CN106814192B - Marker combination and detection kit for liver cancer detection - Google Patents

Abstract

The present invention relates to the marker or combinations thereof detected for liver cancer, for detecting their reagent and kit and their applications in liver cancer detects and mutually distinguishes liver cancer patient and hepatitis B patient, liver cirrhosis patient and/or healthy person.

Description

Marker combinations and detection kits for liver cancer detection

technical field

The present invention relates to markers or combinations thereof for liver cancer detection, reagents and kits for detecting them, and their use in liver cancer detection and in distinguishing liver cancer patients from hepatitis B patients, liver cirrhosis patients and/or healthy persons .

Background technique

Primary liver cancer (PLC, referred to as liver cancer) is one of the most common clinical malignant tumors. It has an insidious onset, rapid invasion and growth, easy recurrence after treatment, and extremely poor prognosis. It is called the "king of cancers". About half of the liver cancer patients in the world are concentrated in China. About 110,000 people die of liver cancer in China every year, ranking second in the death rate of malignant tumors. At present, surgical resection and liver transplantation are the most effective treatment methods for liver cancer, but the vast majority of liver cancer patients are in the middle and late stages when they see a doctor, and cannot be operated on, and the surgical resection rate is only 10-30%. For inoperable cases, the effect of traditional radiotherapy and chemotherapy is not good, and the survival time of patients generally does not exceed half a year. It can be seen that the early diagnosis of liver cancer is very important, and it is the key to clinical diagnosis, treatment and prognosis.

The low detection rate of early liver cancer reflects the limitations of current diagnostic methods for liver cancer. Currently, the diagnosis of liver cancer mainly relies on serological examination and imaging diagnosis. Serological examination mainly detects tumor markers in serum, and currently mainly relies on the detection of alpha-fetoprotein (AFP), but the sensitivity of AFP in small liver cancer is only about 40%. It can be seen that using one tumor marker alone cannot accurately diagnose the occurrence and development of tumors, and the joint detection of multiple tumor markers can improve the accuracy of detection and avoid or reduce missed diagnosis and false positives. Applying new detection techniques, selecting more diagnostically valuable marker combinations, and improving the sensitivity and specificity of existing tumor marker detection are extremely important for the early diagnosis of liver cancer. Currently, commonly used liver cancer markers mainly include: alpha-fetoprotein (AFP), α-L-fucosidase (AFU), hepatocyte growth factor (HGF), heparan sulfate proteoglycan 3 (GPC3), glutamyl transferase isoenzyme Ⅱ (GGT Ⅱ) and so on.

Alpha-fetoprotein (α-fetoprotein, AFP): AFP is currently the most commonly used diagnostic marker for HCC. AFP is a special protein contained in human blood during the embryonic period. The content in normal adult serum is very small, and hepatocellular carcinoma can be significantly raised.

α-L-fucosidase (alpha-L-fucosidase, AFU): In 1984, Deugnier first discovered that the activity of serum AFU in patients with HCC was significantly increased, and reported the effect on normal people, patients with liver cirrhosis and secondary metastatic liver cancer The measurement results of serum AFU in patients showed that its diagnostic sensitivity in primary liver cancer was 75%, and its specificity was 90%. It is considered that AFU may become a useful indicator for diagnosing HCC.

Hepatocyte growth factor (HGF): HGF is a polypeptide growth factor that can stimulate stem cell proliferation. HGF receptor (Met) is mainly distributed in the epithelial cells of various organs and tumor tissues. In severe hepatitis, tumor and other diseases, the level of HGF in serum can change significantly, and it is expected to become a diagnostic marker for HCC.

Heparan sulfate proteoglycan 3 (glypican-3, GPC3): GPC3 is located on the surface of the cell membrane and is a multifunctional co-receptor on the cell surface. Hsu et al. used differential display technology to discover the abnormal expression of GPC3 gene in HCC tissue for the first time. GPC3 has now become one of the important reference markers for the identification of benign and malignant liver diseases in liver biopsy specimens.

Glutamyltransferase isoenzyme 2 (γ-glutamyltransferase isoenzyme2, GGT2): GGT2 is considered to be the best liver cancer marker other than AFP. Currently, GGT2 is considered to be a carcinoembryonic protein produced by liver cells, and the sugar chain structure of GGT produced by liver cells changes. This difference can appear in the pre- and early stages of liver cancer, so it has high predictive value and early diagnostic value.

Contents of the invention

The invention discloses a group of marker combinations for serological detection of liver cancer, including alpha-fetoprotein (AFP), α-L-fucosidase (AFU), hepatocyte growth factor (HGF), heparan sulfate proteoglycan 3 ( GPC3), glutamyl transferase isoenzyme 2 (GGT2), can effectively distinguish liver cancer patients from hepatitis B patients, liver cirrhosis patients or healthy people. Detection of HGF by enzyme-linked immunosorbent assay (ELISA) alone or in combination with one or more of the other four markers can greatly improve the sensitivity and specificity of serological detection of full-stage liver cancer and early liver cancer. It is superior to the AFP single index commonly used in clinical detection of liver cancer. The invention provides a reliable method for serological detection of liver cancer and a detection kit.

In one aspect, the present invention relates to markers for liver cancer detection selected from the group consisting of alpha-fetoprotein (AFP), alpha-L-fucosidase (AFU), hepatocyte growth factor (HGF), heparan sulfate proteoglycan 3 (GPC3) and glutamyl transferase isozyme 2 (GGT2/GGT II), preferably HGF.

In another aspect, the present invention relates to a marker combination for liver cancer detection, wherein said marker combination comprises two or more (three, four or five) markers selected from the group consisting of: Alpha-fetoprotein (AFP), α-L-fucosidase (AFU), hepatocyte growth factor (HGF), heparan sulfate proteoglycan 3 (GPC3) and glutamyl transferase isozyme 2 (GGT2/GGT II).

