CN106589128B

CN106589128B - Preparation and application of liver cancer marker monoclonal antibody

Info

Publication number: CN106589128B
Application number: CN201510682917.9A
Authority: CN
Inventors: 张宁; 罗艺
Original assignee: Shaanxi University of Science and Technology
Current assignee: Shaanxi University of Science and Technology
Priority date: 2015-10-20
Filing date: 2015-10-20
Publication date: 2020-03-03
Anticipated expiration: 2035-10-20
Also published as: CN106589128A

Abstract

The present invention relates to antibodies or fragments thereof (particularly single chain antibodies) against human liver cancer markers, methods for their preparation, detection reagents and kits comprising them, and uses thereof.

Description

Preparation and application of liver cancer marker monoclonal antibody

Technical Field

Background

Primary liver cancer (PLC, liver cancer for short) is one of the most common malignant tumors in clinic, has hidden onset, rapid invasion and growth, easy relapse after treatment and extremely poor prognosis, and is called 'king of cancer'. About half of the liver cancer patients worldwide are concentrated in China, and about 11 thousands of people die of liver cancer every year in China and are the second place of the death rate of malignant tumors. At present, surgical resection and liver transplantation are the most effective treatment methods for liver cancer, but most liver cancer patients have a diagnosis period of middle and late stage and cannot be operated, and the surgical resection rate is only 10-30%. For the case of inoperable patients, the traditional radiotherapy and chemotherapy has poor effect, and the life cycle of the patients is generally not more than half a year. Therefore, the early diagnosis of liver cancer is very important and is the key of clinical diagnosis and prognosis.

The current situation that the detection rate of early liver cancer is low reflects that the current diagnosis method of liver cancer has a large limitation, the current diagnosis of liver cancer mainly depends on serological examination and imaging diagnosis, the serological examination mainly detects tumor markers in serum, and the detection mainly depends on AFP at present, but the sensitivity of AFP in small liver cancer is only about 40 percent, therefore, the occurrence and the development of tumors cannot be accurately diagnosed by singly using one tumor marker, the detection accuracy can be improved only by jointly detecting a plurality of tumor markers, the missed diagnosis and false positive are avoided or reduced, a new detection technology is applied, a marker combination with higher diagnosis value is selected, the detection sensitivity and the specificity of the existing tumor markers are improved, and the detection sensitivity and the specificity of the existing tumor markers are very important for the early diagnosis of liver cancer.

The monoclonal antibody can recognize and specifically bind to a single epitope of various bioactive substances such as proteins, polysaccharides, lipoproteins, neuropeptides, nucleic acids, etc., and is widely used in the field of life science research make internal disorder or usurp due to its homogeneous nature and easy mass production. The single chain antibody (scFv) is the smallest functional fragment of the antibody molecule that retains the antigen-binding portion, has a molecular weight of about 1/6 of the whole antibody, and is a recombinant protein formed by connecting the variable regions of the heavy chain and the light chain of the antibody through a connecting peptide by using a genetic engineering method. The single-chain antibody has the advantages that the single-chain antibody can be expressed in a large amount through an inclusion body, is particularly easy to construct antibody fusion protein, and has the characteristics of small molecule, low immunogenicity, no Fc end, difficult combination with target cells with Fc receptors, strong penetrating power to tumor tissues and the like. The monoclonal antibody and the single-chain antibody are used for detecting early liver cancer, and have incomparable advantages.

Disclosure of Invention

The present invention includes antibodies or fragments thereof that specifically bind to human liver cancer markers selected from the group consisting of alpha-fetoprotein (AFP), glutamyltransferase isozyme II (GGT II), α -L-fucosylglycase (AFU), Hepatocyte Growth Factor (HGF), and heparin sulfate proteoglycan 3(GPC 3).

Preferably the antibody is a monoclonal antibody characterized by comprising a light chain variable domain and a heavy chain variable domain, wherein

a) For antibodies against AFP, the light chain variable domain comprises the CDR1 region of SEQ ID NO:21, the CDR2 region of SEQ ID NO:22 and the CDR3 region of SEQ ID NO:23, and the heavy chain variable domain comprises the CDR1 region of SEQ ID NO:24, the CDR2 region of SEQ ID NO:25 and the CDR3 region of SEQ ID NO: 26;

b) for antibodies against AFU, the light chain variable domain comprises the CDR1 region of SEQ ID NO. 27, the CDR2 region of SEQ ID NO. 28 and the CDR3 region of SEQ ID NO. 29, and the heavy chain variable domain comprises the CDR1 region of SEQ ID NO. 30, the CDR2 region of SEQ ID NO. 31 and the CDR3 region of SEQ ID NO. 32;

c) for antibodies against GGT II, the light chain variable domain comprises the CDR1 region of SEQ ID NO 33, the CDR2 region of SEQ ID NO 34 and the CDR3 region of SEQ ID NO 35, and the heavy chain variable domain comprises the CDR1 region of SEQ ID NO 36, the CDR2 region of SEQ ID NO 37 and the CDR3 region of SEQ ID NO 38;

d) for antibodies against HGF, the light chain variable domain comprises the CDR1 region of SEQ ID NO:39, the CDR2 region of SEQ ID NO:40 and the CDR3 region of SEQ ID NO:41, and the heavy chain variable domain comprises the CDR1 region of SEQ ID NO:42, the CDR2 region of SEQ ID NO:43 and the CDR3 region of SEQ ID NO: 44; and

e) for antibodies against GPC3, the light chain variable domain comprises the CDR1 region of SEQ ID NO:45, the CDR2 region of SEQ ID NO:46 and the CDR3 region of SEQ ID NO:47, and the heavy chain variable domain comprises the CDR1 region of SEQ ID NO:48, the CDR2 region of SEQ ID NO:49 and the CDR3 region of SEQ ID NO: 50.

Preferably, the antibody or fragment thereof is characterized by comprising:

a) SEQ ID NO: 1 and the light chain variable domain of SEQ ID NO: 2;

b) SEQ ID NO:3 and the light chain variable domain of SEQ ID NO: 4;

c) SEQ ID NO:5 and the light chain variable domain of SEQ ID NO: 6;

d) SEQ ID NO: 7 and the light chain variable domain of SEQ ID NO: 8; or

e) SEQ ID NO: 9 and the light chain variable domain of SEQ ID NO: 10.

Preferably, the antibody or fragment thereof is characterized in that the antibody is of the human IgG2 subclass, of the human IgG1 subclass, or of the human IgM subclass.

Preferably, the fragment is a single chain antibody (scFv).

Another embodiment of the present invention is an assay or diagnostic kit or reagent comprising the antibody or fragment thereof of the present invention.

Preferably, the kit or reagent is used in an ELISA assay, Western Blotting assay, immunohistochemical assay or immunofluorescence assay, in particular for diagnosing liver cancer.

Another embodiment of the invention is the use of an antibody or fragment thereof according to the invention for the preparation of a kit or reagent for assays or diagnostics, preferably for ELISA assays, Western Blotting assays, immunohistochemical assays or immunofluorescence assays, in particular for the diagnosis of liver cancer.

Another embodiment of the invention is a nucleic acid encoding an antibody or fragment thereof according to the invention.

Another embodiment of the invention is an expression vector comprising a nucleic acid of the invention, preferably said vector is for expressing an antibody or fragment thereof that specifically binds to a human hepatoma marker selected from the group consisting of alpha-fetoprotein (AFP), glutamyltransferase isozyme II (GGT II), α -L-fucosidase (AFU), Hepatocyte Growth Factor (HGF), and heparin sulfate proteoglycan 3(GPC3) in a prokaryotic or eukaryotic host cell.

Another embodiment of the invention is a prokaryotic or eukaryotic host cell comprising a vector according to the invention.

The invention also comprises a method for producing a recombinant human or humanized antibody according to the invention, characterized in that a nucleic acid according to the invention is expressed in a prokaryotic or eukaryotic host cell and the antibody is recovered from the cell or the cell culture supernatant. The invention also includes antibodies obtainable by such recombinant methods.

The antibodies or fragments thereof according to the invention are particularly useful in ELISA assays, Western Blotting assays, immunohistochemical assays or immunofluorescence assays, in particular for the diagnosis of liver cancer.

Detailed Description

The invention discloses a monoclonal antibody and a single-chain antibody aiming at five liver cancer markers, a preparation method thereof and scientific research medical application, belonging to the technical field of cell engineering. The preparation method comprises the steps of taking AFP sold in the market and HGF, AFU, GGTII and GPC3 fusion proteins prepared in a laboratory as antigens, immunizing a Balb/c mouse to obtain B cells secreting antibodies, fusing the B cells with myeloma cells, and screening fusion cells secreting monoclonal antibodies; the prepared monoclonal antibody can be well applied to detection of corresponding antigens by ELISA, WesternBlotting, immunohistochemistry, immunofluorescence and the like; in addition, the invention also utilizes a genetic engineering method to respectively prepare the single-chain antibody (ScFv) only containing the heavy chain variable region and the light chain variable region of each antibody, and the constructed single-chain antibody has the characteristics of small molecule, low immunogenicity, no Fc end, strong penetrating power to tumor tissues and the like. The antibody is combined, so that an effective kit applied to early serological diagnosis of liver cancer patients is expected to be developed.

Specifically, the present invention provides the following:

1. the monoclonal antibodies comprise five monoclonal antibodies, namely HGF, AFP, AFU, GGT II and GPC3, and are characterized in that the monoclonal antibodies are prepared by taking commercially available AFP and self-prepared fusion proteins of HGF, AFU, GGT II and GPC3 as antigens, immunizing a Balb/c mouse to obtain B cells secreting the antibodies, fusing the B cells with myeloma cells, screening the fused cells secreting the monoclonal antibodies, injecting the fused cells into the abdominal cavity of the mouse to obtain ascites containing the antibodies, and purifying to obtain the monoclonal antibodies, wherein the HGF antibodies are IgG2B and IgG2a, the AFP antibodies are IgG2a, the AFU antibodies are IgM, the GPC3 antibodies are IgG2B, and the GGT II antibodies are IgG2B and IgG 1.

