阅读说明：本技术 一种抗hsp90单克隆抗体及试剂盒 (anti-HSP 90 monoclonal antibody and kit ) 是由 刘晗青 屠志刚 于 2019-09-11 设计创作，主要内容包括：本发明涉及生物工程技术领域,具体涉及一种抗HSP90单克隆抗体及试剂盒。本发明利用细胞融合技术筛选出能够稳定分泌抗HSP90单克隆抗体的杂交瘤细胞株,大量生产抗HSP90单克隆抗体。得到的抗HSP90单克隆抗体具有高效价、高特异性、可大量生产的特点,抗HSP90单克隆抗体能够特异性的结合HSP90蛋白,减少了可能的交叉反应,试验结果可信度更大,制备的蛋白酶联免疫双抗体夹心法试剂盒适用于检测血清中的HSP90表达量,可用于细胞的免疫学检测,具有广阔的市场前景,在制备以HSP90为靶点的体外诊断试剂中具有重要的意义。(The invention relates to the technical field of bioengineering, and particularly relates to an anti-HSP 90 monoclonal antibody and a kit. The invention screens out hybridoma cell strains capable of stably secreting the anti-HSP 90 monoclonal antibody by using a cell fusion technology, and produces the anti-HSP 90 monoclonal antibody in large quantities. The obtained anti-HSP 90 monoclonal antibody has the characteristics of high titer, high specificity and mass production, the anti-HSP 90 monoclonal antibody can be specifically combined with HSP90 protein, the possible cross reaction is reduced, the reliability of the test result is higher, the prepared protein enzyme-linked immune double-antibody sandwich method kit is suitable for detecting the expression quantity of HSP90 in serum, can be used for immunological detection of cells, has wide market prospect and has important significance in preparing in-vitro diagnostic reagents taking HSP90 as targets.)

1. The monoclonal antibody against HSP90 is characterized in that the amino acid sequence of a heavy chain variable region is shown as SEQ.ID.NO.1, and the amino acid sequence of a light chain variable region is shown as SEQ.ID.NO. 2.

2. Use of the monoclonal antibody of claim 1 for the preparation of a biological diagnostic reagent targeting HSP 90.

3. Use of the monoclonal antibody according to claim 1 for the preparation of a reagent for the detection of the HSP90 antigen.

4. An enzyme-linked immunosorbent assay double-antibody sandwich kit of HSP90 protein, which is characterized by comprising the anti-HSP 90 monoclonal antibody, an HRP-labeled HSP90 rabbit polyclonal antibody, a recombinant HSP90 protein standard, a diluent, a developing solution and a stop solution, wherein the anti-HSP 90 monoclonal antibody is described in claim 1.

5. The double antibody sandwich kit according to claim 4, wherein the anti-HSP 90 monoclonal antibody is mixed with the HSP90 rabbit polyclonal antibody, and specifically binds to the HSP90 protein in a recombinant HSP90 protein standard or a test sample; adding HRP-labeled HSP90 rabbit polyclonal antibody, stopping after color development of color development liquid, and reading the absorbance value of each sample to be detected.

6. The double antibody sandwich kit according to any one of claims 4 or 5, wherein the concentration of the anti-HSP 90 monoclonal antibody is 0.1 ~ 0.3.3 μ g/mL.

7. The double antibody sandwich kit according to claim 6, wherein the concentration of the anti-HSP 90 monoclonal antibody is 0.1 μ g/mL.

8. The double antibody sandwich kit according to any one of claims 4 or 5, wherein the concentration of the HRP-labeled rabbit polyclonal antibody is 1-3 μ g/mL.

9. The double antibody sandwich kit according to claim 8, wherein the concentration of the HRP-labeled rabbit polyclonal antibody is 1 μ g/mL.

10. The double antibody sandwich kit according to claim 4, wherein the detection range of the kit is 0.04 ~ 625 pg/μ L.

Technical Field

The invention relates to the technical field of monoclonal antibodies, in particular to an anti-HSP 90 monoclonal antibody and a kit.

