JP2002503813A - Methods and compositions for diagnosis of hepatome - Google Patents

Abstract

  (57) [Summary] The present invention provides methods for hepatome screening, diagnosis and prognosis, for monitoring the effects of hepatome treatment, and for drug administration. Hepatome diagnostic features (HF) have been described that can be detected by two-dimensional electrophoresis from serum or plasma. The present invention further provides hepatome diagnostic protein isoforms detected in human serum or plasma, formulations containing the isolated HPI, immunospecific antibodies to the HPI, and kits containing the foregoing.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] 1. The present invention relates to the identification of proteins and protein isoforms associated with hepatocellular carcinoma (hepatome) and their use for screening, diagnosis, prognosis, therapy, and drug administration.

[0002] 2. BACKGROUND OF THE INVENTION Hepatome is an increasingly prevalent cancer that affects hundreds of thousands of patients worldwide. The methods of treatment are still relatively poor, and the main expectation for more effective treatments is centered on earlier and more accurate diagnosis.

[0003] Serum alpha fetoprotein (AFP) is widely used for the diagnosis of hepatome. AFP is a convenient marker when levels are extremely elevated, but has limited practical application due to low specificity for hepatome at low levels, low positive predictive value (PPV) (Johnson et al., 1997, Br. J. of Cancer 75: 236-240). Certain lectins bind specifically to AFP obtained from hepatome serum as compared to AFP in benign conditions (
Aoyagi et al., 1985, Biochim. Biophys. Acta 83
0: 217-223; Aoyagi et al., 1993, Br. J. Cancer 6
7: 486-492). However, these methods have not gained broad clinical consent (Johnson et al., As above). More recently, isoelectric focusing has been used to detect isoforms of AFP that appear to be relatively specific for hepatome (Johnson et al., As described above).
These reports indicate that the need for identification of markers in serum for the diagnosis of hepatome is increasing.

[0004] 3. SUMMARY OF THE INVENTION The present invention provides methods and compositions for screening, diagnosing, and prognosing hepatome, observing the efficacy of hepatome treatment, and administering drugs.

[0005] A first aspect of the present invention relates to a method for detecting at least one hepatome diagnostic feature (HF), such as a plasma sample or serum to detect the level of HF selected from the group of HFs disclosed in the present invention. It is an object of the present invention to provide a method for diagnosing hepatome, comprising analyzing a sample by two-dimensional electrophoresis. These methods are also suitable for screening, prognosis, observation of treatment results, and drug administration.

[0006] A second aspect of the present invention is to determine the level of at least one hepatome diagnostic protein isoform (HPI) in a plasma or serum sample, eg, an HPI selected from the group of HPIs disclosed in the present invention. It is to provide a method for diagnosis of hepatome, including detecting. These methods are also suitable for screening, prognosis, observation of treatment results, and drug administration.

A third aspect of the present invention is to provide a monoclonal antibody and a polyclonal antibody capable of immunospecifically binding to an HPI, for example, the HPI disclosed in the present invention.

[0008] A fourth aspect of the present invention provides an isolated HPI, a drug comprising HPI separated from a protein or protein isoform that has a significantly different isoelectric point or a significantly different apparent molecular weight from the HPI. It is in.

[0009] 5. DETAILED DESCRIPTION OF THE INVENTION The present invention, described in detail below, provides methods and compositions for screening, diagnosing, and prognosing hepatomes in mammalian subjects, methods for observing hepatome treatment results, and for administering drugs. Including methods. The subject is preferably a human, and more preferably an adult human.

To clarify the disclosure without limitation, the invention will be described with reference to the analysis of serum samples. However, as one of ordinary skill in the art will appreciate, the assays and techniques described in the present invention are applicable to other types of patient samples, including body fluids (eg, plasma, urine, bile, ascites), Samples of tissue suspected of containing material derived from the hepatome (eg, biopsies such as liver biopsies or biopsies of suspicious sites) are included.

5.1. Hepatome Diagnostic Features (HF) In a first aspect of the invention, one or more of the following may be used for screening or diagnosing a hepatome, determining the prognosis of a hepatome patient, observing the effects of hepatome treatment, or administering a drug. Two-dimensional electrophoresis is used to analyze serum obtained from a subject for the purpose of measuring the amount of two or more hepatome diagnostic features (HF) generated. "2D electrophoresis (2D electrophoresis) used in the present invention"
"" Means denaturing electrophoresis.
A technique consisting of isoelectric focusing according to s), which produces a two-dimensional gel (2D gel) containing a large number of separated proteins. In the denaturing electrophoresis step, polyacrylamide electrophoresis (SDS-P) in the presence of sodium dodecyl sulfate was used.
AGE). The very precise and automatable methods and apparatus described in U.S. patent application Ser. No. 08 / 980,574 and WO 98/23950 (incorporated herein by reference in their entireties) ("The Pre-
ferred Technology ") is particularly preferred. Briefly, a "preferred technique" provides an efficient computer-assisted method and apparatus for identifying, selecting, and characterizing biomolecules in a biological sample. Two-dimensional arrays are created by separating biomolecules in a two-dimensional gel according to the electrophoretic mobility and isoelectric point of the biomolecules. A computer-generated digital profile of the sequence is generated, representing the identity, apparent molecular weight, isoelectric point and relative abundance of a number of biomolecules detected in the two-dimensional array, thereby providing a diverse It allows computer-mediated comparison of profiles of biological samples and computer-assisted isolation of isolated target proteins.

The term “hepatome diagnostic feature (HF)” as used in the present invention relates to a feature (eg, a point in a 2D gel) detected by two-dimensional electrophoresis of a biological sample, ie, A sample that is different in one compared sample, such as a sample in serum from a subject with a hepatome, and a sample in serum from a subject without a hepatome. It means something that is different when compared. In the present invention, a method for detecting a feature or a protein isoform (eg, two-dimensional electrophoresis or an immunoassay) comprises:
When it is found that the abundance of a feature (or protein isoform) is relatively different, the feature (or protein isoform) is "differently present" in the first sample relative to the second sample. " If the abundance of the feature measured in the first sample as compared to the second sample is higher, the feature, i.e., the protein isoform, is "increased" in the first sample as compared to the second sample. I do. Conversely, if the abundance of the feature measured in the first sample as compared to the second sample is lower, the feature, ie, the protein isoform, is "decreased."

Preferably, the relative abundance of the features in the two samples is measured in two stages. First, the signal obtained in detecting the feature in the sample is analyzed by suitable background parameters, such as total protein in the sample being analyzed (eg, all proteins injected into the gel), or invariant features, That is, a feature whose abundance in the compared samples is known to be similar,
For example, ERFs (ERFs: Expression Reference) disclosed below
Normalization is performed with reference to one or more expression reference characteristics, such as Sense Features, or all signals detected from all proteins in the sample.

Second, the signal normalized to a feature in one sample or sample set is compared to the signal normalized to the same feature in another sample or sample set, and the second compared to the second. Identify "differentially present" features in one sample (or sample set).

[0015] Two HF groups were identified by the "preferred technique" method and apparatus. The first group consists of reduced HF in the serum of subjects with hepatome as compared to the serum of subjects without hepatome (eg, subjects with cirrhosis). These HFs can be indicated by apparent molecular weight (MW) and isoelectric point (pI) as follows:

[0016]

[Table 1]

A second group is subjects without a hepatome (eg, subjects with cirrhosis)
Consists of increased HF in the serum of subjects with hepatome compared to serum of These HFs can be indicated by apparent molecular weight (MW) and isoelectric point (pI) as follows:

[0018]

[Table 2]

[0019]

[Table 3]

For any HF, the normalized signal obtained by analyzing the serum of a subject with a hepatome and the standardized signal obtained by analyzing the serum of a subject without a hepatome Will depend on the particular analytical rules and detection means used. Thus, in the present invention, as is customary in diagnostic technology, each laboratory establishes a reference range for each HF in subjects without hepatome, according to the analytical rules and detection means used. That is taken into account. At least one positive reference serum sample obtained from a subject known to have a hepatome, or at least one obtained from a subject known to have no hepatome
It is preferred to include in each batch of test samples analyzing two negative reference serum samples (more preferably at least one positive reference serum sample and at least one negative reference serum sample).

As will be readily appreciated by those skilled in the art, the given characteristics, ie, the measured MW and pI of a Protein Isoform, are determined by strict rules for each step of 2D electrophoresis and for agreement of the indices. Somewhat different.
As used herein, “MW” and “pI” refer to the apparent molecular weight, respectively, and the characteristics determined in accordance with the experimental rules set forth in Section 6 (“Reference Rules”) below, ie, protein isoforms and the like. It is defined as indicating an electric point. According to the rules of the reference, the variation in the mean pi measurement of HF or HPI is typically less than 1% when the samples are duplicated or repeated in duplicate,
The variation in the average MW measurement of HF or HPI is generally less than 5%. If one of skill in the art desires to deviate from the reference rules, (a)
And (b) Due to the off-rule, a calibration experiment should be performed to compare each HF detected, ie the MW and pI of the Protein Isoform.

