culturing in an incubator. HT medium was changed for continued culture on day 4 after fusion. And (4) when the colony of the fused cells grows to 1/4 of the culture hole and the culture medium turns yellow slightly, detecting the antibody.
4. Selection of hybridoma positive clones and cloning of cells
The purpose of the selective culture is to screen the fused hybridoma cells using HAT selective medium. In HAT selective media, unfused myeloma cells are not able to die by DNA synthesis using salvage pathways due to the lack of hypoxanthine-guanine-phosphoribosyl transferase. Unfused lymphocytes have hypoxanthine-guanine-phosphoribosyl transferase, but gradually die because they cannot survive in vitro for a long period of time. Only fused hybridoma cells survive and proliferate in HAT-selective medium due to the hypoxanthine-guanine-phosphoribosyl transferase obtained from spleen cells and the immortalized nature of myeloma cells.
The indirect ELISA is used for screening positive hybridoma cells, and the method comprises the following steps:
(1) Coating known antigens: diluting the purified coating antigen to 1-10 mg/mL by using a coating buffer solution; adding 100 μl of the solution into each of the microwells, shaking gently, and standing at 4deg.C overnight or 37deg.C for 1 hr; throwing away the liquid in the hole (taking the liquid in the hole as dry as possible); washing for 3 times for 2-3 minutes each time.
(2) Blocking the positions of the enzyme-labeled wells not coated with antigen: 200. Mu.L of blocking solution (5% skimmed milk powder or 0.1% BSA) was added to each well, gently shaken and treated at 37℃for 1h; throwing away the liquid in the hole; washing the buffer solution Kong Jiaman, standing for 2-3 min, throwing away the liquid in the hole, beating to dry, and washing 3 times with the washing buffer solution.
(3) Sample adding: 50 mu L of supernatant liquid is taken from each hole of the hybridoma to be detected, sequentially added into the enzyme-labeled holes, gently shaken, treated for 1h at 37 ℃, washed and patted dry.
(4) Adding enzyme-labeled anti-antibody: diluting the enzyme-labeled secondary antibody to a proper working concentration according to instructions by using a diluent, adding 100 mu L of the enzyme-labeled secondary antibody into each hole, gently shaking the mixture, and treating the mixture at 37 ℃ for 1h; then washing and beating to dry.
(5) Adding a color development liquid: each well was added with 100. Mu.L of freshly prepared color development solution, gently shaken well, at 37℃for 10min.
(6) Terminating the reaction: 50. Mu.L of stop solution was added to each well.
(7) Determination result: using enzyme label instrument on OD 450 Readings were taken 3 times larger than the negative wells and were judged positive.
Cloning the positive hybridoma cells obtained by screening by adopting a limiting dilution method, wherein the method comprises the following specific steps of:
preparing a mouse feeder cell layer before cloning; gently blowing the hybridoma cells to be cloned from the culture hole, and counting living cells by using a blood cell counting plate; diluting cells to 10 cells/mL with complete medium;
the cell suspensions at the above concentrations were added to 96-well plates containing feeder cells, 100. Mu.L/well, respectively, so that 1 cell was contained in each well. Culturing until 4 days of fluid infusion is one drop, carefully observing and recording the growth of cells in each hole on 5-6 days;
detection of specific antibodies: detecting the specific antibody when the cell clone grows to 1/3-1/2 field of view on the 7 th to 9 th days after cloning; cells in the positive holes can be moved to a 24-hole culture plate, and when the cells in the 24-hole culture plate grow well, the mice can be inoculated in the abdominal cavity to collect ascites.
Two monoclonal antibodies which specifically bind to the human HSPB1 protein and hybridoma cell strains which produce the monoclonal antibodies are obtained through screening, and the monoclonal antibodies are named monoclonal antibodies 9F8 and 20G5 and hybridoma cell strains 9F8 and 20G5.