2. The monoclonal antibody according to the above 1, characterized in that the preparation method thereof comprises the steps of:

1) respectively immunizing Balb/c mice with HGF, AFP, AFU, GGTII and GPC3 antigen proteins, measuring the potency by an ELISA method, and taking the spleen of the mice to obtain B cells capable of secreting corresponding antibodies;

2) fusing a mouse spleen cell with a myeloma cell line SP2/0 by using a cell fusion technology, and screening by using an infinite dilution method, a finite dilution method and an ELISA (enzyme-linked immunosorbent assay) method to obtain a hybridoma with the function of secreting a monoclonal antibody;

3) injecting hybridoma cells into Balb/c mouse abdominal cavity injected with pristine in advance to obtain mouse ascites, dialyzing by salting out method and dialysis bag, and purifying monoclonal antibody by using HiTrap Protein G HP column;

4) and (4) eluting the combined purification column by using an AKTA protein separation and purification instrument to obtain the monoclonal antibody.

3. The monoclonal antibody according to the above 1, characterized in that the prepared HGF monoclonal antibody can be well applied to the detection of HGF by enzyme-linked immunosorbent assay (ELISA) and Western Blotting; the prepared AFP monoclonal antibody can be well applied to detection of AFP by ELISA, Western Blotting and Immunohistochemistry (IHC); the prepared AFU monoclonal antibody can be well applied to detection of AFU by Western Blotting; the prepared GGTII monoclonal antibody can be well applied to detection of GGTII by ELISA, Western Blotting and Immunofluorescence (IF); the prepared GPC3 monoclonal antibody can be well applied to detection of GPC3 by ELISA, Western Blotting and IF.

4. The monoclonal antibody use according to the above 3, characterized in that it comprises the steps of:

1) ELISA method: coating the prepared monoclonal antibody on an enzyme label plate, detecting serum of an HCC patient and normal human serum, incubating a goat anti-mouse secondary antibody, developing OPD, and detecting absorbance;

2) western Blotting method: loading different liver cancer cell proteins (HepG2, Hep3b, 7721, 7702 and the like) to an electrophoresis transfer membrane, incubating the prepared monoclonal antibody as a primary antibody, incubating a secondary antibody, performing ECL color development, and exposing in a dark room;

3) immunohistochemistry (IHC): using a paraffin section of liver cancer tissue, performing dewaxing, antigen retrieval and endogenous peroxidase inactivation, incubating the prepared monoclonal antibody as a primary antibody, incubating a secondary antibody, performing DAB color development, and performing counterstaining and dehydration steps and then mounting and photographing;

4) immunofluorescence (IF): the hepatoma carcinoma cells HepG2 were plated on a twelve-well plate with cover glass, fixed and punched, the monoclonal antibodies prepared were incubated as primary antibodies, secondary antibodies were incubated, and the cells were mounted for photography after nuclear staining with DAPI.

5. The five single-chain antibodies are characterized in that the five single-chain antibodies are prepared by performing the steps of Overlap PCR, plasmid construction, protein expression and purification and the like according to the sequences of the heavy chain variable region and the light chain variable region of the prepared HGF, AFP, AFU, GGTII and GPC3 monoclonal antibodies respectively, and have the characteristics of small molecules, low immunogenicity, no Fc end, strong penetrating power to tumor tissues and the like.

6. The single-chain antibody according to 5 above, characterized by the respective base sequences, is described in detail in the patent specification.

7. The single-chain antibody according to the above 5, characterized in that the preparation method comprises the steps of:

1) extracting RNA of hybridoma cell, reverse transcribing, and PCR amplifying to obtain variable region V_HLight chain variable region V_LThe fragments were ligated with a vector and transformed into competent DH5 α, selecting V_HAnd V_LAnd (4) positive cloning.

2) Respectively amplifying V of corresponding monoclonal antibodies by using PCR method_HAnd V_LThen V was ligated by the Overlap PCR method_H、V_LAnd (3) fragment.

3) Payz-HGF (AFP, AFU, GGTII, GPC3) ScFv plasmid is constructed by recombinant technology, expressed in large amount in Escherichia coli 16C9, and single-chain antibody is purified by HitrapPF affinity chromatography column.

According to the specific, the invention provides five types of antibodies aiming at different liver cancer marker antigens, namely an AFP monoclonal antibody and a single-chain antibody, an AFU monoclonal antibody and a single-chain antibody, a GGTII monoclonal antibody and a single-chain antibody, an HGF monoclonal antibody and a single-chain antibody, and a GPC3 monoclonal antibody and a single-chain antibody.

The antigen AFP of the five monoclonal antibodies is commercially available, the purity is more than 95%, and the HGF, AFU, GGTII and GPC3 antigens are prepared in the laboratory, expressed in escherichia coli and are fusion proteins expressed by pronucleus.

The preparation of the five monoclonal antibodies of the invention comprises the following steps:

The application steps of the monoclonal antibody prepared by the invention are as follows:

1) ELISA method: coating the prepared monoclonal antibody on an enzyme label plate, detecting serum and normal human serum of an HCC patient, incubating the biotin-labeled monoclonal antibody and HRP-labeled avidin, developing OPD, and detecting absorbance;

4) immunofluorescence (IF): spreading liver cancer cells HepG2 on a twelve-well plate with a cover glass, fixing and punching, incubating the prepared monoclonal antibody as a primary antibody, incubating a secondary antibody and staining nuclei with DAPI, and mounting and photographing;

the 5 antibodies screened by the invention have very good stability, and hybridoma cell strains maintain the characteristic of strong antibody secretion after multiple identifications. The method of directly sucking cell strains under a mirror is adopted in the preparation process of the antibody, so that the probability of obtaining monoclonal strains is greatly improved, and the sorting time is effectively shortened. The antigen adopted by the invention has high purity and strong immunogenicity, and the quality of the prepared antibody is ensured from the source.

The HGF monoclonal antibody prepared by the invention can be well applied to detection of HGF by ELISA and Western Blotting; the prepared AFP monoclonal antibody can be well applied to detection of AFP by ELISA, Western Blotting and IHC; the prepared AFU monoclonal antibody can be well applied to detection of AFU by ELISA and Western Blotting; the prepared GGTII monoclonal antibody can be well applied to detection of GGTII by ELISA, Western Blotting and IF; the prepared GPC3 monoclonal antibody can be well applied to detection of GPC3 by ELISA, Western Blotting and IF.

The single chain antibody (scFv) is the minimum functional fragment of the antibody molecule with the antigen binding part preserved, has molecular weight about 1/6 of a complete antibody, is a recombinant protein formed by connecting the variable regions of the heavy chain and the light chain of the antibody by a section of connecting peptide by using a genetic engineering method, and has the characteristics of small molecule, low immunogenicity, no Fc end, strong penetrating power to tumor tissues and the like. In order to better apply the liver cancer marker antibody to liver cancer detection, the invention prepares corresponding single-chain antibodies according to the heavy chain variable region and light chain variable region sequences of the prepared HGF, AFP, AFU, GGTII and GPC3 monoclonal antibodies respectively.

The preparation of the five single-chain antibodies comprises the following steps:

1) extracting RNA of hybridoma cell, reverse transcribing, and PCR amplifying to obtain variable region V_HLight chain variable region V_LThe fragments were ligated with the vector and transformed into DH5 α, selecting V_HAnd V_LAnd (4) positive cloning.

2) Payz-HGF (AFP, AFU, GGTII, GPC3) ScFv plasmid is constructed by recombinant technology, expressed in large amount in Escherichia coli 16C9, and single-chain antibody is purified by HitrapPF affinity chromatography column.

Definition of

The term "antibody" encompasses various forms of antibody structures, including, but not limited to, intact antibodies and antibody fragments. The antibodies according to the invention are preferably humanized, chimeric or otherwise genetically engineered antibodies, as long as the characteristic properties according to the invention are still retained.

The "class" of antibodies refers to the type of constant domain or constant region that the heavy chain has. There are five main classes of antibodies: IgA, IgD, IgE, IgG and IgM, and some of these may be further divided into subclasses (isotypes), e.g., IgG₁，IgG₂，IgG₃，IgG₄，IgA₁And IgA₂The heavy chain constant domains corresponding to different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively.

An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab ', Fab ' -SH, F (ab ')₂(ii) a A diabody; a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules consisting of a single amino acid.

"variable Domain" (light chain (V)_L) Of (2), the heavy chain (V)_H) Variable domains of (a) variable light and heavy chains have the same general structure and each domain comprises 4 Framework (FR) regions, the sequences of which are generally conserved, which are linked by 3 "hypervariable regions" (or complementarity determining regions, CDRs).

As used herein, the term "antigen-binding portion of an antibody" refers to the amino acid residues of an antibody that are responsible for antigen binding. The antigen-binding portion of an antibody includes amino acid residues from "complementarity determining regions" or "CDRs". The "framework" or "FR" regions are those variable domain regions other than the hypervariable region residues defined herein. Thus, the light and heavy chain variable domains of an antibody comprise, from N-terminus to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4. In particular, the CDR3 of the heavy chain is the region that is most conducive to antigen binding and defines the properties of the antibody. The CDR and FR regions, and/or those residues from the "hypervariable loops", are determined according to the standard definition of the protein sequence of Immunological Interest (Sequences of Proteins of Immunological Interest), 5 th edition, Public Health services, National Institutes of Health, Bethesda, Md. (1991), and National Health research institute.

The term "nucleic acid" or "nucleic acid molecule", as used herein, is intended to include both DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA.

The term "amino acid" as used herein refers to the group of naturally occurring carboxy α amino acids, which includes alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all of these designations include progeny. Thus, the words "transformant" and "transformed cell" include the primary test cell and the culture derived therefrom, regardless of the number of transfers. It is also understood that the DNA content of all progeny may not be exactly the same due to deliberate or inadvertent mutations. Variant progeny selected for the same function or biological activity in the originally transformed cell are included.

The antibodies according to the invention may be produced by recombinant means. Thus, one aspect of the invention is a nucleic acid encoding an antibody according to the invention, and another aspect is a cell comprising said nucleic acid encoding an antibody according to the invention. Methods for recombinant production are widely known in the art and involve protein expression in prokaryotic and eukaryotic cells, followed by antibody isolation and often purification to pharmaceutical purity. For expression of the foregoing antibodies in a host cell, the nucleic acids encoding the respective modified light and heavy chains are inserted into the expression vector by standard methods. Expression is carried out in suitable prokaryotic or eukaryotic host cells such as CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells, PER. C6 cells, yeast, or E.coli cells, and the antibody is recovered from the cells (supernatant or lysed cells). General methods for recombinant production of antibodies are well known in the art and are described, for example, in Makrides, S.C., protein Expr. Purif.17(1999) 183-202; geisse, S., et al, Protein Expr. Purif.8(1996) 271-; kaufman, R.J., molecular biology techniques (mol.Biotechnol.)16(2000) 151-161; werner, R.G., J.drug Res (J.Pharmacopeia.). 48(1998)870-880 for review.

