CN109897109B - Monoclonal antibody of antitumor marker protein and application thereof - Google Patents

Abstract

The invention provides an anti-tumor marker protein monoclonal antibody and application thereof, and particularly the inventor successfully obtains a monoclonal antibody of a specific anti-14-3 eta protein in the research process, and the experimental result shows that the monoclonal antibody can be specifically combined with the 14-3-3 eta protein, and has strong affinity and high sensitivity. The antibody can be used for detecting 14-3-3 eta protein in fresh liver cancer tissues and can also be used for detecting 14-3-3 eta protein in a trace amount in serum. Has important significance for laboratory research and clinical research.

Description

Monoclonal antibody of anti-tumor marker protein and application thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to an anti-tumor marker protein 14-3-3 eta protein monoclonal antibody and application thereof.

Background

14-3-3 is a phosphoserine/threonine binding protein family, including seven subtypes, α/β, γ, ε, σ, ξ, θ/τ, and η. Research has shown that: the 5 14-3-3 protein subtypes (α/β, γ, ε, σ and ξ) are involved in tumor progression.

Tumor (tumor) refers to a disease caused by abnormal proliferation of cells due to the loss of normal regulation of their growth under the action of tumorigenic factors. Tumors can be divided into two major groups, benign and malignant. The former has slow growth, clear boundary with surrounding tissues, no transfer and little harm to human health. The latter grows rapidly, can be transferred to other parts of the body, can also produce harmful substances, damage normal organ structures, cause organism dysfunction and threaten life.

Malignant tumors, also called cancers (cancer), are currently the most serious group of diseases that endanger human health. In the united states, mortality from malignancies is second only to cardiovascular disease. Early diagnosis of cancer is of great interest in the treatment of cancer. In addition, in the treatment of cancer, a more reasonable treatment scheme needs to be established by detecting tumor marker proteins and further evaluating the molecular types of the cancer.

Therefore, those skilled in the art are dedicated to develop a detection technique for tumor marker proteins with higher accuracy, higher sensitivity and more convenient use.

Disclosure of Invention

The invention aims to provide an anti-14-3-3 eta protein monoclonal antibody and application thereof.

In a first aspect of the invention, there is provided an anti-14-3-3 eta protein monoclonal antibody,

in a first aspect of the invention, there is provided an antibody having:

(1) a heavy chain variable region; and/or

(2) A light chain variable region;

wherein the heavy chain variable region comprises the following three Complementarity Determining Regions (CDRs):

SEQ ID NO. 1 shows the CDR1,

CDR2 shown in SEQ ID NO. 2, and

a CDR3 shown in SEQ ID NO. 3;

the light chain variable region comprises the following three complementarity determining regions CDRs:

CDR1' shown in SEQ ID NO. 5,

CDR2' as shown in SEQ ID NO. 6, and

CDR3' as shown in SEQ ID NO. 7.

In another preferred embodiment, the heavy chain variable region has the amino acid sequence shown in SEQ ID No. 4.

In another preferred embodiment, the light chain variable region has the amino acid sequence shown in SEQ ID No. 8.

In another preferred embodiment, the antibody further comprises a heavy chain constant region.

In another preferred embodiment, the heavy chain constant region is of human, murine, or rabbit origin.

In another preferred embodiment, the antibody further comprises a light chain constant region.

In another preferred embodiment, the light chain constant region is of human, murine, or rabbit origin.

In another preferred embodiment, the antibody is specific to 14-3-3 eta protein.

In another preferred embodiment, the antibody comprises: single chain antibodies, diabodies, monoclonal antibodies, chimeric antibodies (e.g., human murine chimeric antibodies), murine antibodies, rabbit antibodies, or humanized antibodies.

In a second aspect of the invention, there is provided a polynucleotide encoding an antibody according to the first aspect of the invention.

In a third aspect of the invention, there is provided a vector comprising a polynucleotide according to the second aspect of the invention.

In another preferred embodiment, the carrier comprises: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

In a fourth aspect of the invention, there is provided a genetically engineered host cell comprising a vector according to the third aspect of the invention or a genome into which has been integrated a polynucleotide according to the second aspect of the invention.

In a fifth aspect of the invention, there is provided an immunoconjugate comprising:

(a) an antibody according to the first aspect of the invention; and

(b) a coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

In another preferred embodiment, the conjugate is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles, and the like.

In a sixth aspect of the present invention, there is provided a pharmaceutical composition comprising:

(i) an antibody according to the first aspect of the invention, an immunoconjugate according to the fifth aspect of the invention; and

(ii) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is in the form of injection.

In another preferred embodiment, the pharmaceutical composition is used for preparing a medicament for treating tumors selected from the group consisting of: liver cancer, stomach cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, endometrial cancer, penis cancer, adrenal gland tumor, or bladder tumor.

In a seventh aspect of the invention, there is provided the use of an antibody according to the first aspect of the invention, an immunoconjugate according to the fifth aspect of the invention, for the manufacture of a medicament, a reagent, a test panel or a kit;

the reagent, assay plate or kit is for:

(1) detecting 14-3-3 eta protein in the sample; and/or

(2) Detecting endogenous 14-3-3 eta protein in the tumor cells; and/or

(3) Detecting tumor cells expressing 14-3-3 η protein;

the medicament is used for treating or preventing tumors expressing 14-3-3 eta protein.