In a preferred embodiment, the marker combination involved in the present invention comprises HGF and one or more (two, three or four) markers selected from the group consisting of: alpha-fetoprotein (AFP) , α-L-fucosidase (AFU), heparan sulfate proteoglycan 3 (GPC3) and glutamyl transferase isozyme 2 (GGT2/GGT II).

In a particularly preferred embodiment, the marker combination involved in the present invention is selected from the group consisting of: HGF+AFP; HGF+AFU; HGF+GPC3; HGF+GGT2; HGF+AFP+AFU; HGF+AFP HGF+AFP+GGT2; HGF+AFU+GPC3; HGF+GPC3+GGT2; HGF+AFP+GPC3+GGT2; HGF+AFP+AFU+GGT2; and HGF+AFP+GPC3+GGT2+AFU.

In another aspect, the present invention relates to the use of a reagent or combination of reagents capable of detecting a marker according to the present invention or a combination of markers according to the present invention in the preparation of a diagnostic agent or kit for the detection of liver cancer, preferably the liver cancer is Full-stage liver cancer or early liver cancer. Preferably, the age and/or gender of the subject is also considered when the diagnostic agent or kit is used.

In another aspect, the present invention relates to a reagent or combination of reagents capable of detecting a marker according to the present invention or a combination of markers according to the present invention for use in differentiating liver cancer patients from hepatitis B patients, liver cirrhosis patients and/or healthy individuals The application in the diagnostic agent or kit, preferably, the liver cancer is full-stage liver cancer or early liver cancer. Preferably, the age and/or gender of the subject is also considered when the diagnostic agent or kit is used.

In a preferred embodiment of the above aspects, the reagent or reagent combination comprises one or more (1, 2, 3, 4 or 5) antibodies or fragments thereof that specifically bind to human liver cancer markers, said Human liver cancer markers are selected from the group consisting of alpha-fetoprotein (AFP), glutamyltransferase isozyme II (GGT II or GGT2), alpha-L-fucosidase (AFU), hepatocyte growth factor (HGF) and heparan sulfate proteoglycan 3 (GPC3), preferably the reagent or reagent combination comprises 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 One or more (1, 2, 3 or 4) of these.

Preferably, said antibody is a polyclonal antibody or a monoclonal antibody of human IgG2 subclass, human IgG1 subclass or human IgM subclass, preferably said fragment is a single chain antibody (scFv), preferably wherein said antibody or fragment thereof is used In ELISA assay, WesternBlotting assay, immunohistochemistry assay or immunofluorescence assay.

In another aspect, the present invention relates to a kit comprising a reagent or a combination of reagents capable of detecting the marker of the present invention or the combination of markers of the present invention, the kit is used for detecting liver cancer or for comparing liver cancer patients with hepatitis B patients , liver cirrhosis patients and/or healthy persons, preferably the liver cancer is full-stage liver cancer or early liver cancer, more preferably the kit also considers the age and/or sex of the subject when used, preferably the kit Comprising one or more (1, 2, 3, 4 or 5) antibodies or fragments thereof that specifically bind to said marker, more preferably said fragments are single chain antibodies (scFv).

Detailed description of the invention

In order to improve the low detection rate of clinical liver cancer and the low sensitivity of existing serological detection methods, the present invention provides a combination of liver cancer markers, AFP, AFU, HGF, GPC3 and GGT2. Single-marker detection or combined application of HGF and other markers has greatly improved the sensitivity and specificity of serological detection of full-stage liver cancer and early-stage liver cancer, providing a reliable method for the detection of liver cancer.

Specifically, the present invention provides the following:

1. A combination of markers for liver cancer detection, including alpha-fetoprotein (AFP), α-L-fucosidase (AFU), hepatocyte growth factor (HGF), heparan sulfate proteoglycan 3 (GPC3), gluten Aminoacyltransferase isozyme 2 (GGT2);

2. According to the marker combination described in the above 1, it is characterized in that among the five markers of AFP, AFU, HGF, GPC3 and GGT2, HGF single-index detection is performed, and HGF is combined with other markers for double-index, three-index, and four-index detection. When detecting indicators and five indicators, it can be distinguished from patients with hepatitis B, patients with liver cirrhosis and healthy people to detect liver cancer;

3. The method for detecting liver cancer, which is characterized in that the content of five markers of AFP, AFU, HGF, GPC3 and GGT2 in the serum of the same patient is measured, and it is determined that he has liver cancer by statistically analyzing the amounts of the five markers possibility of

4. According to the method described in the above 3, the determination of the contents of the five markers is carried out by enzyme-linked immunosorbent assay (ELISA);

5. According to the method described in 3 above, when screening liver cancer in the whole population (liver cancer, hepatitis B, liver cirrhosis and normal people), screening liver cancer in patients with hepatitis B, and screening liver cancer in patients with liver cirrhosis, HGF alone The index detection of full-stage liver cancer and early liver cancer has high sensitivity and specificity. Detecting HGF alone, including the age and sex of the subjects when necessary, can meet the needs of clinical liver cancer diagnosis. HGF is a better marker of liver cancer than AFP thing;

6. According to the method described in the above 3, when detecting two indicators to screen liver cancer in the whole population, screen liver cancer in patients with hepatitis B, and screen liver cancer in patients with liver cirrhosis, after one of age or sex is included, HGF and AFP, AFU, GPC3, and GGT2 combined with one of the four markers can achieve high sensitivity and specificity in the detection of full-stage liver cancer and early liver cancer, which is of great significance for the diagnosis of primary liver cancer patients, especially The combination of HGF and AFP is the best combination for the serological diagnosis and early diagnosis of liver cancer patients, indicating that the combined detection of HGF and one of the other four markers can be used as a single index detection of HGF if necessary, considering the age or gender of the subject Supplementary to meet the needs of clinical liver cancer diagnosis;