HF can be used for hepatome detection, prognosis, diagnosis or observation, or drug administration. In one embodiment of the present invention, the serum obtained from the subject is H
It was analyzed by 2D electrophoresis for the quantitative detection of one or more HFs selected from the group consisting of F-1 to HF-14 and HF-57 to HF-59, but in this group hepatome Decrease in the amount of HF generated in sera from subjects as compared to sera (eg, a reference sample or a predetermined reference range) obtained from subjects (or groups of subjects) that do not have It indicates the presence of hepatome. In addition, it is preferable that it is one or two or more HF selected from the group consisting of HF-1 to HF-59. In another embodiment of the present invention, the serum obtained from the subject has a quantitative content of one or more HF selected from the group consisting of HF-15 to HF-56 and HF-60 to HF-61. In this group analyzed by 2D electrophoresis for detection, but compared to serum (eg, a reference sample or a predetermined reference range) obtained from a subject (or group of subjects) without a hepatome. Increased the amount of HF generated in the serum obtained from E. coli, indicating the presence of hepatome. In addition, HF-17,
HF-18, HF-20, HF-21, HF-23, HF-24, HF-25,
HF-28, HF-29, HF-31, HF-33, HF-34, HF-36,
HF-39, HF-44, HF-51, HF-52, HF-54 and HF-5
And preferably one or more HF selected from the group consisting of
-17, HF-18, HF-20, HF-23, HF-24, HF-25, HF
More preferably, it is selected from the group consisting of -29, HF-33, HF-34, HF-39, HF-44 and HF-55.

5.2. Hepatome Diagnostic Protein Isoform (HPI) In another aspect of the present invention, serum obtained from a subject is used for screening or diagnosing hepatome, for measuring the prognosis of a hepatome patient, for observing the effect of hepatome treatment, Alternatively, for drug administration, it is analyzed for quantitative detection of one or more hepatome diagnostic protein isoforms (HPI). The `` hepatome diagnostic protein isoform '' used in the present invention refers to a protein isoform that is differentially present in serum obtained from a subject having a hepatome, as compared to serum obtained from a subject without a hepatome. Say. As is well known in the art, the protein product of a single gene may include variants (isoforms), (a) differentially altered post-translational modifications (eg, glycosylation, phosphorylation, or acylation). Results are different, and therefore proteins of the same amino acid sequence differ in pI, MW, or both, and / or (b) differ in amino acid composition (eg, alternative).
which differ as a result of mRNA splicing or restriction degradation of the protein). The differential presence of a Protein Isoform will not require differential expression of the gene encoding the protein in question.

A group of HPIs has been identified by the complementary amino acid sequence of HF using “preferred technology” methods and equipment. The first group consisted of HPI reduced in the serum of subjects with hepatome compared to the serum of subjects without hepatome and was highly differentially present (p ≦ 0.001). The MW of these HPIs, p
I and the complementary amino acid sequence are shown below in Table 3.

[0025]

[Table 4]

The second group consisted of HPI increased in the serum of subjects with hepatome compared to the serum of subjects without hepatome and was highly differentially present (p ≦ 0.001). ). The MW, pI and complementary amino acid sequences of these HPIs are shown below in Table 4.

[0027]

[Table 5]

In one embodiment of the present invention, serum obtained from the subject is analyzed for quantitative detection of one or two HF selected from the group consisting of HF-1 and HF-23, In this group, the amount of HF generated in the serum obtained from the subject is reduced compared to the serum obtained from the subject without the hepatome or from the group of subjects (eg, a reference sample or a predetermined reference range). , Which indicates the presence of a hepatome.

In another embodiment of the invention, the serum obtained from the subject is HF-2 to H
A subject (or subject group) that is analyzed by 2D electrophoresis for quantitative detection of one or more HFs selected from the group consisting of F-22 and HF-24 and has no hepatome ), The amount of HF generated in the serum obtained from the subject is increased as compared to the serum obtained from the subject (for example, a reference sample or a predetermined reference range), which indicates the presence of hepatome.

As indicated above, the HPIs described in the present application include previously unknown isoforms of proteins in addition to known isoforms of isoforms associated with the hepatome. . For each HPI, the present invention further provides an agent comprising an isolated HPI or a fragment thereof, and provides an antibody that binds to the HPI, the fragment or both the HPI and the fragment. is there. "Isolated" H, as used herein
PI refers to an HPI without a protein or protein isoform having a pI or MW that is substantially distinct from the “isolated” HPI as observed by 2D electrophoresis. As used herein, a “substantially distinct” pI or MW is
PI of contaminating protein isoforms disappearing from HPI on 2D electrophoresis
Or MW.

[0031] In one embodiment, an isolated protein is provided, wherein the protein is H
Consist of a peptide having the amino acid sequence specified in Table 3 or 4 for PI, wherein said protein is within 10% (preferably within 5%, more preferably within 1%) of the value specified in Tables 3 and 4 for HPI Is a protein having a pI and a MW.

The HPI of the present invention can be analyzed by any method known to those skilled in the art.
In one embodiment, HPIs are separated in 2D gels by their MW and pI and visualized by coloring the gel.

Alternatively, the HPI can be detected by analysis, such as an immunoassay, for detection, prognosis, diagnosis or observation of hepatome, or for drug administration. In one embodiment, the immunoassay involves contacting a sample obtained from the subject to be tested with an anti-HPI antibody under conditions that cause immunospecific binding, and determining the amount of all immunospecific binding by the antibody. Is carried out by a method of detecting or measuring. Preferably, the anti-HPI antibody binds preferentially to HPI over other isoforms of the same protein. In a preferred embodiment, the anti-HPI antibody binds HPI with at least 2 times greater binding to other isoforms of the same protein, more preferably at least 5 times greater binding,
More preferably, the binding is at least 10 times greater.

In one embodiment, antibody binding in a tissue fragment can be used to detect abnormal localization of HPI or abnormal (eg, high, low, absent) levels of HPI. In specific embodiments, serum samples are analyzed for antibodies to HPI in a patient's tissue (eg, a biopsy of the liver), or to confirm the presence of HPI at sites where abnormal levels of HPI suggest a hepatome. Can be used to analyze As used herein, “abnormal levels” refers to levels that appear in a body part without a hepatome, or in a similar sample obtained from a subject, ie, levels that are increased or decreased relative to normal levels.

[0035] Immunoassays that can be used include Western blot, radioimmunoassay, ELISA
A (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation analysis, sedimentation reaction, gel diffusion sedimentation reaction, immunodiffusion analysis, agglutination analysis, complement binding analysis, immunoradiometric assay, fluorescent immunoassay, protein Includes, but is not limited to, competitive or non-competitive analytical systems using various techniques such as A immunoassay.

If desired, HPI can be detected by a two-step sandwich analysis. At this time, the HPI expresses a special glycoform of the protein, and the first step can use an anti-HPI antibody (which can be optionally immobilized on a solid phase) to capture the HPI. In the second step, a directly or indirectly labeled lectin can be used to capture the HPI. To this end, HPIs are more likely to bind to (a) other glycoforms that have the same nuclear protein as HPI, or (b) other isoforms that share an antigenic determinant identified by the antibody. Any lectin that binds preferentially can be used. In a preferred embodiment, the lectin selected is relative to the other glycoforms that have the same nuclear protein as the HPI, or that share the same antigenic determinant identified by the antibody. It binds to HPI with at least 2 times greater binding, more preferably at least 5 times greater binding, and even more preferably at least 10 times greater binding. Lectins suitable for detecting a given HPI can be readily identified using known techniques, for example, Smar et al., Lec.
tis as Indicators of Disease-Associa
ted Glycoforms, In: Gabius H-J and Gabiu
s S (eds.), 1993, Lectins and Glycobiol.
ogy, p. 158-174 p. One or more lectins listed in Table 1 at 158-159 (incorporated herein by reference in their entirety).

If desired, the gene encoding the HPI, including the complementary sequence, a related gene,
And related nucleic acid sequences, and their complementary sequences, can be used in hybridization assays. Nucleotides encoding HPI, or at least 8
Its complementary sequence of nucleotides (or its complement) can be used as a hybridization probe. Hybridization analysis of HP in specific hepatomes or relapse of hepatome after surgical or other treatment
It can be used for detection, diagnosis, prognosis, and observation of a condition, disease, or disease state associated with an abnormal change in I gene expression. In a special embodiment,
Such hybridization analysis is performed by a method comprising the steps of contacting a patient sample containing nucleotides with DNA or RNA encoding HPI under conditions under which hybridization can occur, and detecting or measuring the resulting hybridization. .

[0038] The present invention also provides a diagnostic kit comprising an anti-HPI antibody in one or more containers. In addition, the kit may optionally include one or more of the following: (1) instructions for using anti-HPI antibodies for diagnosis, prognosis, observation of therapy, drug administration, or any combination of these applications; (2)
Enforcement rules, i.e. prescribed forms, or forms approved by governmental organizations that dictate the method of production, use or sale of drugs or biological products that reflect the organization's approval of the method of production, human experimentation or control Rules for use or sale for (3) spouses that bind to indexed antibodies, (4) anti-HPI
A solid phase (such as a reagent strip) on which the antibody is immobilized. If no mating is provided that binds to the indexed antibody, the anti-HPI antibody itself can be indexed with a detectable marker, such as a chemiluminescent, enzymatic, fluorescent, or radioactive moiety.