The antibodies according to the invention are suitably isolated from the culture medium by conventional immunoglobulin purification methods such as, for example, protein a-sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. DNA and RNA encoding the monoclonal antibodies are readily isolated and sequenced using conventional methods. Hybridoma cells can serve as a source of the DNA and RNA. Once isolated, the DNA may be inserted into an expression vector which is then transfected into host cells that do not otherwise produce immunoglobulins, such as HEK293 cells, CHO cells, or myeloma cells, to obtain synthesis of recombinant monoclonal antibodies in the host cells.

The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It will be appreciated that modifications may be made without departing from the spirit of the invention.

Description of amino acid and nucleotide sequences

SEQ ID NO: 1 amino acid sequence of the light chain variable domain of an anti-AFP monoclonal antibody

SEQ ID NO:2 amino acid sequence of the variable domain of the heavy chain of an anti-AFP monoclonal antibody

SEQ ID NO:3 amino acid sequence of light chain variable domain of anti-AFU monoclonal antibody

SEQ ID NO:4 amino acid sequence of heavy chain variable domain of anti-AFU monoclonal antibody

SEQ ID NO:5 amino acid sequence of light chain variable domain of anti-GGT II monoclonal antibody

SEQ ID NO: 6 amino acid sequence of heavy chain variable domain of anti-GGT II monoclonal antibody

SEQ ID NO: 7 amino acid sequence of light chain variable domain of anti-HGF monoclonal antibody

SEQ ID NO: 8 amino acid sequence of heavy chain variable domain of anti-HGF monoclonal antibody

SEQ ID NO: 9 amino acid sequence of light chain variable domain of anti-GPC 3 monoclonal antibody

SEQ ID NO: amino acid sequence of the variable domain of the heavy chain of the 10 anti-GPC 3 monoclonal antibody

SEQ ID NO: 11 nucleotide sequence encoding the light chain variable domain of an anti-AFP monoclonal antibody

SEQ ID NO: 12 nucleotide sequence encoding the heavy chain variable domain of an anti-AFP monoclonal antibody

SEQ ID NO: 13 nucleotide sequence encoding the light chain variable domain of an anti-AFU monoclonal antibody

SEQ ID NO: 14 nucleotide sequence encoding the heavy chain variable domain of an anti-AFU monoclonal antibody

SEQ ID NO: 15 nucleotide sequence encoding the light chain variable domain of an anti-GGT II monoclonal antibody

SEQ ID NO: 16 nucleotide sequence encoding the heavy chain variable domain of an anti-GGT II monoclonal antibody

SEQ ID NO: 17 nucleotide sequence encoding the light chain variable domain of an anti-HGF monoclonal antibody

SEQ ID NO: 18 nucleotide sequence encoding the heavy chain variable domain of an anti-HGF monoclonal antibody

SEQ ID NO: 19 nucleotide sequence encoding the light chain variable domain of an anti-GPC 3 monoclonal antibody

SEQ ID NO: 20 nucleotide sequence encoding the heavy chain variable domain of an anti-GPC 3 monoclonal antibody

SEQ ID NO:21 CDR1 sequence of the light chain variable domain of an anti-AFP monoclonal antibody

SEQ ID NO:22 CDR2 sequence of the light chain variable domain of an anti-AFP monoclonal antibody

SEQ ID NO: CDR3 sequence of the light chain variable domain of a 23 anti-AFP monoclonal antibody

SEQ ID NO: CDR1 sequence of the variable domain of the heavy chain of a monoclonal antibody against AFP

SEQ ID NO:25 CDR2 sequence of the variable domain of the heavy chain of an anti-AFP monoclonal antibody

SEQ ID NO:26 CDR3 sequence of the variable domain of the heavy chain of an anti-AFP monoclonal antibody

SEQ ID NO: CDR1 sequence of the light chain variable domain of a 27 anti-AFU monoclonal antibody

SEQ ID NO: CDR2 sequence of light chain variable domain of 28 anti-AFU monoclonal antibody

SEQ ID NO: CDR3 sequence of the light chain variable domain of 29 anti-AFU monoclonal antibodies

SEQ ID NO: CDR1 sequence of the variable domain of the heavy chain of a 30 anti-AFU monoclonal antibody

SEQ ID NO: CDR2 sequence of the variable domain of the heavy chain of a 31 anti-AFU monoclonal antibody

SEQ ID NO: CDR3 sequence of the variable domain of the heavy chain of a 32 anti-AFU monoclonal antibody

SEQ ID NO: CDR1 sequence of the light chain variable domain of 33 anti-GGT II monoclonal antibody

SEQ ID NO: CDR2 sequence of light chain variable domain of 34 anti-GGT II monoclonal antibody

SEQ ID NO: CDR3 sequence of the light chain variable domain of 35 anti-GGT II monoclonal antibody

SEQ ID NO: CDR1 sequence of the variable domain of the heavy chain of a 36 anti-GGT II monoclonal antibody

SEQ ID NO: CDR2 sequence of the variable domain of the heavy chain of a 37 anti-GGT II monoclonal antibody

SEQ ID NO:38 CDR3 sequence of the variable domain of the heavy chain of an anti-GGT II monoclonal antibody

SEQ ID NO:39 CDR1 sequence of the light chain variable domain of an anti-HGF monoclonal antibody

SEQ ID NO: CDR2 sequence of light chain variable domain of 40 anti-HGF monoclonal antibody

SEQ ID NO:41 CDR3 sequence of light chain variable domain of anti-HGF monoclonal antibody

SEQ ID NO: CDR1 sequence of the variable domain of the heavy chain of a 42 anti-HGF monoclonal antibody

SEQ ID NO:43 CDR2 sequence of the variable domain of the heavy chain of an anti-HGF monoclonal antibody

SEQ ID NO: CDR3 sequence of the heavy chain variable domain of 44 anti-HGF monoclonal antibody

SEQ ID NO: CDR1 sequence of the light chain variable domain of the 45 anti-GPC 3 monoclonal antibody

SEQ ID NO: CDR2 sequence of the light chain variable domain of the anti-GPC 3 monoclonal antibody

SEQ ID NO: CDR3 sequence of the light chain variable domain of 47 anti-GPC 3 monoclonal antibody

SEQ ID NO: CDR1 sequence of the variable domain of the heavy chain of the 48 anti-GPC 3 monoclonal antibody

SEQ ID NO: CDR2 sequence of the variable domain of the heavy chain of a 49 anti-GPC 3 monoclonal antibody

SEQ ID NO: CDR3 sequence of the variable domain of the heavy chain of a 50 anti-GPC 3 monoclonal antibody

Drawings

FIG. 1. HGF monoclonal antibody purification pattern in example 3;

FIG. 2. AFP monoclonal antibody purification map in example 3;

FIG. 3 is a purification map of GGTII monoclonal antibody in example 3;

FIG. 4 detection of HGF expression in different cell lines by HGF H1 and H2 monoclonal antibodies (H1 is IgG2a type, H2 is IgG2b type, the first one was sequenced) in example 7;

FIG. 5 detection of AFP expression by AFP monoclonal antibody in example 7 in different cell lines;

FIG. 6 detection of GGTII expression in different cell lines by the GGTII monoclonal antibody of example 7;

FIG. 7 detection of GPC3 monoclonal antibody in example 7 on expression of GPC3 in different cell lines;

FIG. 8 detection of HGF content in human serum by ELISA method in example 6;

FIG. 9 immunohistochemistry with monoclonal antibody AFP in example 8 detects AFP expression in liver cancer tissue;

FIG. 10 shows the detection of GGTII expression of hepatoma cells by the immunofluorescence assay using a monoclonal antibody against GGTII in example 9;

FIG. 11 shows that the expression of GPC3 of hepatocarcinoma cells is detected by GPC3 monoclonal antibody immunofluorescence in example 9;

FIG. 12 SDS-PAGE of HGF single-chain antibody in example 5;

FIG. 13. electrophoretogram of overlap PCR joining AFP VH and AFP VL fragments in example 4;

FIG. 14 shows that the HGF monoclonal antibody immunohistochemistry method in example 8 detects the expression of AFP in liver cancer tissue (conclusion: the antibody can be applied to human hepatocyte liver cancer immunohistochemistry);

FIG. 15, AFU monoclonal antibody immunohistochemistry in example 8 detected AFP expression in liver cancer tissue (conclusion: antibodies can be applied to human hepatocyte liver cancer immunohistochemistry);

FIG. 16. amino acid sequence of each of the immunogenic proteins in example 1 (Note: the AFP antigen used is a commercially available protein);

FIG. 17 is a statistical graph of the serum content of AFP, AFU, HGF, GPC3 and GGT2 in example 10 in four groups of subjects;

FIG. 18. ROC plot of AFP, AFU, HGF, GPC3, and GGT2 alone in whole cohorts screened for liver cancer in whole-age liver cancer patients sera;

FIG. 19 ROC graph of HGF single index included in serum of patients with age and sex detection of patients with liver cancer at the whole population screening for liver cancer in example 10;

FIG. 20. ROC graph of sera from patients with liver cancer at the full age and early stage with dual index inclusion in subjects for gender detection in the full population screening for liver cancer in example 10;

FIG. 21 is a ROC plot of sera from patients with liver cancer at the full age and early stage of subjects tested by dual index inclusion in the cohort screening for liver cancer;

FIG. 22 is a ROC plot of sera from patients with liver cancer at the third index, in example 10, in screening liver cancer in the whole population, in the whole period and in the early stage;

FIG. 23 is a ROC plot of sera from patients with liver cancer at the whole population, four indices, in example 10, in screening for liver cancer;

FIG. 24 is a ROC plot of sera from patients with liver cancer at the full stage and early stage of combined five-index detection in example 10 when screening a whole population for liver cancer;

FIG. 25 is a ROC graph of the age-gender of subjects with HGF single index and HGF when screening for liver cancer in hepatitis patients, showing that the patients have liver cancer at the full stage and the early stage;

FIG. 26 ROC graph of serum from patients with liver cancer at the time of screening for liver cancer in patients with hepatitis, including subjects with dual markers for gender detection of patients with liver cancer at the whole stage and in early stages;