7. According to the method described in 3 above, detect the three indicators to screen liver cancer in the whole population. When detecting full-stage liver cancer, HGF+AFP+AFU is used in combination, HGF+AFP+GPC3 is used in combination, HGF+AFP+GGT2 is used in combination When used in combination with HGF+AFU+GPC3, it can achieve a sensitivity of over 91.8% and a specificity of over 90.2%. When detecting early liver cancer, the combination of HGF+AFP+AFU, HGF+AFP+GPC3, and HGF When +AFP+GGT2 is used in combination, the sensitivity can reach more than 86.3% and the specificity can reach more than 85.5%. When screening liver cancer in patients with hepatitis B, HGF+AFP+AFU, HGF+AFP+GPC3, HGF+AFP+GGT2 , combined application of HGF+AFU+GPC3 and HGF+GPC3+GGT2, the sensitivity of detection of full-stage liver cancer can reach more than 92.3%, the specificity can reach more than 93.5%, and the detection of early liver cancer is also good, especially HGF+AFP The combination of +AFU has a sensitivity of 90.2% and a specificity of 98.4%. When screening liver cancer in patients with liver cirrhosis, HGF combined with the other two markers can also achieve satisfactory results, indicating that HGF and the other two markers can also achieve satisfactory results. The combined detection of multiple markers can meet the needs of clinical serological diagnosis and early diagnosis of liver cancer;

8. According to the method described in the above 3, the sensitivity and specificity of the detection of four indicators for screening liver cancer are improved. When screening liver cancer in the whole population, the combination of HGF, AFP, GPC3 and GGT2 can detect the whole stage of liver cancer The sensitivity of HGF, AFP, AFU and GGT2 is 91.3%, the specificity is 92.0%, the sensitivity for detecting early liver cancer is 86.3%, and the specificity is 90.5%. The sensitivity of liver cancer is 91.8%, the specificity is 98.4%, the sensitivity of detecting early liver cancer is 92.2%, and the specificity is 98.4%. Higher sensitivity and specificity can be achieved;

9. According to the method described in 3 above, the combined application of the five markers can further improve the sensitivity and specificity. When screening liver cancer in the whole population, the sensitivity of detecting liver cancer at all stages can reach 91.2%, and the specificity can reach 91.2%. The sensitivity of detecting early liver cancer is 84.3%, and the specificity can reach 87.2%. When screening liver cancer in patients with hepatitis B, the sensitivity of detecting full-stage liver cancer is 91.8%, and the specificity is 98.4%. The sensitivity is 92.2%, and the specificity is 98.4%. When screening liver cancer in patients with liver cirrhosis, the sensitivity for detecting full-stage liver cancer is 91.3%, the specificity is 76.2%, and the sensitivity for detecting early liver cancer is 92.2%. The specificity is 66.7%;

10. A kit for serological detection of liver cancer, comprising one or more monoclonal antibodies of AFP, AFU, HGF, GPC3 and GGT2, which can be used for ELISA detection of patient serum.

More specifically, the present invention provides a method for the joint application of AFP, AFU, HGF, GPC3 and GGT2, which includes the steps of:

1) Collect serum from different groups of people, including liver cancer patients, hepatitis B patients, liver cirrhosis patients and healthy people;

2) Detect the contents of five markers, AFP, AFU, HGF, GPC3 and GGT2 in each case of serum by enzyme-linked immunosorbent assay (ELISA). ;

3) Perform single-indicator, two-indicator, three-indicator, four-indicator and five-indicator joint ROC curve analysis on the content of the five markers, and further statistical analysis of the results;

4) According to the case data, liver cancer patients were staged according to the Barcelona standard, and the effects of single markers and combinations of two, three or more markers in early (A), middle (B) and late (C+D) stages were analyzed. Sensitivity and Specificity.

5) Model the detection values of the five markers in serum and analyze their accuracy in predicting liver cancer.

The liver cancer marker combination described in the present invention, that is, AFP, AFU, HGF, GPC3 and GGT2, the content of the five indicators in the serum of patients with liver cancer is significantly higher than that of healthy people (p<0.0001); the levels of AFP, HGF and GGT2 in liver cancer The serum levels of patients were higher than those of hepatitis B patients and liver cirrhosis patients, while the serum levels of AFU in liver cancer patients were lower than those of hepatitis B patients and higher than those of liver cirrhosis patients, and the serum levels of GPC3 in liver cancer patients were higher than those of hepatitis B patients and lower than those of liver cirrhosis patient.

The liver cancer serological detection method of the present invention uses a single HGF index to screen liver cancer in all populations (liver cancer, hepatitis B, liver cirrhosis and normal people), and its sensitivity for detecting full-stage liver cancer is 90.3%, and its specificity is 90.5%. %, in patients with early stage (A stage) liver cancer in the Barcelona stage, the sensitivity of the detection was 90.2%, the specificity was 84.1%, and the sensitivity of HGF in full-stage liver cancer was 91.3% %, the specificity is 90.9%, the sensitivity in early liver cancer is 86.3%, and the specificity is 90.5%; in the screening of liver cancer in patients with hepatitis B, the sensitivity of HGF single-index detection of full-stage liver cancer is 94.4%, and the specificity is 91.8%, the sensitivity of detecting early liver cancer is 90.2%, and the specificity is 91.9%. In the screening of liver cancer in patients with liver cirrhosis, the sensitivity and specificity of HGF single-index detection of full-stage liver cancer and early liver cancer can reach 72% and 73%, which are significantly better than those commonly used in clinical practice. AFP, indicating that the detection of HGF content in serum alone, if necessary, combined with the subject's age and sex can meet the needs of clinical diagnosis of liver cancer.

