阅读说明：本技术 一种用于检测人tim-3表达水平的试剂盒 (Kit for detecting human TIM-3 expression level ) 是由 熊浩 于 2019-07-25 设计创作，主要内容包括：本发明提供了一种用于检测人TIM-3表达水平的试剂盒,本发明提供的试剂盒中的人源化单克隆抗体能够高强度、特异性与TIM-3抗原结合,具有高效检测TIM-3相关疾病的辅助诊断作用,Tim-3是1型免疫的负调节因子,Tim-3信号传导是诱导抗原特异性耐受所必需的,而Tim-3阻断促进了自发性自身免疫的发生发展。(The invention provides a kit for detecting the expression level of human TIM-3, wherein a humanized monoclonal antibody in the kit can be combined with a TIM-3 antigen in high strength and specificity and has the auxiliary diagnosis effect of efficiently detecting TIM-3 related diseases, Tim-3 is a negative regulatory factor of type 1 immunity, Tim-3 signaling is necessary for inducing antigen specific tolerance, and Tim-3 blocking promotes the generation and development of spontaneous autoimmunity.)

1. A kit for detecting the expression level of human TIM-3, wherein a TIM-3 antibody is coated onto a plate to form a solid phase antibody, said TIM-3 antibody comprising:

CDR-L1 comprising the amino acid sequence of SEQ ID NO:01, and/or

CDR-L2 comprising the amino acid sequence of SEQ ID NO 02, and/or

03, and/or CDR-L3 comprising the amino acid sequence of SEQ ID NO

04, and/or CDR-H1 comprising the amino acid sequence of SEQ ID NO:04

CDR-H2 comprising the amino acid sequence of SEQ ID NO. 05, and/or

CDR-H3 comprising the amino acid sequence of SEQ ID NO: 06.

2. The kit of claim 1, wherein the method of using the kit comprises:

sequentially adding a sample into antibody-coated micropores, adding an HRP-labeled enzyme labeling solution to form an antibody-antigen-enzyme-labeled compound, completely washing, and adding TMB for color development;

step two, TMB is blue under the catalysis of HRP enzyme, and is yellow after the termination solution is added, and the color depth is positively correlated with the content of TIM-3 antigen;

step three, determining the OD of the solution_450nmAnd quantitatively calculating the content of the TIM-3 protein according to a standard curve.

3. Kit according to claim 1, characterized in that said antibody is selected from the group consisting of monoclonal antibodies, antibody fragments specifically binding to human TIM-3, fusion proteins comprising antibody fragments specifically binding to human TIM-3, murine antibodies, humanized antibodies, murine monoclonal antibodies, preferably said antibodies are humanized monoclonal antibodies.

4. The kit of claim 1, wherein the antibody comprises an antibody heavy chain variable region amino acid sequence comprising a VH sequence as set forth in SEQ ID No. 7 or having at least 99% sequence identity to the amino acid sequence of SEQ ID No. 10.

5. The kit of claim 1, wherein the antibody comprises an antibody light chain variable region amino acid sequence comprising a VL sequence as set forth in SEQ ID No. 8 or having at least 99% sequence identity to the amino acid sequence of SEQ ID No. 12.

6. The kit of claim 1, wherein the antibody heavy chain constant region amino acid sequence is set forth in SEQ ID NO. 7 and the antibody light chain constant region amino acid sequence is set forth in SEQ ID NO. 8.

7. The kit of any one of claims 1 to 6, wherein the antibody production step comprises culturing a host cell comprising a nucleotide sequence that expresses the antibody of claims 1 to 6 in a culture medium under suitable culture conditions, and recovering and purifying the antibody and/or antigen-binding fragment from the culture medium and/or the host cell by conventional methods.

8. The kit of claim 7, further comprising an enzyme-labeled coating plate, a standard buffer, an enzyme labeling solution, a sample diluent, a TMB color developing agent, a washing solution and a stop solution.

9. The kit according to claims 1-8, wherein the detection kit is used for assisting in diagnosing the types of diseases with high TIM-3 protein expression in lung cancer pancreatic cancer, non-small cell lung cancer, diabetes, nephropathy and other diseases.

Technical Field

The invention relates to the field of in-vitro detection, in particular to a kit for detecting the expression level of human TIM-3.

Background

Tim-3 is a negative regulator of type 1 immunity, Tim-3 signaling is necessary for inducing antigen-specific tolerance, and Tim-3 blockade promotes the development of spontaneous autoimmunity, and C-type lectin Galectin-9 is a Tim-3 ligand, which consolidates the inhibitory function of Tim-3, because triggering Tim-3 by Galectin-9 induces cell death in Tim-3+ Th1 cells and improves EAE.

Another Tim-3 ligand that affects the innate immune response is the high mobility group protein B1(HMGB1), HMGB1 binds to DNA released by cells that are about to die and facilitates the delivery of innate immune cells by binding to advanced glycation end products (RAGE) and Toll-like receptors (TLRs), triggering activation of innate immune cells and the production of proinflammatory cytokines, and the binding of Tim-3 to HMGB1 can interfere with this process, inhibiting the activation of the innate immune response.

TIM-3 was found to be associated with various diseases, TIM-3 positively correlated with CD 4- + T cell expression level and CD 8- + T cell in prostate cancer patients, and the immunohistochemical detection analysis shows that TIM-3 does not stain or stains weakly in prostate hyperplasia epithelial cells, but stains strongly in prostate cancer. Another study showed that TIM-3 expression correlates with the presence or absence of lymph node metastasis, the extent of adenocarcinoma cell differentiation, clinical staging and prognosis (P < 0.05).

