JP2009103681A - Effect prediction method for interferon therapy and prediction kit of interferon therapy - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique capable of predicting an effect of interferon therapy to hepatitis C virus (HCV) persistently-infected person. <P>SOLUTION: A subject is anticipated whether the subject will show excellent response (sustained virological response: SVR) or will show relapse of HCV by making concentration of a substance in a body fluid acquired from the subject being a HCV persistently-infected person specific a marker. As an instance of a body fluid, blood is usually pointed out. The marker substance is captured on a carrier such as a substrate on which an ion exchanger or metal chelator has been immobilized. The concentration of the marker substance can be measured by a mass spectrometry. A kit capable of easily performing this method is also provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an interferon therapy effect prediction method and a prediction kit. The method for predicting the effect of interferon therapy according to the present invention includes a hepatitis C virus persistently infected patient whose hepatitis C virus relapses after completion of interferon therapy, and a hepatitis C virus persistent infection whose effect continues without relapsed. Can be discriminated from the person.

  Hepatitis C is viral hepatitis caused by infection with hepatitis C virus (hereinafter abbreviated as “HCV”). Hepatitis C progresses gradually without any special symptoms, and if left untreated, it often shifts to cirrhosis and further to liver cancer over 10 to 30 years.

  As a method for treating hepatitis C, administration of interferon (interferon therapy) has been conventionally performed. Interferon has an action of suppressing the growth of HCV, and if treatment is successful, HCV disappears from the body. However, it has been found that there are limited hepatitis C patients for whom interferon monotherapy (interferon monotherapy) is effective. Therefore, a combination administration of interferon and another antiviral agent ribavirin (interferon / ribavirin combination therapy) has been developed, and is highly effective compared to interferon monotherapy. However, there are still many patients with hepatitis C that are not treated with interferon / ribavirin combination therapy.

  Interferon is one of physiologically active substances produced in vivo, and is known to have antiviral action and anticancer action. Interferon has main subtypes of α, β, and γ. Among them, α and β are used for the treatment of hepatitis C. On the other hand, ribavirin (1-beta-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a kind of nucleic acid analog with purine skeleton, and is an antiviral agent for internal use such as influenza, herpes, measles, etc. It has long been used for treatment. However, administration of ribavirin alone has little effect on the treatment of hepatitis C, and ribavirin exhibits the effect of enhancing the HCV elimination effect when used in combination with interferon.

  When treating hepatitis C by interferon therapy, administration over a long period of about 40 weeks is necessary. However, interferon has side effects such as fever, general malaise, and joint pain, and the physical burden on patients due to long-term administration is large. Furthermore, interferon is an expensive medicine and has a large economic burden. In addition, when interferon / ribavirin combination therapy is performed, ribavirin administration for the same period as that of interferon administration is necessary. However, ribavirin also has a side effect of causing hemolysis, which further increases the physical and economic burden on the patient. And for hepatitis C patients who cannot obtain the therapeutic effect by interferon therapy or interferon-ribavirin combination therapy, these therapies only cause side effects and have no benefit. Therefore, it is desirable to predict the effects of interferon therapy or interferon / ribavirin combination therapy for hepatitis C patients before the start of treatment, and exclude patients who cannot expect therapeutic effects from these therapies.

  Whether the interferon therapy is successful or not is said to involve two factors, the virus side and the patient side. As factors on the virus side, the involvement of the HCV genotype and the amount of HCV has been pointed out. That is, HCV has a plurality of genotypes. Interferon is less effective for HCV with genotypes 1a and 1b, and interferon is more effective for other HCV with genotypes 2a and 2b, for example. Moreover, interferon is less effective as the amount of HCV increases. And the method of predicting the effect of interferon therapy by performing the typing of the infected HCV (Patent Document 1) and the mutation detection of the core region (Patent Document 2) has been proposed. On the other hand, as a technique focusing on the factors on the patient side, single nucleotide polymorphism (SNP) (Patent Documents 3 and 4), expression level of interferon receptor in liver (Patent Document 5), concentration of a specific protein in blood (patent A method for predicting the effect of interferon therapy using literature 6) as an index has been proposed.

  On the other hand, the effect determination of interferon therapy is performed based on a finer classification (Non-Patent Document 1). That is, regarding the virological effect, HCV-RNA is measured at three stages at the 12th week after the start of treatment, at the end of the treatment, and 24 weeks after the end of the treatment. Then, negative HCV (early virological response; EVR) at the 12th week of treatment, negative HCV (end-of-treatment response; ETR) at the end of treatment, and negative HCV at 24 weeks after the end of treatment Assess for sustained virological response (SVR). Here, the evaluation at 24 weeks after the end of the treatment is considered to be most important, and HCV-RNA negative (SVR) at this stage is judged as “highly effective”, and positive is judged as invalid. Furthermore, invalid cases are classified into “relapse” and “non-response” (NR). “Relapse” is an ineffective case in which HCV-RNA was negative at the end of treatment, and “No response” (NR) is an ineffective case in which HCV-RNA was positive at the end of treatment. Patients classified as “non-responsive” will not be suitable for interferon therapy.

  A patient who exhibits “significant effect” (SVR) by interferon therapy and a patient whose HCV “relapses” are both suitable as subjects of interferon therapy, but a treatment plan suitable for each is required. For patients who exhibit “significant efficacy”, it is effective to receive interferon therapy positively, thereby suppressing the incidence of liver cancer. On the other hand, for patients whose HCV “relapses”, it is effective to administer interferon for a long time without interruption, thereby making it possible to suppress the incidence of liver cancer.

  However, there is no technology for predicting whether a patient with hepatitis C is “remarkably effective” by interferon therapy or who “relapses” HCV before starting treatment. It is an obstacle. For example, interferon administration is interrupted after a certain period of time for patients with relapsed HCV, and as a result, HCV relapses, contributing to a decrease in treatment results. All of the conventional techniques discriminate between patients who are “non-responsive” (NR) and other patients with respect to interferon therapy, and patients who show “significant” (SVR) and HCV are “relapsed” ( It does not discriminate from relapsed patients.

  In the techniques disclosed in Patent Documents 1 to 4, it is necessary to prepare DNA and RNA from a sample collected from a patient, and the operation is complicated. In general, when making a determination based on the type or amount of HCV, only a rough determination can be made as to whether or not interferon is effective. Moreover, in the technique disclosed in Patent Document 5, it is necessary to collect liver tissue as a specimen, which involves invasion.

  Interferon therapy is effective not only for patients with hepatitis C, but also for people with persistent hepatitis C virus infection (HCV carriers). That is, if interferon therapy is successful, HCV disappears from a person with persistent hepatitis C virus infection.

JP 2001-238687 A JP 2007-43985 A JP 2003-339380 A JP 2004-298011 A JP 2001-149076 A International Publication No. 2006/107078 Pamphlet The Japan Society of Hepatology edited by Chronic Hepatitis Treatment Guide 2006, published on January 11, 2006, p. 28

  An object of the present invention is to provide a series of techniques capable of discriminating between a hepatitis C virus persistently infected person who is highly effective by interferon therapy and a hepatitis C virus persistently infected person whose HCV relapses after completion of interferon therapy. There is.

One aspect of the present invention is an interferon therapy for a patient with persistent hepatitis C virus infection, using as an index the concentration of at least one of the following marker substances (A1) to (A4) in a body fluid collected from the patient with persistent hepatitis C virus infection: This is a method for predicting the effect of interferon therapy.
(A1) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 7780 when subjected to mass spectrometry;
(A2) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 9300 when subjected to mass spectrometry;
(A3) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 51300 when subjected to mass spectrometry;
(A4) A protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 125000 when subjected to mass spectrometry.

  The method for predicting the effect of interferon therapy in this aspect uses the concentration of a marker substance in the body fluid of a person with persistent hepatitis C virus infection as an index. Then, at least one of the marker substances (A1) to (A4) is used as the marker substance. According to the method of this aspect, whether the subject's hepatitis C virus persistent infection is a person who shows a remarkable effect (SVR) by interferon therapy or a person whose HCV relapses after completion of interferon therapy. Can be predicted. In addition, the method of this aspect is simple and quick because the body fluid collected from the patient is used as the test material in the same manner as in general clinical tests.

  As used herein, “interferon therapy” refers to antiviral therapy that aims to eliminate HCV by administering interferon to persons with persistent hepatitis C virus infection. “Interferon therapy” includes interferon monotherapy (interferon monotherapy), as well as interferon and other drugs, for example, interferon / ribavirin combination therapy (interferon / ribavirin co-administration). In addition to the natural interferon, the administered interferon includes derivatives such as interferon to which polyethylene glycol is added (PEG interferon).

  The mass / charge ratios of “about 7780”, “about 9300”, “about 51300”, “about 125000” and the like are values in consideration of the error range of the measurement value in mass spectrometry, and about 7780 is approximately 7780 ± 0. 2%, about 9300 is about 9300 ± 0.2%, about 51300 is about 51300 ± 0.2%, and about 125000 is about 125000 ± 0.2%. In addition, these marker substances are mainly proteins present in blood. The concentrations of the marker substances (A1), (A2), and (A3) are high in the body fluid of a patient with persistent hepatitis C virus infection that is expected to relapse HCV after completion of interferon therapy. It shows a low value in the body fluid of a person with persistent hepatitis C virus infection that is expected to be shown. On the other hand, the marker substance (A4) shows a low value in the former body fluid and a high value in the latter body fluid.

Preferably, at least one of the following (1) to (3) is satisfied.
(1) The marker substance (A1) is platelet factor 4 or a modified form thereof.
(2) The marker substance (A2) is connective tissue activating peptide 3 or a modified form thereof.
(3) The marker substance (A3) is a vitamin D binding protein or a modified form thereof.

  Platelet Factor 4, Connective tissue-activating peptide 3, and Vitamin D Binding Protein are all well known for their physicochemical properties. Therefore, according to this preferable aspect, the marker substances (A1), (A2), and (A3) can be easily analyzed.

  In this specification, the term “modified protein” is used in various meanings. A representative example of “modified protein” is a protein in which at least one of the amino acid residues constituting the protein is modified. “Modification” includes not only addition of a compound or functional group (eg, phosphorylation) but also elimination (eg, dephosphorylation). The “protein or a modified form thereof” includes an isoform of the protein. Further, “protein or a modified form thereof” includes substantially the same protein in which several amino acid residues are deleted, substituted or added in the primary structure of the protein. Furthermore, “protein or a modified product thereof” includes a protein fragment derived from the protein that has been cleaved by a protease or the like. In the case of a complex protein, “protein or a modified product thereof” includes its subunits. Conversely, in the case of monomeric proteins, “proteins or modifications thereof” include multimers such as dimers thereof.

Another aspect of the present invention is the above-mentioned hepatitis C virus persistence, using as an index at least one concentration of a marker substance belonging to any of the following (C1) to (C3) in a body fluid collected from a hepatitis C virus persistently infected person: This is a method for predicting the effect of interferon therapy, characterized by predicting the effect of interferon therapy on an infected person.
(C1) platelet factor 4 or a modified form thereof,
(C2) connective tissue activating peptide 3 or a modified form thereof,
(C3) Vitamin D binding protein or a modified form thereof.

  In the method for predicting the effect of interferon therapy of this aspect, at least one of marker substances belonging to any one of the above (C1) to (C3) is used. The method of this aspect also predicts whether the subject's persistently infected hepatitis C virus is highly effective (SVR) due to interferon therapy, or is HCV relapsed after completion of interferon therapy can do. The method of this aspect is also simple and quick because the body fluid collected from the patient is used as the test material in the same manner as in general clinical tests. Furthermore, since platelet factor 4 and connective tissue activating peptide 3 and vitamin D binding protein are all well known in physicochemical properties, the marker substance can be easily analyzed according to this preferred aspect. . The “modified protein” is as described above.

Preferably, at least one concentration of the following marker substances (B1) to (B11) in the body fluid is further used as an index.
(B1) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and generates an ion peak with a mass / charge ratio of about 3360 when subjected to mass spectrometry,
(B2) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 3960 when subjected to mass spectrometry;
(B3) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 4160 when subjected to mass spectrometry;
(B4) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and generates an ion peak with a mass / charge ratio of about 6190 when subjected to mass spectrometry,
(B5) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 6510 when subjected to mass spectrometry;
(B6) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 8610 when subjected to mass spectrometry;
(B7) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 8710 when subjected to mass spectrometry;
(B8) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and generates an ion peak with a mass / charge ratio of about 13900 when subjected to mass spectrometry;
(B9) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 17300 when subjected to mass spectrometry;
(B10) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 17400 when subjected to mass spectrometry;
(B11) A protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M and produces an ion peak with a mass / charge ratio of about 28100 when subjected to mass spectrometry.

  In this preferable aspect, the effect of interferon therapy is predicted using at least one concentration of marker substances (B1) to (B11) as an index. Marker substances (B1) to (B11) are all discriminated from those with persistent hepatitis C virus infection who are predicted to be “no response” (NR) to interferon therapy and those with other hepatitis C virus persistent infection It is a marker substance that can be. According to the method of this aspect, the hepatitis C virus persistently infected who is predicted to be “non-responsive” (NR) to interferon therapy can be excluded from the subject, and thus the prediction accuracy is high.

  In addition, hepatitis C virus non-responders who are “non-responding” (NR) to interferon therapy and who are persistently infected with hepatitis C virus eventually become HCV negative (ETR) at the end of interferon therapy. It is a person with persistent hepatitis C virus infection. Such persistently infected hepatitis C virus includes both those who show “significant effect” (SVR) by interferon therapy and those who “relapse” HCV after the end of interferon therapy.