Background

Heat Shock Proteins (HSPs) are highly conserved protein molecules, also known as stress proteins, that are synthesized under stress (viral infection, hypoxia, uv irradiation, etc.) conditions and are widely present in prokaryotes and eukaryotes. Many of them are involved in the processes of intracellular assembly, folding, protein degradation and intracellular transport of related proteins in the form of molecular chaperones. In recent years, the compound has become a hot spot of antitumor treatment due to a close relationship with the occurrence and development of drug resistance of tumors and microorganisms. According to the molecular mass, the protein can be divided into HSP100, HSP90, HSP70, HSP60 and small HSP five major families. HSP90 is one of the most important HSPs and attracts more and more attention, HSP90 is expressed in all eukaryotic cells, but the composition expression of the HSP90 in tumor cells is 2-10 times higher than that of corresponding normal cells. The physiological functions are mainly embodied in the following aspects: (1) maintaining the stability of cell protein. The protein has to be unfolded before transmembrane transport and refolded to form a mature type after transmembrane transport, and the HSP90 has the function of unfolding enzyme as a molecular chaperone, can recognize and combine with a partially exposed hydrophobic surface after the protein is unfolded, and can prevent the protein from interacting to generate aggregation until transmembrane transport. (2) Improving the tolerance of the cells to stress. Under stress conditions, the expression and up-regulation of HSP90 allows the body to respond correctly by transmitting signals. (3) Enhancing antioxidant effect to maintain normal physiological function of cells.

HSP90 is one of the most active molecular chaperones in the cell and plays an important role in the survival of cells under physiological, pathological and stress conditions. In response to stress, HSP90 undergoes a conformational change in itself due to environmental stimuli, ensuring proper folding of the protein and preventing its nonspecific aggregation, thereby maintaining the normal activity of the cell. In addition to physiological functions, the role of HSP90 in tumor tissues has recently been highlighted. HSP90 can bind to a corresponding substrate to play a role in the formation and development of tumors. The substrates combined with the protein have certain selectivity, and most of the substrates are certain transcription factors and protein kinases related to cell signal transduction. During tumor formation, the large number of encoded HSPs act as molecular chaperones to regulate and stabilize this abnormal proliferative process. It can be seen that HSP90 can be one of the biomarkers for tumor diagnosis, the expression of antagonistic HSP90 has potential application value for preventing and treating tumorigenesis and development, and how to quickly and accurately detect the protein expression of local HSP90 in blood or tissues is a problem to be continuously solved.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a high-titer and high-specificity anti-HSP 90 monoclonal antibody and a kit containing the antibody. The monoclonal antibody is reactive to the 201 th to 300 th amino acid sequences of the HSP90 peptide fragment of human.

The invention relates to a method for preparing HSP 90-resistant monoclonal antibody, which comprises the steps of immunizing a BALB/c female mouse with 6 ~ weeks old by using prokaryotic expression recombinant HSP90 protein as immunogen, fusing spleen cells of the mouse successfully immunized with myeloma cells of the mouse in vitro under the action of polyethylene glycol by adopting a cell fusion technology, screening a hybridoma cell strain positively producing the HSP 90-resistant monoclonal antibody by a limited dilution method and an indirect enzyme-linked immunosorbent assay (ELISA) method, then inoculating the hybridoma cell strain into an abdominal cavity of the BALB/c mouse sensitized by liquid paraffin in advance, producing a large amount of HSP 90-resistant monoclonal antibody, preparing a high-purity HSP 90-resistant monoclonal antibody by a protein purification method, and performing titer determination and specificity identification on the HSP 90-resistant monoclonal antibody.

The heavy chain variable region amino acid sequence of the monoclonal antibody resisting HSP90 is shown as SEQ.ID.NO.1, and the light chain variable region amino acid sequence thereof is shown as SEQ.ID.NO. 2.

The term "antibody" as used herein should be construed to encompass any specific binding member having a binding domain with the desired specificity. The monoclonal antibodies of the invention may be, for example, monovalent or single chain antibodies, diabodies, chimeric antibodies, humanized antibodies, and derivatives, functional equivalents and homologs of the foregoing, including antibody fragments and any polypeptides comprising an antigen binding domain.