FIG. 27 ROC graph of serum from patients with liver cancer, tested for liver cancer at the full stage and early stage by the age of the subjects when screening for liver cancer in hepatitis patients, with dual index inclusion;

FIG. 28 is a ROC plot of sera from patients with liver cancer at the whole stage and early stage of the three-index detection in example 10 when screening patients with hepatitis for liver cancer;

FIG. 29 ROC graph of serum from patients with liver cancer at the whole stage and early stage in example 10, when patients with hepatitis are screened for liver cancer;

FIG. 30 is a ROC plot of sera from patients with liver cancer at the full stage and early stage according to the five indicators in example 10 when screening for liver cancer in hepatitis patients;

FIG. 31 ROC graph of single index of HGF and HGF included in age and sex detection of subjects for liver cancer in example 10 when screening for liver cancer in patients with cirrhosis;

FIG. 32 ROC graph of sera from patients with liver cancer tested by both index into subjects for either gender or age in screening for liver cancer in patients with liver cirrhosis;

FIG. 33 is a ROC graph of serum from patients with liver cancer in the whole stage and early stage of liver cancer in the screening of liver cancer in patients with liver cirrhosis in example 10;

FIG. 34 ROC graph of serum from patients with liver cancer in the whole stage and early stage of liver cancer detected by four markers when liver cancer was screened in patients with liver cirrhosis in example 10;

FIG. 35 ROC graph of sera from patients with liver cancer at full stage and early stage according to the five indicators when screening for liver cancer in patients with liver cirrhosis in example 10; and

FIG. 36 model validation of the values of AFP, AFU, HGF, GPC3, and GGT2 in serum of patients with liver cancer, serum of patients with hepatitis B disease, serum of patients with liver cirrhosis, and serum of normal persons in example 10.

Detailed Description

The present invention is described in detail below by way of examples, which are provided for further illustration only, and are not to be construed as limiting the scope of the present invention, and the following insubstantial modifications and adaptations of the present invention will occur to those skilled in the art based on the foregoing description of the present invention.

Example 1: preparation of HGF antibody animal immunization and determination of immune potency (in the case of HGF, the remaining antibody steps are the same or similar)

1) 3 female Balb/c mice of 7 weeks in size were immunized for the first time with 100. mu.g of HGF fusion protein (optionally e.g.HGF protein with a tag protein such as His) per mouse, added with an equal volume of Freund's complete adjuvant, emulsified and mixed by a three-way to form a water-in-oil chyle, injected subcutaneously in multiple spots.

2) After three weeks, the antigen dose is halved, and then the Freund's incomplete adjuvant and normal saline are added and mixed evenly, and the mixture is injected into the abdominal cavity. The same method is adopted for immunization for four times, and then venous blood is collected for measuring the titer.

3) Coating HGF protein, 4 ng/ml. Overnight at 4 ℃;

4) after washing with PBS once, blocking with 0.2% BSA at room temperature for 1 h;

5) the serum is diluted to 1:200, 1:400, 1:800, 1:2000, 1:4000, 1:8000 and 1:20000 respectively. PBS was used as blank control and nonimmunized mouse serum was used as negative control. Incubating at 37 ℃ for 2 h;

6) after PBS is washed for three times, the goat anti-mouse secondary antibody is incubated for 1h at the temperature of 37 ℃ at the ratio of 1: 5000;

7) washing with PBS for five times, and developing OPD for 5-l0 min;

8) the stop solution was stopped and the absorbance was measured at 492 nm.

Example 2: cell fusion and screening of fused cells

1) 2 Balb/c mice with the size of 7 weeks are sacrificed, and macrophages are taken as feeder cells and are paved in 6 pieces of 96-well plates;

2) three days after the venous shock, the mice were sacrificed and the spleens were collected and fused with myeloma cells SP2/0 and then spread in 6 96-well plates by an infinite dilution method. The culture conditions are 1 XHAT DMEM/F12 culture solution containing 20% FBS;

3) after 10 days, the colonies were observed for growth, during which time the serum-containing 1 XHAT DMEM/F12 culture medium was supplemented twice;

4) the hybridoma cells are observed under a microscope, and the specificity can be detected when the round and bright grape bunch grows. Screening out positive holes capable of being specifically combined with the antigen by an ELISA method for subcloning;

5) subcloning by limiting dilution method: several tens of cells were plated as culture cells in 6 96-well plates. The number of positive well cells was adjusted to 1X 10³About/ml, adding 1 × HT RPMI1640 culture medium containing serum to make the number of cells per well be 2/well, 1/well and 0.5/well respectively;

6) observing after 4-5 days, carrying out antibody detection on the supernatant by using an ELISA method, obtaining positive holes, carrying out expanded culture, and cloning until monoclonal antibody cells are obtained;

7) and performing amplification culture on the screened positive monoclonal strains, freezing and storing the positive monoclonal strains in a liquid nitrogen tank, and periodically recovering and checking the cell activity and the stability of the secreted antibody. The 5 antibodies screened by the invention have very good stability, and hybridoma cell strains maintain the characteristic of strong antibody secretion after multiple identifications.

Example 3: preparation and purification of ascites

1) 5 female Balb/c mice about 10 weeks old were prepared and intraperitoneally injected with 500. mu.l of prime per mouse, and after 7-21 days, 2X 10 injections were administered intraperitoneally to each mouse⁶The mice were closely observed after 10 days for several hybridoma cells in logarithmic growth phase, and the mice were sacrificed when ascites became as much as possible and the ascites were removed. Centrifuging at 3000rpm for 15min to remove impurities and remove oil.

2) Precipitation of immunoglobulins by salting out: ascites was diluted one time with PBS, and saturated ammonium sulfate was added to make the final concentration 33%. Shaking was carried out while dropping. Standing at 4 deg.C for more than 4 hr. 12000rpm, centrifugation for 15min and supernatant discarded. The precipitate was dissolved in PBS, placed in a dialysis bag and dialyzed overnight at 4 ℃.

3) Taking out the dialysate, loading the liquid into the column by using a swimming pump, wherein the immunoglobulin can be specifically combined with Protein G, loading the remained liquid into the column again to ensure that the combination is more complete, continuously passing the column by using PB buffer, and eluting the impure Protein;

4) and (3) loading the combined column on an AKTA protein separation and purification instrument, balancing PB buffer, then replacing the mobile phase with 0.1M glycine eluent, and observing a UV absorption peak. The eluate tube was kept on ice to avoid protein degradation;

5) the eluate was transferred to a 50ml concentration tube (100KD), centrifuged at 2000rpm at 4 ℃ for 4 hours. Continuously adding PBbuffer for replacement until about 1ml of protein solution is left, sucking out to an EP tube, detecting the OD value, and determining the concentration;

6) diluting monoclonal antibody to 1mg/ml, adding glycerol, packaging, and storing at-20 deg.C.

Example 4: construction of HGF Single chain antibody vector (in the case of HGF, the remaining antibody steps are the same or similar)

1) 2X 10 hybridoma cells are selected and are in logarithmic growth phase and can continuously secrete monoclonal antibodies⁷RNA was extracted using TRIZOL lysate.

2) The RNA was diluted with DEPC water to 300 ng/. mu.l, reverse transcription of the RNA was performed using a reverse transcription kit, and the resulting cDNA was used for PCR amplification.

3) PCR amplification of HGF V_H、HGF V_LFragment (b): the HGF cDNA synthesized by reverse transcription is used as a template, and P1, P2, P3 and P4 are used as primers to amplify the heavy chain and light chain variable region fragment, and the reaction system is 50 mu l.

Degenerate primers for amplification of heavy chain light chain variable region:

p1: heavy chain upstream 5' -SAGGTGMAGCTKCASSARTCWGG

P2: downstream of the heavy chain 5' -TGGGGSTGTYGTTTTGGCTGMRGAGACRGTGA

P3 kappa upstream 5' -GACATTGTKMTSACMCAGYMKYCM

P4 kappa downstream 5' -GGATACAGTTGGTGCAGCATCATCAGCCCGTTT

4) The reaction product is separated by 1.2 percent agarose gel electrophoresis, and purified and recovered by a gel cutting recovery kit.

5) HGF V to be recovered_HDNA、HGF V_LAnd (3) connecting the DNAs with pMD 19T Vector respectively, transforming the connection products into escherichia coli DH5 α competent cells respectively, selecting positive clones by blue-white screening, and extracting plasmids after sequencing is correct.

6) HGF V is amplified respectively by PCR method by taking sequencing positive clone as template_HAnd HGF V_LHGF V by overlap PCR_HAnd HGF V_LLigation was performed by a GlyGlyGlySer (G4S) gene fragment.

7) HGF V digestion by using restriction enzymes Mlu I and Sph I_H-V_LThe gene fragment was ligated to Payzvector digested with the same enzyme. The ligation product was transformed into 16C9 competent bacteria, positive clones were selected by colony PCR and used for expression of the fusion protein after correct sequencing.

The amino acid sequences of the light chain and heavy chain variable domains of the monoclonal antibody obtained by the invention and the coding nucleotide sequences thereof are shown in the following sequence table.