  Here, the mass / charge ratios of “about 3360”, “about 8610”, “about 13900”, “about 28100”, and the like are values in consideration of error ranges of measured values in mass spectrometry, and about 3360 is approximately 3360. ± 0.2%, about 8610 is about 8610 ± 0.2%, about 13900 is about 13900 ± 0.2%, and about 28100 is about 28100 ± 0.2%. In addition, these marker substances are mainly proteins present in blood. In addition, marker substances (B1), (B2), (B3), (B4), (B6), and (B8) are predicted to be “non-responsive” to interferon therapy. It shows a low value in other body fluids and a high value in other body fluids of persistently infected hepatitis C virus. On the other hand, the marker substances (B5), (B7), (B9), (B10), and (B11) show high values in the former body fluid and low values in the latter body fluid.

Preferably, at least one of the following (4) to (13) is satisfied.
(4) The marker substance (B1) is apolipoprotein A1 or a modified form thereof.
(5) The marker substance (B3) is a C1 inhibitor or a modified form thereof.
(6) The marker substance (B4) is apolipoprotein A1 or a modified form thereof.
(7) The marker substance (B5) is apolipoprotein C1 or a modified form thereof.
(8) The marker substance (B6) is complement C4 or a modified form thereof.
(9) The marker substance (B7) is apolipoprotein A2 or a modified form thereof.
(10) The marker substance (B8) is transthyretin or a modified form thereof.
(11) The marker substance (B9) is apolipoprotein A2 or a modified form thereof.
(12) The marker substance (B10) is apolipoprotein A2 or a modified form thereof.
(13) The marker substance (B11) is apolipoprotein A1 or a modified form thereof.

  Apolipoprotein A1 (Apolipoprotein A1), C1 inhibitor (Plasma Protease C1 Inhibitor), apolipoprotein C1 (Apolipoprotein C1), complement C4 (Complement C4), apolipoprotein A2 (Apolipoprotein A2), and transthyretin (Transthyretin) , Since physicochemical properties are well known, according to this preferred aspect, the marker substances (B1), (B3), (B4), (B5), (B6), (B7), (B8) , (B9), (B10), and (B11) are easy to analyze.

Preferably, at least one concentration belonging to any of the following (D1) to (D6) in the body fluid is further used as an index.
(D1) Apolipoprotein A1 or a modified product thereof,
(D2) C1 inhibitor or a modified form thereof,
(D3) Apolipoprotein C1 or a modified product thereof,
(D4) complement C4 or a modified product thereof,
(D5) Apolipoprotein A2 or a modified product thereof,
(D6) Transthyretin or a modified product thereof.

  In this preferable aspect, the effect of interferon therapy is predicted using at least one concentration belonging to any of the above (D1) to (D6) as an index. The marker substances belonging to (D1) to (D6) are all persistently infected with hepatitis C virus that is predicted to be “no response” (NR) to interferon therapy, and other persistently infected hepatitis C viruses. It is a marker substance that can discriminate. According to the method of this aspect, the patient with persistent hepatitis C virus infection who is predicted to be “non-responsive” (NR) to interferon therapy can be excluded from the subject, and thus the prediction accuracy is high. Furthermore, since apolipoprotein A1, C1 inhibitor, apolipoprotein C1, complement C4, apolipoprotein A2, and transthyretin are all well known for their physicochemical properties, according to this preferred aspect, the marker substance Is easy to analyze. The “modified protein” is as described above.

Another aspect of the present invention is an interferon therapy for a patient with persistent hepatitis C virus infection, using as an index the concentration of at least one of the following marker substances (B1) to (B11) in a body fluid collected from the patient with persistent hepatitis C virus infection: This is a method for predicting the effect of interferon therapy.
(B1) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and generates an ion peak with a mass / charge ratio of about 3360 when subjected to mass spectrometry,
(B2) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 3960 when subjected to mass spectrometry;
(B3) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 4160 when subjected to mass spectrometry;
(B4) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and generates an ion peak with a mass / charge ratio of about 6190 when subjected to mass spectrometry,
(B5) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 6510 when subjected to mass spectrometry;
(B6) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 8610 when subjected to mass spectrometry;
(B7) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 8710 when subjected to mass spectrometry;
(B8) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and generates an ion peak with a mass / charge ratio of about 13900 when subjected to mass spectrometry;
(B9) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 17300 when subjected to mass spectrometry;
(B10) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 17400 when subjected to mass spectrometry;
(B11) A protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M and produces an ion peak with a mass / charge ratio of about 28100 when subjected to mass spectrometry.

  In the method for predicting the effect of interferon therapy in this aspect, the effect of interferon therapy is predicted using at least one concentration of marker substances (B1) to (B11) as an index. According to the method of this aspect, it is possible to predict whether or not the subject who is persistently infected with hepatitis C virus is “non-responsive” (NR) to interferon therapy. The method of this aspect is, for example, whether the subject who is persistently infected with hepatitis C virus is a person who exhibits a remarkable effect (SVR) by interferon therapy or a person whose HCV relapses after completion of interferon therapy. This is useful for preliminary judgment before prediction, that is, primary screening.

Preferably, at least one of the following (4) to (13) is satisfied.
(4) The marker substance (B1) is apolipoprotein A1 or a modified form thereof.
(5) The marker substance (B3) is a C1 inhibitor or a modified form thereof.
(6) The marker substance (B4) is apolipoprotein A1 or a modified form thereof.
(7) The marker substance (B5) is apolipoprotein C1 or a modified form thereof.
(8) The marker substance (B6) is complement C4 or a modified form thereof.
(9) The marker substance (B7) is apolipoprotein A2 or a modified form thereof.
(10) The marker substance (B8) is transthyretin or a modified form thereof.
(11) The marker substance (B9) is apolipoprotein A2 or a modified form thereof.
(12) The marker substance (B10) is apolipoprotein A2 or a modified form thereof.
(13) The marker substance (B11) is apolipoprotein A1 or a modified form thereof.

  Apolipoprotein A1, C1 inhibitor, apolipoprotein C1, complement C4, apolipoprotein A2, and transthyretin are all well known for their physicochemical properties. Therefore, according to this preferred aspect, the marker substance (B1 ), (B3), (B4), (B5), (B6), (B7), (B8), (B9), (B10), and (B11) are easy to analyze.

Another aspect of the present invention is the above-mentioned hepatitis C virus persistence, using as an index at least one concentration of a marker substance belonging to any of the following (D1) to (D6) in a body fluid collected from a hepatitis C virus persistently infected person: This is a method for predicting the effect of interferon therapy, characterized by predicting the effect of interferon therapy on an infected person.
(D1) Apolipoprotein A1 or a modified product thereof,
(D2) C1 inhibitor or a modified form thereof,
(D3) Apolipoprotein C1 or a modified product thereof,
(D4) complement C4 or a modified product thereof,
(D5) Apolipoprotein A2 or a modified product thereof,
(D6) Transthyretin or a modified product thereof.

  In the method for predicting the effect of interferon therapy in this aspect, the effect of interferon therapy is predicted using at least one concentration of the marker substance belonging to any of (D1) to (D6) as an index. Also by the method of this aspect, it is possible to predict whether or not the subject's persistently infected hepatitis C virus is “non-responsive” (NR) to interferon therapy. In the method of this aspect, for example, whether the subject who is persistently infected with hepatitis C virus shows a remarkable effect (SVR) by interferon therapy, or whether HCV relapses after completion of interferon therapy. This is useful for preliminary judgment before prediction, that is, primary screening.

  Preferably, the body fluid is blood.

  According to this preferable aspect, since the test material can be easily collected, the effect of interferon therapy in a person with persistent hepatitis C virus infection can be predicted more easily and quickly.

  Preferably, the bodily fluid or bodily fluid component is brought into contact with a carrier on which a substance having affinity for the marker substance is immobilized to capture the marker substance in the body fluid on the carrier, and the amount of the trapped marker substance Based on the above, the concentration of the marker substance in the body fluid is calculated.

  According to this preferable aspect, since the marker substance captured on the carrier is the measurement object, the influence of the contaminant substance contained in the measurement sample can be reduced. As a result, the concentration of the marker substance can be measured with higher sensitivity and higher accuracy. Examples of the body fluid component include serum or plasma when the body fluid is blood.

  Preferably, the carrier has a flat portion, and the substance having affinity for the marker substance is immobilized on a part of the flat portion.

  According to this preferable aspect, the substance having affinity for the marker substance can be spot-fixed at a plurality of locations on the carrier. As a result, it is possible to simultaneously process a plurality of measurement samples with one carrier, and simultaneously measure the concentrations of a plurality of marker substances with one carrier, and work efficiency is improved. Furthermore, by reducing the area of each spot, the concentration of the marker substance can be measured even from a very small amount of measurement sample. An example of the carrier having a planar portion is a substrate such as a chip.

  Preferably, the substance having affinity for the marker substance is an ion exchanger, a metal chelate or an antibody.

  In this preferable aspect, an ion exchanger, a metal chelate body, or an antibody is used as a substance having affinity for the marker substance, and the marker substance in the measurement sample is captured on the carrier via these substances. When the substance is an ion exchanger or a metal chelate, various substances are easily available, and a carrier for capturing the marker substance can be easily prepared. Further, when the substance is an antibody, the marker substance can be captured more specifically. Examples of the method for measuring the amount of the captured marker substance include mass spectrometry and immunoassay (in the case of an antibody).

  Still another aspect of the present invention is a kit for use in the method for predicting the effect of interferon therapy according to the present invention, comprising a carrier on which a substance having affinity for the marker substance is immobilized. This is an effect prediction kit.

  The kit for predicting the effect of interferon therapy in this aspect includes a carrier on which a substance having affinity for a marker substance is immobilized. According to the kit of this aspect, it is not necessary to prepare the carrier separately when measuring the concentration of the marker substance, and the concentration of the marker substance can be measured very simply.

  Preferably, the substance having affinity for the marker substance is an ion exchanger, a metal chelate or an antibody.

  According to this preferable aspect, the marker substance can be captured on the carrier more reliably.

  According to the method for predicting the effect of interferon therapy according to the present invention, it is predicted whether the subject who is persistently infected with hepatitis C virus is highly effective by interferon therapy or HCV relapses after completion of interferon therapy. can do. As a result, it becomes possible to give more appropriate interferon therapy to the subject's hepatitis C virus persistently infected person.

  According to the kit for predicting the effect of interferon therapy of the present invention, it is possible to more easily and quickly discriminate whether or not a person who shows a remarkable effect by interferon therapy or a person who relapses after HCV relapses. Can do.

One aspect of the present invention is an interferon therapy for a patient with persistent hepatitis C virus infection, using as an index the concentration of at least one of the following marker substances (A1) to (A4) in a body fluid collected from the patient with persistent hepatitis C virus infection: This is a method for predicting the effect of interferon therapy.
(A1) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 7780 when subjected to mass spectrometry;
(A2) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 9300 when subjected to mass spectrometry;
(A3) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 51300 when subjected to mass spectrometry;
(A4) A protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 125000 when subjected to mass spectrometry.

  These marker substances (A1) to (A4) are all persistently infected with hepatitis C virus, which is predicted to relapse HCV after completion of interferon therapy, and hepatitis C virus persistence, which is predicted to show a significant effect by interferon therapy. It is a protein whose concentration in body fluid is significantly different from that of infected persons. The marker substances (A1), (A2) and (A3) show high values in the former body fluid and low values in the latter body fluid. On the other hand, the marker substance (A4) shows a low value in the former body fluid and a high value in the latter body fluid.

In one embodiment, at least one of the following (1) to (3) is satisfied.
(1) The marker substance (A1) is platelet factor 4 or a modified form thereof.
(2) The marker substance (A2) is connective tissue activating peptide 3 or a modified form thereof.
(3) The marker substance (A3) is a vitamin D binding protein or a modified form thereof.

Another aspect of the present invention is the above-mentioned hepatitis C virus persistence, using as an index at least one concentration of a marker substance belonging to any of the following (C1) to (C3) in a body fluid collected from a hepatitis C virus persistently infected person: This is a method for predicting the effect of interferon therapy, characterized by predicting the effect of interferon therapy on an infected person.
(C1) platelet factor 4 or a modified form thereof,
(C2) connective tissue activating peptide 3 or a modified form thereof,
(C3) Vitamin D binding protein or a modified form thereof.

  Examples of protein modifications include methylation, acetylation, adenylylation, myristylation, etc. of N-terminal α-amino group and lysine ε-amino group; addition of sugar or sugar chain to serine / threonine / asparagine; serine / threonine / Examples include phosphorylation of tyrosine, arginine, and histidine; cysteine cysteinylation, homocysteinylation, sulfonylation, etc .; γ-carboxylation of glutamic acid; conversion of N-terminal glutamic acid to pyroglutamic acid, and the like. Further, elimination of these modifications (demethylation, elimination of sugars or sugar chains, dephosphorylation, etc.) is also included in “modification”.

  The “protein or a modified form thereof” includes an isoform of the protein. Isoforms include proteins generated by alternative splicing in addition to the various modifications described above. Furthermore, the “protein or a modified form thereof” includes substantially the same protein in which several amino acid residues are deleted, substituted or added in the primary structure of the protein. Furthermore, “protein or a modified product thereof” includes a protein fragment derived from the protein that has been cleaved by a protease. For example, a protein fragment having a length that can be recognized as derived from the protein, for example, a protein fragment comprising 20 or more amino acid residues, a protein fragment having a molecular weight of 2,000 or more, and the like can be mentioned. In the case of a complex protein, the “protein or a modified product thereof” includes its subunits. Conversely, in the case of a monomeric protein, “protein or a modified product thereof” includes a multimer thereof.