The hybridoma cell strain 9F8 is preserved in China general microbiological culture collection center (CGMCC for short, address: north Xielu No.1, 3 of the area of the Korean of Beijing, and the institute of microbiology, postal code 100101 of the national academy of sciences) at 8 months 2 of 2023, and is classified and named as a mouse anti-human HSPB1 monoclonal antibody hybridoma cell strain, and the preservation number is CGMCC No. 45646.
The hybridoma cell strain 20G5 is preserved in China general microbiological culture collection center (CGMCC for short, address: north Xielu No.1, 3 of the area of Charpy, beijing, and the institute of microbiology, post code 100101) at 8 months 2 of 2023, and is classified and named as mouse anti-human HSPB1 monoclonal antibody hybridoma cell strain with a preservation number of CGMCC No. 45647.
5. Monoclonal antibodies 9F8 and 20G5 variable region sequencing
Collecting hybridoma cells: collecting hybridoma cells secreting monoclonal antibodies 9F8 and 20G5 to a number greater than 10 6 Extracting total RNA of two cell lines by using a total RNA extraction kit (Soy Bao technology Co., ltd.) according to the operation of the instruction book; cDNA was synthesized according to the reverse transcription kit (Soy Bao technology Co., ltd.)A first strand; and (5) sending the sample to the Optimus to carry out subsequent construction and sequencing.
Sequencing results show that the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of a heavy chain variable region of the monoclonal antibody 9F8 are respectively shown as SEQ ID NO.1, 2 and 3, the amino acid sequence of complementarity determining region CDR1 of a light chain variable region is shown as SEQ ID NO.4, the amino acid sequence of CDR2 is WAS, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 5; the amino acid sequence of the heavy chain variable region (122 amino acids) is shown as SEQ ID NO.11, the amino acid sequence of the light chain variable region (113 amino acids) is shown as SEQ ID NO.12, the nucleotide sequence of the heavy chain variable region coding gene (366 bp) is shown as SEQ ID NO.15, and the nucleotide sequence of the light chain variable region coding gene (339 bp) is shown as SEQ ID NO. 16.
The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region of monoclonal antibody 20G5 are shown as SEQ ID NO.6, 7 and 8 respectively, the amino acid sequence of complementarity determining region CDR1 of the light chain variable region is shown as SEQ ID NO.9, the amino acid sequence of CDR2 is YAS, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 10. The amino acid sequence of the heavy chain variable region (116 amino acids) is shown as SEQ ID NO.13, the amino acid sequence of the light chain variable region (107 amino acids) is shown as SEQ ID NO.14, the nucleotide sequence of the heavy chain variable region coding gene (348 bp) is shown as SEQ ID NO.17, and the nucleotide sequence of the light chain variable region coding gene (321 bp) is shown as SEQ ID NO. 18.
6. Mass production of monoclonal antibodies 9F8 and 20G5
The hybridoma cells after the strain establishment were injected into the abdominal cavity of the mouse, ascites were collected for about 7 days, antibodies were purified by Protein G affinity chromatography, and the purified antibodies were detected by SDS-PAGE, and the results are shown in FIG. 2.
Example 2 affinity detection of monoclonal antibodies 9F8 and 20G5
The affinity of monoclonal antibodies 9F8 and 20G5 for binding to human HSPB1 protein (relative affinity constant assay) was tested as follows:
antigen (human HSPB1 protein) was coated onto the elisa plate and blocked. After PBST washing, monoclonal antibodies 9F8 and 20G5 were diluted to saturation concentrations respectively and added to the ELISA plate at 100. Mu.L/well, and incubated at room temperature for 2h. After PBST plate washing, 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 mol/L NaSCN solution 60 mu L/hole is added in sequence, and the mixture is stood at room temperature for 15min for incubation. After PBST plate washing, HRP-labeled goat anti-mouse IgG is added, and the plate is incubated at room temperature for 45min for chromogenic detection. The concentration of sodium thiocyanate corresponding to the decrease of OD value at 450nm after elution to 50% without elution is the relative affinity constant of the antibody, which is expressed in mol/L. The results show (Table 1) that the relative affinity constants of the monoclonal antibodies 9F8 and 20G5 are far greater than 2.5mol/L, and the affinity is good.