The present invention also provides a kit comprising, in one or more containers, a nucleic acid probe capable of hybridizing to RNA encoding another HPI.
In a specific embodiment, the kit is suitable for use under the suitable reaction conditions of at least a portion of the nucleic acid encoding the HPI, for example, the polymerase chain reaction (eg, Inni
s. , 1990, PCR Protocols, Academic Pres.
s, Inc. , San Diego, CA), a ligase chain reaction using Qβ replicase (see EP 320,308), a cyclic probe reaction, or a pair of primers capable of initiating amplification, such as other known techniques (eg, Each in the size range of 6 to 30, more preferably 10 to 20)
Alternatively, it can be contained in two or more containers.

Kits are also provided that allow for the detection of multiple HPIs or nucleic acids encoding multiple HPIs. The kit can optionally further comprise a predetermined amount of isolated HPI, eg, used as a standard or control, or nucleic acid encoding the HPI.

5.3. Use in Clinical Studies The diagnostic methods and compositions of the invention can assist in processing or observing clinical studies, for example, to test agents that treat hepatome. In one embodiment, the candidate molecule restores the level of HF or HPI in a patient with a hepatome to a level in a subject without a hepatome or in a treated patient (eg, post-operative). , Or HF or HPI levels are maintained at or near hepatome levels. One or more HF and HP
The level of I can be analyzed.

In another embodiment, when there are screening candidates for clinical research, the methods and compositions of the invention are used to identify individuals with a hepatome, and such individuals are included in the study. Or be excluded or in separate cohorts for treatment or analysis. If desired, candidates are hepatitis B and / or
Alternatively, screening can be performed simultaneously to identify individuals having hepatitis C, and known techniques can be used for such screening, for example, antibodies against one or more hepatitis B or hepatitis C antigens. And PCR studies to identify one or more oligonucleotide sequences of the hepatitis B or C genome.

5.4. Purification of HPI In a particular aspect, the invention relates to an isolated HPI consisting of a determinant of antigenicity (ie, identified by an antibody), or a functionally active, as well as a nucleic acid sequence encoding the foregoing. , Preferably human HPI, and fragments and derivatives thereof. The term “functionally active” as used in the present invention refers to a substance exhibiting one or more known functional activities associated with full-length HPI (wild type), for example, an HPI bound to an HPI substrate or a mate. Binding, antigenicity (bound to anti-target antibody), immunogenicity, and the like.

In particular embodiments, the invention provides for a fragment of the HPI consisting of at least 6 amino acids, 10 amino acids, 50 amino acids, or at least 75 amino acids. Fragments or proteins lacking some or all of the HPI sites are also provided. Also provided are nucleic acids encoding the foregoing.

Once a recombinant nucleic acid representing an HPI gene sequence has been identified, the gene product can be analyzed. This is done by analysis based on the physical or functional characteristics of the substance, and includes analysis by gel electrophoresis after adding a radioactive index to the product, immunoassay, and the like.

Once the HPI is identified, it can be used for general methods, including chromatography (eg, ion exchange chromatography, affinity chromatography, sizing column chromatography), centrifugation, differential solubility, or protein purification. It can be isolated and purified by other commonly used techniques.

Alternatively, once the HPI produced by the recombinant nucleic acid has been identified, the entire amino acid sequence of the HPI can be deduced from the nucleic acid sequence of the chimeric gene contained in the recombinant. As a result, known general chemical techniques (eg, Hunkap)
iller et al. , 1984, Nature 310: 105-111).

[0048] In another alternative embodiment, native HPI can be purified from natural sources by general methods as described above (eg, immunoaffinity purification).

[0049] In a preferred embodiment, US application no. 0, incorporated herein by reference.
HPI can be isolated by the "preferred technique" described in 8 / 980,574 and WO 98/23950. For preparation-scale implementation, Westminster, 1993, Electrophoresis in
Practice (VCH, Weinheim, Germany) pp. 1
Using a narrow range "zoom gel" having a pH range of one or two pH units for the isoelectric focusing step according to the method described in 97-209 (incorporated herein by reference in its entirety). Is preferred. Such a change allows for injecting large amounts of the target protein into the gel, thus also increasing the amount of isolated HPI that can be removed from the gel. The amount of HPI isolated in a single run is typically 100 ng in the "preferred technique", but can provide 1000 ng when the method is used in a preparative scale run. One skilled in the art will recognize that zoom gels can be used in any separation method using gel isoelectric focusing.

In a preferred embodiment of the present invention, such an HPI is a recombinant DNA
In addition to fragments, other derivatives, analogs thereof, and homologous proteins, whether by technology, by chemical synthesis, or by purification of natural proteins, all or all of the amino acid sequence of HPI (But not limited thereto).

5.5. Generation of Antibodies to HPI In accordance with the present invention, HPI, fragments or other derivatives thereof, and analogs thereof, may be used as immunizing antigens to generate antibodies that immunospecifically bind to the immunizing antigen. Such proteins, fragments, derivatives, analogs are:
Isolation can be by any of the conventional methods described in the above sections of the present application. The antibodies generated are polyclonal, monoclonal, chimeric, single chain,
Fab fragments, including but not limited to Fab expression libraries. In a specific embodiment, antibodies to human HPI are generated. In other embodiments, antibodies to certain regions of the HPI are generated. In a specific embodiment, a hydrophilic fragment of HPI is used as an immunizing antigen for antibody production.

Various known means can be used for the production of polyclonal antibodies to HPIs or derivatives and analogs thereof. In a particular embodiment, a rabbit polyclonal antibody against an HPI epitope, or a portion thereof, is obtained.
For the production of antibodies, native HPIs, or synthetic variants, can be introduced into various host animals.
In addition, immunization can be performed by injecting a derivative (eg, a fragment) thereof, and includes, but is not limited to, rabbits, mice, rats, horses, goats, chickens, and the like. Various adjuvants may be used depending on the host species to increase the immunological response, complete Freund's adjuvant and incomplete Freund's adjuvant, inorganic gels such as aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, peptides, Includes but is not limited to oil emulsions, keyhole limpet hemocyanin, dinitrophenol, potentially available human adjuvants such as BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum.

For the production of monoclonal antibodies directed to the HPI sequence or an analog thereof, any technique provided for the production of antibody molecules by persistent cell lines in culture may be used. For example, trioma technology, human B cell hybridoma technology (Kozbor et al., 1983, Immunology Toda).
y 4:72), EBV-hybridoma technology for producing human monoclonal antibodies (Cole et al., 1985, Monoclonal Antibodies a).
nd Cancer Therapy, Alan R .; Liss, Inc. , P
p. 77-96), as well as hybridoma technology originally developed by Kohler and Milstein (1975, Nature 256: 495-49).
7). In an additional embodiment of the present invention, PCT / US90 / 0
Monoclonal antibodies can also be produced in sterile animals, as described in 2545, and are incorporated herein by reference. According to the present invention, human antibodies may be used, and human antibodies can be obtained by using human hybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci. USA8).
0: 2026-2030), or human B with the EBV virus in vitro.
By transforming cells (cole et al., 1985, Monoclonal
Antibodies and Cancer Therapy, Alan R
. Liss, Inc. Pp. 77-96). Indeed, according to the present invention, a "chimeric antibody" that splices a gene obtained from a mouse antibody molecule specific for HPI with a gene obtained from a human antibody molecule having suitable biological activity. (Morrison et al., 1984,
Proc. Natl. Acad. Sci. USA 81: 6851-6855; N
Euberger et al., 1984, Nature 312: 604-608; Takeda et al., 1985, Nature 314: 452-454), and such antibodies are within the scope of the present invention. (Each of the above references is incorporated by reference into the present invention.) In accordance with the present invention, the described techniques for the production of single chain antibodies (US Patent 4, incorporated herein by reference). 946,778), HP
It can be used to generate I-specific single chain antibodies. In an additional embodiment of the invention, a Fab expression library (Huse et al., 1989 Science 246).
1275-1281) can be used to rapidly and easily identify monoclonal Fab fragments with the desired specificity for HPIs, derivatives and analogs.

[0054] Antibody fragments which contain the idiotype of the molecule can be generated using known techniques. For example, such fragments include F (ab ') ₂ fragments produced by pepsin digestion of antibody molecules, Fab' fragments produced by reducing disulfide bridges of F (ab ') ₂ fragments, antibody molecules And Fv fragments generated by treating E. coli with papain and a reducing agent, and Fv fragments.

In the production of an antibody, screening for a desired antibody can be performed by a known technique such as ELISA. For example, to select antibodies that identify certain specific regions of the HPI, the resulting hybridomas may be analyzed against products that specifically bind to HPI fragments that include such regions. First HP
To select antibodies that bind specifically to the I homolog but not to another HPI homolog, bind positively to the first HPI homolog and bind to the second HPI homolog Selection can be based on lack of gender. Similarly, HPIs are positive upon selection for antibodies that specifically bind to HPI but do not specifically bind to different isoforms of the same protein (eg, different glycoforms having the same nuclear protein as HPI). And can be selected based on lack of binding to different isoforms (eg, glycoforms).

[0056] Antibodies specific to a region of the HPI may also be provided.

The antibodies relate to the localization and activity of the HPIs of the invention, eg, in diagnostic methods, imaging these proteins, measuring their levels in suitable physiological samples, etc. Can be used in a method using a known technique.

5.6 Isolation of HPI-encoding DNA Specific embodiments of the cloning of the HPI gene are provided below by way of example, but not by way of limitation.