In a specific embodiment, the invention also specifically discloses a preparation method of the anti-HSP 90 monoclonal antibody, which comprises the following process steps:

(1) expressing and purifying recombinant HSP90 protein;

(2) detecting the immunity and the titer of a BALB/c mouse;

(3) fusing and screening hybridoma cells;

(4) production and purification of monoclonal antibody of HSP 90.

The invention also provides a hybridoma cell strain capable of producing the monoclonal antibody.

The invention also provides application of the anti-HSP 90 monoclonal antibody in preparing a diagnostic reagent taking HSP90 as a target.

The invention also provides application of the anti-HSP 90 monoclonal antibody in preparing a reagent for detecting the HSP90 antigen.

The invention also provides an enzyme-linked immune double antibody sandwich method kit for the HSP90 protein. The kit is characterized in that an anti-HSP 90 monoclonal antibody and an HSP90 rabbit polyclonal antibody are combined in a matched mode, the anti-HSP 90 monoclonal antibody is coated on a solid phase carrier, and specifically binds to a recombinant HSP90 protein standard substance or HSP90 protein in a sample to be detected; adding a Horse Radish Peroxidase (HRP) -labeled HSP90 rabbit polyclonal antibody to form a solid phase antibody-antigen-enzyme labeled detection antibody complex, stopping developing the substrate by a developing solution, reading the absorbance value of each sample to be detected at the wavelength of 450nm, and comparing with a standard curve to obtain the content of the HSP90 protein in the sample to be detected.

The invention also provides a reagent or a chip, which comprises the anti-HSP 90 monoclonal antibody.

Compared with the prior art, the invention has the beneficial effects that:

the invention utilizes protein immunogen expressed by 201 th to 300 th amino acid sequences of HSP90 peptide segment to immunize BALB/c mice, utilizes cell fusion technology to fuse spleen cells of the mice which are successfully immunized with myeloma cells of the mice, screens out hybridoma cell strains capable of stably secreting anti-HSP 90 monoclonal antibody, produces anti-HSP 90 monoclonal antibody in large quantities, obtains high-purity anti-HSP 90 monoclonal antibody after purification, has the characteristics of high titer, high specificity and mass production, and the titer of the obtained antibody reaches 1 x 10⁵The above. The protein enzyme-linked immune double antibody sandwich method kit is suitable for detecting the expression level of HSP90 in serum, and has high detection sensitivity and accuracy and good precision. Detection ofThe method is simple, easy to operate, low in detection cost, low in requirement on operators and relatively small in harm to human bodies.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of fractions eluted from recombinant protein HSP90, wherein the fractions eluted from HSP90 are in sequence from left to right;

FIG. 2 is a SDS-PAGE electrophoresis of fractions of purified anti-HSP 90 monoclonal antibody;

FIG. 3 is a graph showing the results of measurement of the immunological titer of anti-HSP 90 monoclonal antibody at various dilutions;

FIG. 4 shows the specificity and cross-reaction identification of the anti-HSP 90 monoclonal antibody;

FIG. 5 shows the result of specificity identification of HSP90 genetically engineered antibody;

FIG. 6 is a graph showing the detection results of different enzyme-linked immune double antibody sandwich kits on HSP90 protein; in the figure, a picture A is a picture of a detection result of an HSP90 enzyme-linked immunosorbent assay kit prepared by the application, a picture B is a picture of a detection result of a kit of a tobacco platform Progji biotechnology company, a picture C is a picture of a detection result of a kit of a Thermo Fisher company, and a picture D is a picture of a detection result of a kit of a Protein Tech company; .

Detailed Description

The invention discloses an anti-HSP 90 monoclonal antibody and a detection kit, and a person skilled in the art can realize the detection by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention. The methods, devices and materials used in the examples which follow, if not specifically indicated, are all conventional and commercially available methods, devices and materials used in the art.