Thus, there is a need to develop more sensitive, faster methods for animal models or patient biological samples than existing ELISAs to aid diagnosis and prognosis.

Disclosure of Invention

In order to solve the above technical problems, the present invention provides a kit for detecting the expression level of human TIM-3.

The invention is realized by the following technical scheme:

a kit for detecting the expression level of human TIM-3, comprising a TIM-3 antibody coated plate as a solid phase antibody, said TIM-3 antibody comprising:

CDR-L1 comprising the amino acid sequence of SEQ ID NO:01, and/or

CDR-L2 comprising the amino acid sequence of SEQ ID NO 02, and/or

03, and/or CDR-L3 comprising the amino acid sequence of SEQ ID NO

04, and/or CDR-H1 comprising the amino acid sequence of SEQ ID NO:04

CDR-H2 comprising the amino acid sequence of SEQ ID NO. 05, and/or

CDR-H3 comprising the amino acid sequence of SEQ ID NO: 06.

Further, methods of using the kit include:

sequentially adding a sample into antibody-coated micropores, adding an HRP-labeled enzyme labeling solution to form an antibody-antigen-enzyme-labeled compound, completely washing, and adding TMB for color development;

step two, TMB is blue under the catalysis of HRP enzyme, and is yellow after the termination solution is added, and the color depth is positively correlated with the content of TIM-3 antigen;

and step three, determining the OD450nm of the solution, and quantitatively calculating the TIM-3 protein content according to a standard curve.

Further, the antibody is selected from a monoclonal antibody, an antibody fragment specifically binding to human TIM-3, a fusion protein comprising an antibody fragment specifically binding to human TIM-3, a murine antibody, a humanized antibody, a murine monoclonal antibody, preferably, the antibody is a humanized monoclonal antibody.

Further, the antibody comprises an antibody heavy chain variable region amino acid sequence comprising a VH sequence set forth as SEQ ID NO. 7 or having at least 99% sequence identity to the amino acid sequence of SEQ ID NO. 10.

Further, the antibody comprises an antibody light chain variable region amino acid sequence comprising a VL sequence as set forth in SEQ ID NO 8 or having at least 99% sequence identity to the amino acid sequence of SEQ ID NO 12.

Preferably, the amino acid sequence of the antibody heavy chain constant region is shown as SEQ ID NO. 7, and the amino acid sequence of the antibody light chain constant region is shown as SEQ ID NO. 8.

Further, the antibody production step comprises culturing a host cell containing a nucleotide sequence expressing the above antibody in a medium and under suitable culture conditions, and recovering and purifying the antibody and/or antigen-binding fragment from the medium and/or the host cell by a conventional method.

Furthermore, the kit also comprises an enzyme-labeled coated plate, a standard buffer, an enzyme-labeled solution, a sample diluent, a TMB color developing agent, a washing solution and a stop solution.

Preferably, the detection kit assists in diagnosing the types of diseases with high TIM-3 protein expression in lung cancer pancreatic cancer, non-small cell lung cancer, diabetes, nephropathy and other diseases.

The humanized monoclonal antibody in the kit provided by the invention can be well and specifically combined with the TIM-3 antigen, and has an auxiliary diagnosis effect of efficiently detecting TIM-3 related diseases.

Detailed Description

Definition of

The term "antibody" is used in the broadest sense of the present invention to include a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies, bispecific antibodies, and antibody fragments, so long as they exhibit the specified antigen binding activity, and the term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds, and antibody fragments including, but not limited to, Fv, Fab', bispecific antibodies, linear antibodies, single chain antibody molecules (e.g., scFv), and/or multispecific antibodies formed from antibody fragments.

The class of antibodies refers to the type of constant domain or constant region that the heavy chain has. There are five main types of antibodies: IgA, IgD, IgE, IgG and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA 2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

The term "IgG" of the present invention is an abbreviation for immunoglobulin G (immunoglobulin G). The term "humanized" of the present invention refers to a chimeric antibody comprising amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the HVRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.

The binding specificity and avidity of an antibody are determined primarily by the CDR sequences, and variants with similar biological activity can be obtained by readily altering the amino acid sequence of the non-CDR regions according to well-established and well-known techniques of the art. It is well known in the art that an antigen binding domain refers to a region that can specifically interact with a target molecule, such as an antigen, with a high degree of selectivity of action, and that sequences recognizing one target molecule are generally unable to recognize other molecular sequences.

The term "binding" of the present invention refers to a non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, the term "binding force" as used herein refers to the intrinsic binding affinity of a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can be generally expressed by the dissociation constant (KD), and can be measured by common methods known in the art.

The terms "identity", "similarity", and "similarity" of the present invention are the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art.

Studies have shown that TIM-3 is a key factor in tumor formation. TIM-3 concentration has high correlation with diseases such as diabetes, nephropathy, tumor and the like. Numerous studies have been performed in the day ahead to show a strong correlation between circulating TIM-3 levels and tumor burden, and suggest that TIM-3 levels are a potential prognostic marker, and accurate measurement of TIM-3 is of potential importance in understanding its role in many biological processes. The ability to measure endogenous TIM-3 levels depends on the availability of sensitive and specific assays. Enzyme-linked immunosorbent assays (ELISAs) based on colorimetric, chemiluminescent, and fluorescent assays have been reported for TIM-3.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below.