Kit for detecting V-domain immunosuppressive factor activated by T cells
阅读说明：本技术 用于检测t细胞活化的v域免疫抑制因子的试剂盒 (Kit for detecting V-domain immunosuppressive factor activated by T cells ) 是由 郑权 于 2019-07-25 设计创作，主要内容包括：本发明提供了用于检测T细胞活化的V域免疫抑制因子(VISTA)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VISTA抗原结合,具有高效检测VISTA表达相关疾病的辅助诊断作用。(The invention provides a kit for detecting a V-domain immunosuppressive factor (VISTA) activated by T cells, and the humanized monoclonal antibody in the kit provided by the invention can be combined with a VISTA antigen in high strength and specificity and has an auxiliary diagnosis effect of efficiently detecting VISTA expression-related diseases.)
1. A kit for detecting a V-domain immunosuppressive factor activated by T cells, characterized in that a VISTA antibody coated plate is prepared as a solid phase antibody, and the VISTA antibody comprises:
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 the 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; TMB is blue under the catalysis of HRP enzyme, and is yellow after being added with a termination solution, and the color depth is positively correlated with the content of VISTA antigen; determination of solution OD450nmAnd quantitatively calculating the content of VISTA protein according to a standard curve.
3. The kit of claim 1, wherein said antibody is selected from the group consisting of a monoclonal antibody, an antibody fragment that specifically binds human VISTA, a fusion protein comprising an antibody fragment that specifically binds human VISTA, a murine antibody, a humanized antibody, a murine monoclonal antibody, and preferably said antibody is a humanized monoclonal antibody.
4. The kit of claim 1, wherein the antibody comprises an antibody heavy chain variable region amino acid sequence comprising a VH sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 7.
5. The kit of claim 1, wherein the antibody comprises an antibody light chain variable region amino acid sequence comprising a VL sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 8.
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 of claims 1-8, wherein the detection kit aids in diagnosing lung cancer, pancreatic cancer, breast cancer, ovarian cancer, endometrial cancer, melanoma, and other disease types with high expression of VISTA protein.
Technical Field
The present invention relates to the field of in vitro assays, in particular to kits for detecting V domain immunosuppressive factors of T cell activation.
Background
The V-domain immunosuppressive factor of T-cell activation (VISTA) is a novel inhibitory immune checkpoint protein. The expression of VISTA on tumor cells and its associated regulatory mechanisms are not known. VISTA has been shown to be highly expressed in human ovarian and endometrial cancers. Up-regulation of VISTA in endometrial cancer is associated with VISTA promoter methylation status. VISTA inhibits T cell proliferation and cytokine production in tumor cells and reduces tumor-infiltrating CD8+ T cells in vivo. anti-VISTA antibodies prolong the survival of tumor-bearing mice.
Recently, the university of Texas, MD Anderson cancer center, published a paper "Complex of animal infilterates in melanoma and cultural cancer high hlights VISTA as exogenous target in cultural cancer", at the PNAS journal. The research panel found that VISTA is preferentially expressed at high levels in pancreatic cancer compared to melanoma, and the researchers' studies also provided detailed analysis of primary and metastatic pancreatic cancer immune infiltrates compared to melanoma, which may further help guide immunotherapy strategies for pancreatic cancer treatment. Another study showed that VISTA 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 a V-domain immunosuppressive factor activated by T cells.
The invention is realized by the following technical scheme:
A kit for detecting a V-domain immunosuppressive factor activated by T cells, wherein a VISTA antibody coated plate is prepared into a solid-phase antibody, and the VISTA antibody comprises:
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 the 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; TMB is blue under the catalysis of HRP enzyme, and is yellow after being added with a termination solution, and the color depth is positively correlated with the content of VISTA antigen; and measuring OD 450nm of the solution, and quantitatively calculating the content of VISTA protein according to a standard curve.
Further, the antibody is selected from a monoclonal antibody, an antibody fragment specifically binding to human VISTA, a fusion protein comprising an antibody fragment specifically binding to human VISTA, 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 having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7.
Further, the antibody comprises an antibody light chain variable region amino acid sequence comprising a VL sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8.
Furthermore, 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 antibody of claims 1-6 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.
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.
Further, the detection kit assists in diagnosing lung cancer, pancreatic cancer, breast cancer, ovarian cancer, endometrial cancer, melanoma and other disease types with high expression of VISTA protein.
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.
The term "specifically binds" refers to an antibody or antigen-binding fragment thereof "specifically binding" a region of another molecule, specifically, reacting or binding to the epitope more frequently, more rapidly, with a longer duration, and/or with greater affinity or avidity relative to the other epitope. In some embodiments, an antibody or antigen-binding fragment thereof of the invention is present in an amount of at least 10-7Affinity of M for binding antigen, e.g. 10-8M、10-9M、10-10M、10-11M or higher. Preferably, the antibody or antigen-binding fragment thereof binds under physiological conditions (e.g., in vivo). Thus, specifically binding to an antigen refers to the ability of the antibody or antigen-binding fragment thereof to bind to an antigen with the above-described specificity and/or under such conditions, and methods suitable for determining such binding are known in the art.
The term "combined" is generally meant to correspond to about 10-6M or less, that is at least 10-fold, such as at least 100-fold, at least 1,000-fold less than the affinity of the antibody for binding to a non-specific antigen other than the designated antigen or a closely related antigen.
As used herein, the term "kd" (sec-1 or 1/s) refers to the off-rate constant for a particular antibody-antigen interaction, a value also known as koffThe value is obtained.
As used herein, the term "ka" (M-1x sec-1 or 1/Msec) refers to the binding rate constant for a particular antibody-antigen interaction, a value also known as konThe value is obtained. .
As used herein, the term "KD" (M) refers to the dissociation equilibrium constant for a particular antibody-antigen interaction and is obtained by dividing KD by ka.
As used herein, the term "KA" (M-1 or 1/M) refers to the binding equilibrium constant for a particular antibody-antigen interaction and is obtained by dividing KA by kd.
Studies have shown VISTA to be a key factor in tumor formation. VISTA concentration tumor and other diseases are highly correlated. Many studies have been made in the past to show that circulating VISTA levels are strongly correlated with tumor burden, and suggest that VISTA levels are potential prognostic markers, and accurate measurement of VISTA is of potential importance in understanding its role in many biological processes. The ability to measure endogenous VISTA levels depends on the availability of sensitive and specific assays. Enzyme-linked immunosorbent assays (ELISA) based on colorimetric, chemiluminescent, and fluorescent assays have been reported for VISTA.
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.