TABLE 1
Example 3 establishment and evaluation of double antibody sandwich ELISA method
1. Biotin labelling of monoclonal antibodies
Diluting the monoclonal antibody 20G5 purified by Protein G affinity chromatography to 2-20 mg/mL by using a carbonate buffer solution (pH 9.5) with the concentration of 0.1M; the antibody was extensively dialyzed with 0.1M carbonate buffer (pH 9.5) at 4 ℃. NHS-D-Biotin was dissolved in DMSO to prepare a 5mg/mL solution. The prepared NHS-D-Biotin solution was added to the antibody solution dialyzed overnight, protected from light at room temperature, and gently stirred for 4 hours. The reacted solution was thoroughly dialyzed against 0.01M PBS (pH 7.2 to 7.4) at 4 ℃.
The labeled monoclonal antibody 20G5 was removed from the dialysis bag, placed in an antibody tube, and stored at-20 ℃.
2. Preparation of monoclonal antibody coated ELISA plate
Using 96-hole flat-bottom polystyrene ELISA plate as solid phase carrier, diluting Protein G affinity chromatography purified monoclonal antibody 9F8 to 2 mug/mL with antibody coating liquid, adding diluted antibody into micropores of ELISA plate, 100 mug/hole, sealing with sealing plate membrane, coating at 4deg.C overnight, taking out coated ELISA plate, discarding liquid in the hole, washing the plate 3 times with ELISA washing liquid, adding sealing liquid containing 2% BSA, 250 mug/hole, sealing with sealing plate membrane, placing in room temperature for 2h, discarding liquid in the plate hole, and placing in drying room for drying for 16-18 h. Placing into aluminum foil bag containing drying agent, vacuum sealing, and preserving at 4deg.C.
3. Establishment of double antibody sandwich ELISA method
And taking out the ELISA plate coated with the monoclonal antibody 9F8 30 min before the experiment, recovering to room temperature, washing the plate for 3 times and spin-drying. Human HSPB1 standard was subjected to multiple dilution (5000, 2500, 1250, 625, 312.5, 156.25, 78.125 pg/mL) and then added to the ELISA plate at 100. Mu.L/well, with blank wells; incubate for 2h at room temperature (25.+ -. 2 ℃) and then wash. Diluting the biotin-labeled monoclonal antibody 20G5 to a working concentration, and adding into each reaction well of the ELISA plate to obtain 100 mu L/well; incubate at room temperature (25.+ -. 2 ℃) for 1h and then wash. Diluting the enzyme conjugate to a working concentration, and adding the enzyme conjugate into each reaction hole of the ELISA plate to obtain 100 mu L/hole; incubate at room temperature (25.+ -. 2 ℃) for 30 min and then wash. Adding TMB chromogenic substrate in each reaction well, 100 μl/well; standing at room temperature (25+/-2 ℃) for color development for 10-20 min. Finally, a stop solution was added to each reaction well at a concentration of 50. Mu.L/well, the stop reaction was performed within 5 minutes, the detection was performed at two wavelengths using an ELISA reader, the OD at the maximum absorption wavelength of 450nm and the reference wavelength of 630 nm was determined, and the OD at 630 nm was subtracted from the OD at 450 nm. The detection range of the method for detecting the human HSPB1 protein is 78.125-5000pg/mL, R is shown by drawing a standard curve (using four-parameter fitting) by ELISA Calc software with the concentration of the human HSPB1 standard substance as the abscissa and the absorbance OD value as the ordinate 2 0.99948 (fig. 3).