The nucleotide sequences of the present invention include DNA and RNA, including HPI-encoding sequences or fragments or analogs thereof, and can be synthesized by methods well known in the art. Examples include conventional chemical approaches or polymerase chain reaction (PCR) amplification of overlapping oligonucleotides. The array also
Provides identification and cloning of HPI genes from any species, e.g., cDN
For screening A library, genomic library or expression library.

[0060] Nucleotide sequences, including sequences encoding the HPIs of the present invention, are useful for their ability to selectively form replicating molecules using the complementary extension of other protein genes. Depending on the application, various hybridization conditions are used to achieve various sequence identities.

Due to the high selectivity, relatively harsh conditions are used for replica formation, for example low salt or high temperature conditions. Here, "highly strict conditions" means that the temperature is
Sodium hydrogen phosphate, 7% sodium dodecyl sulfate (SDS), 1 mM ED
Hybridize to filter-bound DNA in TA and add
Means washing in 1 × SSC / 0.1% (Ausubel FM.
etc. , Eds. , 1989, Current Protocols in Mo.
See, Molecular Biology, Vol. I, Green Publisher
ng Associates, Inc. , And John Wiley and Sons, Inc. , New York, at p. 2.10.3; all of which are incorporated by reference). In some applications, moderately stringent hybridization conditions are required. Here, “moderately strict conditions” means 42
Means washing in 0.2 × SSC / 0.1% SDS at 20 ° C. (Ausub
el et al. , 1989, as described above). Hybridization conditions can also make hybrid replication destabilized and more stringent by adding increasing amounts of formamide. Thus, a particular hybridization is easily operable and is generally selected depending on the desired result. For example, the hybridization temperature in the presence of conventional 50% formamide is:
42 ° C. for probes that are 95-100% homologous to the HPI gene fragment, 37 ° C. for 90-95% homology, 70-90%
32 ° C. in case of homology.

In preparing a genomic library, DNA fragments are generated, some of which encode some or all of the HPI. The DNA can be cleaved at specific sites using various restriction enzymes. Alternatively, one fragments the DNA using a DNAse in the presence of manganase, or the DNA is physically fragmented, such as by sonication. The DN
The A fragments are then separated according to size by standard techniques including, but not limited to, agarose and polyacrylamide gel electrophoresis, column cloning and sucrose gradient centrifugation. The DNA fragments are then, but are not limited to, plasmids, cosmids are inserted into suitable vectors containing the bacteriophage lambda or T _4, and yeast artificial chromosome (YAC) (Sambrook, etc., 1989, Molecular Cloni
^{ng, A Laboratory Mannual, 2 nd} Ed. , Cold
Spring Harbor Laboratory Press, Cold
Springs Harbor, New York; Glover, D .; M. (
ed. ), 1985, DNA Cloning: A Practical Ap.
proach, MRL Preess, Ltd. Oxford, U.S.A. K. vo
l. Ausubel F. I, II; M. et al., Eds. , 1989, Curren
t Protocols in Molecular Biology, Vol
. I, Green Publishing Associates, Inc. ,
and John Wiley & Sons, Inc. , New York)
, But is not limited to these. Genomic libraries can be screened by performing nucleotide hybridization with a labeled probe. (Benton and Dabis, 1977, Science
e 196: 180; Grunstein and Hogness, 1975, P.
rc. Natl. Acad. Sci. ScL USA 72 : 3961).

The genomic library can be prepared using appropriate approaches well known in the art.
The PI can be screened using a labeled degenerate oligonucleotide probe corresponding to the amino acid sequence of any peptide. Preferably, any probe used is at least 10 nucleotides in length (more preferably, 1 nucleotide).
5 nucleotides, even more preferably 20 nucleotides).

As shown in Tables III and IV, some of the HPIs disclosed herein correspond to previously identified proteins whose sequences are encoded by publicly known genes. To screen such a gene, any probe that is complementary to the gene or its complement is used; preferably the probe is 10 or more nucleotides, more preferably 15 or more nucleotides. These are the nucleotides. National Center for Biotechnology Information (National Center for Biotechnology)
technology Information (NCBI)) (http : / entry Let held by accessible) in /Www.Ncbi.Nlm.Nih.Gov/ (Entrez) database (although, HPI-16
NCBI does not have a sufficient database), which provides the gene sequences for these HPIs under the accession numbers below, each of which is incorporated herein by reference.

[0065]

[Table 6]

For each of HPI-13 and HPI-21, a degenerate set of probes has been provided and is shown below: (a) Probe for HPI-21

[0067]

Embedded image

[0068]

Embedded image

[0069]

Embedded image

(B) Probe for HPI-13

[0071]

Embedded image

A library clone with inserted DNA encoding HPI or a fragment thereof may contain one or more degenerate oligonucleotide probes (
Or their complementary strands). Hybridization of such an oligonucleotide probe to a genomic library,
This is done using methods known in the art. For example, hybridization using the above-described degenerate set of oligonucleotide probes or one of its complements (or some of such sets, or their complements),
Performed under the highly stringent or moderately stringent conditions described above, or 2 × at 50 ° C.
Performed in SSC, 1.0% SDS and washed under similar conditions.

In yet another aspect, clones of a nucleotide sequence encoding part or all of an HPI or HPI-derived polypeptide are also obtained by a screening expression library. For example, DNA from the relevant source is isolated, random fragments are prepared, and ligated into an expression vector (eg, a bacteriophage, plasmid, phagemid, or cosmid), the expression vector comprising an insertion sequence in the vector. Then, it can be expressed by the introduced host cell. Various sequence analyzes are then used to select HPI or HPI-derived polypeptides. In one embodiment, the various anti-HPI antibodies of the present invention are used to identify a desired clone using methods known in the art. For example, Harlow and Lane,
1988, Antibodies: A Laboratory Manual,
Cold Springs Harbor Laboratory Press
, Cold Springs Harbor, NY, Appendix IV. Clones or plaques from the library are used for binding to the antibody,
These binding clones are identified.

In certain embodiments, colonies or plaques carrying DNA encoding the HPI or HPI-derived polypeptide are isolated from the 29th IC, such as Olswick, et al.
AAC, Houston, Tex. 1989 (incorporated herein by reference).
Is detected using DYNA beads. Anti-HPI antibody is tosylated DYN
A beads M208, which are then crosslinked to HPI or HP
Used to incorporate into colonies or plaques expressing the I-derived polypeptide. Colonies or plaques expressing HPI or HPI-derived polypeptide are identified as any binding beads.

Alternatively, the anti-HPI antibody is non-specifically immobilized on a suitable support such as, for example, silica or Celite ^™ resin. This material is then used to incorporate a bacterial clone expressing the HPI protein or HPI-derived polypeptide described in the previous section.

In another aspect, PCR amplification is used to produce substantially pure DNA encoding some or all of the HPI from genomic DNA. Oligonucleotide primers, degenerates or others related to known HPI sequences are used as primers.

PCR is performed, for example, on a Perkin-Elmer Cetus thermal cycler and, for example, on Thermus aqua
ticus) DNA polymerase (Gene Amp ^™ or AmpliTaq® DNA polymerase). For use in PCR reactions, several different degenerate primers can be selected for synthesis. The stringency of the hybridization conditions used to prime the PCR reaction can also be varied, thereby increasing or decreasing the nucleotide sequence homology between the degenerate primer and its homologous sequence in DNA. Can be. After successful amplification of a segment of the HPI-encoding sequence, the segment is molecularly cloned and sequenced and used as a probe to isolate a complete genomic clone. The determination of the complete nucleotide sequence of the gene as described below, the analysis of its expression, and the production of its protein product for functional analysis are performed sequentially.

[0078] The HPI gene can also be identified by mRNA selection by nucleic acid hybridization with in vitro translation. In this method, the fragments are used to separate complementary mRNAs by hybridization.
Such DNA fragments may be used in other species (eg, mouse, human)
May represent purified HPI DNA. The mRNA is identified by in vitro immunoprecipitation or functional analysis (eg, in vitro agglutination; binding to receptor) of the translation products of the separated mRNA in vitro. The complementary DNA fragment containing is identified. In addition, specific mRNA is
Polysomes separated from cells can be selected by incorporation into immobilized antibodies specifically immunized against HPI. Radiolabeled HPI cDNA can be synthesized using the selected mRNA (from the incorporated polysome) as a template. The radiolabeled mRNA or cDNA is then used for other genomic D
Used as a probe to identify HPI DNA fragments from NA fragments.

Alternative methods for isolating HPI genomic DNA include, but are not limited to, chemically synthesizing the gene sequence itself from a known sequence, or
And methods for producing cDNA for mRNA encoding For example,
DNA for cDNA cloning of the HPI gene is isolated from cells expressing the HPI. Other methods are possible within the scope of the present invention.

[0080] Any eukaryotic cell can potentially be used as a nucleic acid source for molecular cloning of the HPI gene. The nucleic acid sequence encoding the HPI can be isolated from vertebrates, mammals, humans, pigs, cows, cats, birds, horses, dogs, as well as primates, insects, plants and the like. The DNA is obtained from DNA cloned by standard methods well known in the art (eg, a DNA “library”) by chemical synthesis, cDNA cloning, or cloning of genomic DNA or fragments thereof purified from the desired cells (eg, Sambrook et al., 1989, Molec.
ullar Cloning, A Laboratory Manual, 2nd
Ed. , Cold Spring Harbor Laboratory P
less, Cold Spring Harbor, New York; Glo.
ver, D .; M. (Ed.), 1985, DNA Cloning: A Pra.
physical Approach, MRL Press, Ltd. , Oxfor
d. U. K. Vol. I, II. See). Clones obtained from genomic DNA may contain control genes and intron DNA regions in addition to the coding region; clones obtained from cDNA contain only exon sequences. Whatever the source, the HPI gene should be molecularly cloned into a suitable vector for propagation.