In the liver cancer serological detection method of the present invention, when the two indicators are used to screen liver cancer in the whole population, only the gender of the subject is included for statistics, and the sensitivity of the combination of HGF and AFP to detect liver cancer in the whole stage is 90.8%. The sensitivity was 93.1%, the sensitivity of HGF and AFU combined to detect full-stage liver cancer was 87.2%, and the specificity was 94.6%. The sensitivity of HGF and GPC3 combined to detect full-stage liver cancer was 91.8%, and the specificity was 90.6%. The sensitivity of combined with GGT2 to detect full-stage liver cancer is 89.3%, and the specificity is 93.1%; after only including the age of the subjects, the sensitivity of combined HGF and AFP to detect full-stage liver cancer is 92.3%, and the specificity is 90.9% , the sensitivity of HGF combined with AFU to detect full-stage liver cancer is 90.8%, and the specificity is 91.6%. The sensitivity of combined HGF and GPC3 to detect full-stage liver cancer is 90.3%, and the specificity is 90.9%. The sensitivity to detect full-stage liver cancer was 89.3%, and the specificity was 92.3%. In the detection of early liver cancer, the combination of HGF and AFP, HGF and AFU, HGF and GPC3, HGF and GGT2 respectively, after including age or gender, can achieve a sensitivity of more than 86% and a specificity of more than 84%. sex.

When screening liver cancer in hepatitis B patients, only the gender of the subjects was included for statistics. The sensitivity of the combination of HGF and AFP to detect the full-stage liver cancer was 92.9%, and the specificity was 95.2%. The combination of HGF and AFU to detect the full-stage liver cancer The sensitivity is 93.4%, the specificity is 93.5%, the sensitivity of the combination of HGF and GPC3 to detect the full-stage liver cancer is 93.4%, the specificity is 93.5%, the sensitivity of the combination of HGF and GGT2 to detect the full-stage liver cancer is 92.3%, the specificity was 93.5%. ; After only including the subject’s age for statistics, the sensitivity of the combination of HGF and AFP to detect full-stage liver cancer is 92.3%, and the specificity is 93.5%. The sensitivity of HGF and AFU to detect full-stage liver cancer is 93.9%, and the specificity is The combined use of HGF and GPC3 has a sensitivity of 93.9% and a specificity of 93.4% for the detection of full-stage liver cancer. The combined use of HGF and GGT2 has a sensitivity of 93.4% and a specificity of 95.1% for the detection of full-stage liver cancer. In the detection of early liver cancer, the combination of HGF and AFP, HGF and AFU, HGF and GPC3, HGF and GGT2 respectively, after including age or gender, can achieve a sensitivity of more than 86.3% and a specificity of more than 83.9% sex. When screening liver cancer in patients with liver cirrhosis, after including one of age or gender, HGF is combined with the other four markers respectively, and the detection of full-stage liver cancer and early liver cancer can achieve high sensitivity and specificity. It shows that the combined detection of HGF and other four indicators alone, combined with the age or gender of the subjects, has a significant significance for the diagnosis of patients with primary liver cancer, especially the combination of HGF and AFP is a good choice for serological diagnosis and early diagnosis of liver cancer patients. best combination.

The serological detection method of liver cancer described in the present invention detects three indicators to screen liver cancer in the whole population. When detecting full-stage liver cancer, HGF+AFP+AFU is used in combination, HGF+AFP+GPC3 is used in combination, and HGF+AFP+GGT2 is used in combination. When used in combination, it can achieve a sensitivity of more than 91.8% and a specificity of more than 90.2%. Both can reach a sensitivity of more than 86.3% and a specificity of more than 85.5%. When screening liver cancer in patients with hepatitis B, the combined application of HGF+AFP+AFU, HGF+AFP+GPC3, HGF+AFU+GPC3 and HGF+GPC3+GGT2 can detect all stages of liver cancer with a sensitivity of more than 92.3%. The sensitivity can reach more than 93.5%, and the detection of early liver cancer also has a good effect, especially the combination of HGF+AFP+AFU, the sensitivity reaches 90.2%, and the specificity reaches 98.4%. When screening liver cancer in patients with liver cirrhosis, HGF combined with the other two markers can also achieve satisfactory results. It shows that the combined detection of HGF and the other two markers can meet the needs of clinical serological diagnosis and early diagnosis of liver cancer.

The serological detection method for liver cancer of the present invention has improved sensitivity and specificity when detecting four indicators for screening liver cancer. When screening liver cancer in the whole population, the combination of HGF, AFP, GPC3 and GGT2 has a sensitivity of 91.3% and a specificity of 92.0% for the detection of full-stage liver cancer, and a sensitivity of 86.3% and a specificity of 90.5% for the detection of early liver cancer ; When screening liver cancer in patient B, the combination of HGF, AFP, AFU and GGT2 has a sensitivity of 91.8% and a specificity of 98.4% for the detection of full-stage liver cancer, and a sensitivity of 92.2% and a specificity of 98.4% for the detection of early liver cancer %; When screening liver cancer in patients with liver cirrhosis, when HGF is used in combination with the other three indicators, they can achieve high sensitivity and specificity.

The serological detection method for liver cancer described in the present invention uses five markers in combination. When screening liver cancer in the whole population, the sensitivity of detecting liver cancer in the whole stage can reach 91.2%, and the specificity can reach 93%. It can detect early liver cancer The sensitivity is 88.2%, and the specificity can reach 87.2%. When screening liver cancer in patients with hepatitis B, the sensitivity of detecting full-stage liver cancer is 91.8%, the specificity is 98.4%, and the sensitivity of detecting early liver cancer is 92.2%. The specificity was 98.4%. When screening liver cancer in patients with liver cirrhosis, the sensitivity for detecting full-stage liver cancer was 91.3%, and the specificity was 76.2%. The sensitivity for detecting early liver cancer was 92.2%, and the specificity was 66.7%. Significant It is superior to the current gold standard AFP for clinical liver cancer detection.