The base sequences of the five single-chain antibodies are as follows:

1)HGF SCFV(IgG2b)

GAGCTCGATATTCAGATGATACAGTCTCCATCCTCCGTGGAGGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTGAGGATATTAGTAGTAATTTAGCCTGGTGTCAGCAGAAACCAGGACAGCCTCCCAAGCTCCTGATCTATGGTGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTATACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTGCAGGCGGTTATAGTCGTGGTAGTGATACTTTTGCTTTCGGCGTGGGGACCAAGCTGGAAATAAAAGGTGGTTCCTCTAGATCTTCCCTCGAGGTGAAGCTGATGGAATCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACGCTGTCCTGCAAGGCTTCGGGCTACACATTTACTGACTATGAAATGCACTGGGTGAAGCAGACACCTGTGCATGGCCTGGAATGGATTGGAGCTATTGATCCTGAAACTGGTGGTACTGCCTACAGTCAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCTTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTACGCTAAGGATTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGAGAGTCAGTCCTTCCCAAATGTCACTAGTGGAGGTGGAGGTAAAGGAGGTGGAGGTGGCCAGGCCGGCCAGCACCATCACCATCACCATGGCGCATACCCGTACGACGTTCCGGACTACGCTTCTTAG

2)AFP SCFV(IgG2a)

GCGC

ACGCTGAGGTGCAGCTGCAGCAGTCAGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAACTACTATATGTACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAATCCTAGCAATGGTGATACTAACTTCAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCATACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGATGGAGGGCAACTCGGACTATAGGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACAGCCCCAGACATTGTGCTCACCCAGTCGCCCGCCTCCCTATCTGTATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTGAATTAACATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATGTTGCAAGAAACTTAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAATATTCCCTCAAGATCAACAGCCTGCAGTCTGAAGATTTTGGAATTTATTACTGTCAACATTTTTGGGGTACTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCC

CATCACCATCACCATCATTGA

GCGC

3)AFU SCFV(IgM)

GAGCTCGATATTAAGATAACCCAGTCTCCATCTTATCTTGCTGCATCTCCTGGAGAAACCATTACTATTAATTGCAGGGCAAGTAAGAGCATTAGCAAATATTTAGCCTGGTATCAAGAGAAACCTGGGAAAACTAATAAGCTTCTTATCTACTCTGGATCCACTTTGCAATCTGGAATTCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGATTTCACTCTCACCATCAGTAGCCTGGAGCCTGAAGATTTTGCAATGTATTACTGTCAACAGCATAATGAATACCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGCTGAAAGGTGGTTCCTCTAGATCTTCCCTCGAGGTGAAGCTTGAGGAGTCTGGGGCTGAGCTGGTGAGGCCTGGAGTCTCAGTGAAGATTTCCTGCAAGGGTTCTGGCTACACGCTCACTGATTATGCTATGCACTGGGTGAGGCAGAGTCCTGCAAAGAGTCTAGAGTGGATTGGAGTTATTAGTACATACTATGGTGATGCTAACTACAACCAGAAGTTCAAGGGCAAGGCCACAATGACTGTTGACAGACCCTCCAGCACAGCCTATATGGAACTTGCCAGACTGACATCTGAGGATTCTGCCATCTATTACTGTGCAAGAGATGGTTACGACGTCTTCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGAGAGTCAGTCCTTCCCAAATGTCACTAGTGGAGGTGGAGGTAAAGGAGGTGGAGGTGGCCAGGCCGGCCAGCACCATCACCATCACCATGGCGCATACCCGTACGACGTTCCGGACTACGCTTCTTAG

4)GGT Ⅱ SCFV(IgG2b)

GAGCTCGACATTTTGATGACTCAGACTCCACTCTCCCTGTCTGTCAGtCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAATCGGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGCTGAAAGGTGGTTCCTCTAGATCTTCCCTCGAGGTGCAGCTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGAGTCTCAGTGAAGATTTCCTGCAAGGGTTCTGGCTACACGTTCACTGATTATGCTATGCACTGGGTGAGGCAGAGTCCTGCAAAGAGTCTAGAGTGGATTGGAGTTATTAGTACATACTATGGTGATGCTAACTACAACCAGAAGTTCAAGGGCAAGGCCACAATGACTGTTGACAGATCCTCCAGCACAGCCTATATGGAACTTGCCAGACTGACATCTGAGGATTCTGCCATCTATTACTGTGCAAGAGATGGTTACGACGTCTTCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACAACAGCCCCATCTGTCACTAGTGGAGGTGGAGGTAAAGGAGGTGGAGGTGGCCAGGCCGGCCAGCACCATCACCATCACCATGGCGCATACCCGTACGACGTTCCGGACTACGCTTCTTAG

5)GPC3 SCFV(IgG2b)

GAGCTCGACATTGTGTTGACACAGTCTCCAGCCACCCTGTCTGTGTCTCCTGGGGAAACAGCCACCCTCTCCTGCTGGGCCAGTCAGAGTATTGGCACCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTACGGTGCATTCACCAGGGCCGCTGGTGTCCCAGACAGGTTCACTGGCAGTGGCTCTGGGACACTCTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAATTTATTATTGTCAGCAGTTTAATAACTGGCCTCGGACGTTCGGCCGGGGGACCAAGCTGGAAATCAAAGGTGGTTCCTCTAGATCTTCCCTCGAGGTGCAGCTGAAGGAGTCGGGACCTGGCCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTGATTATGGCTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAGTGGATGGGCTACATAAGCTACAGTGGTGGCACTAGCTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTTCTGTGCAAGAGATCCTGGGGAATATTACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCCCAGCCAAAACGACACCCCCATCTGTCACTAGTGGAGGTGGAGGTAAAGGAGGTGGAGGTGGCCAGGCCGGCCAGCACCATCACCATCACCATGGCGCATACCCGTACGACGTTCCGGACTACGCTTCTTAG

variable region amino acid sequences of heavy and light chains of AFP and nucleotide sequences encoding the same, wherein the bold and underlined parts are CDR region sequences: light chain:

GGATACAGTTGGTGCAGCATCAGCCCGTTTGATTTCCAGCTTGGTGCCTCCACCGAACGTCCACGGAGTACCCCAAAAATGTTGACAGTAATAAATTCCAAAATCTTCAGACTGCAGGCTGTTGATCTTGAGGGAATATTGTGTGCCTGATCCACTGCCACTGAACCTTGATGGCACACCATCTGCTAAGTTTCTTGCAACATAGACCAGGAGCTGAGGAGATTTTCCCTGTTTCTGCTGATACCATGTTAATTCACTGTAAATATTCTCACTTGCTCGACATGTGATGGTGACAGTTTCTCCCACAGATACAGATAGGGAGGCGGGCGACTGGGTGAGCACAATGTC

amino acid sequence:

DIVLTQSPASLSVSVGETVTITCRASENIYSELTWYQQKQGKSPQLLVYVARNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGIYYCQHFWGTPWTFGGGTKLEIKRADAAPTVS

heavy chain:

GAGGTGCAGCTGCAGCAGTCAGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAACTACTATATGTACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAATCCTAGCAATGGTGATACTAACTTCAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCATACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGATGGAGGGCAACTCGGACTATAGGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACAGCCCCA

amino acid sequence:

EVQLQQSGAELVKPGASVKLSCKASGYTFTNYYMYWVKQRPGQGLEWIGEINPSNGDTNFNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCTRWRATRTIGFAYWGQGTLVTVSAAKTTAP

the variable region amino acid sequences of the heavy chain and the light chain of AFU and the coding nucleotide sequences thereof:

light chain

GAGCTCGATATTAAGATAACCCAGTCTCCATCTTATCTTGCTGCATCTCCTGGAGAAACCATTACTATTAATTGCAGGGCAAGTAAGAGCATTAGCAAATATTTAGCCTGGTATCAAGAGAAACCTGGGAAAACTAATAAGCTTCTTATCTACTCTGGATCCACTTTGCAATCTGGAATTCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGATTTCACTCTCACCATCAGTAGCCTGGAGCCTGAAGATTTTGCAATGTATTACTGTCAACAGCATAATGAATACCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGCTGAAA

Amino acid sequence:

ELDIKITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPYTFGGGTKLELK

heavy chain

CTCGAGGTGAAGCTTGAGGAGTCTGGGGCTGAGCTGGTGAGGCCTGGAGTCTCAGTGAAGATTTCCTGCAAGGGTTCTGGCTACACGCTCACTGATTATGCTATGCACTGGGTGAGGCAGAGTCCTGCAAAGAGTCTAGAGTGGATTGGAGTTATTAGTACATACTATGGTGATGCTAACTACAACCAGAAGTTCAAGGGCAAGGCCACAATGACTGTTGACAGACCCTCCAGCACAGCCTATATGGAACTTGCCAGACTGACATCTGAGGATTCTGCCATCTATTACTGTGCAAGAGATGGTTACGACGTCTTCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGAGAGTCAGTCCTTCCCAAATGTCACTAGTGGAGGTGGAGGTAAAGGAGGTGGAGGT

Amino acid sequence:

LEVKLEESGAELVRPGVSVKISCKGSGYTLTDYAMHWVRQSPAKSLEWIGVISTYYGDANYNQKFKGKATMTVDRPSSTAYMELARLTSEDSAIYYCARDGYDVFYAMDYWGQGTSVTVSSESQSFPNVTSGGGGKGGGG

heavy and light chain variable region amino acid sequences of GGT2 and nucleotide sequences encoding the same:

light chain

GAGCTCGACATTTTGATGACTCAGACTCCACTCTCCCTGTCTGTCAGtCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAATCGGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGCTGAAA

Amino acid sequence:

ELDILMTQTPLSLSVSLGDQASISCRSNRSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLELK

heavy chain

CTCGAGGTGCAGCTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGAGTCTCAGTGAAGATTTCCTGCAAGGGTTCTGGCTACACGTTCACTGATTATGCTATGCACTGGGTGAGGCAGAGTCCTGCAAAGAGTCTAGAGTGGATTGGAGTTATTAGTACATACTATGGTGATGCTAACTACAACCAGAAGTTCAAGGGCAAGGCCACAATGACTGTTGACAGATCCTCCAGCACAGCCTATATGGAACTTGCCAGACTGACATCTGAGGATTCTGCCATCTATTACTGTGCAAGAGATGGTTACGACGTCTTCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACAACAGCCCCATCTGTCACTAGTGGAGGTGGAGGTAAAGGAGGTGGAGGT

Amino acid sequence:

LEVQLQQSGAELVRPGVSVKISCKGSGYTFTDYAMHWVRQSPAKSLEWIGVISTYYGDANYNQKFKGKATMTVDRSSSTAYMELARLTSEDSAIYYCARDGYDVFYAMDYWGQGTSVTVSSAKTTAPSVTSGGGGKGGGG

the variable region amino acid sequences of HGF heavy chain and light chain and the coding nucleotide sequences thereof:

light chain

GAGCTCGATATTCAGATGATACAGTCTCCATCCTCCGTGGAGGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTGAGGATATTAGTAGTAATTTAGCCTGGTGTCAGCAGAAACCAGGACAGCCTCCCAAGCTCCTGATCTATGGTGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTATACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTGCAGGCGGTTATAGTCGTGGTAGTGATACTTTTGCTTTCGGCGTGGGGACCAAGCTGGAAATAAAA

Amino acid sequence:

ELDIQMIQSPSSVEAAVGGTVTIKCQASEDISSNLAWCQQKPGQPPKLLIYGASTLASGVPSRFKGSGSGTQYTLTISDVQCDDAATYYCAGGYSRGSDTFAFGVGTKLEIK

heavy chain

CTCGAGGTGAAGCTGATGGAATCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACGCTGTCCTGCAAGGCTTCGGGCTACACATTTACTGACTATGAAATGCACTGGGTGAAGCAGACACCTGTGCATGGCCTGGAATGGATTGGAGCTATTGATCCTGAAACTGGTGGTACTGCCTACAGTCAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCTTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTACGCTAAGGATTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGAGAGTCAGTCCTTCCCAAATGTCACTAGTGGAGGTGGAGGTAAAGGAGGTGGAGGT