In another preferred embodiment, the tumor comprises: gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, endometrial cancer, penis cancer, small cell lung cancer, melanoma or head and neck tumor, or adrenal gland tumor.

In another preferred embodiment, the reagent comprises a chip and immune microparticles coated with antibodies.

In an eighth aspect of the invention, there is provided a method of detecting 14-3-3 η protein in a sample, said method comprising the steps of:

(1) contacting the sample with an antibody according to the first aspect of the invention;

(2) detecting the formation of an antigen-antibody complex, wherein the formation of the complex is indicative of the presence of 14-3-3 eta protein in the sample.

In another preferred example, the detection is performed by ELISA method in step (2).

In another preferred example, the sample comprises: human or animal tissue samples, tumor resection samples, exfoliated cell samples.

In another preferred embodiment, the method is used for non-diagnostic purposes.

In a ninth aspect of the present invention, there is provided a kit comprising:

a first container comprising an antibody according to the first aspect of the invention.

In another preferred embodiment, the kit further comprises:

a second container comprising a cell lysis reagent.

In another preferred embodiment, the antibody in the first container is detectably labeled.

In a tenth aspect of the present invention, there is provided a method for producing a recombinant polypeptide, the method comprising:

(a) culturing the host cell of the fourth aspect of the invention under conditions suitable for expression;

(b) isolating a recombinant polypeptide from the culture, wherein the recombinant polypeptide is an antibody according to the first aspect of the invention.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the result of staining 110M01 monoclonal antibody in hepatocarcinoma tissue.

FIG. 2 shows the result of staining 110M01 monoclonal antibody in hepatocarcinoma tissue.

FIG. 3 shows the staining results of the 110M01 monoclonal antibody in normal hepatocytes.

FIG. 4 shows the staining results of the 110M01 monoclonal antibody in normal hepatocytes.

FIG. 5 shows the electrophoresis result of the serum sample 14-3-3 eta of the liver cancer patient detected by the 110M01 monoclonal antibody WB.

FIG. 6 shows the result of electrophoresis of the serum sample 14-3-3. eta. for the detection of hepatoma patient with the 102M07 monoclonal antibody WB.

FIG. 7 shows the electrophoresis result of detecting 14-3-3 eta of the serum sample of the liver cancer patient by the 101M26 monoclonal antibody WB.

Detailed Description

The inventor successfully obtains a monoclonal antibody (110M01 monoclonal antibody) with specificity for resisting 14-3-3 eta protein through intensive research and a large number of screens, and experimental results show that the monoclonal antibody can specifically bind 14-3-3 eta protein, and has strong affinity and high sensitivity. The antibody can be used for detecting 14-3-3 eta protein in fresh tissues and can also be used for detecting a very small amount of target protein in serum, so that the antibody has extremely high detection sensitivity. On the basis of this, the present invention has been completed.

14-3-3 is a phosphoserine/threonine binding protein family, including seven subtypes, α/β, γ, ε, σ, ξ, θ/τ, and η. Research has shown that: 5 14-3-3 protein subtypes (alpha/beta, gamma, epsilon, sigma and xi) participate in the liver cancer process. The inventor researches and discovers that: compared with the reported subtypes, 14-3-3 eta has distribution in tumor cells and vascular endothelial cells in liver cancer tissues, and the expression level of the 14-3-3 eta is gradually increased along with the progress of liver cancer. Further found that: 14-3-3 eta in liver cancer and vascular endothelial cells can form a positive feedback loop with p-ERK1/2, so that the progress of the liver cancer is promoted, and the high expression of the tissue 14-3-3 eta of a liver cancer patient is related to sorafenib resistance and poor prognosis.

The amino acid sequence of 14-3-3 eta is as follows:

the inventor unexpectedly finds that the 14-3-3 eta protein in the serum of a liver cancer patient is obviously increased in the research process, so the protein can be used as a liver cancer marker protein. The 14-3-3 eta plasmalography detection has important value as potential liver cancer early-stage discovery and tumor prognosis prediction.

However, 14-3-3 eta in the tumor tissue of a liver cancer patient is inconvenient to detect, the inventor tries to detect the level of 14-3-3 eta in serum/plasma, but because the molecular weight of 14-3-3 eta is relatively small, about 28KD, which is close to the size of a protein light chain, the detection result cannot observe the difference of the level of 14-3-3 eta in the serum/plasma sample of the tumor patient and the serum/plasma sample of a normal human.

After further removing high-abundance proteins (such as IgG, IgM, IgA, albumin, alpha 1-acid glycoprotein and the like) in plasma/serum, the inventor successfully detects 14-3-3 eta in a serum/plasma sample by using a modified immunoblotting detection method, and unexpectedly finds that the 14-3-3 eta level in the serum/plasma sample of a tumor patient and the 14-3-3 eta level in a normal human serum/plasma sample have significant difference. Furthermore, through comparative analysis of a large sample of plasma samples of liver cancer patients and normal plasma samples of the liver cancer patients, it is found that 14-3-3 eta in the plasma can be used as a liver cancer marker and can be used for potential early detection of liver cancer, prognosis prediction of tumors and the like.

Specifically, the invention adopts recombinant 14-3-3 eta protein to immunize BALB/C mice, and 14-3-3 eta protein recombinant protein is used as a screening antigen to screen and obtain a positive monoclonal hybridoma cell strain. Finally, 5 cell strains capable of specifically binding to the 14-3-3 eta protein are obtained.