In order to verify the accuracy of the detection results, the present invention establishes a model for the detection values of five markers in liver cancer patient serum, normal human serum and hepatitis B patient serum, and arranges the risk coefficients (RS) of all research objects from small to large, Divided into high-risk group (RS≥0.5) and low-risk group (RS<0.5), most liver cancer patients were correctly classified into high-risk group, and the trend of scatter plot was obvious, indicating that the model results were in line with the facts. This model can be used to analyze the correlation between the detected values of AFP, AFU, HGF, GPC3, and GGT2 five markers and liver cancer.

Through a large number of sample verification and statistical analysis, the present invention proves that HGF can be used as a liver cancer marker alone for screening and early diagnosis of liver cancer, and its detection sensitivity and specificity are much better than the clinical gold standard AFP. The combined use of multiple indicators can be used as a supplement to HGF detection to further improve the sensitivity and specificity of serological diagnosis of liver cancer.

The present invention also provides an ELISA kit for serological detection of liver cancer, comprising one or more pairs of monoclonal antibodies among HGF, AFP, AFU, GPC3, and GGT2, which can be simultaneously tested in one experiment by the double-antibody sandwich method. Determining the content of one or more indicators in the subject's serum is expected to be developed into a powerful tool for serological clinical diagnosis of liver cancer.

definition

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

The "class" of an antibody refers to the type of constant domain or region that its heavy chain possesses. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), for example, IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ . The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

"Antibody fragment" refers to a molecule other than an intact antibody that comprises the portion of an intact antibody that binds the 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 ; diabodies; linear antibodies; single chain antibody molecules (e.g. scFv); Sexual antibodies.

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

"Variable domain" (variable domain of light chain (V _L ), variable domain of heavy chain (V _H )) as used herein means each pair of light chain and heavy chain domain. The domains of the variable light and heavy chains have the same general structure and each domain includes 4 framework (FR) regions whose sequences are generally conserved and which are passed through 3 "hypervariable regions" (or complementary Determining regions, CDRs) are connected. The framework regions adopt a β-sheet conformation and the CDRs can form loops connecting the β-sheet structures. The CDRs in each chain are held in their three-dimensional structure by the framework regions and together with the CDRs from the other chain form the antigen-binding site. Antibody heavy and light chain CDR3 regions play a particularly important role in the binding specificity/affinity of the antibodies according to the invention and thus provide another object of the invention.

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." "Framework" or "FR" regions are those regions of the variable domain other than the hypervariable region residues as 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 FR4. In particular, CDR3 of the heavy chain is the region that contributes most to antigen binding and defines the properties of the antibody. According to Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991 )) Standard definitions identify CDR and FR regions, and/or those residues from "hypervariable loops".

Antibodies according to the invention can be produced recombinantly. 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 isolation and usually purification of the antibody to pharmaceutically acceptable purity. For expression of the foregoing antibodies in host cells, nucleic acids encoding the respective modified light and heavy chains are inserted into expression vectors by standard methods. Express in suitable prokaryotic or eukaryotic host cells such as CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast, or E. coli cells, and extract the antibody from the cells ( recovered from the supernatant or lysed cells). General methods for the 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 ) 271-282; Kaufman, R.J., Molecular Biotechnology (Mol.Biotechnol.) 16 (2000) 151-161; Werner, R.G., J.DrugRes (Journal of Pharmaceutical Research). 48 (1998) 870-880 in the review article describe.

Antibodies according to the invention are suitably isolated from the culture medium by conventional immunoglobulin purification methods such as, for example, protein A-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity and chromatography. DNA and RNA encoding the monoclonal antibodies are readily isolated and sequenced using conventional methods. Hybridoma cells can serve as the source of the DNA and RNA. Once isolated, the DNA can be inserted into expression vectors that are then transfected into host cells that do not otherwise produce immunoglobulins, such as HEK 293 cells, CHO cells, or myeloma cells, thereby obtaining in the host cells Synthesis of recombinant monoclonal antibodies.

The following Examples, Sequence Listing and Figures are provided to aid in the understanding of the invention, the true scope of which is set forth in the appended claims. It is to be understood that changes may be made in the invention without departing from the essence of the invention.

Description of drawings

Figure 1. Statistical diagram of AFP, AFU, HGF, GPC3 and GGT2 in serum of four groups of subjects;

Figure 2. The ROC curve of AFP, AFU, HGF, GPC3 and GGT2 alone in the serum of patients with liver cancer at all stages in the screening of liver cancer in the whole population;

Figure 3. The ROC curve of the serum of patients with full-stage liver cancer and early-stage liver cancer when the HGF single index is included in the age and sex of the subjects in the screening of liver cancer in the whole population;

Figure 4. When screening liver cancer in the whole population, the ROC curve of the serum of patients with full-stage liver cancer and early-stage liver cancer detected by double-indicator gender;

Figure 5. When screening liver cancer in the whole population, the ROC curve of the serum of patients with full-stage liver cancer and early liver cancer detected by double-indicator age;

Figure 6. When screening liver cancer in the whole population, the ROC curves of the serum of patients with full-stage liver cancer and early liver cancer detected by three indicators;

Figure 7. When screening liver cancer in the whole population, the ROC curves of the serum of patients with full-stage liver cancer and early liver cancer detected by four indicators;

Figure 8. When screening liver cancer in the whole population, the ROC curve of the five-indicator joint detection of the serum of patients with full-stage and early-stage liver cancer;