Amino acid sequence:

LEVKLMESGAELVRPGASVTLSCKASGYTFTDYEMHWVKQTPVHGLEWIGAIDPETGGTAYSQKFKGKATLTADKSSSTAYMELRSLTSEDSAVYYCTLRIAYWGQGTLVTVSAESQSFPNVTSGGGGKGGGG

the variable region amino acid sequences of the heavy chain and the light chain of GPC3 and the coding nucleotide sequences thereof:

light chain

GAGCTCGACATTGTGTTGACACAGTCTCCAGCCACCCTGTCTGTGTCTCCTGGGGAAACAGCCACCCTCTCCTGCTGGGCCAGTCAGAGTATTGGCACCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTACGGTGCATTCACCAGGGCCGCTGGTGTCCCAGACAGGTTCACTGGCAGTGGCTCTGGGACACTCTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAATTTATTATTGTCAGCAGTTTAATAACTGGCCTCGGACGTTCGGCCGGGGGACCAAGCTGGAAATCAAA

Amino acid sequence:

ELDIVLTQSPATLSVSPGETATLSCWASQSIGTYLAWYQQKPGQAPRLLIYGAFTRAAGVPDRFTGSGSGTLFTLTISSLQSEDFAIYYCQQFNNWPRTFGRGTKLEIK

heavy chain

CTCGAGGTGCAGCTGAAGGAGTCGGGACCTGGCCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTGATTATGGCTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAGTGGATGGGCTACATAAGCTACAGTGGTGGCACTAGCTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTTCTGTGCAAGAGATCCTGGGGAATATTACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCCCAGCCAAAACGACACCCCCATCTGTCACTAGTGGAGGTGGAGGTAAAGGAGGTGGAGGT

Amino acid sequence:

LEVQLKESGPGLVKPSQSLSLTCTVTGYSITSDYGWNWIRQFPGNKLEWMGYISYSGGTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYFCARDPGEYYYAMDYWGQGTSVTVSPAKTTPPSVTSGGGGKGGGG

example 5: expression and purification of Single chain antibody (in the case of HGF, the remaining antibody steps are the same)

1) Recombinant fusion protein expression vector Payz-HGF V_H-V_LColi 16C9 was transformed, and single colonies with the correct sequencing were inoculated into 15ml of 2 XYT medium containing 100. mu.g/ml ampicillin and cultured overnight at 37 ℃ in a thermostatted shaking flask with shaking at 200 rpm. Transferring the cultured small amount of bacteria liquid into 1000ml 2 XYT culture medium containing ampicillin 100 μ g/ml, performing amplification culture, centrifuging at 4 deg.C and 8000rpm for 10min, and collecting thallus.

2) Repeatedly freezing and thawing the collected thalli for several times between room temperature and minus 20 ℃, so as to increase the permeability of the bacterial wall and facilitate the lysis; according to the culture medium: adding 50ml of bacteria periplasmic cavity lysate into the thawed thalli at the lysate volume ratio of 40:1, shaking gently and mixing uniformly, and placing at 4 ℃ for shaking gently for 1 h; centrifuging at 4 deg.C and 12000rpm for 10min, and collecting supernatant;

3) putting the supernatant containing the target protein into a dialysis bag which is pretreated (after EDTA and sodium bicarbonate are mixed and boiled for 5min and then EDTA is boiled for 5min), dialyzing with 1 × PBS for 24h, changing the dialysate every 2h in the first 4h, and gradually prolonging the dialysate changing interval in the last 20h until the overnight;

4) the dialyzed periplasmic cavity protein solution was filtered through a 0.22 μm filter and purified by a HitrapPF affinity column. Eluting, concentrating protein, packaging, and storing at-20 deg.C to obtain HGF single chain antibody.

5) And detecting the purified single-chain antibody by SDS-PAGE gel electrophoresis.

Example 6: ELISA method for verifying the prepared monoclonal antibody (HGF is taken as an example, the steps of the other antibodies are the same)

1) The HGF monoclonal antibody is coated by an ELISA plate and stays overnight at 4 ℃;

2) after washing with PBS once, blocking with 0.2% BSA at room temperature for 1 h;

3) diluting 30 HCC patient serum and 30 normal human serum respectively at a ratio of 1:2, adding into an enzyme label plate micropore with PBS as a blank pair, and incubating for 2h at 37 ℃;

4) adding biotin-labeled HGF antibody for 1:500 dilution, and incubating for 1h at 37 ℃;

4) washing with PBS for three times, adding avidin-horseradish peroxidase (Advin-HRP), and incubating at 37 deg.C for 1 h;

5) washing with PBS for five times, and developing OPD for 5-l0 min;

6) the stop solution was stopped and the absorbance was measured at 492 nm.

Example 7: western Blotting method for verifying prepared monoclonal antibody

1) The cultured liver cancer cells HepG2(ATCC HB-8065), Hep3b (ATCC HB-8064), SMMC-7721 (commercially available from Shanghai Fuxiang Biotech Co., Ltd.), HL-7702 (commercially available from Tongpai Biotech Co., Ltd.) and the like were subjected to protein extraction using SDS lysate, and the protein concentration was measured by BCA method and then subjected to sampling or storage at-80 ℃.

2) 10% SDS-PAGE gels and 5% concentrated gels were prepared.

3) Loading 30-50 μ g protein, adding 5 × loading buffer before loading, and denaturing at 95 deg.C for 5 min; protein marker of appropriate molecular weight was chosen as a scale.

4) Electrophoresis: the constant voltage is 60V at the beginning, after the strip enters the separation gel, the voltage is adjusted to 120V, electrophoresis is stopped until bromophenol blue runs to the bottom of the gel, and Coomassie brilliant blue dyeing or film transfer is carried out.

5) Coomassie brilliant blue staining: soaking the gel in at least 5 times of Coomassie brilliant blue staining solution, incubating on a shaking table for 3-4h, removing the staining solution by using a decolorizing solution until the target band is clear, and scanning the gel.

6) And if not, performing film transfer. The membrane transferring clamp is internally provided with sponge, filter paper, a PVDF membrane and glue (a layer of sponge pad is firstly arranged in the positive direction, then three layers of filter paper are arranged, the PVDF membrane is arranged on the filter paper, the glue, three layers of filter paper, the sponge pad and the negative electrode), the PVDF membrane is arranged in a membrane transferring instrument, the constant current of 250mA is carried out, and the membrane is transferred for l-3h according to the molecular weight of target protein.

7) After the membrane transfer was completed, the PVDF membrane was washed once with TBST and incubated with 5% nonfat dry milk or BSA at room temperature for 1h to block the antigen.

8) Adding a primary antibody: antibodies were diluted to the appropriate concentration with skim milk powder, his-tag (l:1000), monoclonal culture supernatant or ascites fluid (1:1000-1:5000) were added to the membrane, incubated overnight at 4 ℃ and washed three times in TBST for 10min each.

9) The secondary antibody was incubated at room temperature for 1h, diluted to appropriate concentration (1:5000-1:10000) with 1% nonfat dry milk or 1% BSA, and washed three times with TBST for 10min each.

10) Chemiluminescence (ECL) color development, dark room exposure.

Example 8: immunohistochemistry method for verifying prepared monoclonal antibody

1) Dewaxing the paraffin section of the liver cancer tissue: drying and baking at 65 ℃ overnight-xylene I20 min-xylene II20 min-absolute ethanol I5-10 min-absolute ethanol II 5-10 min-95% ethanol 2 min-85% ethanol 2 min-75% ethanol 2 min-60% ethanol 2 min-distilled water cleaning.

2) Antigen heat repair: adding 400ml PBS +8ml antigen repairing solution into the slices, using a pressure cooker to maintain the temperature at 140 ℃ for 2min, and naturally cooling at room temperature.

3) Inactivation of endogenous peroxidase: throwing and wiping the slices to be dry with 3% of H₂O₂And (4) keeping the temperature away from light for 10min (operating in a wet plate, the liquid amount of each sheet is 50-60 mu l).

4) Incubation primary antibody (monoclonal antibody): washing with PBS for 3 times and 2 min; monoclonal antibodies were diluted to the appropriate concentration (1: 500-1: 2000) with 3% BSA and added to the sections, and after incubation for half an hour at room temperature, overnight at 4 ℃.

5) Incubation of secondary antibody: the sections taken out of the chamber at 4 ℃ are first incubated for half an hour at room temperature and washed 3 times with PBS for 2 min; the secondary antibody is diluted to a suitable concentration (1:500) with 3% BSA and added to the sections and incubated in an incubator at 37 ℃ for 30-40 min.

6) And (5) DAB color development.

7) And (3) counterstaining, wherein ① hematoxylin is washed by tap water for 3-5 min, ② hydrochloric acid alcohol is washed by tap water for about 30 seconds, purple-deep red-light red (observed color) is washed by the tap water, ③ 0.5.5% ammonia water is washed by the tap water for 1-2 min, cell staining is observed under a mirror, differentiation time (hydrochloric acid alcohol) is slightly longer when staining is deep, and staining is carried out in hematoxylin when staining is light.

8) And (3) dehydrating: 70% ethanol for 3min, 85% ethanol for 3min, 95% ethanol for 3min, absolute ethanol I for 5-10 min, absolute ethanol II for 5-10 min, xylene I for 1 hour, and xylene II for 1 hour (time is indefinite).

9) Sealing with neutral resin, storing and taking pictures.

Example 9: immunofluorescence method for verifying prepared monoclonal antibody

1) The day before the experiment will contain 2-3X 10⁴A single cell suspension of cells (HepG2) was plated in one well of a twelve-well dish with a coverslip (to ensure single cells). And (4) making two attaching holes under each condition, and after the cells are attached to the wall for 12-24 hours, using the attaching holes for experimental analysis.

2) Cells were washed 3 times with PBS and fixed with 1 ml/well of 4% PFA for 10min at room temperature. PFA was aspirated, washed 3 times with PBS, 1ml of 50mM ammonium chloride was added and incubated at room temperature for 10min, ammonium chloride was aspirated, and PBS was washed 3 times.