5 monoclonal cell strains are subjected to mouse ascites antibody preparation, formalin fixation and paraffin-embedded liver cancer tissue sections are detected, and finally, a monoclonal antibody which can specifically identify 14-3-3 eta protein of the paraffin tissue sections and can be detected by target protein in serum is obtained.

As used herein, the term "antibody" or "immunoglobulin" is an heterotetrameric glycan protein of about 150000 daltons with the same structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable region (VH) followed by a plurality of constant regions. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Particular amino acid residues form the interface between the variable regions of the light and heavy chains.

As used herein, the term "variable" means that certain portions of the variable regions of an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, which are in a substantially β -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain are held close together by the FR region and form the antigen binding site of the antibody with the CDRs of the other chain (see Kabat et al, NIH Publ. No.91-3242, Vol I, 647-669 (1991)). The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.

The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two distinct classes (termed kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be assigned to different classes based on the amino acid sequence of their heavy chain constant regions. There are mainly 5 classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA 2. The heavy chain constant regions corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

As used herein, the term "monoclonal antibody (mab)" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are directed against a single antigenic site with high specificity. Moreover, unlike conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the rabbit monoclonal antibodies herein are obtained by constructing full-length rabbit monoclonal antibody gene expression vectors by molecular biology methods after screening phage libraries, transferring the vectors into eukaryotic expression systems, and harvesting cell supernatants after culture, and are free from contamination by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

The invention also includes monoclonal antibodies having the corresponding amino acid sequences of said monoclonal antibodies against 14-3-3 eta protein, monoclonal antibodies having the variable region chains of said monoclonal antibodies against 14-3-3 eta protein, as well as other proteins or protein conjugates and fusion expression products having these chains. Specifically, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having light and heavy chains with hypervariable regions (complementarity determining regions, CDRs) so long as the hypervariable regions are identical or at least 90% homologous, preferably at least 95% homologous to the hypervariable regions of the light and heavy chains of the invention.

As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines (cytokines), radionuclides, enzymes, and other diagnostic or therapeutic molecules are conjugated to the 14-3-3 η protein monoclonal antibody or fragment thereof. The invention also includes a cell surface marker or antigen conjugated with the anti-14-3-3 eta protein monoclonal antibody or the fragment thereof.

The invention includes not only intact monoclonal antibodies, but also immunologically active antibody fragments, such as Fab or (Fab') ₂ A fragment; an antibody heavy chain; the light chain of the antibody.

As used herein, the terms "heavy chain variable region" and "V _H "may be used interchangeably.

As used herein, the term "variable region" is used interchangeably with "Complementary Determining Region (CDR)".

In a preferred embodiment of the invention, the heavy chain variable region of the antibody comprises the following three complementarity determining regions CDRs:

a CDR1 having an amino acid sequence of TFSGQYTFATS (SEQ ID No.: 1);

CDR2 having the amino acid sequence LQYGA (SEQ ID No.: 2);

CDR3 having an amino acid sequence of TYAGSVKGPFNL (SEQ ID No.: 3).

In another preferred embodiment, the amino acid sequence of the heavy chain variable region is:

EVQLKQSTPGSVKPEWSLKMSCKASTFSGQYTFATSADSKGRQTWVSLVEWIGSILQYGATAYDMSVGVRGTISYDRARLINYQGMSSTRSQDTAMYFCARTYAGSVKGPFNLQWGEGTSVTVSS(SEQ ID NO.:4)。

in a preferred embodiment of the invention, the heavy chain of the antibody comprises the above-described heavy chain variable region and a heavy chain constant region, which may be murine, human or rabbit.

As used herein, the terms "light chain variable region" and "V _L "may be used interchangeably.

In a preferred embodiment of the invention, the light chain variable region of the antibody according to the invention has complementarity determining regions CDRs selected from the group consisting of:

CDR1' having an amino acid sequence of PGVTFAGKFEPTVI (SEQ ID No.: 5);

CDR2' having an amino acid sequence of QGVGSRHEF (SEQ ID No.: 6);

CDR3', the amino acid sequence of which is ETDLQPVM (SEQ ID No.: 7);

in another preferred embodiment, the amino acid sequence of the light chain variable region is:

GIVLFYYCVSSLAVQTGQAEISYPGVTFAGKFEPTVIWYSGQKPGQPVMLIYQGVGSRHEFQGKDRFYIMPQWGRTDFTLTIEFEAEDQAVPCETDLQPVMVTPETKENDGQR(SEQ ID NO.:8)。

in a preferred embodiment of the present invention, the light chain of the antibody comprises the light chain variable region and the light chain constant region, and the light chain constant region may be of murine, human or rabbit origin.

In the present invention, the terms "antibody of the present invention", "protein of the present invention", or "polypeptide of the present invention" are used interchangeably and refer to an antibody that specifically binds to a 14-3-3 η protein. They may or may not contain the initial methionine.

In another preferred embodiment, the antibody is a murine or human murine chimeric monoclonal antibody directed against the 14-3-3 eta protein, and the heavy chain constant region and/or the light chain constant region thereof may be humanized. More preferably, the humanized heavy or light chain constant region is that of human IgG1, IgG2, or the like.