4. Specific detection of double-antibody sandwich ELISA method
And taking out the ELISA plate coated with the monoclonal antibody 9F8 30 min before the experiment, recovering to room temperature, washing the plate for 3 times and spin-drying. Human HSPB1 standard was diluted by a multiple ratio (5000, 2500, 1250, 625, 312.5, 156.25, 78.125 pg/mL) and added to the ELISA plate at 100. Mu.L/well; simultaneously, each recombinant protein shown in Table 2 was added to the ELISA plate at 100. Mu.L/well; setting blank control holes and negative control holes at the same time; incubate for 2h at room temperature (25.+ -. 2 ℃) and then wash. Diluting the biotin-labeled monoclonal antibody 20G5 to a working concentration, and adding into each reaction well of the ELISA plate to obtain 100 mu L/well; incubate at room temperature (25.+ -. 2 ℃) for 1h and then wash. Diluting the enzyme conjugate to a working concentration, and adding the enzyme conjugate into each reaction hole of the ELISA plate to obtain 100 mu L/hole; incubate at room temperature (25.+ -. 2 ℃) for 30 min and then wash. Adding TMB chromogenic substrate in each reaction well, 100 μl/well; standing at room temperature (25+/-2 ℃) for color development for 10-20 min. Finally, a stop solution was added to each reaction well at a concentration of 50. Mu.L/well, the stop reaction was performed within 5 minutes, the detection was performed at two wavelengths using an ELISA reader, the OD at the maximum absorption wavelength of 450nm and the reference wavelength of 630 nm was determined, and the OD at 630 nm was subtracted from the OD at 450 nm. The results are shown in Table 2, and no significant cross-reaction was detected for each recombinant protein except human HSPB1, indicating higher specificity.
TABLE 2
5. Stability detection of double-antibody sandwich ELISA method
The acceleration stability test was performed by placing the ELISA plate coated with monoclonal antibody 9F8, biotin-labeled monoclonal antibody 20G5 and standard human HSPB1 at 37℃for 13 days (approximately equivalent to 19.5 months at 4 ℃). Then taking out for detection, wherein the detection method comprises the following steps: and washing the ELISA plate for 3 times and spin-drying. Human HSPB1 standard was diluted by a multiple ratio (5000, 2500, 1250, 625, 312.5, 156.25, 78.125 pg/mL) and added to the ELISA plate at 100. Mu.L/well; meanwhile, adding the sample to be detected into the ELISA plate at a concentration of 100 mu L/hole; setting blank control holes and negative control holes at the same time; incubate for 2h at room temperature (25.+ -. 2 ℃) and then wash. Diluting the biotin-labeled monoclonal antibody 20G5 to a working concentration, and adding into each reaction well of the ELISA plate to obtain 100 mu L/well; incubate at room temperature (25.+ -. 2 ℃) for 1h and then wash. Diluting the enzyme conjugate to a working concentration, and adding the enzyme conjugate into each reaction hole of the ELISA plate to obtain 100 mu L/hole; incubate at room temperature (25.+ -. 2 ℃) for 30 min and then wash. Adding TMB chromogenic substrate in each reaction well, 100 μl/well; standing at room temperature (25+/-2 ℃) for color development for 10-20 min. Finally, a stop solution was added to each reaction well at a concentration of 50. Mu.L/well, the stop reaction was performed within 5 minutes, the detection was performed at two wavelengths using an ELISA reader, the OD at the maximum absorption wavelength of 450nm and the reference wavelength of 630 nm was determined, and the OD at 630 nm was subtracted from the OD at 450 nm. Standard curves (using four parameter fitting) were plotted using ELISA Calc software with standard concentration on the abscissa and absorbance OD on the ordinate, and equations were established. The results are shown in Table 3, and the OD CV of the standard curve is less than 10%, which indicates that the stability of the kit is good.