3) Adding 1ml of 0.2% Triton X-100 for 10 min; suction Triton, PBS wash 3 times;

4) adding 1ml of 3% BSA for blocking, and shaking on a shaking table at room temperature for 1 h; BSA was aspirated and washed with PBS for 10 min.

5) Plus primary antibody (monoclonal antibody), dilution ratio 1: 200-1: 500, antibodies (20. mu.l for double stain and 40. mu.l for single stain) were applied to the preservative film or sealing film, the coverslips removed from the twelve-well plate, blotted to remove excess water, and the side with cells exposed to the antibodies at room temperature >1h or overnight at 4 ℃.

6) The slide was replaced into the twelve-well plate with the cell side facing up, and washed 3 times with PBS for 10 min/time; adding secondary antibody, and diluting at a ratio of 1:400, and reacting at room temperature for 1h in the dark.

7) The slide was replaced in the twelve-well plate with the cell side facing up and washed 3 times with PBS for 10 min/time. DAPI was added to stain nuclei, typically at a dilution ratio of 1:1000, 20. mu.l of each cover glass, protected from light at room temperature for 10 min.

8) The slide was replaced in the twelve-well plate with the cell side facing up and washed 2 times with PBS for 10 min/time. The encapsulated tablets (mowoil) were dropped onto a glass slide, 15. mu.l each, with the cell side facing down, without air bubbles. After the sealing agent is dried, the sealing agent is placed at 4 ℃ for about 30min and then is stored in the dark.

9) The staining results were observed under a fluorescent microscope.

As can be seen from the attached drawing, the HGF monoclonal antibody prepared by the invention can be well applied to the detection of HGF by ELISA and Western Blotting; the prepared AFP monoclonal antibody can be well applied to ELISA, WesternBlotting and IHC for detecting AFP; the prepared AFU monoclonal antibody can be well applied to detection of AFU by ELISA and WesternBlotting; the prepared GGTII monoclonal antibody can be well applied to detection of GGTII by ELISA, Western Blotting and IF; the prepared GPC3 monoclonal antibody can be well applied to detection of GPC3 by ELISA, Western Blotting and IF.

Example 10 use of the antibodies of the invention or combinations thereof for diagnosing liver cancer

In Tianjin medical university tumor hospital, 60-300 cases of peripheral blood were collected from liver cancer patients, hepatitis B patients and normal patients, respectively. 288 serum of liver cancer patients, 87 serum of hepatitis B patients, 80 serum of cirrhosis patients and 241 serum of normal patients are collected in the experiment.

Collecting peripheral blood within 1 hr, centrifuging at 3000g for 5-10 min, transferring serum into new centrifuge tube, and storing in refrigerator at-80 deg.C.

By using the monoclonal antibodies or single chain antibodies prepared in the above examples, the concentrations of AFP, AFU, GPC3, GGT2, and HGF in the serum to be tested were measured by ELISA.

1) Diagnostic test evaluation index and selection

And (3) evaluating the diagnosis effect of the index to be detected by adopting Sensitivity (true positive rate, Sensitivity), Specificity (true negative rate, Specificity), Positive Predictive Value (PPV) and Negative Predictive Value (NPV):

sensitivity TP/(TP + FN). times.100%

Specificity TN/(TN + FP). times.100%

Positive predictive value TP/(TP + FP) × 100%

Negative predictive value TN/(TN + FN). times.100%

The correct index (Youden index) — (sensitivity + specificity) -100%

Wherein, TN (true negative); fp (false positive); tp (true positive); fn (false negative).

The correlation between sensitivity and specificity was shown by calculating all possible cut points using Receiver Operating Curves (ROC). By changing the tangent point, a plurality of pairs of true positive rate (namely sensitivity) and false positive rate (1-specificity) values are obtained, the false positive rate is used as a horizontal coordinate, the sensitivity is used as a vertical coordinate, and an ROC curve is drawn, so that the efficiency of the diagnostic system is reflected dynamically and objectively. The area under the ROC curve (AUC) represents the degree of overlap of the positive and negative diagnostic result distributions in the diagnostic system, reflecting the magnitude of the diagnostic system's ability to distinguish between positive and negative diagnostic results, i.e., the magnitude of the value of the diagnostic test. And determining a critical value of the diagnostic index by finding a maximum point of a correct index on an ROC curve, and randomly sampling for 1000 times by a Bootstrap method to determine a 95% credible interval of the critical value.

2) Construction of statistical models

Single-index and multi-index combined diagnosis test HCC is realized through a logistic regression model, and the HCC is respectively applied to healthy normal people, HBV infected people and liver cirrhosis patients, and ROC analysis is carried out on three groups of people and sick people. And establishing a logistic regression model according to the disease state, taking the formed prediction probability or the combined prediction factor as an analysis index, and establishing an ROC curve by combining a nonparametric model and a binormal model.

Logistic regression model:

and is provided with

Constant term β₀Natural logarithm of the ratio of probability of an individual developing disease (HCC) to probability of not developing disease (HBV-infected, liver cirrhosis patient, healthy person, or the sum of the three) at an exposure dose of 0. regression coefficient β₀，β₁，…β_nIndicating that the argument xi changes by one unit

The amount of change in (c). For the study variable (5 proteins) at a certain continuous cut-off point P_kβ Y is more than or equal to g (P)_k)，Y_ik＝1；

Y

_ik0. The method is the same as the diagnostic test combining multiple indexes, and the graph of the method is a plane so as to obtain the sensitivity and specificity to construct an ROC curve.

See the figure for analysis and statistics.

Results and discussion:

the current situation of low detection rate of early liver cancer reflects that the current diagnosis method of liver cancer has great limitation. The current diagnosis of liver cancer relies mainly on serological examination and imaging diagnosis. Serological examination mainly detects tumor markers in serum, and currently mainly depends on the detection of AFP, but the sensitivity of AFP in small liver cancer is only about 40%.

The antibody or the single-chain antibody fragment thereof specifically binding to the human liver cancer marker, which is prepared by the invention, can be distinguished from hepatitis B patients, cirrhosis patients and healthy patients to detect liver cancer when HGF single index detection is carried out in five markers of AFP, AFU, HGF, GPC3 and GGT2, and when HGF is combined with other markers to carry out double index, three index, four index and five index detection.

When liver cancer is screened in all people (liver cancer, hepatitis B, cirrhosis and normal people), liver cancer is screened in hepatitis B patients, and liver cancer is screened in cirrhosis patients, the HGF single index has high sensitivity and specificity in detecting the whole-stage liver cancer and the early-stage liver cancer. By using the antibody or the fragment thereof specifically binding to HGF prepared by the invention, by using the serological detection method of hepatoma, the single index of HGF is used to screen hepatoma in all populations (hepatoma, hepatitis B, cirrhosis and normal persons), the sensitivity of detecting the whole-stage hepatoma is 90.3%, the specificity is 90.5%, the sensitivity of detection in early stage (stage A) hepatoma patients in the Barcelona stage is 90.2%, the specificity is 84.1%, after the patients are aged and sexed, the sensitivity of HGF in the whole-stage hepatoma is 91.3%, the specificity is 90.9%, the sensitivity in the early stage hepatoma is 86.3%, and the specificity is 90.5%; screening liver cancer in hepatitis B patients, wherein the sensitivity of detecting the whole liver cancer by HGF single index is 94.4%, the specificity is 91.8%, the sensitivity of detecting the early liver cancer is 90.2%, the specificity is 91.9%, after the liver cancer is introduced into the age and sex of a subject, the sensitivity of detecting the whole liver cancer is 90.8%, the specificity is 95.2%, the sensitivity of detecting the early liver cancer is 92.2%, and the specificity is 90.2%; the sensitivity and specificity of detecting the whole-stage liver cancer and the early-stage liver cancer by the HGF single index can reach more than 72 percent and 73 percent when the liver cancer is screened from a liver cirrhosis patient, and the sensitivity and specificity are obviously superior to those of the AFP commonly used in clinic, which indicates that the HGF content in serum is independently detected, and the age and the sex of a subject can meet the requirements of clinical liver cancer diagnosis when necessary.

When two indexes are detected to screen liver cancer in all people, liver cancer is screened in hepatitis B patients, and liver cancer is screened in cirrhosis patients, HGF and one of AFP, AFU, GPC3 and GGT2 are respectively combined, so that high sensitivity and specificity can be achieved for detecting the whole-stage liver cancer and the early-stage liver cancer, the diagnosis of primary liver cancer patients is significant, and particularly, the combination of HGF and AFP is the optimal combination of serological diagnosis and early-stage diagnosis of the liver cancer patients.

Particularly, when the whole population is screened for liver cancer, only after the gender of a subject is included for statistics, the sensitivity of the combination of HGF and AFP for detecting the full-term liver cancer is 90.8 percent, the specificity is 93.1 percent, the sensitivity of the combination of HGF and AFU for detecting the full-term liver cancer is 87.2 percent, the specificity is 94.6 percent, the sensitivity of the combination of HGF and GPC3 for detecting the full-term liver cancer is 91.8 percent, the specificity is 90.6 percent, the sensitivity of the combination of HGF and GGT2 for detecting the full-term liver cancer is 89.3 percent, and the specificity is 93.1 percent; after the test subjects were aged, the sensitivity of the combination of HGF and AFP for detecting the full-term liver cancer was 92.3%, the specificity was 90.9%, the sensitivity of the combination of HGF and AFU for detecting the full-term liver cancer was 90.8%, the specificity was 91.6%, the sensitivity of the combination of HGF and GPC3 for detecting the full-term liver cancer was 90.3%, the specificity was 90.9%, and the sensitivity of the combination of HGF and GGT2 for detecting the full-term liver cancer was 89.3%, and the specificity was 92.3%. When detecting early liver cancer, HGF and AFP, HGF and AFU, HGF and GPC3, HGF and GGT2 are respectively combined together, and after the liver cancer is aged or sexed, the sensitivity can reach more than 86 percent and the specificity can reach more than 84 percent.