The invention also provides other proteins or fusion expression products having an antibody of the invention. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having heavy and light chains with variable regions, provided that the variable regions are identical or at least 90% homologous, preferably at least 95% homologous, to the variable regions of the heavy and light chains of the antibody of the invention.

In general, the antigen binding properties of an antibody can be described by 3 specific regions in the heavy and light chain variable regions, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, and the β -sheets formed by the FRs between them are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type.

The variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest, since at least some of them are involved in binding to an antigen. Thus, the invention includes those molecules having the light and heavy chain variable regions of a monoclonal antibody with CDRs that are more than 90% (preferably more than 95%, most preferably more than 98%) homologous to the CDRs identified herein.

The invention includes not only complete monoclonal antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.

As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as an antibody of the invention. A polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g. a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.

The antibody of the present invention refers to a polypeptide having 14-3-3 eta protein binding activity, which comprises the above-mentioned CDR region. The term also includes variants of the polypeptides comprising the above CDR regions that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA capable of hybridizing to DNA encoding the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antisera raised against the antibody of the present invention.

The invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof. In addition to almost full-length polypeptides, the invention also encompasses fragments of the antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of an antibody of the invention.

In the present invention, "conservative variant of the antibody of the present invention" means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are substituted by amino acids having similar or similar properties as compared with the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservative variants are preferably produced by amino acid substitutions according to Table A.

TABLE A

Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.

The present invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃; or (2) denaturant is added during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 ℃ and the like; or (3) hybridization only when the identity between two sequences is at least 90% or more, preferably 95% or more.

The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be obtained by a PCR amplification method, a recombinant method, or an artificial synthesis method. One possibility is to use synthetic methods to synthesize the sequence of interest, especially when the fragment length is short. Typically, long fragments are obtained by first synthesizing a plurality of small fragments and then ligating them together. In addition, the coding sequence of the heavy chain and an expression tag (e.g., 6His) can be fused together to form a fusion protein.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules in an isolated form.

At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; CHO, COS7, 293 cells, etc.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl ₂ Methods, the steps used are well known in the art. Another method is to use MgCl ₂ . If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for the growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

The antibodies of the invention may be used alone or in combination or conjugated with detectable labels (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties or combinations of any of the above.

Detectable labels for diagnostic purposes include, but are not limited to: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product.

Therapeutic agents that may be conjugated or conjugated to the antibodies of the invention include, but are not limited to: 1. radionuclides (Koppe et al, 2005, Cancer metastasis reviews (Cancer metastasis) 24, 539); 2. biotoxicity (Chaudhary et al, 1989, Nature 339, 394; Epel et al, 2002, Cancer Immunology and Immunotherapy)51, 565); 3. cytokines such as IL-2 and the like (Gillies et al, 1992, Proc. Natl. Acad. Sci. USA (PNAS)89, 1428; Card et al, 2004, Cancer Immunology and Immunotherapy)53, 345; Halin et al, 2003, Cancer Research 63, 3202); 4. gold nanoparticles/nanorods (Lapotko et al, 2005, Cancer letters 239, 36; Huang et al, 2006, Journal of the American Chemical Society 128, 2115); 5. viral particles (Peng et al, 2004, Gene therapy 11, 1234); 6. liposomes (Mamot et al, 2005, Cancer research 65, 11631); 7. nano magnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents (e.g., cisplatin) or nanoparticles in any form, and the like.

The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the above-described antibody or active fragment thereof or fusion protein thereof, and a pharmaceutically acceptable carrier. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.

The pharmaceutical composition of the present invention can be directly used for binding 14-3-3 eta protein molecules, and thus can be used for preventing and treating tumors. In addition, other therapeutic agents may also be used simultaneously.

The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the monoclonal antibody (or conjugate thereof) of the present invention as described above and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.

In the case of pharmaceutical compositions, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 1 mg/kg body weight. Of course, the particular dosage will also take into account such factors as the route of administration, the health of the patient, and the like, which are within the skill of the skilled practitioner.

Preparation of monoclonal antibodies

The antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. For example, the antigens of the invention can be administered to an animal to induce the production of monoclonal antibodies. For Monoclonal Antibodies, they can be prepared using hybridoma technology (see Kohler et al, Nature 256; 495, 1975; Kohler et al, Eur. J. Immunol.6:511,1976; Kohler et al, Eur. J. Immunol.6:292,1976; Hammerling et al, In Monoclonal Antibodies and T Cell hybrids, Elsevier, N.Y.,1981), phage display technology or can be prepared using recombinant DNA methods (U.S. Pat. No.4,816,567).

Representative myeloma cells are those that fuse efficiently, support stable high-level production of antibody by selected antibody-producing cells, and are sensitive to medium (HAT medium matrix), including myeloma Cell lines, such as murine myeloma Cell lines, including those derived from MOPC-21 and MPC-11 mouse tumors (available from Salk Institute Cell Distribution Center, san diego, california, usa), and SP-2, NZ0, or X63-Ag8-653 cells (available from American Type Culture Collection, rockwell, maryland, usa). Human myeloma and mouse-human heterozygous myeloma cell lines have also been described for the production of human monoclonal antibodies [ Kozbor, j.immunol., 133: 3001 (1984); brodeur et al, Techniques for the Production and use of Monoclonal Antibodies (Monoclonal Antibodies Production Techniques and Applications), pp 51-63 (Marcel Dekker, Inc., New York, 1987).