The identified or isolated gene or cDNA can then be inserted into a suitable cloning vector. Many art-known vector-host systems can be used. Possible vectors include, but are not limited to, plasmids or modified viruses, and the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as, for example, lambda derivatives, or plasmids, such as PBR322 or pUC plasmid derivatives, or Bluescript vectors (Stratagene). Insertion into a cloning vector is achieved, for example, by ligating the DNA fragment to a vector having ends that complementarily bind. However, if the complementary reaction site used to fragment the DNA is not present in the cloning vector, the ends of the DNA molecule can be modified enzymatically. Alternatively, any desired sites can be created by ligating nucleotide sequences (linkers) onto the ends of the DNA; these ligated linkers are used to bind specific chemically synthesized oligos encoding a restriction endonuclease recognition sequence. Contains nucleotides. In an alternative method, the cleaved vector and the HPI gene are homopolymerized (
It can be modified by homopolymeric tailing. The recombinant molecule is introduced into a host cell via transformation, transfection, infection, electroporation, etc., to produce many copies of the gene sequence.

In a specific embodiment, the isolated HPI gene, cDNA or synthetic DN
Transformation of a host cell with a recombinant DNA molecule linked to the A sequence allows for the production of multiple copies of the gene. Thus, a large amount of genes can be obtained by growing the transformant, separating the recombinant DNA molecule from the transformant, and recovering the inserted gene from the separated recombinant DNA as necessary.

The HPI sequences obtained according to the present invention include nucleotide sequences encoding amino acid sequences substantially similar to those found in wild-type HPI, nucleotide sequences encoding amino acid sequences having functionally the same amino acids, and others. Nucleotide sequences encoding the target derivatives or classes of the same.

5.7. Expression of DNA encoding HPI The nucleotide sequence encoding HPI or a functionally active analog or fragment or other derivative thereof can be inserted into a suitable expression vector, ie, encodes the inserted protein A vector containing elements necessary for the transcription and translation of a sequence. The necessary transcription and translation signals are also available in native HP
Provided by the I gene or its flanking region. A variety of host-vector systems are used to express the protein-encoding sequence. These include, but are not limited to, mammalian cell lines infected with a virus (eg, vaccinia virus, adenovirus, etc.); virus (eg, baculovirus)
An insect cell line infected with a microorganism; for example, a microorganism such as a yeast comprising a yeast vector, or a bacterium, DNA, plasmid DNA or cosmid DNA infected with a bacteriophage. The expression elements of vectors vary in their strength and specificities. Depending on the host-vector system used, any one of the appropriate transcription and translation elements may be used. In a specific embodiment, the human HPI
A gene or sequence encoding a functionally active site of human HPI is expressed.
In yet another embodiment, a target fragment comprising the domain of the HPI is expressed.

The method for inserting a DNA fragment into a vector described above can be used to construct an expression vector containing a chimeric gene consisting of appropriate transcription and translation control signals and protein coding sequences. These methods include in vitro recombinant DNA and synthetic techniques and in vivo recombinant DNA (genetic recombination). Expression of a nucleic acid sequence encoding an HPI or peptide fragment can be controlled by a second nucleic acid sequence, whereby the HPI or peptide sequence is expressed in a host transformed with the recombinant DNA molecule.
For example, HPI gene expression vector The expression of the HPI gene can be controlled by any promoter or enhancer element known in the art. Promoters used to control HPI gene expression include, but are not limited to, S
V40 early promoter region (Bernoist and Chambon, 1
981, Nature 290 : 304-310), 3 'of Rous sarcoma virus.
Promoters contained in long terminal repeats (Yamamoto et al., 1980, Cell
22 : 787-797), the herpes thymidine kinase promoter (Wagn).
er et al. , 1981, Proc. Natl. Acad. Sci. USA 78 :
1441-1445), the regulatory sequence of the metallothionein gene (Brinster
Et al., 1982, Nature 296 : 39-42); Expression of Prokaryotes such as the β-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. Natl. Acad. Scad. USA.
75: 3727-3731, or the tac promoter (DeBoer et al.
1983. Proc. Natl. Acad. Sci. USA 80 : 21-2
Prokaryotic expression vectors such as 5); and "Useful proteins from recombinant bacteria (
Useful proteins from recombinant bac
teria) "Scientific American, 1980, 242:
74-94; the nopaline synthase promoter region (Herrera-Estrella et al., Nature 303: 209-213) or the cauliflower mosaic virus 35S RNA promoter (Ga
rdner et al., 1981, Nucl. Acids Res. 9 : 2891) and the promoter of the photosynthetic enzyme ribulose diphosphate carboxylase (Herr).
era-Estrella et al., 1984, Nature 310; 115-120); for example, yeast such as Gal4 promoter, ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter. Promoter elements from other fungi, as well as the following animal transcription control regions, which are tissue-specific and used in transgenic animals; elastase I gene control regions active in pancreatic gland cells (Swift et al., 1984, Cell 38: 639
-646; Ornits et al., 1986, Cold Spring Harbor.
Symp. Quant. Biol. 50: 399-409; MacDonal
d, 1987, Hepatology 7: 425-515); an insulin gene regulatory region active in pancreatic beta cells (Hanahan, 1985, Nat.
315: 115-122); immunoglobulin gene regulatory regions active in lymphocytes (Grosschedl et al., 1984, Cell 38: 647).
-658; Adames et al., 1985, Nature 318: 533-538.
Alexander et al., 1987, Mol. Cell. Biol. 7: 143
6-1444), a mouse mammary adenocarcinoma virus control region active in testis, breast, lymphatic system and mast cells (Leder et al., 1986, Cell 45: 485-495).
, An albumin gene regulatory region active in liver (Pinkert et al., 1987, Ge
nes and Level. 1: 268-276), alpha-active in liver
Fetoprotein gene regulatory region (Krumlauf et al., 1985, Mol.
Cell. Biol. 5: 1639-1648; Hammer et al., 1987, S.
Science 235: 53-58); Alpha-1-antitrypsin gene regulatory region active in liver (Kelsey et al., 1987. Genes and Dave).
l. 1: 161-171), a beta-globulin gene regulatory region active in bone marrow cells (Mogram et al., 1985, Nature 315: 338-340; Ko).
llias et al. 1986, Cell 46: 89-94); a myelin basic protein gene regulatory region active in oligodendrocytes in the brain (Readhead et al., 1).
987, Cell 48: 703-712); Myosin light chain-2 active in skeletal muscle
Gene regulatory regions (Sani, 1985, Nature 314: 283-286)
), And a gonadotropin-releasing hormone gene regulatory region active in the hypothalamus (Mas
on et al., 1986, Science 234 : 1372-1378).

In a specific embodiment, the nucleic acid encoding the HPI, one or more replication origins, and optionally one or more selectable markers (eg, an antibiotic resistance gene) are operably manipulated. A vector containing an associated promoter is used.

In a specific embodiment, expression construction is performed by converting the HPI-encoding gene to three p
EcoRI restriction sites of each GEX vector (glutathione S-transferase expression vector; Smith and Johnson, 1988, Ge
ne 7: 31-40). This allows expression of the HPI product from subcloning in the correct reading frame.

An expression vector containing an HPI gene insert can be prepared by three general approaches: (a) nucleic acid hybridization, (b) the presence or absence of a “marker” gene, and (c) expression of the inserted sequence. Can be identified. In the first approach, the presence of the HPI gene inserted into the expression vector indicates that the inserted HPI gene
It can be detected by nucleic acid hybridization using a probe containing a sequence homologous to the gene. In the second approach, the recombinant vector / host system is used to generate certain "marker" gene functions (eg, thymidine kinase activity, antibiotic resistance, transformed phenotype, baculovirus) resulting from insertion of the HPI gene into the vector. Identification and selection based on the presence or absence of an absorber formation). For example, if the HPI gene is inserted within a marker gene of a vector, a recombinant having the HPI gene insert can be identified by a lack of function of the marker gene. In a third approach, a recombinant expression vector can be identified by analyzing the HPI gene product expressed by the recombinant. Such analysis can be based, for example, on the physical or functional characteristics of the HPI gene product in an in vitro analysis system, for example, based on binding to an anti-HPI antibody.

Once a particular recombinant DNA molecule has been identified and isolated, methods well known in the art can be used to propagate it. Once a suitable host system and growth conditions have been established, the recombinant expression vector can be propagated and prepared in large quantities. As explained above, expression vectors that can be used include, but are not limited to, the following vectors or derivatives thereof: human or animal viruses such as vaccinia virus or adenovirus; insects such as baculovirus Viruses; yeast vectors; bacteriophage vectors (eg, lambda);
And plasmid and cosmid DNA vectors.