When screening for liver cancer in hepatitis B patients, only after the gender of a subject is included for statistics, the sensitivity of the combination of HGF and AFP for detecting the full-stage liver cancer is 92.9 percent, the specificity is 95.2 percent, the sensitivity of the combination of HGF and AFU for detecting the full-stage liver cancer is 93.4 percent, the specificity is 93.5 percent, the sensitivity of the combination of HGF and GPC3 for detecting the full-stage liver cancer is 93.4 percent, the specificity is 93.5 percent, the sensitivity of the combination of HGF and GGT2 for detecting the full-stage liver cancer is 92.3 percent, and the specificity is 93.5 percent. (ii) a After the test subjects were only included in the age group for statistics, the sensitivity of the combination of HGF and AFP for detecting the whole-stage liver cancer was 92.3%, the specificity was 93.5%, the sensitivity of the combination of HGF and AFU for detecting the whole-stage liver cancer was 93.9%, the specificity was 93.4%, the sensitivity of the combination of HGF and GPC3 for detecting the whole-stage liver cancer was 93.9%, the specificity was 93.4%, and the sensitivity of the combination of HGF and GGT2 for detecting the whole-stage liver cancer was 93.4%, and the specificity was 95.1%. When detecting early liver cancer, HGF and AFP, HGF and AFU, HGF and GPC3, HGF and GGT2 are respectively combined together, and after being aged or sexed, the sensitivity can reach more than 86.3 percent and the specificity reaches more than 83.9 percent. When liver cancer is screened in patients with liver cirrhosis, HGF and other four markers are respectively combined after the patients are aged or sexed, and the detection of the whole-stage liver cancer and the early-stage liver cancer can achieve higher sensitivity and specificity. The HGF and other four indexes are separately combined for detection, and after the age or sex of the subject is integrated, the method has significant significance for the diagnosis of the primary liver cancer patient, and particularly the combination of the HGF and the AFP is the optimal combination for serological diagnosis and early diagnosis of the liver cancer patient.

The method is characterized in that liver cancer is screened in all people by detecting three indexes, when the full-period liver cancer is detected, the combined use of HGF + AFP + AFU, HGF + AFP + GPC3, HGF + AFP + GGT2 and HGF + AFU + GPC3 can reach more than 91.8% of sensitivity and more than 90.2% of specificity, when the early-period liver cancer is detected, the combined use of HGF + AFP + AFU, HGF + AFP + GPC3 and HGF + AFP + GGT2 can reach more than 86.3% of sensitivity and more than 85.5% of specificity, when the liver cancer is screened in a hepatitis B patient, the combined use of HGF + AFP + AFU, HGF + AFP + GPC3, HGF + AFP + GGT2, AFF + GPC3 and HGF + GPC3+ GPC AFT 2 can reach more than 92.3% of specificity, when the liver cancer is detected, the sensitivity of the full-period liver cancer can reach more than 93.5%, and the combined use of HGF + AFP + GPC 363% and the sensitivity and the combined use of HGF +; when liver cancer is screened in a cirrhosis patient, the combined use of HGF and the other two markers can achieve a more satisfactory effect, which indicates that the combined detection of HGF and the other two markers can meet the requirements of clinical liver cancer serological diagnosis and early diagnosis.

When the four indexes are used for screening the liver cancer, the sensitivity and the specificity are improved, when the liver cancer is screened in all people, the sensitivity of the combination of HGF, AFP, GPC3 and GGT2 for detecting the whole-stage liver cancer is 91.3 percent, the specificity is 92.0 percent, the sensitivity for detecting the early-stage liver cancer is 86.3 percent and the specificity is 90.5 percent, when the liver cancer is screened in a patient B, the sensitivity of the combination of HGF, AFP, AFU and GGT2 for detecting the whole-stage liver cancer is 91.8 percent, the specificity is 98.4 percent, the sensitivity for detecting the early-stage liver cancer is 92.2 percent and the specificity is 98.4 percent, and when the HGF is used together with the other three indexes for screening the liver cancer in a cirrhosis patient, the high sensitivity and specificity can be.

The five markers are applied together, the sensitivity and the specificity are further improved, when liver cancer is screened in a whole population, the sensitivity of detecting the whole-stage liver cancer can reach 91.2 percent, the specificity can reach 93 percent, the sensitivity of detecting the early-stage liver cancer can reach 84.3 percent, the specificity can reach 87.2 percent, when liver cancer is screened in a hepatitis B patient, the sensitivity of detecting the whole-stage liver cancer is 91.8 percent, the specificity is 98.4 percent, the sensitivity of detecting the early-stage liver cancer is 92.2 percent, the specificity is 98.4 percent, when liver cancer is screened in a liver cirrhosis patient, the sensitivity of detecting the whole-stage liver cancer is 91.3 percent, the specificity is 76.2 percent, the sensitivity of detecting the early-stage liver cancer is 92.2 percent, and the specificity is. Is obviously superior to the gold standard AFP of the current clinical liver cancer detection.

In order to verify the accuracy of detection results, the detection values of five markers in liver cancer patient serum, normal human serum and hepatitis B disease human serum are modeled, the risk coefficients (RS) of all research objects are arranged from small to large and are divided into a high-risk group (RS is more than or equal to 0.5) and a low-risk group (RS is less than 0.5), most liver cancer patients are correctly classified into the high-risk group, the trend of a scatter diagram is obvious, and the model results are in line with the fact. The model can be used for analyzing the correlation between the detection values of five markers including AFP, AFU, HGF, GPC3 and GGT2 and liver cancer.

According to the invention, through a large number of sample verifications and statistical analyses, by utilizing the monoclonal antibody or the single-chain antibody fragment thereof prepared by the invention, HGF can be used as a liver cancer marker to be singly used for screening and early diagnosis of liver cancer, the detection sensitivity and specificity of the monoclonal antibody are greatly superior to those of clinical gold standard AFP, and the HGF can be used together with one or more other indexes to supplement HGF detection, so that the sensitivity and specificity of serological diagnosis of liver cancer are further improved.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and example should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

1. A kit or reagent for determination or diagnosis, characterized by comprising one or more of an antibody or fragment thereof against HGF and an antibody or fragment thereof against a marker selected from the group consisting of AFP, GGT II, AFU and GPC3, respectively,

the antibody is a monoclonal antibody characterized by comprising a light chain variable domain and a heavy chain variable domain, wherein

a) For antibodies against AFP, the light chain variable domain comprises the CDR1 region shown in SEQ ID NO:21, the CDR2 region shown in SEQ ID NO:22 and the CDR3 region shown in SEQ ID NO:23, and the heavy chain variable domain comprises the CDR1 region shown in SEQ ID NO:24, the CDR2 region shown in SEQ ID NO:25 and the CDR3 region shown in SEQ ID NO: 26;

b) for antibodies against AFU, the light chain variable domain comprises the CDR1 region shown in SEQ ID NO:27, the CDR2 region shown in SEQ ID NO:28 and the CDR3 region shown in SEQ ID NO:29, and the heavy chain variable domain comprises the CDR1 region shown in SEQ ID NO:30, the CDR2 region shown in SEQ ID NO:31 and the CDR3 region shown in SEQ ID NO: 32;

c) for antibodies against GGT II, the light chain variable domain comprises the CDR1 region shown in SEQ ID NO. 33, the CDR2 region shown in SEQ ID NO. 34 and the CDR3 region shown in SEQ ID NO. 35, and the heavy chain variable domain comprises the CDR1 region shown in SEQ ID NO. 36, the CDR2 region shown in SEQ ID NO. 37 and the CDR3 region shown in SEQ ID NO. 38;

d) for antibodies against HGF, the light chain variable domain comprises the CDR1 region shown in SEQ ID NO:39, the CDR2 region shown in SEQ ID NO:40 and the CDR3 region shown in SEQ ID NO:41, and the heavy chain variable domain comprises the CDR1 region shown in SEQ ID NO:42, the CDR2 region shown in SEQ ID NO:43 and the CDR3 region shown in SEQ ID NO: 44; and

e) for an antibody against GPC3, the light chain variable domain comprises a CDR1 region shown as SEQ ID NO:45, a CDR2 region shown as SEQ ID NO:46 and a CDR3 region shown as SEQ ID NO:47, and the heavy chain variable domain comprises a CDR1 region shown as SEQ ID NO:48, a CDR2 region shown as SEQ ID NO:49 and a CDR3 region shown as SEQ ID NO:50,

the fragment is Fv, Fab, Fab ', Fab ' -SH or F (ab ')₂。

2. The kit or reagent of claim 1 comprising 2, 3 or 4 of an antibody or fragment thereof against HGF and an antibody or fragment thereof against a marker selected from AFP, GGT II, AFU and GPC3, respectively.

3. The kit or reagent of claim 1 for use in an ELISA assay, a Western Blotting assay, an immunohistochemical assay, or an immunofluorescence assay.

4. The kit or reagent according to claim 3, which is used for diagnosing liver cancer.

5. The kit or reagent of claim 1, wherein:

a) antibodies to AFP comprise an amino acid sequence as set forth in SEQ ID NO: 1 and a light chain variable domain as set forth in SEQ ID NO: 2;

b) an antibody against AFU comprises the amino acid sequence as set forth in SEQ ID NO:3 and a light chain variable domain as set forth in SEQ ID NO: 4;

c) an antibody directed to GGT II comprises the amino acid sequence as set forth in SEQ ID NO:5 and a light chain variable domain as set forth in SEQ ID NO: 6;

d) the antibody to HGF comprises the amino acid sequence as set forth in SEQ ID NO: 7 and a light chain variable domain as set forth in SEQ ID NO: 8; or

e) The antibody to GPC3 comprises the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable domain as set forth in SEQ ID NO: 10.

6. The kit or reagent of claim 1, wherein the antibody is a human IgG2 subclass, human IgG1 subclass, or human IgM subclass monoclonal antibody.

7. Use of a combination of an antibody against HGF, which is a monoclonal antibody characterized by comprising a light chain variable domain and a heavy chain variable domain, or a fragment thereof, and one or more of an antibody or fragment thereof, respectively, against a marker selected from AFP, GGT II, AFU and GPC3, in the preparation of a kit or reagent for assay or diagnosis, wherein

the fragment is Fv, Fab, Fab ', Fab ' -SH or F (ab ')₂。

8. The use of claim 7, wherein the kit or agent comprises 2, 3 or 4 of an antibody or fragment thereof against HGF and an antibody or fragment thereof against a marker selected from AFP, GGT II, AFU and GPC3, respectively.

9. The use according to claim 7, wherein the kit or reagent is for an ELISA assay, a WesternBlotting assay, an immunohistochemical assay or an immunofluorescence assay.

10. The use according to claim 9, wherein the kit or reagent is for diagnosing liver cancer.

11. The use of claim 7, wherein:

12. Use according to claim 7, characterized in that the antibody is a monoclonal antibody of the human IgG2 subclass, of the human IgG1 subclass or of the human IgM subclass.