TABLE 3 Table 3
6. Recovery rate and linear detection of double antibody sandwich ELISA method
And taking out the ELISA plate coated with the monoclonal antibody 9F8 30 min before the experiment, recovering to room temperature, washing the plate for 3 times and spin-drying. Human HSPB1 standard was diluted by a multiple ratio (5000, 2500, 1250, 625, 312.5, 156.25, 78.125 pg/mL) and added to the ELISA plate at 100. Mu.L/well; selecting RPMI-1640 basal medium and serum (human serum 1-4) of healthy volunteers respectively, and adding HSPB1 standard substances with different concentrations, wherein 100 mu L/hole; setting blank control holes and negative control holes at the same time; incubate for 2h at room temperature (25.+ -. 2 ℃) and then wash. Diluting the biotin-labeled monoclonal antibody 20G5 to a working concentration, and adding into each reaction well of the ELISA plate to obtain 100 mu L/well; incubate at room temperature (25.+ -. 2 ℃) for 1h and then wash. Diluting the enzyme conjugate to a working concentration, and adding the enzyme conjugate into each reaction hole of the ELISA plate to obtain 100 mu L/hole; incubate at room temperature (25.+ -. 2 ℃) for 30 min and then wash. Adding TMB chromogenic substrate in each reaction well, 100 μl/well; standing at room temperature (25+/-2 ℃) for color development for 10-20 min. Finally, a stop solution was added to each reaction well at a concentration of 50. Mu.L/well, the stop reaction was performed within 5 minutes, the detection was performed at two wavelengths using an ELISA reader, the OD at the maximum absorption wavelength of 450nm and the reference wavelength of 630 nm was determined, and the OD at 630 nm was subtracted from the OD at 450 nm. And drawing a standard curve (using four-parameter fitting) by ELISA Calc software with the standard concentration as an abscissa and the absorbance OD value as an ordinate, establishing an equation, and calculating the standard adding recovery rate. The results are shown in Table 4, the recovery rate is 80% -120%, and the standard recovery rate is good.
TABLE 4 Table 4
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And taking out the ELISA plate coated with the monoclonal antibody 9F8 30 min before the experiment, recovering to room temperature, washing the plate for 3 times and spin-drying. Human HSPB1 standard was diluted by a multiple ratio (5000, 2500, 1250, 625, 312.5, 156.25, 78.125 pg/mL) and added to the ELISA plate at 100. Mu.L/well; selecting RPMI-1640 basal medium and serum (human serum 1-3) of healthy volunteers respectively, adding an HSPB1 standard substance, performing double dilution, and then adding into an ELISA plate at a concentration of 100 mu L/hole; setting blank control holes and negative control holes at the same time; incubate for 2h at room temperature (25.+ -. 2 ℃) and then wash. Diluting the biotin-labeled monoclonal antibody 20G5 to a working concentration, and adding into each reaction well of the ELISA plate to obtain 100 mu L/well; incubate at room temperature (25.+ -. 2 ℃) for 1h and then wash. Diluting the enzyme conjugate to a working concentration, and adding the enzyme conjugate into each reaction hole of the ELISA plate to obtain 100 mu L/hole; incubate at room temperature (25.+ -. 2 ℃) for 30 min and then wash. Adding TMB chromogenic substrate in each reaction well, 100 μl/well; standing at room temperature (25+/-2 ℃) for color development for 10-20 min; finally, a stop solution was added to each reaction well at a concentration of 50. Mu.L/well, the stop reaction was performed within 5 minutes, the detection was performed at two wavelengths using an ELISA reader, the OD at the maximum absorption wavelength of 450nm and the reference wavelength of 630 nm was determined, and the OD at 630 nm was subtracted from the OD at 450 nm. Standard curves were drawn (using four parameter fitting) with standard concentration on the abscissa and absorbance OD on the ordinate using ELISA Calc software, and equations were established to calculate the linear dilution recovery. As shown in Table 5, the recovery rate was 80% -120%, and the linear dilution recovery rate was good.
TABLE 5
Example 4 comparison of detection results with commercially available isotype kit
The performance of the double-antibody sandwich ELISA assay kit using monoclonal antibodies 9F8 and 20G5 of the invention as coating and labeling antibodies, respectively, and the commercial kit were compared by selecting the commercially available human HSPB1 protein double-antibody sandwich ELISA assay kit (SZ kit (Sino Biological SEK 10351) and AY kit (Ai Bokang ab 303741) for analog testing in the manner described in the respective brands of instructions.