The medium in which the hybridoma cells are grown is assayed to detect the production of monoclonal antibodies of the desired specificity, e.g., by an in vitro binding assay, e.g., an enzyme-linked immunosorbent assay (ELISA) or Radioimmunoassay (RIA). The location of the antibody-expressing cells can be detected by FACS. The hybridoma clones can then be subcloned by limiting dilution procedures (subcloned) and grown by standard methods (Goding, Monoclonal Antibodies): Principles and Practice (Principles and Practice), Academic Press (1986) pp 59-103). Suitable media for use in this purpose include, for example, DMEM or RPMI-1640 medium. In addition, hybridoma cells can grow in animals as ascites tumors.

The monoclonal antibodies secreted by the subclones are suitably isolated from the culture medium, ascites fluid or serum by conventional immunoglobulin purification procedures, such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

The phage display technology is a screening technology, exogenous polypeptide or protein and capsid protein of phage are fused and expressed, the fusion protein is displayed on the surface of virus particle, and DNA coding the fusion is located in virus particle, so that direct connection is established between a large number of polypeptides and DNA coding sequences thereof, and polypeptide ligands of various target molecules (antibody, enzyme, cell surface receptor, etc.) can be rapidly identified by panning.

In a preferred embodiment of the present invention, the monoclonal antibody is prepared by culturing hybridoma cells. The supernatant from the hybridoma cell culture was purified by affinity chromatography (Protein A/G-Sepharose).

In a preferred embodiment of the invention, the monoclonal antibody is prepared by a method for producing the monoclonal antibody by Balb/C mouse ascites. The hybridoma cells were inoculated into the abdominal cavity of a sensitized mouse, and the ascites was extracted and purified by an affinity chromatography column (Protein A/G-Sepharose).

In a preferred embodiment of the present invention, the monoclonal antibody is purified by affinity chromatography (Protein A/G-Sepharose) using a recombinant DNA method to construct a eukaryotic expression system, transiently transforming HEK293 cells to express the antibody, and then secreting the antibody into the culture medium.

Method and sample

The present invention relates to methods for detecting cancer in a tissue sample. The method comprises the following steps: obtaining a tissue sample; detecting the level of 14-3-3 η protein in said sample. The samples used in the method of the invention are serum samples, or formalin-fixed, paraffin-embedded tissue sections commonly used in clinical pathology.

Samples (specimens) used in the present invention include tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to the person skilled in the art. Thus biopsies as used in the present invention may comprise e.g. resection samples of tumours, tissue samples prepared by endoscopic methods or needle biopsy of organs.

Samples used in the present invention may include fixed or preserved tissue samples. Tissue samples may be preserved, for example, in standard sample collection, storage or transport media, such as those commercially available preservation media known to those skilled in the art (formalin, Cytyc "PreservCyt" or Tripath Imaging "Cytorich", etc.). Suitable preservation media may include one or more mixtures selected from alcohols, aldehydes, ketones, acids, metal ions or mercury, ethers, and the like, for preserving cellular components. The alcohol comprises methanol, ethanol, (n-or iso-) propanol, (n-, iso-or tert-) butanol or a highly branched or unbranched alcohol. Aldehydes include formaldehyde, acetaldehyde, glutaraldehyde, and the like. Ketones such as acetone may also be used. Acids used in standard sample media include organic acids (acetic, trichloroacetic, salicylic and picric) or inorganic acids such as chromic acid. Standard sample solutions may include metals such as silver, copper, chromium, mercury, osmium, and uranium. Salt solutions such as uranyl acetate, potassium dichromate, ammonium sulfate, and the like may be components of the preservation medium.

Reagent kit

The invention also provides a kit containing only the antibody (or fragment thereof) of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like.

The invention further designs a detection kit for detecting the 14-3-3 eta protein, which comprises an antibody for identifying the 14-3-3 eta protein, general reagents and buffers required for detection, such as various buffers, enzyme-linked labeled secondary antibodies, detection labels, detection substrates and the like. The test kit may be an in vitro diagnostic device.

The main advantages of the invention are:

(1) the antibody aiming at the 14-3-3 eta protein provided by the invention has high specificity and strong affinity, and can be prepared in large scale, and the quality of the monoclonal antibody is easy to control.

(2) The antibody provided by the invention is high in detection sensitivity and can be used for detecting target protein in a serum sample.

(3) The antibody provided by the invention is used in the method for detecting the 14-3-3 eta protein, and the stability is good.

(4) The monoclonal antibody and the detection method provided by the invention are suitable for early diagnosis of related cancers and can be used for assessing prognosis of cancers.

The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures for conditions not specified in detail in the following examples are generally carried out under conventional conditions such as those described in molecular cloning, A laboratory Manual (Huang Petang et al, Beijing: scientific Press, 2002) by Sambrook. J, USA, or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.

EXAMPLE 1 preparation of anti-14-3-3 eta protein monoclonal antibody

1. Immunization of mice

The immunogen was recombinant 14-3-3 eta protein (SEQ ID No.:17), and 4-week-old female Balb/C healthy mice (purchased from Shanghai Spikel company) were immunized. The first immunization: mu.g (250. mu.l) of antigen was mixed with 250. mu.l of complete adjuvant and injected into Balb/c mice at subcutaneous multiple sites and in the ball of the foot. The first immunization was performed three weeks later with the same dose for the second immunization. After two weeks of the second immunization, the same dose was used for the third immunization. After two weeks of the third immunization interval, the fourth immunization was performed at the same dose.