In addition, a host cell type may be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be activated in the presence of certain inducers; that is, the expression of genetically engineered HPI is regulated. Furthermore, different host cells have characteristic and specific mechanisms for translation and post-translational processing and modification (eg, glycosylation, phosphorylation of proteins). Appropriate cell lines or host systems are chosen to confirm the desired modification and processing of the foreign protein expressed. For example, expression in a microbial strain is used to produce an unglycosylated nucleoprotein product. Expression in yeast produces a glycosylated product. Expression in mammalian cells can be used to confirm "native" glycosylation of a heterologous protein. Furthermore,
Different vector / host expression systems can affect processing reactions to different degrees.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, differentially expressed or pathway gene proteins (pathwa)
cell lines that stably express ygene proteins) can be engineered. Rather than using an expression vector containing a viral origin of replication, the host cell is transfected with DNA and a selectable marker that are controlled by appropriate expression control elements (eg, promoters, enhancer sequences, translation ends, polyadenylation sites, etc.). Can be converted. Following the introduction of the foreign DNA, the processed cells (en
grown cells) in nutrient media for 1-2 days, then replace them with selective media. The selectable marker in the recombinant plasmid confers resistance to selection and the cells will stably integrate the plasmid into their chromosomes, grow in order and be cloned and expanded into a cell line.
To form This method can be advantageously used to engineer cells that are differentially expressed or express pathway gene proteins. Such engineered cell lines are particularly useful in screening and evaluating compounds that are differentially expressed or affect the endogenous activity of pathway gene proteins.

Several selection systems have been used, including but not limited to herpes simplex virus kinase (Wigler et al., 1977, Cell 11: 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska).
And Szybalski, 1962, Proc. Natl. Acad. S
ci. USA 48: 2026) and adenine phosphoribosyltransferase (Lowy, etc., 1980.Cell 22: 817) genes, respectively tk ^-, hgprt ^- used as a cell ^- or aprt. Antimetabolite resistance also provides dhfr (Wigler et al., 198), which confers resistance to methotrexate.
0, Proc. Natl. Acad. Sci. USA 77: 3567; OHa
Re et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1
527); gpt that confers resistance to mycophenolic acid (Mulligan and Berg, 1981, Pr.
oc. Natl. Acad. Sci. USA 78: 2072); neo (Colberre-Garapin et al., 1981. J. Mol. Biol. 150: 1), which confers resistance to aminoglycoside G-418; and the hygro gene, which confers resistance to hygromycin.

In another specific embodiment, the HPI, fragment, analog or derivative is a fusion or chimeric protein product (a protein, fragment, analog, or peptide bond via a peptide bond to a heterologous protein sequence (of a different protein)). Derivative). Such a chimeric product can be obtained by ligating desired nucleic acid sequences encoding desired amino acid sequences to each other in an appropriate coding frame by a method well known in the art, and expressing the chimeric product by a general method well known in the art. It is made by letting Alternatively, such chimeric products are made by protein synthesis techniques, for example using a peptide synthesizer.

[0094] Both cDNA and genomic sequences can be cloned and expressed.

6: Example: Proteins from Serum With and Without Hepatome Proteins in Serum From 33 Patients With Hepatome and From 19 Patients With Cirrhosis Using Methods Referred To Below Were separated and analyzed by isoelectric focusing on SDS-PAGE. Each sample was performed in duplicate.

6.1 Sample Preparation Protein analysis was performed using the serum samples provided (Pierce BCA Cat).
# 23225). An amount of serum corresponding to 300 μg of the total protein was collected and an equal amount of 10% (w / v) SDS (Fluka 71729), 2
. 3% (w / v) dithiothreitol (BDH 443852A) was added.
The sample was heated at 95 ° C for 5 minutes and then cooled to 20 ° C. Next, the following buffers were added to the samples.

8M urea (BDH 452043w) 4% CHAPS (Sigma C3023) 65 mM dithiothreitol (DDT) 2% (v / v) Resolites 3.5-10 (BDH)
44338 2x) The mixture was vortexed and centrifuged at 13000 rpm for 5 minutes at 15 ° C, and the supernatant was analyzed by isoelectric focusing.

6.2 Isoelectric Focusing Isoelectric focusing (IEF) is performed using the Immobilin® Dry Strip Kit (Immunobiline DryStrip Kit) (Pharmacia Biotech) according to the manufacturer's instructions. (Instructions for the Immobilin® Dry Strip Kit, Pharmacia, #
18-1038-63, Edition AB (incorporated herein by reference in its entirety).
See Immobilized pH gradient (IPG) strip (18 cm, pH 3-1)
0, non-linear strip; Pharmacia Cat. # 17-1235-01)
8 as described in the Immobilin Dry Strip User's Manual
M urea, 2% (w / v) CHAPS, 10 mM DDT, 2% (v / v) Resolites 3.5 to 10 were rehydrated overnight at 20 ° C in a solution. For the IEF,
50 μl of the supernatant (prepared above) was loaded on the strip and the
The loading unit was on the base side of the strip. The loaded gel is then covered with mineral oil (Pharmacia 17-3335-01),
Pharmacia EPS3500XL Power Supply (Cat 10-3500-
01), the strip was immediately energized according to the following conditions:

Initial voltage = 300 V, 2 hours Linear ramp from 300 V to 500 V over 3 hours Maintain at 3500 V for 19 hours At all stages of the process, the current limit was set to 10 mA for 12 gels and the wattage limit Was 5W. The temperature was maintained at 20 ° C during operation.

6.3 Gel Equilibration and SDS-PAGE After the last 19 hour step, the strip was immediately transferred and immersed in a first solution of the following composition for 10 minutes at 20 ° C .: 6M urea 2% (w / v) DTT; 2% (w / v)
w / v) SDS; 30% (v / v) glycerol (Fluka 49767);
0.05M Tris / HCl (Sigma Cat T-1503), pH 6.8.
The strip was removed from the first solution and added to a second solution of the following composition at 20 ° C. for 10
Soak for 6 minutes: 6M urea; 2% (w / v) iodoacetamide (Sigma I-
6125); 30% (v / v) glycerol; 0.05 M Tris / HCl; p
H6.8. After removal from the second solution, the strip was removed using a Hochstrasser et al. (1988, Analytical B) modified as specified below.
iochemistry 173: 412-423) (hereby incorporated by reference in its entirety) on a supporting gel for SDS-PAGE.

6.4 Preparation of Supporting Gel The gel was cast between two glass plates of the following dimensions: 23c
m width x 24 cm length (back plate); 23 cm width x 24 cm length (front plate) with a 2 cm deep notch at the center 19 cm. To promote the covalent binding of the SDS-PAGE gel, the back plate was treated with γ-methacryl-
Oxypropyltrimethoxysilane (BindSilan ^™ ; Pharmacia)
Cat # 17-1330-01) treated with a 0.4% solution. Front plate is R
Treatment with epelSilan ^™ (Pharmacia Cat # 17-1332-01) reduced gel adhesion. Excess reagent was removed by washing with water and the plate was dried. At this stage, an adhesive barcode was attached to the back plate as a marker to identify the gel and identify the coated surface of the plate so that it did not come into contact with the gel substrate.

The dried plates were mounted in a casting box with a capacity of 13 gel sandwiches. The upper and lower plates of each of the sandwiches were spaced by a 1 mm thick, 2.5 cm wide spacer. The sandwich was sandwiched between acetate sheets to facilitate separation of the sandwich after gel polymerization. Next, casting was performed according to Hochstrasser and the like (same as described above).

[0103] A 9-16% linear polyacrylamide gradient was run on an Angelique gradient casting system.
ystem) (Large Scale B
(iology)) and stretched to a position 2 cm below the notch level in the front plate and cast. The following stock solutions were used.
Acrylamide (40% aqueous solution) was added to Serva (Cat. # 1067).
7). The crosslinker is used at a concentration of 2.6% (w / w) of the total initial monomer content.
DA (BioRad 161-0202) was used. The gel buffer used was 0.375 M Tris / HCl, pH 8.8. The polymerization catalyst is 0.0
5% (v / v) TEMED (Bio-Rad 161-0801) and 0.1% (w / v) APS (Bio-Rad 161-0700) were used as the initiator. SD
S was not contained in the gel, and no gel for concentration was used. The casting gel was polymerized overnight at 20 ° C., then stored at 4 ° C. in a sealed polyethylene bag containing 6 ml of gel buffer and used within 4 weeks.

6.5 SDS-PAGE 0.5% (w / v) agarose (Fluka Cat 05075) solution was added to running buffer (0.025 M Tris, 0.198 M glycine (Fluka 5005)
0), 1% (w / v) SDS, a small amount of bromophenol blue was added). The agarose suspension was heated to 70 ° C. with stirring until the agarose dissolved. The top of the supported 2D D gel was filled with agarose solution, the equilibrated strip was placed in agarose and gently tapped with a palette knife until the gel was in complete contact with the 2D D gel. Ames et al., 1995, Electrophor.
The gel was placed in a second D running tank as described in ess 16: 1255-1267 (incorporated herein by reference). The tank was filled with running buffer (as described above) such that the buffer level was just above the upper region of the second D gel containing polyacrylamide. Running buffer was added to the upper buffer compartment formed by the gel, and then a voltage was immediately applied to the gel using a Consult E-833 power supply. For 1 hour, run the gel at 20 mA /
Run on gel. The wattage limit was set at 150 W for a tank containing six gels, and the voltage limit was set at 600 V. After 1 hour, the gel was run at 40 mA / gel at the same voltage and wattage limit as before until the bromophenol blue line was 0.5 cm from the bottom of the gel. The temperature of the buffer was 10 ° C during electrophoresis.
Was maintained at.