There is no particular limitation on the persistently infected hepatitis C virus who is the subject of the method of these aspects, and all persistently infected hepatitis C viruses including hepatitis C patients can be the subjects. However, if hepatitis C virus persistently infected who is predicted to be “non-responsive” (NR) to interferon therapy is excluded from the subjects, HCV relapses with those who show significant efficacy (SVR) by interferon therapy. The accuracy of discrimination from those who (relapse) can be increased. There is no particular limitation on the method for preliminarily eliminating a hepatitis C virus persistently infected patient who is predicted to be “non-responsive” to interferon therapy, and a known method can be adopted. It is preferable to select at least one concentration of (B1) to (B11) as an index. That is, in a preferred embodiment of the present invention, at least one concentration of the following marker substances (B1) to (B11) in the body fluid is further used as an index.
(B1) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and generates an ion peak with a mass / charge ratio of about 3360 when subjected to mass spectrometry,
(B2) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 3960 when subjected to mass spectrometry;
(B3) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 4160 when subjected to mass spectrometry;
(B4) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and generates an ion peak with a mass / charge ratio of about 6190 when subjected to mass spectrometry,
(B5) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 6510 when subjected to mass spectrometry;
(B6) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 8610 when subjected to mass spectrometry;
(B7) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 8710 when subjected to mass spectrometry;
(B8) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and generates an ion peak with a mass / charge ratio of about 13900 when subjected to mass spectrometry;
(B9) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 17300 when subjected to mass spectrometry;
(B10) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 17400 when subjected to mass spectrometry;
(B11) A protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M and produces an ion peak with a mass / charge ratio of about 28100 when subjected to mass spectrometry.

  These marker substances (B1) to (B11) are all persistently infected with hepatitis C virus that is predicted to be “no response” (NR) to interferon therapy, and other (ETR) hepatitis C virus persistence. It is a protein whose concentration in body fluid is significantly different from that of infected persons. The marker substances (B1), (B2), (B3), (B4), (B6), and (B8) show a low value in the former body fluid and a high value in the latter body fluid. On the other hand, the marker substances (B5), (B7), (B9), (B10), and (B11) show high values in the former body fluid and low values in the latter body fluid.

  In this preferred embodiment, first, a subject who is predicted to be “non-responsive” (NR) to interferon therapy for a subject with persistent hepatitis C virus infection using at least one of (B1) to (B11) as an index (Preliminary determination). Next, with respect to a subject who is determined not to be predicted to be “non-responsive” to interferon therapy (ETR), at least one of (A1) to (A4) is used as an index, and is markedly effective by interferon therapy ( It is determined whether the person is predicted to show (SVR) or the person who is predicted to relapse HCV after completion of interferon therapy (this determination). However, the marker substance concentration measurement need not be performed in this order, and may be appropriately performed in consideration of work efficiency. For example, the preliminary determination and the main determination may be performed by measuring the concentrations of the markers (A1) to (A4) and (B1) to (B11) at a time and comparing the measured values with each other. . In this embodiment, since the target of this determination is narrowed down by preliminary determination in advance, the discrimination accuracy between a person who is predicted to be effective by interferon therapy and a person who is predicted to relapse HCV after completion of interferon therapy is particularly high.

In one embodiment, at least one of the following (4) to (13) is satisfied.
(4) The marker substance (B1) is apolipoprotein A1 or a modified form thereof.
(5) The marker substance (B3) is a C1 inhibitor or a modified form thereof.
(6) The marker substance (B4) is apolipoprotein A1 or a modified form thereof.
(7) The marker substance (B5) is apolipoprotein C1 or a modified form thereof.
(8) The marker substance (B6) is complement C4 or a modified form thereof.
(9) The marker substance (B7) is apolipoprotein A2 or a modified form thereof.
(10) The marker substance (B8) is transthyretin or a modified form thereof.
(11) The marker substance (B9) is apolipoprotein A2 or a modified form thereof.
(12) The marker substance (B10) is apolipoprotein A2 or a modified form thereof.
(13) The marker substance (B11) is apolipoprotein A1 or a modified form thereof.

In another embodiment, at least one concentration belonging to any of the following (D1) to (D6) in the body fluid is further used as an index.
(D1) Apolipoprotein A1 or a modified product thereof,
(D2) C1 inhibitor or a modified form thereof,
(D3) Apolipoprotein C1 or a modified product thereof,
(D4) complement C4 or a modified product thereof,
(D5) Apolipoprotein A2 or a modified product thereof,
(D6) Transthyretin or a modified product thereof.

  Examples of “protein modifications” are as described above. More specifically, for example, a protein fragment (SEQ ID NO: 37) corresponding to the amino acid number 81 to 109 portion of human apolipoprotein A1 (SEQ ID NO: 36) as shown in Example 5 described later is: Included in “Modified Apolipoprotein A1”. In addition, each protein fragment corresponding to the C-terminal side of human apolipoprotein A1, as shown in Example 7 described later, is included in the “modified body of apolipoprotein A1”.

  The protein fragment (SEQ ID NO: 40) corresponding to amino acid numbers 467 to 500 of human C1 inhibitor (SEQ ID NO: 39) as shown in Example 6 described later is included in the “modified form of C1 inhibitor”.

  A protein fragment (SEQ ID NO: 46) lacking two amino acids on the N-terminal side of human apolipoprotein C1 (SEQ ID NO: 45) as shown in Example 8 to be described later is included in the “modified product of apolipoprotein C1”. .

  Complement C4a (SEQ ID NO: 49), which is a fragment of complement C4 (SEQ ID NO: 99), is included in “Modified form of complement C4”. Furthermore, the protein fragment (SEQ ID NO: 50) lacking Cg-terminal Arg of complement C4a as shown in Example 9 described later is included in “modified body of complement C4”. The protein fragment of SEQ ID NO: 50 can also be referred to as “complement of complement C4a”.

  Cysteinylated apolipoprotein A2 in which Cys is added to human apolipoprotein A2 (SEQ ID NO: 57), as shown in Example 10 described later, is included in the “modified product of apolipoprotein A2”. In addition, a protein fragment (SEQ ID NO: 58) lacking the C-terminal Gln of apolipoprotein A2 and a protein fragment added with Cys (fragment of cysteinylated apolipoprotein A2) are “modified products of apolipoprotein A2.” include.

  As shown in Example 12 described later, the dimer of apolipoprotein A2 (SEQ ID NO: 57) is included in the “modified body of apolipoprotein A2”. Furthermore, a dimer composed of a combination of apolipoprotein A2 (SEQ ID NO: 57) and Gln-deficient apolipoprotein A2 (SEQ ID NO: 58) is included in the “modified product of apolipoprotein A2”. Furthermore, a dimer of Gln-deficient apolipoprotein A2 (SEQ ID NO: 58) is included in the “modified body of apolipoprotein A2”.

  For transthyretin, various modified transthyretins in which only the Cys residue is modified have been found (Amareth Lim et al., J. Biol. Chem., Vol. 258, No. 50). , 2003). As shown in Example 11 described later, cysteinylated transthyretin in which Cys is added to the Cys residue of human transthyretin (SEQ ID NO: 65) is included in the “transthyretin modification”.

  The marker substances (B1) to (B11) are useful for preliminary determination when discriminating between a person who is predicted to show a significant effect by interferon therapy and a person who is predicted to relapse HCV after completion of interferon therapy. However, it can also be used for the purpose of discriminating between those who are predicted to be “no response” to the interferon therapy and those who are not. The same applies to each marker substance belonging to any one of (D1) to (D6).

  As the body fluid used in the present invention, blood is preferably used. In particular, it is preferable to use serum or plasma (body fluid component) prepared from blood as a test material (measurement sample). Serum or plasma can be prepared from blood by a known method such as centrifugation.

  In the present invention, as a method for measuring the concentration of a marker substance, a method generally used for protein quantification can be used as it is as long as it can specifically measure the concentration of the marker substance. For example, various immunoassays, mass spectrometry (MS), chromatography, electrophoresis and the like can be used.

  According to the immunoassay, the concentration of the marker substance can be accurately measured even with a sample having a lot of contaminants. Examples of immunoassays include classical methods such as precipitation, agglutination, and hemolysis, which directly or indirectly measure antigen-antibody conjugates, and enzyme immunoassays (EIA) with increased detection sensitivity in combination with labeling methods. , Radioimmunoassay (RIA), fluorescent immunoassay (FIA) and the like. The antibody specific for the marker substance used in these immunoassays may be monoclonal or polyclonal.

  According to mass spectrometry, an ion peak derived from each marker substance can be specified, and the amount (concentration) of each marker substance can be measured with the ion peak intensity. As the ionization method for measuring the concentration of the marker substance by mass spectrometry, either matrix-assisted laser desorption / ionization (MALDI) or electrospray ionization (ESI) can be applied. However, MALDI is preferred because it produces less multivalent ions. In particular, according to MALDI-TOF-MS combined with a time-of-flight mass spectrometer (TOF), an ion peak derived from a marker substance can be identified more accurately.

  When measuring the concentration of the marker substance by electrophoresis, for example, the target sample substance is separated by subjecting the measurement sample to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the gel is prepared with an appropriate dye or fluorescent substance. And the intensity and fluorescence intensity of the band corresponding to the target marker substance may be measured. When the separation of the marker substance is insufficient by SDS-PAGE alone, two-dimensional electrophoresis combined with isoelectric focusing (IEF) can also be used. Furthermore, instead of detecting directly from the gel, Western blotting can also be performed to measure the amount of marker substance on the membrane.

  When measuring the concentration of the marker substance by chromatography, for example, a method by liquid high performance chromatography (HPLC) can be used. That is, the concentration of the marker substance in the sample can be measured by subjecting the sample to HPLC to separate the target marker substance and measuring the peak area of the chromatogram.

  In a preferred embodiment, a marker substance is captured on a carrier, and the captured marker substance is a measurement target. That is, a substance having affinity for the marker substance is immobilized on the carrier, and the marker substance is captured on the carrier via the substance having the affinity. Then, the concentration of the marker substance in the body fluid is calculated based on the amount of the captured marker substance. According to this embodiment, it is possible to reduce the influence of contaminants contained in the sample, and to measure the concentration of the marker substance with higher sensitivity and higher accuracy. As an example of the carrier that can be used in the present embodiment, a carrier having a flat portion such as a substrate can be used in addition to a general one such as beads, metal, glass, resin, and the like. In the case of using a substrate, it is preferable to immobilize a substance having affinity for the marker substance on a part of the planar portion. As an example, there may be mentioned a carrier in which a chip is used as a substrate and a substance having affinity for a marker substance is spot-fixed at a plurality of spots on the surface. Examples of “affinity” include ion binding; affinity between a metal chelate and a histidine residue in a protein; bioaffinity such as an antigen and an antibody, an enzyme and a substrate, a hormone and a receptor; a hydrophobic interaction Such as chemical interaction.

  When capturing the marker substance on the carrier by ionic bonding, the ion exchanger is immobilized on the carrier. In this case, either a cation exchanger or an anion exchanger can be used as the ion exchanger, and further, a strong cation exchanger, a weak cation exchanger, a strong anion exchanger, and a weak anion exchanger. Either of these can be used, but a strong anion exchanger and a weak cation exchanger are preferably used. Examples of strong anion exchangers include quaternary ammonium (trimethylaminomethyl) (QA), quaternary aminoethyl (diethyl, mono-2-hydroxybutylaminoethyl) (QAE), quaternary ammonium (trimethylammonium) ( And those having a strong anion exchange group such as QMA). Examples of weak cation exchangers include those having weak cation exchange groups such as carboxymethyl (CM). Examples of strong cation exchangers include those having a strong cation exchange group such as sulfopropyl (SP). Furthermore, examples of the weak anion exchanger include those having a weak anion exchange group such as dimethylaminoethyl (DE) and diethylaminoethyl (DEAE).

When capturing a marker substance via a metal chelate, for example, a metal chelate such as Cu ²⁺ , Zn ²⁺ , Ni ²⁺ , Co ²⁺ , Al ³⁺ , Fe ³⁺ , Ga ³⁺ is fixed. A fluorinated carrier can be used. Preferably, a copper ion-bonded metal chelate is used.

  When a marker substance is captured on a carrier by an antibody, an antibody specific for the marker substance may be immobilized on the carrier.

  When a marker substance is captured on a carrier by hydrophobic interaction, a substance having a hydrophobic group is immobilized on the carrier. Examples of hydrophobic groups include C4-C20 alkyl groups, phenyl groups, and the like.

  In this embodiment, when an immunoassay is used as a method for measuring a marker substance, it is preferable to use a carrier on which an antibody is immobilized. In this way, an immunoassay system using the antibody immobilized on the carrier as the primary antibody can be easily constructed. For example, two types of antibodies specific to a marker substance and having different epitopes are prepared, one is immobilized on a carrier as a primary antibody, and the other is enzyme-labeled as a secondary antibody to construct a sandwich EIA system. . In addition, immunoassay systems based on binding inhibition methods and competitive methods can be constructed. Further, when a substrate is used as a carrier, immunoassay using an antibody chip is possible. According to the antibody chip, the concentration of a plurality of marker substances can be measured simultaneously, and rapid measurement is possible.