Figure 9. When screening liver cancer in hepatitis patients, the ROC curve of HGF single index and the age and gender of HGF included subjects to detect full-stage liver cancer and early liver cancer;

Figure 10. When screening liver cancer in hepatitis patients, the ROC curve of the serum of patients with full-stage liver cancer and early-stage liver cancer is detected by double-indicator inclusion of subject gender;

Figure 11. When screening liver cancer in patients with hepatitis, the ROC curve of the serum of patients with full-stage liver cancer and early liver cancer detected by double-indicator inclusion age;

Figure 12. When screening liver cancer in patients with hepatitis, the ROC curves of the serum of patients with full-stage liver cancer and early liver cancer detected by three indicators;

Figure 13. When screening liver cancer in patients with hepatitis, the ROC curves of the serum of patients with full-stage liver cancer and early liver cancer detected by four indicators;

Figure 14. When screening liver cancer in hepatitis patients, the five indicators detect the ROC curve of the serum of patients with full-stage liver cancer and early liver cancer;

Figure 15. When screening liver cancer in patients with liver cirrhosis, the ROC curve of HGF single index and the age and gender of HGF included subjects to detect full-stage liver cancer and early liver cancer;

Figure 16. When screening liver cancer in patients with liver cirrhosis, the ROC curve of the serum of patients with full-stage liver cancer and early-stage liver cancer is detected by including the sex or age of the subjects with dual indicators;

Figure 17. When screening liver cancer in patients with liver cirrhosis, the ROC curves of the serum of patients with full-stage liver cancer and early liver cancer detected by three indicators;

Figure 18. When screening liver cancer in patients with liver cirrhosis, the ROC curves of the serum of patients with full-stage liver cancer and early liver cancer detected by four indicators;

Figure 19. When screening liver cancer in patients with liver cirrhosis, the five indicators detect the ROC curve of the serum of patients with full-stage liver cancer and early liver cancer; and

Figure 20. Model validation of AFP, AFU, HGF, GPC3 and GGT2 detection values in liver cancer patient serum, hepatitis B patient serum, liver cirrhosis patient serum and normal human serum.

Detailed ways

The present invention is described in detail by the following examples, they are only used to further illustrate the present invention, can not be interpreted as the limitation of protection scope of the present invention, those skilled in the art make some non-essential improvements and improvements according to the above-mentioned content of the present invention The adjustments all belong to the protection scope of the present invention.

Example 1: Collection and processing of serum from different groups of people

In the Cancer Hospital of Tianjin Medical University, 60-300 cases of peripheral blood were collected from liver cancer patients, hepatitis B patients and normal subjects respectively. In the experiment, the serum of 288 patients with liver cancer, 87 patients with hepatitis B, 80 patients with liver cirrhosis and 241 normal patients were collected.

Within 1 hour of peripheral blood collection, centrifuge at 3000g for 5-10 minutes, transfer the serum into a new centrifuge tube, and store in a -80°C refrigerator.

Example 2: Diagnostic application of markers of the present invention or combinations thereof

The concentrations of AFP, AFU, GPC3, GGT2, and HGF in the serum to be tested were detected by ELISA using a commercially available kit (details are shown below) according to the manufacturer's instructions.

1) Evaluation indicators and selection of diagnostic tests

Sensitivity (true positive rate, Sensitivity), specificity (true negative rate, Specificity), positive predictive value (PPV), negative predictive value (NPV) were used to evaluate the diagnostic effect of the tested indicators:

Sensitivity=TP/(TP+FN)×100%

Specificity=TN/(TN+FP)×100%

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

Negative predictive value = TN/(TN+FN) × 100%

Correct index (Youden index) = (sensitivity + specificity) - 100%

Among them, TN (true negative) = true negative; FP (false positive) = false positive; TP (true positive) = true positive; FN (false negative) = false negative.

The receiver operating characteristic curve (ROC) was used to calculate all possible cut points to show the relationship between sensitivity and specificity. By changing the cut point, multiple pairs of true positive rate (sensitivity) and false positive rate (1-specificity) values are obtained, and the false positive rate is used as the abscissa and the sensitivity as the ordinate to draw the ROC curve, so as to dynamically and objectively Reflect the effectiveness of the diagnostic system. The area under the ROC curve (AUC) represents the overlapping degree of the distribution of positive and negative diagnostic results in the diagnostic system, reflecting the ability of the diagnostic system to distinguish positive and negative diagnostic results, that is, the value of the diagnostic test. The critical value of the diagnostic index was determined by finding the maximum point of the correct index on the ROC curve, and randomly sampled 1000 times by the Bootstrap method to determine the 95% confidence interval of the critical value.

2) Construction of statistical model

The single-index and multi-index combined diagnostic test for HCC was realized through the logistic regression model, and applied to healthy normal people, HBV-infected patients, and liver cirrhosis patients respectively, and ROC analysis was carried out in the three groups and the patient population. The logistic regression model was established according to the disease state, and the formed prediction probability or joint predictor was used as the analysis index, and the ROC curve was established by combining the non-parametric model and the double normal model.

Logistic regression model:

and have

The constant term β ₀ indicates that when the exposure dose is 0, the individual becomes ill (HCC) and does not develop (HBV infection). Quantity (5 proteins) at a certain continuous cut-off point P _k : βY≥g(P _k ), Y _ik =1; βY|g(P _k ), Y _ik =0. Unlike the diagnostic test that combines multiple indicators, the graph is a plane to obtain the sensitivity and specificity to construct the ROC curve.

The analysis and statistical results are shown in the accompanying drawings.

Results and discussion:

The low detection rate of early liver cancer reflects the limitations of current diagnostic methods for liver cancer. Currently, the diagnosis of liver cancer mainly relies on serological examination and imaging diagnosis. Serological examination mainly detects tumor markers in serum, and currently mainly relies on the detection of AFP, but the sensitivity of AFP in small liver cancer is only about 40%.