6.6 Staining After the electrophoresis was completed, the gel was immediately removed for fixation. The upper plate of the gel cassette was carefully removed and the gel attached to the lower plate was removed. The top plate with the attached gel was then placed in a staining device that could accommodate 12 gels. The gel was run with 40% (v / v) ethanol (BDH 2871).
9) Completely immersed in a fixation solution consisting of 10% (v / v) acetic acid (BDH 1000016x), 50% (v / v) water (MilliQ-Millipore) and circulated continuously over the gel. After overnight incubation, the fixative was removed from the tank and the gel was washed with 7.5% (v / v) acetic acid, 0.05% (w / v) SDS, 92.5% (v / v).
v) Preparation was performed by immersing in water for 30 minutes. Next, the charged solution is removed,
The gel was stained by immersing it completely in the staining solution for 4 hours. Fluorescent dye solution was prepared according to the instructions by Sypro Red (Molecular Probes,
Inc., Eugene, Oreg.); The diluted solution was filtered under reduced pressure through a 0.4 μm filter.

6.7 Gel Imaging The computer readable output was prepared as described below with the following modifications.
Storm User's Guide, 1995, version 4.0,
Part No. 149-355, incorporated herein by reference) by imaging fluorescent stained gels with a Storm scanner (Molecular Dynamics, sunnyvale, CA). The gel was removed from the dye, rinsed briefly with water, and imaged on a storm scanner in red fluorescence mode with a PMT setting of 1000 V and a resolution of 200 μm. The gel remains firmly attached to the glass plate so that the gel remains in contact with the scanner stage during imaging. To avoid interference patterns due to non-uniform contact between the gel and the scanner stage, a film of water is introduced under the gel and care is taken to prevent air pockets. Furthermore, in the frame providing two fluorescent buttons that are imaged with the gel and provide relevant points (M1 and M2 are shown) to determine the x, y coordinates of other features detected in the gel. Put the gel on. A matching frame is fitted with an automated gel excizer to maintain the correct linearity of the gel.
isor). After imaging, the gel was sealed in a polyethylene bag containing a small amount of staining solution and stored at 4 ° C.

6.8 Digital Analysis of Data Data is provided in US patent application Ser. No. 08 / 980,574, sections 5.4-5.
. 4 (cited herein by reference), in particular as follows.

6.8.1 Computer Analysis of the Output of the Detector The output from the scanner, as well as the MELANIE® II 2D PAGE analysis program (Release 2.2, 1997, Bio-Rad)
Laboratories, Hercules, CA, Cat. # 170-756
6) to automatically search for registered sites, M1, M2; automatically collect images (ie, outside the gel boundary, eg, a reference frame).
remove the signal generated from the area of the scanned image of the image); remove the artificial image caused by dust; search for quantitative features; create an image file in GIF format. Features were searched using the following parameters:

Smooth = 2 Laplacian threshold 50 Saturation = 100 Peakedness = 0 Minimum perimeter = 10 6.9 Assignment of pI and MW values Image is anomalous Were evaluated by removing high, too low loading or overall image intensity, very poor resolution, or duplicate samples that were not too similar. If one image of a duplicate was removed, the other image of the duplicate was also removed, regardless of image quality. The removed sample was scheduled for repeated analysis.

[0110] Landmark identification was used to determine the pI and MW values of the features detected in the images. This step involves identifying certain proteins that are expected to be found in any given biological sample. Since these common proteins have the same isoelectric point, molecular weight between samples, they are used as standards; this step also corrected for any possible gel diversity or variation.

From the data set of normal serum gels, the gel was arbitrarily selected as the initial master gel. Landmark features were then identified by comparing features previously identified in 2D electrophoresis of normal human serum with features detected in this initial master gel (Bjellqvist et al., 1993, Electrophoresis 14: 1357). -1365, incorporated herein by reference).

The 14 landmark features were assigned PL1 for PL12 and PL15 for PL16 and were identified in the initial master gel. These landmark features were identified in FIG. 1 and were assigned pi and / or MW values as shown in Table 5.

[0113]

[Table 7]

[0114] As many of these landmarks as possible were identified in each gel image in the data set.

Next, all features in the master gel were determined by linear interpolation / extrapolation to the pI values of the two nearest landmarks assigned pI values (MELANI
(Using E II software), by linear interpolation / extrapolation to the MW values of the two closest landmarks assigned pI values and assigned MW values (
MW values were assigned (using MELANIE II software). Each feature was also labeled with a special number known as its molecular cluster index (or MCI).

A second master gel was selected for the cirrhosis gel and the HCC gel. The characteristics of these gels are paired with the common characteristics of the master gel, MELANI
E II 2D PAGE (Release 2.2) User Manual (The Melanie)
The algorithm provided by MELANIE II software was used as described in Section A, pp. 8-10, (Group, Geneva, Switzerland). The paired features are related to the corresponding MCI, and therefore to the pI and MW values. The unpaired features in these second master gels were determined by linear interpolation / extrapolation of landmark pI and MW (MELAN
PI and MW values were assigned (using IE II software). Additional special registrations in the MCI for these features have been made.

6.9.1 Profile Construction All gels in the data set were matched to the first and second master gels,
The paired features were related to the corresponding entries in the molecular cluster index.

The duplicate gel was then linearized through the landmarks and a fitting step was performed so that identical spots on the duplicate gel were paired. This indicates an increase in the assurance that the subsequently measured isoelectric point and molecular weight are accurate, since the paired spots perform the reproduction of the separation and remove artifacts.

Intensity measurements of each protein spot were performed and stored. Each protein spot was assigned an identification code and corresponded to a spot on the master gel.

The end result in this aspect of the analysis is the generation of a digital profile for each duplicate set of gels representing a single serum sample, the digital profile being assigned to each identified spot. On the other hand, (1) arbitrary certification code, (2) x and y coordinates, (3) isoelectric point, (4) molecular weight, (
5) signal value, (6) standard deviation for each of the above measurements, and (7)
A pointer to the MCI of the spot on the master gel to which the spot corresponds.

6.9.2. Cross-matching between samples Once the profile was generated, the analysis was directed to the analysis for interesting proteins. Each key feature in the profile is a metric,
A molecular cluster index "(MCI) is assigned, which identifies the feature for all gels and acts as a pointer to the above feature parameters (1)-(7). Prepared from master gels for the types (ie, hepatome serum and cirrhosis serum) Gels from all other samples of the same type were matched to the relevant first and second master gels, then digitally for each sample The profiles were annotated for each corresponding feature by adding the MCI assigned to the feature in the master profile.

6.9.3. Differential Analysis of Profiles Within each sample set (hepatome serum or cirrhosis serum), profiles were analyzed to identify and select those features present in at least 50% of the profiles. These selected features were then integrated into the hepatome serum feature set and the cirrhosis serum feature set. The corresponding features of each feature set were then compared to identify those features that exhibited at least a two-fold difference in median intensity between hepatome and cirrhosis sera. Features that exist differently
Hepatome-identified as diagnostic features (HFs).

6.9.4 Statistical Analysis of HFs Student's t-test (Student's
Statistical analysis using t-test) was performed to compare the distribution of signal intensities for features in 33 hepatome serum profiles and 19 cirrhosis serum profiles. A p-value was derived for each HF.

6.10. Recovery and Analysis of Selected Proteins Proteins of HF are automatically excised, processed, and
Tryptic peptides were made using the preferred technique described in 980,574, incorporated herein by reference in its entirety (The Preferred Technology). The partial amino acid sequences of these peptides are given de novo (d
e novo) determined by mass spectrometry using sequence analysis.

6.11. Results These initial experiments identified 17 features reduced and 44 features increased in hepatome serum compared to cirrhosis serum. The HFs and p values for each are shown in Tables 1 and 2. Each HF was differently present in hepatome serum compared to non-hepatome serum (p <0.1). Some preferred HFs (HF-1, HF-3, HF-10, HF-15, HF-16,
HF-17, HF-18, HF-19, HF-20, HF-21, HF-23,
HF-24, HF-25, HF-26, HF-28, HF-29, HF-31,
HF-32, HF-33, HF-34, HF-36, HF-37, HF-38,
HF-39, HF-40, HF-42, HF-44, HF-46, HF-47,
HF-48, HF-49, HF-51, HF-52, HF-53, HF-54,
HF-55, HF-56, HF-59, and HF-60), the differences are striking (p <0.01) and certain more preferred HFs (HF-1, HF-1)
7, HF-18, HF-20, HF-21, HF-23, HF-24, HF-2
5, HF-28, HF-29, HF-31, HF-33, HF-34, HF-3
6, HF-39, HF-44, HF-51, HF-52, HF-54, HF-5
5, HF-59 and HF-60), the difference was even more pronounced (p ≦ 0.001).