  On the other hand, when mass spectrometry is used in the present embodiment, for example, in addition to antibodies, ion exchangers, metal chelates, or carriers on which hydrophobic groups are immobilized can be used. Since binding by these substances is not as specific as bioaffinity such as antigens and antibodies, when using a carrier on which these substances are immobilized, substances other than the marker substance can also be captured on the carrier. According to the analysis, there is no problem because it is quantified by the mass spectrometer spectrum reflecting the molecular weight. In particular, using a substrate as a carrier, surface-enhanced laser desorption / ionization-time-of-flight mass spectrometry (hereinafter referred to as "SELDI-TOF-MS") ), The concentration of the marker substance can be measured more accurately. As the types of substrates that can be used, cation exchange substrates, anion exchange substrates, normal phase substrates, reverse phase substrates, metal ion substrates, antibody substrates, etc. can be used, but cation exchange substrates, particularly weak cation exchanges. A substrate, an anion exchange substrate, particularly a strong anion exchange substrate, and a metal ion substrate are preferably used.

  According to the method of the present invention, either the subject who is persistently infected with hepatitis C virus is expected to show a significant effect (SVR) by interferon therapy, or the one who is predicted to relapse HCV after completion of interferon therapy An example of a procedure for actually determining whether or not will be described step by step. In this example, the preliminary determination using at least one of the marker substances (B1) to (B11) as an index and the main determination using at least one of the marker substances (A1) to (A4) as an index are performed. In addition, plasma is used as a test material, and the concentration of each marker is measured by SELDI-TOF-MS.

  First, blood is collected from a subject who is persistently infected with hepatitis C virus, and plasma serving as a test material is prepared. This plasma is applied to a strong anion exchange resin column such as QA under the condition of pH 9.0. Thereafter, the elution is performed sequentially with elution solutions of pH 9.0 (through), pH 8.0, pH 7.0, pH 6.0, pH 5.0, pH 4.0, and pH 3.0, and finally with an organic solvent. Secure each elution fraction.

  The fraction of pH 9.0 is brought into contact with a substrate (weak cation exchange substrate) on which a weak cation exchanger such as CM is immobilized, and then washed under conditions of pH 4.0. At this time, the marker substance (B6) is captured on the substrate. Further, the fraction of pH 9.0 is brought into contact with a substrate (strong anion exchange substrate) on which a strong anion exchanger such as QA is immobilized, and then washed under the condition of pH 9.0. At this time, the marker substance (A4) is captured on the substrate. Further, the fraction of pH 9.0 is brought into contact with a substrate (copper ion substrate) on which a copper ion metal chelate is immobilized, and then washed under conditions of pH 7.0 and 0.5 M NaCl. At this time, the marker substances (B1) and (B4) are captured on the substrate.

  The fraction of pH 5.0 is brought into contact with a weak cation exchange substrate such as CM, and then washed under the condition of pH 4.0. At this time, the marker substance (A1) is captured on the substrate. Further, the fraction having a pH of 5.0 is brought into contact with a strong anion exchange substrate such as QA, and then washed under the condition of pH 9.0. At this time, the marker substances (A2) and (B2) are captured on the substrate.

  The fraction of pH 4.0 is brought into contact with a strong anion exchange substrate such as QA, and then washed under the condition of pH 9.0. At this time, the marker substances (A3), (B5), (B7), (B9), and (B10) are captured on the substrate.

  The fraction of the organic solvent is brought into contact with a weak cation exchange substrate such as CM, and then washed under the condition of pH 4.0. At this time, the marker substance (B3) is captured on the substrate. In addition, the fraction of the organic solvent is brought into contact with the copper ion substrate, and then washed under conditions of pH 7.0 and 0.5 M NaCl. At this time, the marker substances (B8) and (B11) are captured on the substrate.

  Each substrate is subjected to SELDI-TOF-MS, and the intensity of the ion peak corresponding to each marker substance is measured. Each obtained ion peak intensity is compared with a reference value. As reference values, for example, in the case of marker substances (A1) to (A4), plasma before treatment of a patient with persistent hepatitis C virus infection that has been shown to be highly effective by interferon therapy, and after completion of interferon therapy The same SELDI-TOF-MS was performed in advance using plasma before treatment of hepatitis C virus persistently known to have HCV relapsed, and the cut-off value set by comparing both values was adopted. can do. On the other hand, in the case of marker substances (B1) to (B11), they are plasma before treatment of hepatitis C virus persistently infected patients who are known to be unresponsive to interferon therapy, and others It is possible to adopt a cut-off value set in advance by performing similar SELDI-TOF-MS using plasma before treatment of a patient with persistent hepatitis C virus infection, which is known . In this case, for the definite diagnosis of “significant effect” (SVR), “relapse” (relapse), “no response” (NR), and “other than no response” (ETR), for example, based on the plasma HCV concentration Can be done.

  First, preliminary determination is performed based on the measurement results of the marker substances (B1) to (B11). Specifically, in the case of the marker substance (B1), (B2), (B3), (B4), (B6), or (B8), when the ion peak intensity is smaller than the set reference value, The person with persistent hepatitis C virus infection is determined to be “a person who is unresponsive to interferon therapy”. In the case of the marker substance (B5), (B7), (B9), (B10), or (B11), when the ion peak intensity is greater than the set reference value, the hepatitis C virus persistently infected person It is determined that the patient is unresponsive to interferon therapy. Here, about each marker substance of (B1)-(B11), only 1 may be employ | adopted and it may employ | adopt in combination of 2 or more.

  Next, the hepatitis C virus persistently infected person who was determined to be “no responder to interferon therapy” in the preliminary determination is excluded, and the marker substances (A1) to (A4) This determination is made based on the measurement results. Specifically, in the case of the marker substance (A1), (A2), or (A3), when the ion peak intensity is larger than the set reference value, the person with persistent hepatitis C virus infection is “interferon therapy. It is determined that the HCV is a person who relapses after completion. On the other hand, in the case of the marker substance (A4), when the ion peak intensity is smaller than the set reference value, the person with persistent hepatitis C virus infection is determined to be “the person who relapses with HCV after completion of interferon therapy”. To do. Here, about each marker substance of (A1)-(A4), only one may be employ | adopted and you may employ | adopt combining 2 or more.

  For those with persistent hepatitis C virus infection, who were determined to be “persons whose HCV relapses after the completion of interferon therapy” in this determination, HCV is likely to relapse if interferon administration is interrupted, and long-term interferon administration is effective It is predicted. On the other hand, for patients with persistent hepatitis C virus infection, who are determined not to be “persons whose HCV relapses after the completion of interferon therapy” in this determination, interferon therapy is highly likely to be effective. Predicted. In addition, it is necessary to carefully examine whether or not interferon therapy should be performed for those who are persistently infected with hepatitis C virus determined as “no responder to interferon therapy” in the preliminary determination.

  The interferon therapy effect prediction kit of the present invention includes a carrier on which a substance having affinity for the marker substance is immobilized. In a preferred embodiment, the substance having affinity for the marker substance is an ion exchanger, a metal chelate or an antibody. According to the kit of this embodiment, SELDI-TOF-MS etc. can be performed simply. The kit may contain other reagents, for example, standard products of various marker substances, various buffers and the like. Examples of the configuration of the kit of the present invention are given below.

(Kit configuration example)
(1) Weak cation exchange substrate: 1 (2) Strong anion exchange substrate: 1 (3) Copper ion substrate: 1 (4) Substrate cleaning buffer A (pH 4.0): appropriate amount (5) substrate Cleaning buffer B (pH 9.0): appropriate amount (6) Substrate cleaning buffer C (pH 7.0; containing 0.5 M NaCl): appropriate amount (7) Standard product of each marker substance: each appropriate amount

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

1. Search for marker substance using protein chip Collect plasma samples before treatment of hepatitis C patients who received “interferon α-ribavirin combination therapy” by 48-week administration of PEG interferon α2b and ribavirin and follow-up 24 weeks after administration did. The breakdown of patients was 10 persons who showed significant efficacy (SVR) (HCV negative at both end of 48-week administration and 24 weeks after administration), and no response (NR) (at the end of 48-week administration) HCV positive in 10 people and HCV relapsed (HCV negative at the end of 48 weeks administration, HCV positive at 24 weeks after administration) 12 people. Confirmation of HCV negativeity was performed by measuring the number of HCV (genotype 1b) in the blood.

  To 20 μL of each plasma sample, 30 μL of denaturation buffer (9 M urea, 2% CHAPS, 50 mM Tris-HCl (pH 9.0)) was added for pretreatment to denature the protein. Next, each pretreated serum sample was applied to a strong anion exchange resin column (Q Ceramic-Hyper DF, Biosepra). Subsequently, a buffer of pH 9.0 (50 mM Tris-HCl (pH 9.0), 0.1% (w / v) 1-o-N-octyl-β-D-glucopyranoside (hereinafter referred to as “OGP”). ), PH 8.0 buffer (50 mM Tris-HCl (pH 8.0), 0.1% (w / v) OGP), pH 7.0 buffer (50 mM HEPES-NaOH (pH 7.0), 0.1%) (W / v) OGP), pH 6.0 buffer (100 mM sodium phosphate (pH 6.0), 0.1% (w / v) OGP), pH 5.0 buffer (100 mM sodium acetate (pH 5.0)) , 0.1% (w / v) OGP), pH 4.0 buffer (100 mM sodium acetate (pH 4.0), 0.1% (w / v) OGP), pH 3.0 buffer (5 mM sodium citrate (pH 3.0), 0.1% (w / v) OGP), and organic solvent (mixture of 33.3% isopropanol, 16.7% acetonitrile, 0.1% trifluoroacetic acid) Elute in order with 200 μL of each, fraction 1 (pH 9.0, pass through), fraction 2 (pH 8.0), fraction 3 (pH 7.0), fraction 4 (pH 6.0), fraction 5 (pH 5). 0.0), fraction 6 (pH 4.0), fraction 7 (pH 3.0), and fraction 8 (organic solvent), eight crude fractions were obtained.

  10 μL of each of the obtained fractions was diluted 10-fold with a pH 4.0 protein chip binding buffer (100 mM sodium acetate, pH 4.0), and then added to a weak cation exchange chip CM10 (Bio-Rad). Similarly, 10 μL of each of the obtained fractions was diluted 10-fold with a pH 9.0 protein chip binding buffer (50 mM Tris-HCl, pH 9.0), and then added to a strong anion exchange chip Q10 (Bio-Rad). . Similarly, 10 μL of each of the obtained fractions was diluted 10-fold with a protein chip binding buffer (100 mM sodium phosphate, 0.5 M NaCl, pH 7.0) at pH 7.0 and 0.5 M NaCl, and then copper-modified chip IMAC30. (Bio-Rad). Each protein chip was washed 3 times with each binding buffer, then once with deionized water and dried.

  Next, sinapinic acid (SPA-H, SPA-L) or α-cyano-4-hydroxycinnamic acid (CHCA), which is an energy absorbing molecule, is added to each protein chip, and protein chip leader Model PBS IIc (BioRad) SELDI-TOF-MS was carried out. In addition, the measurement molecular weight range (m / z) was performed in the range of 3000-200000. Moreover, the measurement was performed in duplicate and the average value of m / z was calculated. Data analysis was performed using Protein Chip Software, Ciphergen Express Data Manager, and Biomarker Patterns Software (all from Bio-Rad). Specifically, after performing baseline correction, molecular weight calibration, and spectrum normalization processing, single marker analysis and multiflow analysis combining several markers were performed. As a result, a large number of peaks were detected depending on the combination of the type of crude fraction, the type of protein chip, the washing conditions of the chip, and the like. For each peak, a p-value (Mann-Whitney test method), ROC area, and ion peak intensity were calculated. From these results, four types of ion peaks that showed a significant difference in ion peak intensity between those who showed remarkable efficacy (SVR) and those who relapsed HCV were selected, and the corresponding proteins Were named (A1) to (A4). Similarly, 11 types of ion peaks that showed a significant difference in ion peak intensity between those who were not reacted (NR) and those who were not (ETR) were selected, and proteins corresponding to these were selected (B1 ) To (B11).

2. Characteristics of marker substance (A1) Fraction 5 (pH 5.0) is brought into contact with a weak cation exchange chip CM10, washed with a protein chip binding buffer of pH 4.0, and SELDI-TOF-MS (EAM: SPA-H). , An ion peak with a mass / charge ratio of 7777 (average value) was detected. This peak showed a low value in those who showed remarkable efficacy (SVR), and showed a high value in those who relapsed HCV. FIG. 1 (a) shows a dot plot in which the peak intensity is plotted separately for those who showed a remarkable effect and those who relapsed HCV, and FIG. 1 (b) shows the box diagram. The horizontal bar in FIG. 1 (a) is the average value, the upper and lower ends of the basket in FIG. 1 (b) are the maximum and minimum values, respectively, and the upper and lower sides of the box are the third quartile (75th percentile), respectively. In the first quartile (25th percentile), the line in the box is the median (same for FIG. 2 and later). As a result, the p value (SVR vs relapse) of this peak was 0.020, and the ROC area was 0.72. From the above, a protein that produces a peak with a mass / charge ratio of about 7780 when subjected to SELDI-TOF-MS is a protein in blood that has been successfully treated with hepatitis C virus, which is highly effective by interferon therapy, and HCV after completion of interferon therapy. It became clear that it becomes an index of discrimination from the persistently infected hepatitis C virus. For example, when SELDI-TOF-MS under the above-mentioned conditions is performed using plasma before treatment of a patient with persistent hepatitis C virus infection as a test material, and the ion peak intensity with a mass / charge ratio of about 7780 is higher than the reference value, It can be predicted that the persistently infected hepatitis C virus is a person who relapses with HCV after completion of interferon therapy.