The tail of the mouse is cut off before each immunization, 14-3-3 eta protein recombinant protein (5 mu g/ml) is used as a detection antigen, the serum titer of the mouse is detected by an ELISA method, and when the serum titer of the immunized mouse is more than 1:10,000, splenocytes are taken for fusion.

2. Cell fusion and culture

Myeloma cells SP2/0 (purchased from ATCC) in a well-grown logarithmic growth phase were fused with spleen cells of immunized mice collected by a conventional method, and the ratio of the spleen cells: SP2/0 ratio of 10:1 at 2X10 ⁴ Density of Individual cells the fused cells were seeded into 96-well plates at 37 ℃ in 5% CO ₂ Tending in a constant temperature incubator. After 14 days of culture, ELISA detection was performed to screen for 14-3-3 eta protein antibody positive clones.

Screening positive hybridomas by adopting indirect ELISA, taking antigen as immunogen protein mixed solution, wrapping the antigen at 4 ℃ overnight, sealing with 5% skimmed milk powder, acting at 37 ℃ for 2h, washing, patting dry, adding hybridoma supernatant, setting positive (P), negative (N) and blank control, reacting at 37 ℃ for 1h, washing, patting dry, adding goat anti-mouse-HRP (Sigma A2554) secondary antibody, reacting at 37 ℃ for 45-60 min, developing by adopting TMB (Tetramethylbenzidine), and performing 2M H ₂ SO ₄ And (6) terminating. Clones which are positive after two successive detections are amplified.

3. Subcloning of hybridoma cells

Subcloning was performed by limiting dilution. The cell suspension was diluted to 60/ml and 100. mu.l (about 6 cells/well) was added to each well in a 96-well plate. Inoculating 2 rows, diluting the rest cell suspension with culture solution in multiple proportion, and inoculating 2 rows. And repeating the steps once. Placing at 37 ℃ and 5% CO ₂ And (5) incubating in a cell incubator. And replacing 1/2 culture solution every 2-3 days. After approximately 10 days of culture, positive wells from which individual clones grew were selected for secondary screening and subcloning. After three successive subclonings, the stable cell line was determined when the antibody positivity was 100% by ELISA. Obtaining 5 knots in totalStable monoclonal antibody strains 102M07, 102M08, 101M26, 110M01, 112M16 against immunogenic proteins.

4. Preparation of monoclonal antibodies

Injecting Balb/c female mice of 6 weeks old with liquid paraffin for 1 week in advance for sensitization, and then injecting hybridoma cells into abdominal cavity at 1-2 x10 ⁶ Hybridoma cells/hybridoma, 3 per hybridoma were injected. Ascites was extracted 7-10 days later. Ascites titer reached 1 by ELISA detection: purifying by Protein A/G affinity column chromatography of more than 10,000.

EXAMPLE 2 specificity and affinity detection of monoclonal antibodies

ELISA (enzyme-Linked immuno sorbent assay) detection of monoclonal antibody cross reaction, wherein an antigen respectively selects seven subtype recombinant proteins of 14-3-3 including alpha/beta, gamma, epsilon, Sigma, zeta, theta/tau and eta, 2 mug/ml of a coated plate is used for coating at 4 ℃, PBST is washed and dried by patting, 5% skimmed milk powder is added for sealing, the reaction is carried out at 37 ℃ for 2h or 4 ℃ for overnight, 1 mug/ml of monoclonal antibody is added after PBST is washed and dried by patting, the reaction is carried out at 37 ℃ for 1h, a goat anti-mouse-HRP secondary antibody (Sigma A2554) (1:10000) is added after PBST is washed and dried by patting, the reaction is carried out at 37 ℃ for 45-60 min, TMB (Tetramethylbenzidine) is developed and 2M H is carried out after PBST is washed and dried by patting ₂ SO ₄ End, OD450 nm. The results show that: the 102M07, 101M26 and 110M01 monoclonal antibodies can be specifically combined with 14-3-3 eta protein, and have no obvious cross reaction with other subtype proteins; the specificity of the 102M08 and 112M16 monoclonal antibodies is poor, and the eta subtype cannot be specifically recognized.

The affinity of the 102M07, 101M26 and 110M01 monoclonal antibodies is detected, and the results are as follows:

antibody numbering	Affinity constant M
		102M07	1.87E -9
101M26	6.18E -9
		110M01	1.72E -10

Extracting total RNA of the monoclonal antibody hybridoma cells, performing reverse transcription, and performing antibody gene cloning sequencing:

the sequence information for the 110M01 single heavy chain variable region is as follows:

the sequence information of the light chain variable region of the 110M01 monoclonal antibody is as follows:

example 3 detection of 14-3-3 eta protein in liver cancer tissue by immunohistochemical staining

The monoclonal antibodies 102M07, 101M26 and 110M01 are used as primary antibodies to perform immunohistochemical staining on liver cancer tissues and liver normal tissues. Sequentially soaking paraffin tissue slices in xylene and ethanol, adding into 0.01M sodium citrate buffer solution, and boiling; after washing, blocking the mixture for 20 minutes at room temperature by using TBST containing 10% calf serum; adding the antibody to be detected, wherein the concentration of the antibody is 15ug/ml, incubating at room temperature, adding Anti-Mouse IgG-HRP dropwise, and incubating at room temperature for 30min for color development observation.