A partial amino acid sequence has been determined for HPIs that are differentially present in these HFs. Details of these HPIs are shown in Tables 3 and 4. Computer searches of public databases identified at least two HPIs for which no oligonucleotide encoding the peptide sequence, as well as the partial amino acid sequence, was described in the public databases examined. . Table 4 shows that several HPIs are isoforms of the same protein. For example, HPI-2, HPI-6, HPI-8, HPI-14, H
PI-15, HPI-17, and HPI-18 are complementary element 4 isoforms; HPI-4, HPI-5, and HPI-12 are complementary element 3
And HPI-19, HPI-20, HPI-2
1 is an isoform of ceruloplasmin. These isoforms are believed to result from differences in post-translational processing (eg, glycosylation, phosphorylation, acylation or minimal proteolysis).

The present invention is not particularly limited to the scope according to the illustrated embodiments, which are set forth to illustrate one aspect of the invention. Of course, various modifications of the invention in addition to those enumerated herein from the foregoing description and the accompanying drawings will be apparent to those skilled in the art. Such modifications are intended to be made within the scope of the appended claims.

All publications cited herein are incorporated by reference in their entirety.

[Brief description of the drawings]

FIG. 1 is an image obtained from two-dimensional electrophoresis of normal human serum.
PL1 to PL12 and PL15 and PL
16 is shown.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG , KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Palek, Rajesh, Viku, United Kingdom Abingdon, Abingdon Science Park, Burton Lane, The Quadrant 10, Oxford Glyco Sciences (UK) Limited (72) Inventor Patel, Zakolbhai, Pershombombhai United Kingdom, Oxon Auex 14 3 Wyeth, Abingdon, Abingdon Science Park, Burton Rain, The Quadrant 10, Oxford Glycoscience (S) (UK) Limited (72) Inventors Townsend, Robert, Raid Oxon Ox, United Kingdom 14 3 Wyeth, Abingdon, Abingdon Science Park, Burton Lane, The Quadrant 10, Oxford GlycoScience (UK) Limited Ted (72) Inventor Moises, Christopher United Kingdom, Oxon Ox143 3 Wyeth, Abingdon, Abingdon Science Park, Burton Lane, The Quadrant 10, Oxford Glycosciences (UK) Limited (72) Inventor Johnson, Philip, Jay. N. China. tea. , Shatin, Prince of Wales Holpital, Department of Clinical Oncology, The Chinese University of Hong Kong F-term (reference) 2G045 AA26 AA40 CA25 CA26 DA36 DA78 FB02 FB03 FB05 4H045 AA11 BA14

Claims (34)

[Claims] 1. a) analyzing a sample of serum or plasma from a subject by two-dimensional electrophoresis to produce a two-dimensional array of features; and Comparing the intensity of each selected characteristic in the sample with the intensity of the selected characteristic in serum or plasma from one or more humans without a hepatome for at least two selected characteristics that are For the screening, diagnosis or prognosis of a hepatome in a human subject, wherein the intensity of the selected feature in the sample is indicative of the presence or absence of the hepatome in the subject.
Alternatively, a method for monitoring an anti-hepatome agent or therapeutic effect administered to a human subject. 2. The method of claim 1, wherein said selected characteristic does not include alpha fetoprotein (AFP). 3. The method of claim 1, wherein alpha fetoprotein (AFP) is the selected characteristic. 4. (a) analyzing a serum or plasma sample from a subject by two-dimensional electrophoresis to separate a plurality of proteins according to isoelectric point and electrophoretic mobility; (b) the following hepatome diagnostic features: (HF): HF-1, HF-2, HF-3, HF
-4, HF-5, HF-6, HF-7, HF-8, HF-9, HF-10, HF
-11, HF-12, HF-13, HF-14, HF-15, HF-16, HF
-17, HF-18, HF-19, HF-20, HF-21, HF-22, HF
-23, HF-24, HF-25, HF-26, HF-27, HF-28, HF
-29, HF-30, HF-31, HF-32, HF-33, HF-34, HF
-35, HF-36, HF-37, HF-38, HF-39, HF-40, HF
-41, HF-42, HF-43, HF-44, HF-45, HF-46, HF
-47, HF-48, HF-49, HF-50, HF-51, HF-52, HF
Quantitatively detecting at least one of -53, HF-54, HF-55, and HF-56, for screening, diagnosing or prognosing a hepatome in a human subject, or in a human subject. For monitoring an anti-hepatome drug or therapeutic effect administered to a subject. 5. The step (b) comprises the following steps: HF-17, HF-18, HF-
20, HF-21, HF-23, HF-24, HF-25, HF-28, HF-
29, HF-31, HF-33, HF-34, HF-36, HF-39, HF-
5. The method of claim 4, comprising quantitatively detecting at least one of 44, HF-51, HF-52, HF-54 and HF-55. 6. The step (b) comprises the following steps: HF-17, HF-18, HF-
20, HF-23, HF-24, HF-25, HF-29, HF-33, HF-
The method according to claim 5, comprising quantitatively detecting at least one of 34, HF-39, HF-44 and HF-55. 7. Step (a) comprises isoelectric focusing followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE),
A method according to any one of claims 4, 5 or 6. 8. (a) In a serum or plasma sample from a subject, the following hepatome diagnostic protein isoforms (HPI): HPI-1, HPI-
2, HPI-3, HPI-4, HPI-5, HPI-6, HPI-8, HPI-
9, HPI-10, HPI-11, HPI-12, HPI-13, HPI-14
, HPI-15, HPI-16, HPI-17, HPI-18, HPI-19,
Quantitatively detecting at least one of HPI-20, HPI-21 and HPI-22, for screening, diagnosis or prognosis of a hepatome in a subject, or administered to a subject. A method for monitoring an anti-hepatome drug or therapeutic effect. 9. The step of quantitatively detecting comprises testing at least one aliquot of the sample, the step of testing comprising: (a) immunospecifically to the aliquot against a preselected HPI. 9. The method of claim 8, further comprising: contacting with an antibody of interest; and (b) detecting whether binding has occurred between the antibody and at least one of the subsamples. 10. The method according to claim 9, wherein said antibody is a monoclonal antibody. 11. The method of claim 9, wherein the step of quantitatively detecting comprises testing a plurality of subsamples with a plurality of antibodies. 12. The method according to claim 11, wherein said antibody is a monoclonal antibody. 13. The isolated hepatome diagnostic protein (HPI): H
PI-1, HPI-2, HPI-3, HPI-4, HPI-5, HPI-6, H
PI-8, HPI-9, HPI-10, HPI-11, HPI-12, HPI-
13, HPI-14, HPI-15, HPI-16, HPI-17, HPI-1
8, a formulation comprising one of HPI-19, HPI-20, HPI-21 and HPI-22. 14. A kit comprising the one preparation according to claim 13. 15. A kit comprising the plurality of preparations according to claim 13. 16. A formulation comprising an isolated human protein, wherein said protein comprises a peptide having the sequence AETTDF. 17. The protein has an isoelectric point (pI) of about 7.44 and about 6
17. The formulation of claim 16, having an apparent molecular weight (MW) of 3,882. 18. The pI is within 10% of 7.44 and the MW is 63,8.
18. The formulation of claim 17, which is within 10% of 82. 19. The pI is within 5% of 7.44 and the MW is 63,88.
20. The formulation of claim 18, which is within 5% of 2. 20. The pI within 1% of 7.44 and the MW is 63,88.
20. The formulation of claim 19, which is within 1% of 2. 21. A preparation comprising an isolated human protein, wherein the protein has the following sequence: IWIGTTR, IWLGTTR, LWIGTTR or L
A formulation comprising a peptide having one of WLGTTR. 22. The protein has an isoelectric point (pI) of about 5.29 and about 4
22. The formulation of claim 21 having an apparent molecular weight (MW) of 3,541. 23. The pi is within 10% of 5.29 and the MW is about 43,
23. The formulation of claim 22, which is within 10% of 541. 24. The pi is within 5% of 5.29 and the MW is about 43,5.
24. The formulation of claim 23, which is within 5% of 41. 25. The pI within 1% of 5.29 and the MW is about 43,5.
25. The formulation of claim 24 which is within 1% of 41. 26. The following hepatome diagnostic protein isoform: HPI-
1, HPI-2, HPI-3, HPI-4, HPI-5, HPI-6, HPI-
8, HPI-9, HPI-10, HPI-11, HPI-12, HPI-13,
HPI-14, HPI-15, HPI-16, HPI-17, HPI-18, H
An antibody capable of immunospecifically binding to one of PI-19, HPI-20, HPI-21 and HPI-22. 27. A kit comprising the one antibody according to claim 26. 28. A kit comprising the plurality of antibodies according to claim 26. 29. A method for screening, diagnosing or prognosing a hepatome in a human subject, or for monitoring the effect of an anti-hepatome agent.
Use of one or more HFs according to claim 4. 30. A method for screening, diagnosing or prognosing a hepatome in a human subject, or for monitoring the effect of an anti-hepatome agent.
Use of one or more of the HPIs of claim 8. 31. A method for screening, diagnosing, or prognosing a hepatome in a human subject, or for monitoring the effect of an anti-hepatome agent.
Use of a protein according to any one of claims 16 to 25. 32. A method for screening, diagnosing or prognosing a hepatome in a human subject, or for monitoring the effect of an anti-hepatome agent.
Use of one or more antibodies that are immunospecific for one or more of the HPIs of claim 8. 33. The use according to claim 32, wherein said one or more antibodies is as defined in claim 26. 34. The use according to any one of claims 29 to 33 for use in combination with an assay or evaluation for hepatitis B and / or hepatitis C.