3. Characteristics of marker substance (A2) Fraction 5 (pH 5.0) was brought into contact with strong anion exchange chip Q10, washed with a protein chip binding buffer of pH 9.0, and SELDI-TOF-MS (EAM: SPA-H). , An ion peak with a mass / charge ratio of 9298 (average value) was detected. This peak showed a low value in those who showed remarkable efficacy (SVR), and showed a high value in those who relapsed HCV. FIG. 2A shows a dot plot in which peak intensities are plotted separately for those who showed a significant effect and those who relapsed HCV, and FIG. 2B shows the box diagram. As a result, the p value (SVR vs relapse) of this peak was 0.008, and the ROC area was 0.72. From the above, proteins that cause a peak with a mass / charge ratio of about 9300 when subjected to SELDI-TOF-MS as a protein in blood are HCV persistently infected with hepatitis C virus that shows a significant effect by interferon therapy and HCV after completion of interferon therapy. It became clear that it becomes an index of discrimination from the persistently infected hepatitis C virus. For example, when SELDI-TOF-MS under the above conditions is performed using plasma before treatment of a patient with persistent hepatitis C virus infection as a test material, and the ion peak intensity with a mass / charge ratio of about 9300 is higher than a reference value, It can be predicted that the persistently infected hepatitis C virus is a person who relapses with HCV after completion of interferon therapy.

4). Characteristics of marker substance (A3) Fraction 6 (pH 4.0) was brought into contact with strong anion exchange chip Q10, washed with a protein chip binding buffer of pH 9.0, and then SELDI-TOF-MS (EAM: SPA-H). , An ion peak with a mass / charge ratio of 51321 (average value) was detected. This peak showed a low value in those who showed remarkable efficacy (SVR), and showed a high value in those who relapsed HCV. FIG. 3A shows a dot plot in which peak intensities are plotted separately for those who showed a remarkable effect and those who relapsed HCV, and FIG. 3B shows the box diagram. As a result, the p value (SVR vs relapse) of this peak was 0.045, and the ROC area was 0.70. From the above, a protein that produces a peak with a mass / charge ratio of about 51300 when subjected to SELDI-TOF-MS is a protein in blood that has been successfully treated with hepatitis C virus, which is highly effective by interferon therapy, and HCV after completion of interferon therapy. It became clear that it becomes an index of discrimination from the persistently infected hepatitis C virus. For example, when SELDI-TOF-MS under the above conditions is performed using plasma before treatment of a patient with persistent hepatitis C virus infection as a test material, and the ion peak intensity with a mass / charge ratio of about 51300 is higher than the reference value, It can be predicted that the persistently infected hepatitis C virus is a person who relapses with HCV after completion of interferon therapy.

5). Characteristics of Marker Substance (A4) Fraction 1 (pH 9.0) is contacted with strong anion exchange chip Q10, washed with a protein chip binding buffer of pH 9.0, and then SELDI-TOF-MS (EAM: SPA-H) , An ion peak having a mass / charge ratio of 125350 (average value) was detected. This peak showed a high value in those who showed remarkable efficacy (SVR), and showed a low value in those who relapsed HCV. FIG. 4A shows a dot plot in which peak intensities are plotted separately for those who showed a significant effect and those who relapsed HCV, and FIG. 4B shows the box diagram. As a result, the p value (SVR vs relapse) of this peak was 0.003, and the ROC area was 0.75. From the above, a protein that produces a peak with a mass / charge ratio of about 125,000 when subjected to SELDI-TOF-MS is a protein in blood that has been successfully treated with hepatitis C virus that exhibits a significant effect by interferon therapy and HCV after completion of interferon therapy. It became clear that it becomes an index of discrimination from the persistently infected hepatitis C virus. For example, when SELDI-TOF-MS under the above conditions is performed using plasma before treatment of a patient with persistent hepatitis C virus infection as a test material, and the ion peak intensity with a mass / charge ratio of about 125000 is lower than the reference value, It can be predicted that the persistently infected hepatitis C virus is a person who relapses with HCV after completion of interferon therapy.

6). Characteristics of Marker Substance (B1) Fraction 1 (pH 9.0) was contacted with a copper-modified chip IMAC30, washed with a protein chip binding buffer of pH 7.0 and 0.5 M NaCl, and SELDI-TOF-MS (EAM: CHCA). ), An ion peak having a mass / charge ratio of 3356 (average value) was detected. This peak was low in those who did not respond (NR) to interferon therapy, and high in those who did not (ETR). FIG. 5A shows a dot plot in which peak intensities are plotted separately for those who have not reacted and others, and FIG. 5B shows the box diagram. As a result, the p value (NR vs ETR) of this peak was 0.028, and the ROC area was 0.67. Based on the above, proteins that cause a peak with a mass / charge ratio of about 3360 when subjected to SELDI-TOF-MS with proteins in blood are hepatitis C virus persistently unresponsive to interferon therapy and other C It was found to be an index for discrimination from people with persistent hepatitis B virus infection. For example, when SELDI-TOF-MS under the above-described conditions is performed using plasma before treatment of a patient with persistent hepatitis C virus infection as a test material, and the ion peak intensity with a mass / charge ratio of about 3360 is lower than the reference value, It can be predicted that the hepatitis C virus persistently infected person is unresponsive to interferon therapy. In this way, for those with persistent hepatitis C virus infections other than those who were predicted to be unresponsive, those who showed significant effects using at least one of the marker substances (A1) to (A4) as an index, and It is possible to predict which HCV will relapse.

7). Characteristics of marker substance (B2) Fraction 5 (pH 5.0) was brought into contact with strong anion exchange chip Q10, washed with a protein chip binding buffer of pH 9.0, and SELDI-TOF-MS (EAM: SPA-L) , An ion peak with a mass / charge ratio of 3962 (average value) was detected. This peak was low in those who did not respond (NR) to interferon therapy, and high in those who did not (ETR). FIG. 6A shows a dot plot in which peak intensities are plotted separately for those who have not reacted and others, and FIG. 6B shows the box diagram. As a result, the p value (NR vs ETR) of this peak was 0.00002, and the ROC area was 0.83. Based on the above, proteins that cause a peak with a mass / charge ratio of about 3960 when subjected to SELDI-TOF-MS among proteins in blood are those with persistent hepatitis C virus infection unresponsive to interferon therapy and other C It was found to be an index for discrimination from people with persistent hepatitis B virus infection. For example, when SELDI-TOF-MS under the above-mentioned conditions is performed using plasma before treatment of hepatitis C virus persistently infected as a test material, and the ion peak intensity with a mass / charge ratio of about 3960 is lower than the reference value, It can be predicted that the hepatitis C virus persistently infected person is unresponsive to interferon therapy. In this way, for those with persistent hepatitis C virus infections other than those who were predicted to be unresponsive, those who showed significant effects using at least one of the marker substances (A1) to (A4) as an index, and It is possible to predict which HCV will relapse.

8). Characteristics of Marker Substance (B3) Fraction 8 (organic solvent) was brought into contact with a weak cation exchange chip CM10, washed with a pH 4.0 protein chip binding buffer and subjected to SELDI-TOF-MS (EAM: CHCA). In some cases, an ion peak having a mass / charge ratio of 4156 (average value) was detected. This peak was low in those who did not respond (NR) to interferon therapy, and high in those who did not (ETR). FIG. 7A shows a dot plot in which peak intensities are plotted separately for those who have not reacted and others, and FIG. 7B shows the box diagram. As a result, the p value (NR vs ETR) of this peak was 0.031, and the ROC area was 0.68. Based on the above, proteins that cause a peak with a mass / charge ratio of about 4160 when subjected to SELDI-TOF-MS with proteins in blood are those who are persistently infected with hepatitis C virus that are unresponsive to interferon therapy, and other C It was found to be an index for discrimination from people with persistent hepatitis B virus infection. For example, when SELDI-TOF-MS under the above conditions is performed using plasma before treatment of a patient with persistent hepatitis C virus infection as a test material, and the ion peak intensity with a mass / charge ratio of about 4160 is lower than the reference value, It can be predicted that the hepatitis C virus persistently infected person is unresponsive to interferon therapy. In this way, for those with persistent hepatitis C virus infections other than those who were predicted to be unresponsive, those who showed significant effects using at least one of the marker substances (A1) to (A4) as an index, and It is possible to predict which HCV will relapse.

9. Characteristics of Marker Substance (B4) Fraction 1 (pH 9.0) was brought into contact with a copper-modified chip IMAC30, washed with a protein chip binding buffer of pH 7.0 and 0.5 M NaCl, and SELDI-TOF-MS (EAM: CHCA). ), An ion peak having a mass / charge ratio of 6194 (average value) was detected. This peak was low in those who did not respond (NR) to interferon therapy, and high in those who did not (ETR). FIG. 8A shows a dot plot in which peak intensities are plotted separately for those who have not reacted and others, and FIG. 8B shows the box diagram. As a result, the p value (NR vs ETR) of this peak was 0.006, and the ROC area was 0.70. From the above, proteins that cause a peak with a mass / charge ratio of about 6190 when subjected to SELDI-TOF-MS among proteins in blood are hepatitis C virus persistently unresponsive to interferon therapy and other C It was found to be an index for discrimination from people with persistent hepatitis B virus infection. For example, when SELDI-TOF-MS under the above-mentioned conditions is performed using plasma before treatment of hepatitis C virus persistently infected as a test material, and the ion peak intensity with a mass / charge ratio of about 6190 is lower than the reference value, It can be predicted that the hepatitis C virus persistently infected person is unresponsive to interferon therapy. In this way, for those with persistent hepatitis C virus infections other than those who were predicted to be unresponsive, those who showed significant effects using at least one of the marker substances (A1) to (A4) as an index, and It is possible to predict which HCV will relapse.

10. Characteristics of marker substance (B5) Fraction 6 (pH 4.0) was brought into contact with strong anion exchange chip Q10, washed with a protein chip binding buffer of pH 9.0, and SELDI-TOF-MS (EAM: SPA-H). , An ion peak with a mass / charge ratio of 6511 (average value) was detected. This peak was low in those who were not (ETR), and high in those who were unresponsive (NR) to interferon therapy. FIG. 9A shows a dot plot in which peak intensities are plotted separately for those who have not reacted and others, and FIG. 9B shows the box diagram. As a result, the p value (NR vs ETR) of this peak was 0.049, and the ROC area was 0.64. Based on the above, proteins that cause a peak with a mass / charge ratio of about 6510 when subjected to SELDI-TOF-MS with proteins in blood are those who are persistently infected with hepatitis C virus that are unresponsive to interferon therapy and other C It was found to be an index for discrimination from people with persistent hepatitis B virus infection. For example, when SELDI-TOF-MS under the above conditions is performed using plasma before treatment of a patient with persistent hepatitis C virus infection as a test material, and when the ion peak intensity with a mass / charge ratio of about 6510 is higher than the reference value, It can be predicted that the hepatitis C virus persistently infected person is unresponsive to interferon therapy. In this way, for those with persistent hepatitis C virus infections other than those who were predicted to be unresponsive, those who showed significant effects using at least one of the marker substances (A1) to (A4) as an index, and It is possible to predict which HCV will relapse.

11. Characteristics of Marker Substance (B6) Fraction 1 (pH 9.0) is contacted with a weak cation exchange chip CM10, washed with a protein chip binding buffer of pH 4.0, and SELDI-TOF-MS (EAM: SPA-L) , An ion peak with a mass / charge ratio of 8608 (average value) was detected. This peak was low in those who did not respond (NR) to interferon therapy, and high in those who did not (ETR). FIG. 10A shows a dot plot in which peak intensities are plotted separately for those who have not reacted and others, and FIG. 10B shows the box diagram. As a result, the p value (NR vs ETR) of this peak was 0.030, and the ROC area was 0.66. Based on the above, proteins that cause a peak with a mass / charge ratio of about 8610 when subjected to SELDI-TOF-MS among proteins in blood are hepatitis C virus persistently unresponsive to interferon therapy and other C It was found to be an index for discrimination from people with persistent hepatitis B virus infection. For example, when SELDI-TOF-MS under the above conditions is performed using plasma before treatment of a patient with persistent hepatitis C virus infection as a test material, and the ion peak intensity with a mass / charge ratio of about 8610 is lower than the reference value, It can be predicted that the hepatitis C virus persistently infected person is unresponsive to interferon therapy. In this way, for those with persistent hepatitis C virus infections other than those who were predicted to be unresponsive, those who showed significant effects using at least one of the marker substances (A1) to (A4) as an index, and It is possible to predict which HCV will relapse.

12 Characteristics of marker substance (B7) Fraction 6 (pH 4.0) was brought into contact with strong anion exchange chip Q10, washed with a protein chip binding buffer of pH 9.0, and SELDI-TOF-MS (EAM: SPA-L). , An ion peak with a mass / charge ratio of 8713 (average value) was detected. This peak was high in those who did not respond (NR) to interferon therapy, and low in those who did not (ETR). FIG. 11 (a) shows a dot plot in which peak intensities are plotted separately for those who have not reacted and others, and FIG. 11 (b) shows the box diagram. As a result, the p value (NR vs ETR) of this peak was 0.0008 and the ROC area was 0.75. Based on the above, proteins that cause a peak with a mass / charge ratio of about 8710 when subjected to SELDI-TOF-MS among proteins in blood are hepatitis C virus persistently unresponsive to interferon therapy and other C It was found to be an index for discrimination from people with persistent hepatitis B virus infection. For example, when SELDI-TOF-MS under the above conditions is performed using plasma before treatment of a patient with persistent hepatitis C virus infection as a test material, and the ion peak intensity with a mass / charge ratio of about 8710 is higher than the reference value, It can be predicted that the hepatitis C virus persistently infected person is unresponsive to interferon therapy. In this way, for those with persistent hepatitis C virus infections other than those who were predicted to be unresponsive, those who showed significant effects using at least one of the marker substances (A1) to (A4) as an index, and It is possible to predict which HCV will relapse.