The 110M01 monoclonal antibody has obvious staining in liver cancer tissue cells (figure 1 and figure 2), weak staining in normal liver cells (figure 3 and figure 4), clean staining background and obvious differentiation, so the experimental result shows that the 110M01 monoclonal antibody can specifically identify liver cancer tumor cells. The monoclonal antibodies 102M07 and 101M26 are weakly stained in liver cancer tissues and are not obviously different from normal liver cells. Therefore, the experimental result shows that only the 110M01 monoclonal antibody can specifically recognize the liver cancer tumor cells.

Example 4WB assay blood samples

In this example, the 14-3-3 eta protein in a blood sample was detected using a monoclonal antibody.

1. Preparation of serum samples

Firstly, placing a serum sample on ice, and if the serum sample is a frozen sample, thawing the serum sample in water bath at 25 ℃ until the serum sample is completely thawed, and placing the serum sample on the ice;

collecting 200ul serum sample, centrifuging at 4 deg.C and 3000g for 10 min;

transferring the supernatant to a new centrifugal tube, and placing on ice;

2. exosome extraction

Taking 200ul of centrifuged serum sample, adding 50ul of extraction reagent A, and uniformly swirling;

② mixing the solution at 4 ℃, standing and incubating for 30 min;

③ after the incubation is finished, centrifuging the mixed solution for 10min at room temperature and 3000g, removing the supernatant, and enabling the bottom of the tube to be visible for precipitation;

fourthly, the obtained precipitate is centrifuged briefly, and residual supernatant is removed;

fifthly, resuspending the precipitate with 200ul 1 × sterilized PBS, and repeatedly and uniformly blowing;

sixthly, adding 50ul of reagent B into the heavy suspension, and uniformly swirling;

seventhly, standing the mixed solution at 4 ℃, and incubating for 30 min;

after the incubation is finished, centrifuging at room temperature and 3000g for 10min, and removing the supernatant;

ninthly, centrifuging the obtained precipitate briefly, and removing residual supernatant;

the precipitate in red (R) is re-suspended with 50-120ul of sterilized PBS and blown to be even repeatedly.

3. Protein extraction

Firstly, the RIPA lysate is melted and mixed evenly. An appropriate amount of lysate was taken and PMSF was added several minutes before use.

② adding 50 mul of lysis solution according to the cell volume.

And thirdly, fully oscillating on the oscillator for a plurality of times.

Fourthly, after full cracking, centrifuging at 12000rpm for 5 minutes, and taking the supernatant.

Taking part of the supernatant protein, and determining the protein concentration by using a BCA protein concentration determination kit according to the following steps:

sixthly, mixing the solution A and the solution B according to the specification of the kit, wherein the ratio of 50: 1 to prepare working solution;

seventhly, taking 2000 mu g/ml BSA standard substance, diluting the BSA standard substance with PBS (pH 7.4) to 9 concentration gradients of 2000 mu g/ml, 1500 mu g/ml, 1000 mu g/ml, 750 mu g/ml, 500 mu g/ml, 250 mu g/ml, 125 mu g/ml, 25 mu g/ml and 0 mu g/ml;

eighthly, taking 10 mu l of protein extracting solution, and diluting the protein extracting solution by 5 times by using PBS;

ninthly, adding 25 mu l of protein standard substance or diluted tissue protein extracting solution into each tube, adding 200 mu l of the working solution, incubating for 30min at 37 ℃, measuring absorbance at 562nm, and recording OD value;

and (c) drawing a standard curve according to the OD value and the concentration of the protein standard substance, and calculating the total protein concentration of the sample.

4. WB (Western blotting) detection of 14-3-3 eta

1) Glue filling

The glass plate is cleaned by distilled water and dried vertically.

Preparing 10ml of 15% separation gel according to the method: 2.3ml of water, 5.0ml of 30% acrylamide, 2.5ml of 1.5M Tris buffer solution, 0.1ml of 10% SDS, 6.4g of urea, 4 mul of TEMED and 100 mul of 10% (w/v) AP are mixed uniformly, then immediately poured into glue, poured to 2-3 mm (marked in advance) of the lower edge of a comb, sealed by isopropanol to remove bubbles and isolate air, and kept stand at room temperature for 45 minutes until the glue is completely polymerized.

Thirdly, after the separation gel is completely polymerized, pouring the deionized water on the top of the separation gel, and sucking the water by using filter paper.

Preparing 6ml of 5% concentrated glue: 4ml of water, 1ml of 30% acrylamide, 1ml of 1.0M Tris buffer, 80ul of 10% SDS,60 ul of 10% AP and 8ul of TEMED, uniformly mixing, immediately pouring glue, pouring to the top, vertically inserting a Teflon tooth comb, and standing for 20 minutes at room temperature until the glue is polymerized.

Fifthly, after the gel is completely polymerized, the comb is pulled out, the gel is placed in an electrophoresis tank, electrophoresis buffer solution is added, and the sample loading hole is washed by the electrophoresis buffer solution to remove air bubbles.