1. Standard curve comparison: the results show that the kit of the invention and the R of the standard curve of two commercially available kits 2 >0.999, the fitting degree is good; and the kit and AY kit of the invention have excellent background performance (zero Kong Zhi)<0.1 Is superior to SZ kit (zero Kong Zhi)>0.1 (table 6).
TABLE 6
2. Sample measurement result comparison: 17 human serum samples and 19 human plasma samples were randomly selected and assayed simultaneously (in ng/mL) using the above kit. The measurement results show (Table 7) that the CV of the kit and the SZ kit is 0% -31%, and the CV of the kit and the AY kit is 0% -132%, which shows that the measurement results of the kit are consistent with the SZ kit and have larger difference with the AY kit. The analysis of the results shows that the CV of the kit and the AY kit is less than 41% when the sample content is higher than 100ng/mL, and the CV shows an increasing trend when the sample content is lower, and the AY kit is obviously lower than the kit, so that the capture capability of the AY kit on human HSPB1 protein in the sample is presumably poorer when the sample content is lower, and the detection accuracy in the low-concentration sample is obviously lower than that of the antibody composition.
TABLE 7
Example 5 application of monoclonal antibodies 9F8 and 20G5 in detection of HSPB1 content in human serum and plasma
The monoclonal antibodies 9F8 and 20G5 are respectively used as coating antibodies and labeled antibodies, and the double-antibody sandwich ELISA method is adopted to detect the contents of human serum and plasma HSPB1 of a real sample, and the method specifically comprises the following steps:
and taking out the ELISA plate coated with the monoclonal antibody 9F8 30 min before the experiment, recovering to room temperature, washing the plate for 3 times and spin-drying. Human HSPB1 standard was subjected to multiple dilution (5000, 2500, 1250, 625, 312.5, 156.25, 78.125 pg/mL) and then added to the ELISA plate at 100. Mu.L/well, with blank wells; 28 human serum samples and 20 human plasma samples are respectively selected, diluted and added into an ELISA plate, 100 mu L/hole, and incubated for 2 hours at room temperature (25+/-2 ℃) in a standing way, and then washed. Diluting the biotin-labeled monoclonal antibody 20G5 to a working concentration, and adding into each reaction well of the ELISA plate to obtain 100 mu L/well; incubate at room temperature (25.+ -. 2 ℃) for 1h and then wash. Diluting the enzyme conjugate to a working concentration, and adding the enzyme conjugate into each reaction hole of the ELISA plate to obtain 100 mu L/hole; incubate at room temperature (25.+ -. 2 ℃) for 30 min and then wash. Adding TMB chromogenic substrate in each reaction well, 100 μl/well; standing at room temperature (25+/-2 ℃) for color development for 10-20 min. Finally, a stop solution was added to each reaction well at a concentration of 50. Mu.L/well, the stop reaction was performed within 5 minutes, the detection was performed at two wavelengths using an ELISA reader, the OD at the maximum absorption wavelength of 450nm and the reference wavelength of 630 nm was determined, and the OD at 630 nm was subtracted from the OD at 450 nm. Standard curves (using four-parameter fitting) were drawn with standard concentration on the abscissa and absorbance OD on the ordinate using ELISA Calc software, and equations were established to calculate sample concentrations. The results are shown in Table 8, wherein the serum samples had a human HSPB1 content ranging from 0.17 to 169.42 ng/mL and an average content of 34.13. 34.13 ng/mL. The content range of the human HSPB1 in the plasma sample is 5.02-84.60 ng/mL, the average content value is 19.94 ng/mL, and the detection result is consistent with the content of the HSPB1 in human serum/plasma reported in the literature, so that the content of the HSPB1 in human serum and plasma can be accurately measured by utilizing the monoclonal antibody disclosed by the invention.