13. Characteristics of Marker Substance (B8) Fraction 8 (organic solvent) was brought into contact with a copper-modified chip IMAC30, washed with a protein chip binding buffer of pH 7.0 and 0.5 M NaCl, and SELDI-TOF-MS (EAM: SPA- When H) was performed, an ion peak with a mass / charge ratio of 13882 (average value) was detected. This peak was low in those who did not respond (NR) to interferon therapy, and high in those who did not (ETR). FIG. 12A shows a dot plot in which peak intensities are plotted separately for those who have not reacted and others, and FIG. 12B shows the box diagram. As a result, the p value (NR vs ETR) of this peak was 0.037, and the ROC area was 0.66. Based on the above, proteins that cause a peak with a mass / charge ratio of about 13900 when subjected to SELDI-TOF-MS with proteins in blood are those with persistent hepatitis C virus infection unresponsive to interferon therapy and other C It was found to be an index for discrimination from people with persistent hepatitis B virus infection. For example, when SELDI-TOF-MS under the above-mentioned conditions is performed using plasma before treatment of hepatitis C virus persistently infected as a test material, and the ion peak intensity with a mass / charge ratio of about 13900 is lower than the reference value, It can be predicted that the hepatitis C virus persistently infected person is unresponsive to interferon therapy. In this way, for those with persistent hepatitis C virus infections other than those who were predicted to be unresponsive, those who showed significant effects using at least one of the marker substances (A1) to (A4) as an index, and It is possible to predict which HCV will relapse.

14 Characteristics of marker substance (B9) Fraction 6 (pH 4.0) was brought into contact with strong anion exchange chip Q10, washed with a protein chip binding buffer of pH 9.0, and then SELDI-TOF-MS (EAM: SPA-H). , An ion peak with a mass / charge ratio of 17271 (average value) was detected. This peak was low in those who were not (ETR), and high in those who were unresponsive (NR) to interferon therapy. FIG. 13 (a) shows a dot plot in which peak intensities are plotted separately for those who have not reacted and others, and FIG. 13 (b) shows the box diagram. As a result, the p value (NR vs ETR) of this peak was 0.0001, and the ROC area was 0.80. Based on the above, proteins that cause a peak with a mass / charge ratio of about 17300 when subjected to SELDI-TOF-MS among proteins in blood are hepatitis C virus persistently unresponsive to interferon therapy and other C It was found to be an index for discrimination from people with persistent hepatitis B virus infection. For example, when SELDI-TOF-MS under the above conditions is performed using plasma before treatment of a patient with persistent hepatitis C virus infection as a test material, and the ion peak intensity with a mass / charge ratio of about 17300 is higher than the reference value, It can be predicted that the hepatitis C virus persistently infected person is unresponsive to interferon therapy. In this way, for those with persistent hepatitis C virus infections other than those who were predicted to be unresponsive, those who showed significant effects using at least one of the marker substances (A1) to (A4) as an index, and It is possible to predict which HCV will relapse.

15. Characteristics of marker substance (B10) Fraction 6 (pH 4.0) is brought into contact with strong anion exchange chip Q10, washed with a protein chip binding buffer of pH 9.0, and SELDI-TOF-MS (EAM: CHCA) is performed. In this case, an ion peak having a mass / charge ratio of 17424 (average value) was detected. This peak was high in those who did not respond (NR) to interferon therapy, and low in those who did not (ETR). FIG. 14A shows a dot plot in which peak intensities are plotted separately for those who have not reacted and others, and FIG. 14B shows the box diagram. As a result, the p value (NR vs ETR) of this peak was 0.0000001, and the ROC area was 0.90. Based on the above, proteins that cause a peak with a mass / charge ratio of about 17400 when subjected to SELDI-TOF-MS among proteins in blood are hepatitis C virus persistently infected patients unresponsive to interferon therapy and other C It was found to be an index for discrimination from people with persistent hepatitis B virus infection. For example, when SELDI-TOF-MS under the above conditions is performed using plasma before treatment of a patient with persistent hepatitis C virus infection as a test material, and the ion peak intensity with a mass / charge ratio of about 17400 is higher than the reference value, It can be predicted that the hepatitis C virus persistently infected person is unresponsive to interferon therapy. In this way, for those with persistent hepatitis C virus infections other than those who were predicted to be unresponsive, those who showed significant effects using at least one of the marker substances (A1) to (A4) as an index, and It is possible to predict which HCV will relapse.

16. Characteristics of marker substance (B11) Fraction 8 (organic solvent) was brought into contact with a copper-modified chip IMAC30, washed with a protein chip binding buffer of pH 7.0 and 0.5 M NaCl, and SELDI-TOF-MS (EAM: SPA-). In the case of (H), an ion peak having a mass / charge ratio of 28076 (average value) was detected. This peak was low in those who were not (ETR), and high in those who were unresponsive (NR) to interferon therapy. FIG. 15A shows a dot plot in which peak intensities are plotted separately for those who have not reacted and others, and FIG. 15B shows the box diagram. As a result, the p value (NR vs ETR) of this peak was 0.0004, and the ROC area was 0.77. From the above, proteins that cause a peak with a mass / charge ratio of about 28100 when subjected to SELDI-TOF-MS among proteins in blood are hepatitis C virus persistently unresponsive to interferon therapy and other C It was found to be an index for discrimination from people with persistent hepatitis B virus infection. For example, when SELDI-TOF-MS under the above conditions is performed using plasma before treatment of a patient with persistent hepatitis C virus infection as a test material, and the ion peak intensity with a mass / charge ratio of about 28100 is higher than a reference value, It can be predicted that the hepatitis C virus persistently infected person is unresponsive to interferon therapy. In this way, for those with persistent hepatitis C virus infections other than those who were predicted to be unresponsive, those who showed significant effects using at least one of the marker substances (A1) to (A4) as an index, and It is possible to predict which HCV will relapse.

Purification and identification of marker substance (A1) 100 μL of affinity resin NHS-activated Sepharose (GE Healthcare) was packed in a spin column, and 1 mg of rabbit IgG (Sigma) was bound as a ligand, and equilibrated with PBS. 100 μL of human standard serum (CEMICOM) was diluted 2-fold with PBS and added to the equilibrated column. The column was washed with TPBS (400 μL × 3 times), further washed with PBS (400 μL × 2 times), and then eluted with 100 μL of 50% acetonitrile / 0.5% TFA. The purity of the collected elution fraction was confirmed by SELDI-TOF-MS.

  A portion of the collected fraction was subjected to acetone precipitation, and then subjected to SDS-PAGE using a polypeptide separating gel (15-20%) NTH-5AOT (DRC). FIG. 16 shows the result. In FIG. 16, lanes 1 and 2 are both recovered fractions, and M is a molecular weight marker. The separated band of about 7.8 kDa (arrow in FIG. 16) is cut out, protein is extracted, SELDI-TOF-MS analysis is performed, and it is equivalent to the mass / charge ratio (average value: 7777) of the marker substance (A1) A peak indicating the mass / charge ratio was confirmed (arrow in FIG. 17).

  A similar SDS-PAGE was performed again to cut out a band of about 7.8 kDa, and after reduction alkylation treatment, 0.01 μg / μL of a trypsin solution (dissolved in 50 mM ammonium bicarbonate (pH 8.0)) was allowed to act. Digested in the gel. 1 μL of the digested sample was dropped on a metal plate, 0.4 μL of a saturated CHCA solution was further dropped and dried, and then measured with a mass spectrometer Proteomics Analyzer 4700 (Applied Biosystems). At least 6 peaks And their exact masses were calculated as “944.53”, “157774”, “1905.00”, “203.10”, “1333.71”, and “146.80”. . Based on these data, a known protein was searched by Mascot database (Matrix Science), and peptide mass fingerprinting was performed. These peptides were consistent with peptides having amino acid sequences represented by SEQ ID NOs: 1 to 6, respectively. The target protein was identified as “platelet factor 4” (SEQ ID NO: 7) with a probability of 99% or more. Table 1 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide.

Purification and identification of marker substance (A2) Spin column packed with 1 mL of strong cation exchange resin SP-Sepharose HP (GE Healthcare) was equilibrated with equilibration buffer (100 mM sodium acetate buffer (pH 5.5)). . 1.25 mL of human standard serum (CEMICOM) was diluted 10-fold with the equilibration buffer, filtered through Steriflip HV 0.45 μm, and added to the equilibrated column. The equilibration buffer (2.5 mL × 1), 50 mM CAPS-NaOH buffer (pH 10.0) (2.5 mL × 4), and 50 mM potassium phosphate buffer (pH 11.0) (2.5 mL × 1) ) And the column was eluted with 2.5 mL of 50 mM potassium phosphate buffer (pH 11.0). The purity of the collected elution fraction was confirmed by SELDI-TOF-MS.

  A portion of the collected fraction was subjected to acetone precipitation, and then subjected to SDS-PAGE using a polypeptide separating gel (15-20%) NTH-5AOT (DRC). The results are shown in FIG. In FIG. 18, lanes 1 to 3 are all recovered fractions, and M is a molecular weight marker. The separated band of about 9.3 kDa (arrow in FIG. 18) is cut out, protein is extracted, SELDI-TOF-MS analysis is performed, and it is equivalent to the mass / charge ratio (average value: 9298) of the marker substance (A2) A peak indicating the mass / charge ratio of was confirmed (arrow in FIG. 19).

  The same SDS-PAGE was performed again to cut out a band of about 9.3 kDa, and when measured with a mass spectrometer Proteomics Analyzer 4700 (Applied Biosystems) in the same manner as in Example 2, at least 13 peaks were detected. , Their precise masses are “2151.08”, “1724.83”, “2433.12”, “1548.73”, “1100.63”, “2651.42”, “297795”, “ 1569.80 "," 1897.95 "," 2026.05 "," 1184.61 "," 1056.52 ", and" 1297.69 ". Based on these data, a known protein was searched by Mascot database (Matrix Science), and peptide mass fingerprinting was performed. These peptides were consistent with peptides having amino acid sequences represented by SEQ ID NOs: 8 to 20, respectively. The target protein was identified as “Platelet Basic Protein” (SEQ ID NO: 21) with a probability of 99% or more. Furthermore, from the charge / mass ratio (average value: 9298), the marker substance (A2) is one of platelet basic protein fragments, “connective tissue activating peptide 3” (SEQ ID NO: 22, theoretical molecular weight from amino acid sequence). : 9292). Table 2 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide.

Purification and identification of marker substance (A3)
Hi Trap Blue HP 1mL (GE Healthcare) was equilibrated with 20 mM phosphate buffer (pH 7.0). 500 μL of human standard serum (CEMICOM) was centrifuged at 20,000 G for 10 minutes at 4 ° C., and the supernatant was collected. 450 μL of the collected supernatant was diluted 10-fold with 20 mM phosphate buffer (pH 7.0), filtered through Millex HV 0.45 μm, and then added to the equilibrated column. The column was washed with 20 mM phosphate buffer (pH 7.0) to secure a flow-through fraction and a washed fraction. The fraction having a high absorbance at 280 nm was collected, and the degree of purification was confirmed by SELDI-TOF-MS.

  Hi Trap Q HP 1mL (GE Healthcare) was equilibrated with 20 mM phosphate buffer (pH 7.0) / 50 mM Tris-HCl. 4.5 mL of the collected fraction was diluted 2-fold with 50 mM Tris-HCl and added to the equilibrated column. The column was washed with 20 mM phosphate buffer (pH 7.0) / 50 mM Tris-HCl (5 CV), 50 mM Tris-HCl (pH 7.0), 50 mM MES-NaOH (pH 6.0), 50 mM sodium acetate buffer ( Washed sequentially at pH 5.0). Elution was performed with 50 mM sodium acetate (pH 4.0), and a fraction having a high absorbance at 280 nm was collected, and the degree of purification was confirmed by SELDI-TOF-MS.

  A part of the collected fraction was subjected to acetone precipitation, and then subjected to SDS-PAGE using 7.5% polyacrylamide gel. The results are shown in FIG. In FIG. 20, lanes 1 to 6 are all collected fractions, and M is a molecular weight marker. The isolated band of about 51.3 kDa (arrow in FIG. 20) is cut out, protein is extracted, SELDI-TOF-MS analysis is performed, and it is equivalent to the mass / charge ratio (average value: 51321) of the marker substance (A3) A peak indicating the mass / charge ratio was confirmed (arrow in FIG. 21).

  The same SDS-PAGE was performed again to cut out a band of about 51.3 kDa, and when measured with a mass spectrometer Proteomics Analyzer 4700 (Applied Biosystems) in the same manner as in Example 2, at least 11 peaks were detected. , Their precise masses are “232.16”, “1529.28”, “1694.90”, “2518.00”, “2707.18”, “1254.71”, “1277.56”, “ 1388.68 "," 915.46 "," 1326.75 ", and" 2092.82 ". Based on these data, a known protein was searched by Mascot database (Matrix Science), and peptide mass fingerprinting was performed, and these peptides were consistent with peptides having amino acid sequences represented by SEQ ID NOs: 23 to 33, respectively. The target protein was identified as “vitamin D binding protein” (SEQ ID NO: 34) with a probability of 99% or more. Table 3 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide.