2) Electrophoresis

Taking each processed histone extract, adjusting the protein concentration, and mixing with an equal volume of 5 multiplied sample buffer solution to obtain a sample solution.

Boiling the sample solution in boiling water at 100 deg.c for 5min to denature protein, quenching in ice and centrifuging at 3000 rpm for 1 min.

③ 10ul of sample liquid is added into each hole, and 10 mul of pre-dyed Marker is added into one hole. Filling up an electrophoresis buffer solution, covering a tank cover, switching on a power supply, performing constant voltage electrophoresis at 70v for about 30min, performing constant voltage electrophoresis at 90v after an indicator bromFinland enters separation gel, stopping electrophoresis when an electrophoresis strip of a25 Kd Marker reaches the middle position of the separation gel, switching off the power supply, and taking out a gel plate.

3) Trans-proteins and immunoassays

Firstly, just before the end of electrophoresis, the PVDF membrane is soaked in methanol for 15s in advance and then is treated with ddH ₂ Rinsing for 2min with O, soaking in transfer buffer for 5min, and starting subsequent operation.

Prying the glue in water, soaking the glue in a transfer buffer solution for balancing for 15min after glue repairing.

Preparing a transfer film sandwich in the sequence of black surface (negative electrode) → sponge → filter paper → glue → PVDF film (0.2um) → filter paper → sponge → red surface (positive electrode), and spreading each layer, removing bubbles and then spreading the other layer. The sandwich is prepared in a transfer buffer to avoid the generation of air bubbles.

And fourthly, connecting the positive electrode and the negative electrode, putting the transfer box into the electrotransfer instrument according to the direction from the membrane to the positive electrode, and adding a membrane transfer buffer solution.

Fifthly, placing the electric rotating instrument in ice water, and constantly rotating the membrane for 70min at 200 mA.

Sixthly, after the membrane transfer is finished, quickly taking out the PVDF membrane, and putting 5% BSA for sealing for 2 hours at room temperature.

Seventhly, taking out the membrane, and washing the membrane for 5min multiplied by 3 times on a shaking table.

Adding an anti-14-3-3 eta antibody diluted by TBST into the incubation bag, and incubating overnight at 4 ℃.

Ninthly, TBST washes the membrane for 5min multiplied by 3 times, and horseradish peroxidase (HRP) labeled goat anti-mouse secondary antibody is incubated for 2h at room temperature.

Wash membrane 10min X3 times in TBST. Reacting the film with a chemiluminescence detection reagent (reagent A: reagent B ═ 1: 1) for 2min, taking out the film, throwing off excessive liquid, covering a PVDF film with a preservative film, and carrying out light sensation, development and fixation on the PVDF film by an X film in a dark room.

The experimental results are shown in the figure. The immunoblotting results of the 110M01, 102M07 and 101M26 monoclonal antibodies in FIGS. 5, 6 and 7, respectively. (wherein 1-6 is serum sample of liver cancer patient, protein loading amount is 10ug, antibody dilution is 1: 2500.7-8 is 14-3-3 eta human recombinant protein, N segment is added with Flag tag protein, and the molecular weight of human recombinant protein is slightly larger than that of natural 14-3-3 eta)

From the results, 110M01 showed a distinct single band at 28Kd, whereas 102M07, 101M26 showed bands at 28Kd and 55Kd, which were not very specific. Therefore, 110M01 can be used as a specific antibody for detecting 14-3-3 eta in serum.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

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Shanghai Zhuoli Biotech Co Ltd

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Claims (10)

1. An anti-14-3-3 eta protein monoclonal antibody, which antibody has:

(1) a heavy chain variable region; and

(2) a light chain variable region;

wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.4, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8.

2. A polynucleotide encoding the antibody of claim 1.

3. A vector comprising the polynucleotide of claim 2.

4. A genetically engineered host cell comprising the vector or genome of claim 3 having integrated therein the polynucleotide of claim 2.

5. An immunoconjugate, comprising:

(a) the antibody of claim 1; and

(b) a coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, or enzyme.

6. A pharmaceutical composition comprising:

(i) the antibody of claim 1, the immunoconjugate of claim 5; and

(ii) a pharmaceutically acceptable carrier.

7. Use of the antibody of claim 1, the immunoconjugate of claim 5, for the manufacture of a medicament, a reagent, a test panel, or a kit;

the reagent, assay plate or kit is for:

(1) detecting 14-3-3 eta protein in the sample; and/or

(2) Detecting endogenous 14-3-3 eta protein in the tumor cells; and/or

(3) Detecting tumor cells expressing 14-3-3 η protein;

the medicament is used for treating or preventing tumors expressing 14-3-3 eta protein.

8. A method for the detection of 14-3-3 η protein in a sample for non-diagnostic purposes, said method comprising the steps of:

(1) contacting a sample with the antibody of claim 1;

(2) detecting the formation of an antigen-antibody complex, wherein the formation of the complex is indicative of the presence of 14-3-3 eta protein in the sample.

9. A kit, comprising:

a first container comprising the antibody of claim 1 therein.

10. A method for producing a recombinant polypeptide, the method comprising:

(a) culturing the host cell of claim 4 under conditions suitable for expression;

(b) isolating a recombinant polypeptide from the culture, said recombinant polypeptide being the antibody of claim 1.

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