TABLE 8
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (13)

1. An antibody or antigen binding fragment thereof of small heat shock protein HSPB1 is characterized in that the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of a heavy chain variable region of the antibody or antigen binding fragment thereof are shown as SEQ ID NO.6, 7 and 8 respectively, the amino acid sequence of complementarity determining region CDR1 of a light chain variable region is shown as SEQ ID NO.9, the amino acid sequence of CDR2 is YAS, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 10.
2. An antibody or antigen-binding fragment thereof of small heat shock protein HSPB1 according to claim 1, wherein the heavy chain variable region of the antibody or antigen-binding fragment thereof has an amino acid sequence as shown in SEQ ID No.13 or has at least 80% similarity to the sequence shown in SEQ ID No.13, and the light chain variable region has an amino acid sequence as shown in SEQ ID No.14 or has at least 80% similarity to the sequence shown in SEQ ID No. 14.
3. An antibody or antigen-binding fragment thereof to small heat shock protein HSPB1 according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereofFragments are monoclonal antibodies, fab ', F (ab') 2 Fv or single chain antibodies.
4. A nucleic acid molecule encoding the antibody or antigen binding fragment thereof of small heat shock protein HSPB1 of any of claims 1 to 3.
5. A biological material comprising the nucleic acid molecule of claim 4;
the biological material is an expression cassette, a vector or a host cell.
6. An antibody conjugate, which is characterized in that the antibody conjugate is obtained by coupling the antibody or the antigen binding fragment thereof of the small heat shock protein HSPB1 according to any one of claims 1-3 with a label, wherein the label is one or more selected from the group consisting of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label and a radioactive label.
7. An antibody composition for small heat shock protein HSPB1, characterized in that the antibody composition comprises the antibodies in (1) and (2) below:
(1) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown as SEQ ID NO.1, 2 and 3 respectively, the amino acid sequence of complementarity determining region CDR1 of the light chain variable region is shown as SEQ ID NO.4, the amino acid sequence of CDR2 is WAS, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 5;
(2) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown as SEQ ID NO.6, 7 and 8 respectively, the amino acid sequence of complementarity determining region CDR1 of the light chain variable region is shown as SEQ ID NO.9, the amino acid sequence of CDR2 is YAS, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 10.
8. The mouse anti-human HSPB1 monoclonal antibody hybridoma cell strain is characterized in that the hybridoma cell strain is 20G5, and the hybridoma cell strain 20G5 is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No. 45647.
9. A monoclonal antibody produced by the hybridoma cell line of claim 8 and capable of specifically binding small heat shock protein HSPB1.
10. Use of an antibody or antigen binding fragment thereof of small heat shock protein HSPB1 according to any one of claims 1-3 or a nucleic acid molecule according to claim 4 or a biological material according to claim 5 or an antibody conjugate according to claim 6 or an antibody composition of small heat shock protein HSPB1 according to claim 7 or a hybridoma cell line according to claim 8 or a monoclonal antibody according to claim 9 for the preparation of a product for detecting the presence or level of small heat shock protein HSPB1 in a sample.
11. Use of an antibody or antigen binding fragment thereof of small heat shock protein HSPB1 according to any of claims 1-3 or an antibody conjugate of claim 6 or an antibody composition of small heat shock protein HSPB1 according to claim 7 or a monoclonal antibody of claim 9 for detecting the presence or level of small heat shock protein HSPB1 in a sample for non-disease diagnostic purposes.
12. A kit comprising the antibody or antigen binding fragment thereof of small heat shock protein HSPB1 of any of claims 1 to 3, or comprising the antibody conjugate of claim 6, or comprising the antibody composition of small heat shock protein HSPB1 of claim 7, or comprising the monoclonal antibody of claim 9.
13. A medicament comprising the antibody or antigen binding fragment thereof of small heat shock protein HSPB1 of any of claims 1 to 3 or comprising the monoclonal antibody of claim 9.
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