Purification and Identification of Marker Substance (B1) A spin column packed with 500 μL of strong anion exchange resin Q-Sepharose FF (GE Healthcare) was equilibrated with 50 mM Tris-HCl (pH 9.0). 100 μL of human standard serum (CEMICOM) was diluted 5-fold with 50 mM Tris-HCl (pH 9.0) and then added to the equilibrated column. The column was washed with 500 μL of 50 mM Tris-HCl (pH 9.0) and eluted with 50 mM phosphate buffer (pH 7.0) /0.1% OGP. The purity of the collected elution fraction was confirmed by SELDI-TOF-MS.

  A spin column packed with 100 μL of weak cation exchange resin CM-Sepharose FF (GE Healthcare) was equilibrated with 50 mM acetate buffer (pH 5.0). 1 mL of the collected fraction was adjusted to pH 5.0 with 10% acetic acid, and the whole amount was added to the equilibrated column. The non-adsorbed fraction was collected and the degree of purification was confirmed by SELDI-TOF-MS.

  TSK-GEL Super ODS (Tosoh Corporation) was equilibrated with 0.1% TFA. The collected fraction was added to the column. Elution was performed with a linear gradient from solvent A (0.1% TFA) to solvent B (acetonitrile / 0.1% TFA). The purity of the collected fractions was confirmed by SELDI-TOF-MS. The collected fractions were again subjected to reverse phase HPLC under the same conditions. The collected fractions were divided into three (main fraction, reference fractions 1 and 2), and the degree of purification was confirmed by SELDI-TOF-MS (FIGS. 22 (a) to (c)). Peaks having a mass / charge ratio equivalent to that of the marker substance (B1) were confirmed in the main fraction and the reference fraction 1 (arrows in FIGS. 22 (a) and (b)).

  Each fraction was digested with trypsin and subjected to SELDI-TOF-MS. From comparison between the main fraction and the reference fraction 2, a fragment peak (m / z: 1374) unique to the marker substance (B1) was found from the main fraction and the reference fraction 1. The tryptic digest of reference fraction 1 was measured with a mass spectrometer Q-STAR (Applied Biosystems), and a fragment peak (m / z: 1374) unique to the marker substance (B1) was analyzed.

  The sequence tag method was performed based on the MS / MS data of the fragment peak (m / z: 1374) unique to the marker substance (B1), and when a known protein was searched using the Mascot database (Matrix Science), the fragment peak (m The peptide of / z: 1374) matches the peptide of the amino acid sequence represented by SEQ ID NO: 35 (LSPLGEEMRDR), and the target protein was identified as “apolipoprotein A1” (SEQ ID NO: 36) with a probability of 99% or more. Furthermore, from the comparison between the amino acid sequence (SEQ ID NO: 36) of the full length of apolipoprotein A1 and SEQ ID NO: 35, and the mass / charge ratio (average value: 3356) of the marker substance (B1), the marker substance (B1) is apolipoprotein. It was identified as a protein fragment (SEQ ID NO: 37) corresponding to the 81st to 109th positions of A1.

Purification and identification of marker substance (B3) A spin column filled with 1 mL of strong cation exchange resin SP-Sepharose HP (GE Healthcare) was equilibrated with an equilibration buffer (100 mM sodium acetate buffer (pH 5.5)). . 1.25 mL of human standard serum (CEMICOM) was diluted 10-fold with the equilibration buffer, filtered through Steriflip HV 0.45 μm, and added to the equilibrated column. The column was washed with 2.5 mL of the equilibration buffer, and then eluted four times with 2.5 mL of 50 mM CAPS-NaOH buffer (pH 10.0) (total of 10.0 mL). The second elution fraction was collected, and the degree of purification was confirmed by SELDI-TOF-MS.

  A portion of the collected fraction was subjected to acetone precipitation, and then subjected to SDS-PAGE using a polypeptide separating gel (15-20%) NTH-5AOT (DRC). The results are shown in FIG. In FIG. 23, lanes 1 to 4 are all collected fractions, and M is a molecular weight marker. The separated band of about 4.2 kDa (arrow in FIG. 23) is cut out, protein is extracted, SELDI-TOF-MS analysis is performed, and it is equivalent to the mass / charge ratio (average value: 4156) of the marker substance (B3) A peak indicating the mass / charge ratio of was confirmed (arrow in FIG. 24).

  The same SDS-PAGE was performed again to cut out a band of about 4.2 kDa, and when measured with a mass spectrometer Proteomics Analyzer 4700 (Applied Biosystems) in the same manner as in Example 2, at least one peak was detected. The exact mass was calculated as “853.44”. Based on this data, a known protein was searched by Mascot database (Matrix Science), and peptide mass fingerprinting was performed. As a result, this peptide matched the peptide of the amino acid sequence represented by SEQ ID NO: 38 (FPFMFMGR), and the target The protein was identified as “C1 inhibitor” (SEQ ID NO: 39) with a probability of 99% or more. Furthermore, from the charge / mass ratio (average value: 4156), the marker substance (B3) is “a protein fragment corresponding to the 468th to 500th (C-terminal) of SEQ ID NO: 38 (SEQ ID NO: 40, theoretical molecular weight from amino acid sequence). : 4153).

Purification and identification of marker substance (B4) Spin column packed with 2 mL of strong anion exchange resin Q-Sepharose FF (GE Healthcare) was equilibrated with buffer (50 mM Tris-HCl (pH 9.0), 1 M urea, 0. 22% CHAPS). Denaturation buffer (50 mM Tris-HCl (pH 9.0), 9 M urea, 2% CHAPS) was added to 1 mL of human standard serum (CEMICOM) for denaturation treatment. Further, the pH was adjusted to 9.0 using 1.5 M Tris buffer, and then added to the equilibrated column. After collecting 2 mL of the non-adsorbed fraction, the column was washed with 2 mL of 50 mM Tris-HCl (pH 9.0) /0.1% OGP, and the washed fraction was collected. The purity of each fraction was confirmed by SELDI-TOF-MS.

  A spin column packed with 200 μL of weak cation exchange resin CM-Sepharose FF (GE Healthcare) was equilibrated with 100 mM acetate buffer (pH 4.0) /0.1% OGP. The non-adsorbed fraction and the washed fraction were mixed, further adjusted to pH 4.0 with 10% acetic acid, and then added to the equilibrated column. The non-adsorbed fraction was allowed to pass through, and the column was further washed while the NaCl concentration was gradually increased to 0.5 M based on 100 mM acetate buffer (pH 4.0) /0.1% OGP. Elute three times with 200 μL of 33.3% isopropanol / 16.7% acetonitrile / 0.1% TFA. The purity of each collected fraction was confirmed by SELDI-TOF-MS.

  Each collected fraction was mixed. This mixed fraction was subjected to HPLC using a reverse phase column TSK-GEL Super ODS (Tosoh Corporation). Two columns were used, and a linear gradient from solvent A (0.1% TFA) to solvent B (70% acetonitrile / 0.1% TFA) was employed as elution conditions. While confirming the elution position by SELDI-TOF-MS, the test was repeated 8 times. Furthermore, the gradient conditions were changed and repeated 6 times. The degree of purification of the final elution fraction was confirmed by SELDI-TOF-MS. A peak showing a mass / charge ratio equivalent to the mass / charge ratio equivalent to the mass / charge ratio (average value: 6194) of the marker substance (B4) was confirmed (arrow in FIG. 25).

  A part of the collected fractions were subjected to acetone precipitation, and then subjected to SDS-PAGE using 16.2% T-6% C polyacrylamide (Tricine) gel. FIG. 26 shows the result. In FIG. 26, lanes 1 and 2 are both recovered fractions, and M is a molecular weight marker. A band of about 6.2 kDa was detected (arrow in FIG. 26).

  A similar SDS-PAGE was performed again to cut out a band of about 6.2 kDa, and when measured with a mass spectrometer Q-STAR (Applied Biosystems) in the same manner as in Example 2, at least one peak was detected. The exact mass was calculated as “1386.71”. Based on this data, a known protein was searched by Mascot database (Matrix Science) and peptide mass fingerprinting was performed. As a result, this peptide matched the peptide of the amino acid sequence represented by SEQ ID NO: 41 (VSFLSALEEYTKK), The protein was identified as “apolipoprotein A1” (SEQ ID NO: 36) with a probability of 99% or more. Furthermore, from the charge / mass ratio (average value: 6194) and the amino acid sequence of SEQ ID NO: 41, the marker substance (B4) and the marker substance (B4) were considered to be protein fragments on the C-terminal side of apolipoprotein A1. As a possibility, protein fragments corresponding to the 140th to 195th, 141st to 196th, 142th to 197th, 144th to 198th, or 146th to 200th of SEQ ID NO: 36 were considered.

Purification and Identification of Marker Substance (B5) A spin column packed with 1 mL of strong anion exchange resin Q-Sepharose FF (GE Healthcare) was equilibrated with 50 mM Tris-HCl (pH 9.0). Denaturation buffer (50 mM Tris-HCl (pH 9.0), 9 M urea, 2% CHAPS) 750 μL was added to 500 μL of human standard serum (CEMICOM) and then added to the equilibrated column. The non-adsorbed fraction was collected and the degree of purification was confirmed by SELDI-TOF-MS.

  A portion of the collected fraction was subjected to acetone precipitation, and then subjected to SDS-PAGE using a polypeptide separating gel (15-20%) NTH-5AOT (DRC). FIG. 27 shows the result. In FIG. 27, lanes 1 to 3 are all recovered fractions, and M is a molecular weight marker. The separated band of about 6.5 kDa (arrow in FIG. 27) is cut out, protein is extracted, SELDI-TOF-MS analysis is performed, and it is equivalent to the mass / charge ratio (average value: 6511) of the marker substance (B5) A peak indicating the mass / charge ratio of was confirmed (arrow in FIG. 28).

  The same SDS-PAGE was performed again to cut out a band of about 6.5 kDa, and when measured with a mass spectrometer Proteomics Analyzer 4700 (Applied Biosystems) in the same manner as in Example 2, at least three peaks were detected. Their exact masses were calculated as “129.67”, “1488.70”, and “1201.55”. Based on these data, a known protein was searched by Mascot database (Matrix Science), and peptide mass fingerprinting was performed. These peptides were consistent with peptides having amino acid sequences represented by SEQ ID NOs: 42 to 44, respectively. The target protein was identified as “apolipoprotein C1” (SEQ ID NO: 45) with a probability of 99% or more. Furthermore, from the charge / mass ratio (average value: 6511), the marker substance (B5) is a “fragment of apolipoprotein C1” lacking two amino acids on the N-terminal side of SEQ ID NO: 45 (SEQ ID NO: 46, from the amino acid sequence Theoretical molecular weight: 6432). Table 4 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide.

Purification and identification of marker substance (B6) Spin column packed with 500 μL of strong anion exchange resin Q-Sepharose FF (GE Healthcare) was equilibrated with buffer (50 mM Tris-HCl (pH 9.0), 1 M urea, 0. 22% CHAPS). Denaturation buffer (50 mM Tris-HCl (pH 9.0), 9 M urea, 2% CHAPS) 150 μL was added to 100 μL of human standard serum (CEMICOM) for denaturation treatment. Further, 250 μL of the equilibration buffer was added for dilution, and then added to the equilibrated column. 500 μL of the non-adsorbed fraction was collected. Further, elution was performed with 500 μL of 50 mM Tris-HCl (pH 9.0) /0.1% OGP. The non-adsorbed fraction and the eluted fraction were mixed to obtain a target fraction. The degree of purification of the target fraction was confirmed by SELDI-TOF-MS.

  A spin column packed with 100 μL of weak cation exchange resin CM-Sepharose FF (GE Healthcare) was equilibrated with 100 mM phosphate buffer. The target fraction was adjusted to pH 6.0 with 10% acetic acid and then added to the equilibrated column. Non-adsorbed fraction was passed through. The column was washed 3 times with 100 μL of 100 mM phosphate buffer (pH 6.0) / 0, 1% OGP. Each fraction was collected by elution twice with 100 mM phosphate buffer (pH 6.0) / 0, 1% OGP containing 100 μL of 0.1 M NaCl. The purity of each collected fraction was confirmed by SELDI-TOF-MS.

  A non-porous cation exchange column SP-NPR (Tosoh Corporation) was parallelized with 50 mM MES-NaOH, and the elution fraction was added. Elution was performed with a linear concentration gradient from 50 mM MES-NaOH (pH 6.0) to 50 mM MES-NaOH (pH 6.0) containing 500 mM NaCl. The degree of purification of the eluted fraction was confirmed by SELDI-TOF-MS.

  A part of the collected fraction was subjected to acetone precipitation, and then subjected to SDS-PAGE using a 15% polyacrylamide gel. FIG. 29 shows the result. In FIG. 29, lanes 1 and 2 are both recovered fractions, and M is a molecular weight marker. The separated band of about 8.6 kDa (arrow in FIG. 29) is cut out, protein is extracted, SELDI-TOF-MS analysis is performed, and it is equivalent to the mass / charge ratio (average value: 8607) of the marker substance (B6) A peak indicating the mass / charge ratio was confirmed (arrow in FIG. 30).