Using antibodies or single-chain antibody fragments that specifically bind to human liver cancer markers, among the five markers of AFP, AFU, HGF, GPC3 and GGT2, HGF single-index detection, and HGF combined with other markers for dual-index, When three indicators, four indicators and five indicators are detected, it can be distinguished from hepatitis B patients, liver cirrhosis patients and healthy people to detect liver cancer.

When screening liver cancer in the whole population (liver cancer, hepatitis B, liver cirrhosis, and normal people), screening liver cancer in patients with hepatitis B, and screening liver cancer in patients with liver cirrhosis, HGF single-index detection of full-stage liver cancer and early liver cancer is effective. High sensitivity and specificity. By using the antibody specifically binding to HGF or its single-chain antibody fragment, using the serological detection method for liver cancer, using the HGF single indicator to screen liver cancer in all populations (liver cancer, hepatitis B, liver cirrhosis and normal people), which The sensitivity of detecting full-stage liver cancer was 90.3%, and the specificity was 90.5%. In the early stage (A stage) liver cancer patients of the Barcelona stage, the sensitivity of detection was 90.2%, and the specificity was 84.1%. The age of the included subjects The sensitivity of HGF in full-stage liver cancer was 91.3%, and the specificity was 90.9%, and the sensitivity in early liver cancer was 86.3%, and the specificity was 90.5%. The sensitivity of the indicator to detect the full-stage liver cancer is 94.4%, and the specificity is 91.8%, and the sensitivity to detect the early stage liver cancer is 90.2%, and the specificity is 91.9%. 90.8%, 95.2% specificity, 92.2% sensitivity, 90.2% specificity for detecting early liver cancer; screening for liver cancer in patients with liver cirrhosis, the sensitivity and specificity of HGF single index detection of full-stage liver cancer and early liver cancer Both can reach 72% and 73%, which is significantly better than AFP commonly used in clinic, indicating that detecting the content of HGF in serum alone, and combining the age and sex of the subjects when necessary can meet the needs of clinical liver cancer diagnosis.

Detection of two indicators When screening liver cancer in the whole population, screening liver cancer in patients with hepatitis B, and screening liver cancer in patients with liver cirrhosis, HGF is combined with one of the four markers of AFP, AFU, GPC3, and GGT2 to detect Both full-stage liver cancer and early liver cancer can achieve high sensitivity and specificity, which is of great significance for the diagnosis of primary liver cancer patients, especially the combination of HGF and AFP is the best combination for serological diagnosis and early diagnosis of liver cancer patients .

In particular, when the two indicators are used to screen liver cancer in the whole population, only the gender of the subjects is included for statistics. The sensitivity of HGF and AFP to detect full-stage liver cancer is 90.8%, and the specificity is 93.1%. HGF and AFU The sensitivity of combined detection of full-stage liver cancer is 87.2%, and the specificity is 94.6%. The sensitivity of combined use of HGF and GPC3 in the detection of full-stage liver cancer is 91.8%, and the specificity is 90.6%. The combination of HGF and GGT2 in the detection of full-stage liver cancer The sensitivity of HGF and AFP was 89.3%, and the specificity was 93.1%. After only including the subject’s age, the sensitivity of HGF and AFP combined to detect full-stage liver cancer was 92.3%, and the specificity was 90.9%. The sensitivity of HGF and GPC3 combined to detect full-stage liver cancer was 90.8%, and the specificity was 91.6%. The sensitivity of HGF and GPC3 combined to detect full-stage liver cancer was 90.3%, and the specificity was 90.9%. 89.3%, with a specificity of 92.3%. In the detection of early liver cancer, the combination of HGF and AFP, HGF and AFU, HGF and GPC3, HGF and GGT2 respectively, after including age or gender, can achieve a sensitivity of more than 86% and a specificity of more than 84% sex.

When screening liver cancer in hepatitis B patients, only the gender of the subjects was included for statistics. The sensitivity of the combination of HGF and AFP to detect the full-stage liver cancer was 92.9%, and the specificity was 95.2%. The combination of HGF and AFU to detect the full-stage liver cancer The sensitivity is 93.4%, the specificity is 93.5%, the sensitivity of the combination of HGF and GPC3 to detect the full-stage liver cancer is 93.4%, the specificity is 93.5%, the sensitivity of the combination of HGF and GGT2 to detect the full-stage liver cancer is 92.3%, the specificity was 93.5%. ; After only including the subject’s age for statistics, the sensitivity of the combination of HGF and AFP to detect full-stage liver cancer is 92.3%, and the specificity is 93.5%. The sensitivity of HGF and AFU to detect full-stage liver cancer is 93.9%, and the specificity is The combined use of HGF and GPC3 has a sensitivity of 93.9% and a specificity of 93.4% for the detection of full-stage liver cancer. The combined use of HGF and GGT2 has a sensitivity of 93.4% and a specificity of 95.1% for the detection of full-stage liver cancer. In the detection of early liver cancer, the combination of HGF and AFP, HGF and AFU, HGF and GPC3, HGF and GGT2 respectively, after including age or gender, can achieve a sensitivity of more than 86.3% and a specificity of more than 83.9% sex. When screening liver cancer in patients with liver cirrhosis, after including one of age or gender, HGF is combined with the other four markers respectively, and the detection of full-stage liver cancer and early liver cancer can achieve high sensitivity and specificity. It shows that the combined detection of HGF and other four indicators alone, combined with the age or gender of the subjects, has a significant significance for the diagnosis of patients with primary liver cancer, especially the combination of HGF and AFP is a good choice for serological diagnosis and early diagnosis of liver cancer patients. best combination.

Detect three indicators to screen liver cancer in the whole population. When detecting full-stage liver cancer, HGF+AFP+AFU is used in combination, HGF+AFP+GPC3 is used in combination, HGF+AFP+GGT2 is used in combination, and HGF+AFU+GPC3 is used in combination. Both can achieve a sensitivity of more than 91.8% and a specificity of more than 90.2%. When detecting early liver cancer, the combination of HGF+AFP+AFU, HGF+AFP+GPC3, and HGF+AFP+GGT2 can reach More than 86.3% sensitivity and more than 85.5% specificity, when screening liver cancer in hepatitis B patients, HGF+AFP+AFU, HGF+AFP+GPC3, HGF+AFP+GGT2, HGF+AFU+GPC3 and HGF+GPC3 +GGT2 combined application, the sensitivity of detecting full-stage liver cancer can reach more than 92.3%, the specificity can reach more than 93.5%, and the detection of early liver cancer is also good, especially the combination of HGF+AFP+AFU, the sensitivity can reach 90.2% , with a specificity of 98.4%. When screening liver cancer in patients with liver cirrhosis, HGF combined with the other two markers can also achieve satisfactory results. Need for serological diagnosis and early diagnosis.

When detecting the four indicators to screen liver cancer, the sensitivity and specificity are improved. When screening liver cancer in the whole population, the combination of HGF, AFP, GPC3 and GGT2 has a sensitivity of 91.3% and a specificity of 91.3%. 92.0%, the sensitivity of detecting early liver cancer was 86.3%, and the specificity was 90.5%. When screening liver cancer in patient B, the combination of HGF, AFP, AFU and GGT2 detected the sensitivity of full-stage liver cancer was 91.8%, and the specificity The sensitivity of detecting early liver cancer is 92.2%, and the specificity is 98.4%. When screening liver cancer in patients with liver cirrhosis, HGF can achieve high sensitivity and specificity when combined with the other three indicators .

The combined application of the five markers further improves the sensitivity and specificity. When screening liver cancer in the whole population, the sensitivity for detecting full-stage liver cancer can reach 91.2%, and the specificity can reach 93%. The sensitivity for detecting early liver cancer 84.3% and specificity up to 87.2%. When screening liver cancer in patients with hepatitis B, the sensitivity for detecting full-stage liver cancer is 91.8%, and the specificity is 98.4%. The sensitivity for detecting early liver cancer is 92.2%, and the specificity is 98.4%, when screening liver cancer in patients with liver cirrhosis, the sensitivity for detecting full-stage liver cancer is 91.3%, and the specificity is 76.2%, and the sensitivity for detecting early liver cancer is 92.2%, and the specificity is 66.7%. Significantly better than the current gold standard AFP for clinical liver cancer detection.

In order to verify the accuracy of the detection results, the present invention establishes a model for the detection values of five markers in liver cancer patient serum, normal human serum and hepatitis B patient serum, and arranges the risk coefficients (RS) of all research objects from small to large, Divided into high-risk group (RS≥0.5) and low-risk group (RS<0.5), most liver cancer patients were correctly classified into high-risk group, and the trend of scatter plot was obvious, indicating that the model results were in line with the facts. This model can be used to analyze the correlation between the detected values of AFP, AFU, HGF, GPC3, and GGT2 five markers and liver cancer.

Through a large number of sample verification and statistical analysis, the present invention proves that HGF can be used as a liver cancer marker alone for screening and early diagnosis of liver cancer by using monoclonal antibody or its single-chain antibody fragment, and its detection sensitivity and specificity are greatly superior The combination of AFP, HGF and one or more other indicators, which are the gold standard in clinical practice, can be used as a supplement to HGF detection to further improve the sensitivity and specificity of serological diagnosis of liver cancer.

Although the foregoing invention has been described in some detail by means of figures and examples for clarity of understanding, the description and examples should not be considered as limiting the scope of the invention. The disclosures of all patent and scientific documents cited herein are expressly incorporated by reference in their entirety.

Claims (8)

1. being able to detect hepatocyte growth factor (HGF), alpha-fetoprotein (AFP) and the sweet enzyme of α-L-fucose (AFU) marker group Reagent or the reagent combination of conjunction are in preparation in hepatitis B patient in the diagnosticum or kit of early liver cancer Serologic detection Application.

2. application according to claim 1, the diagnosticum or kit further include detection glutamyl transferase isodynamic enzyme 2 The reagent or reagent of (GGT2/GGT II) and/or heparan sulfate proteoglycan 3 (GPC3) combine.

3. being able to detect hepatocyte growth factor (HGF), alpha-fetoprotein (AFP) and the sweet enzyme of α-L-fucose (AFU) marker group Reagent or the reagent combination of conjunction are preparing serodiagnosis agent or examination for mutually distinguishing early liver cancer patient with hepatitis B patient Application in agent box.

4. application according to claim 3, the diagnosticum or kit further include detection glutamyl transferase isodynamic enzyme 2 The reagent or reagent of (GGT2/GGT II) and/or heparan sulfate proteoglycan 3 (GPC3) combine.

5. application according to claim 1 or 3, wherein the diagnosticum or kit also consider subject's in application Age and/or gender.

6. application according to claim 1 or 3, wherein the reagent or reagent combination include one or more specificity knots Antibody or its segment together in human liver cancer marker, wherein the segment is Fv, Fab, Fab ', Fab '-SH, F (ab ')₂, it is dual anti- Body, linear antibodies or single-chain antibody (scFv).

7. application according to claim 6, wherein the antibody is polyclonal antibody or human IgG2's subclass, human IgG1's subclass Or people's IgM subclass monoclonal antibody.

8. application according to claim 7, wherein the antibody or its segment are for ELISA measurement, Western Blotting measurement, Immunohistochemistry or immunofluorescence assay.