The same SDS-PAGE was performed again to cut out a band of about 8.6 kDa, and when measured with a mass spectrometer Q-STAR (Applied Biosystems) in the same manner as in Example 2, at least two peaks were detected. Their exact masses were calculated as “749.40” and “1035.50”. Based on these data, a known protein was searched by Mascot database (Matrix Science), and peptide mass fingerprinting was performed. These peptides were matched with peptides having amino acid sequences represented by SEQ ID NOs: 47 and 48, respectively. The protein of interest was identified as “complement C4a” (SEQ ID NO: 49) with a probability of 99% or higher.
Furthermore, from the charge / mass ratio (average value: 8607), the marker substance (B6) is a fragment of complement C4a “complement C4a des Arg” (SEQ ID NO. It was suggested that the theoretical molecular weight was 8608). Table 5 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide.

Purification and identification of marker substance (B7) A spin column packed with 1 mL of strong anion exchange resin Q-Sepharose FF (GE Healthcare) was equilibrated with 50 mM Tris-HCl (pH 9.0). Denaturation buffer (50 mM Tris-HCl (pH 9.0), 9 M urea, 2% CHAPS) 750 μL was added to 500 μL of human standard serum (CEMICOM) and then added to the equilibrated column. The non-adsorbed fraction was collected and the degree of purification was confirmed by SELDI-TOF-MS.

  A portion of the collected fraction was subjected to acetone precipitation, and then subjected to SDS-PAGE using a polypeptide separating gel (15-20%) NTH-5AOT (DRC). FIG. 31 shows the result. In FIG. 31, lanes 1 to 3 are all recovered fractions, and M is a molecular weight marker. The separated band of about 8.7 kDa (arrow in FIG. 31) is cut out, protein is extracted, SELDI-TOF-MS analysis is performed, and it is equivalent to the mass / charge ratio (average value: 8700) of the marker substance (B7) A peak indicating the mass / charge ratio was confirmed (arrow in FIG. 32).

  The same SDS-PAGE was performed again to cut out a band of about 8.7 kDa, and when measured with a mass spectrometer Proteomics Analyzer 4700 (Applied Biosystems) in the same manner as in Example 2, at least 6 peaks were detected. Their exact masses were calculated as "26777.29", "2350.11", "2982.44", "1156.69", "2513.31", and "2385.25". Based on these data, a known protein was searched by Mascot database (Matrix Science), and peptide mass fingerprinting was performed. These peptides were consistent with peptides having amino acid sequences represented by SEQ ID NOs: 51 to 56, respectively. The target protein was identified as “apolipoprotein A2” (SEQ ID NO: 57) with a probability of 99% or more. Table 6 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide.

  It is known that apolipoprotein A2 exists as a dimer with a disulfide bond, and that there is a deletion of the N-terminal Gln residue (Brewer et al., Proc. Natl. Acad. Sci. , 1972, 69, 1304-1308). Therefore, the protein extracted from the gel was reduced with On-Chip, and the ion peak profiles were compared under reducing and non-reducing conditions. As a result, a peak shift corresponding to the mass of Cys was observed. From this result and the charge / mass ratio (average value: 8700), the marker substance (B7) is a modified form of apolipoprotein A2 that is cysteinylated apolipoprotein A2 and lacks its C-terminal Gln. It was thought that there was. The sequence of the apolipoprotein A2 fragment lacking the C terminal Gln is shown in SEQ ID NO: 58.

Purification and identification of marker substance (B8) Spin column packed with 1 mL of strong anion exchange resin Q-Sepharose HP (GE Healthcare) was equilibrated with buffer (50 mM Tris-HCl (pH 9.0), 1 M urea, 0. 22% CHAPS). 750 μL of denaturation buffer (50 mM Tris-HCl (pH 9.0), 9M urea, 2% CHAPS) was added to 500 μL of human standard serum (Cosmo Bio), and denaturation treatment was performed at 4 ° C. for 20 minutes. The denatured sample was added to the equilibrated column. The column was washed successively with 50 mM Tris-HCl (pH 9.0), 100 mM sodium acetate buffer (pH 5.0), and 50 mM sodium citrate (pH 3.0). Elution was performed with 50% acetonitrile / 0.1% TFA. The degree of purification of the eluted fraction was confirmed by SELDI-TOF-MS.

  A portion of the collected fraction was subjected to acetone precipitation, and then subjected to SDS-PAGE using a polypeptide separating gel (15-20%) NTH-5AOT (DRC). FIG. 33 shows the result. In FIG. 33, lanes 1 to 6 are all collected fractions, and M is a molecular weight marker. The separated band of about 13.9 kDa (arrow A in FIG. 33) was cut out, protein was extracted, SELDI-TOF-MS analysis was performed, and the mass / charge ratio (average value: 13882) of the marker substance (B8) A peak showing an equivalent mass / charge ratio was confirmed (arrow in FIG. 34).

  The same SDS-PAGE was performed again to cut out a band of about 13.9 kDa, and when measured with a mass spectrometer Proteomics Analyzer 4700 (Applied Biosystems) in the same manner as in Example 2, at least 6 peaks were detected. Their exact masses were calculated as “1522.72”, “1394.62”, “2455.57”, “3140.53”, “2451.20”, and “2607.31”. Based on these data, a known protein was searched by Mascot database (Matrix Science), and peptide mass fingerprinting was performed. These peptides were consistent with peptides having amino acid sequences represented by SEQ ID NOs: 59 to 64, respectively. The protein of interest was identified as “transthyretin” (SEQ ID NO: 65) with a probability of 99% or higher. Table 7 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide.

  As described above, various modified transthyretins in which only one Cys residue is modified have been found for transthyretin. From the charge / mass ratio (average value: 13882), the marker substance (B8) was considered to be cysteinylated transthyretin in which Cys was added to the Cys residue.

Purification and identification of marker substances (B9) and (B10)
Hi Trap Blue HP 1mL (GE Healthcare) was equilibrated with 20 mM phosphate buffer (pH 7.0). 1 mL of human standard serum (Cosmo Bio) was diluted 10-fold with 20 mM phosphate buffer (pH 7.0), filtered through Millex HV 0.45 μm, and added to the equilibrated column. The column was washed with 20 mM phosphate buffer (pH 7.0) to secure a flow-through fraction and a washed fraction. The fraction having a high absorbance at 280 nm was collected, and the degree of purification was confirmed by SELDI-TOF-MS. Four sets of this purification were performed.

  Hi Trap Q HP 1mL (GE Healthcare) was equilibrated with 20 mM phosphate buffer (pH 7.0). 20 mL of collected fractions (4 sets) were added to the equilibrated column. The column was washed with 20 mM phosphate buffer (pH 7.0). Furthermore, it wash | cleaned one by one by each 20 mM phosphate buffer (pH 7.0) containing 100 mM, 125 mM, or 160 mM NaCl. Elution was performed with 20 mM phosphate buffer (pH 7.0) containing 200 mM NaCl. The fraction having a high absorbance at 280 nm was collected, and the degree of purification was confirmed by SELDI-TOF-MS.

  A part of the collected fraction was subjected to acetone precipitation, and then subjected to SDS-PAGE using a 15% polyacrylamide gel. FIG. 35 shows the result. In FIG. 35, lanes 1 and 6 are all collected fractions, and M is a molecular weight marker. The separated band of about 17.4 kDa (arrow in FIG. 35) is cut out, protein is extracted, SELDI-TOF-MS analysis is performed, and the mass / charge ratio (B9) of both marker substances (B9) and (B10) is analyzed. (Average value of 17274, average value of B10: 17407) were confirmed (arrows in FIG. 36).

  The same SDS-PAGE was performed again to cut out about 17.4 bands, and when measured with a mass spectrometer Proteomics Analyzer 4700 (Applied Biosystems) in the same manner as in Example 2, at least 6 peaks were detected. Their exact masses were calculated as “1199.68”, “1853.96”, “1626.79”, “1156.68”, “1284.78”, and “1069.65”. Based on these data, a known protein was searched by Mascot database (Matrix Science), and peptide mass fingerprinting was performed. These peptides were consistent with peptides having amino acid sequences represented by SEQ ID NOs: 66 to 71, respectively. The target protein was identified as “apolipoprotein A2” (SEQ ID NO: 57) with a probability of 99% or more. Table 8 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide.

  As described above, it is known that apolipoprotein A2 exists as a dimer with a disulfide bond, and that a Cln terminal Gln residue is deleted (SEQ ID NO: 58). Furthermore, it is also known that the SH group of the Cys residue can be blocked to become cystine. (A) a dimer of apolipoprotein A2 (SEQ ID NO: 57), (b) a dimer consisting of apolipoprotein A2 (SEQ ID NO: 57) and one lacking the Cln terminal Gln residue (SEQ ID NO: 58), (C) Since the theoretical molecular weights of the two apolipoproteins A2 (SEQ ID NO: 58) lacking the C-terminal Gln residue are 17380, 17252, and 17123, respectively, the marker substance (B9) was considered to be the dimer of (b), and the marker substance (B10) was considered to be the dimer of (a).

Purification and identification of marker substance (B11) In SDS-PAGE performed in Example 11, the separated band of about 28.1 kDa (arrow B in FIG. 33) was cut out to extract the protein. The extracted protein was subjected to SELDI-TOF-MS analysis, and a peak having a mass / charge ratio equivalent to the mass / charge ratio (average value: 28120) of the marker substance (B11) was confirmed (arrow in FIG. 37).

  The same SDS-PAGE was performed again to cut out a band of about 28.1 kDa, and when measured with a mass spectrometer Proteomics Analyzer 4700 (Applied Biosystems) in the same manner as in Example 2, at least 28 peaks were detected. The exact masses are “1650.84”, “181.85”, “1400.67”, “1612.78”, “2202.11”, “2888.74”, “1932.94”, “ 3237.46 "," 2020.99 "," 1252.62 "," 1380.71 "," 1283.58 "," 869.52 "," 1706.92 "," 1466.79 "," 1723. " 95 ”,“ 873.45 ”,“ 896.48 ”,“ 1031.52 ”,“ 131.64 ”,“ 781.44 ”,“ 1301.65 ”,“ 831 ” .43 "," 1012.58 "," 1230.71 "," 1386.71 ", and" 1514.81 ". Based on these data, a known protein was searched by Mascot database (Matrix Science), and peptide mass fingerprinting was performed. These peptides were consistent with peptides having amino acid sequences represented by SEQ ID NOs: 72 to 99, respectively. The target protein was identified as “apolipoprotein A1” (SEQ ID NO: 36) with a probability of 99% or more. Table 9 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide.

(A) is the dot plot about the ion peak whose mass / charge ratio is 7777 (average value), (b) is the box diagram. (A) is a dot plot for an ion peak having a mass / charge ratio of 9298 (average value), and (b) is a box diagram. (A) is the dot plot about the ion peak whose mass / charge ratio is 51321 (average value), (b) is the box diagram. (A) is the dot plot about the ion peak whose mass / charge ratio is 125350 (average value), (b) is the box diagram. (A) is the dot plot about the ion peak whose mass / charge ratio is 3356 (average value), (b) is the box diagram. (A) is a dot plot for an ion peak with a mass / charge ratio of 3962 (average value), and (b) is a box diagram. (A) is the dot plot about the ion peak whose mass / charge ratio is 4156 (average value), (b) is the box diagram. (A) is the dot plot about the ion peak whose mass / charge ratio is 6194 (average value), (b) is the box diagram. (A) is the dot plot about the ion peak whose mass / charge ratio is 6511 (average value), (b) is the box diagram. (A) is the dot plot about the ion peak whose mass / charge ratio is 8608 (average value), (b) is the box diagram. (A) is the dot plot about the ion peak whose mass / charge ratio is 8713 (average value), (b) is the box diagram. (A) is the dot plot about the ion peak whose mass / charge ratio is 13882 (average value), (b) is the box diagram. (A) is the dot plot about the ion peak whose mass / charge ratio is 17271 (average value), (b) is the box diagram. (A) is the dot plot about the ion peak whose mass / charge ratio is 17424 (average value), (b) is the box diagram. (A) is the dot plot about the ion peak whose mass / charge ratio is 28076 (average value), (b) is the box diagram. 6 is a photograph showing the results of SDS-PAGE performed in Example 2. FIG. 6 is an ion peak diagram showing the results of SELDI-TOF-MS performed in Example 2. FIG. 6 is a photograph showing the results of SDS-PAGE performed in Example 3. FIG. 6 is an ion peak diagram showing the results of SELDI-TOF-MS performed in Example 3. FIG. 6 is a photograph showing the results of SDS-PAGE performed in Example 4. It is an ion peak figure showing the result of SELDI-TOF-MS performed in Example 4. It is an ion peak figure showing the result of SELDI-TOF-MS performed in Example 5, (a) is the main fraction, (b) is the reference fraction 1, and (c) is the reference fraction 2. 6 is a photograph showing the results of SDS-PAGE performed in Example 6. FIG. 6 is an ion peak diagram showing the results of SELDI-TOF-MS performed in Example 6. FIG. 10 is an ion peak diagram showing a result of SELDI-TOF-MS performed in Example 7. FIG. 6 is a photograph showing the results of SDS-PAGE performed in Example 7. 6 is a photograph showing the results of SDS-PAGE performed in Example 8. 10 is an ion peak diagram showing a result of SELDI-TOF-MS performed in Example 8. FIG. 6 is a photograph showing the results of SDS-PAGE performed in Example 9. 10 is an ion peak diagram showing a result of SELDI-TOF-MS performed in Example 9. FIG. 10 is a photograph showing the results of SDS-PAGE performed in Example 10. 10 is an ion peak diagram showing the results of SELDI-TOF-MS performed in Example 10. FIG. 2 is a photograph showing the results of SDS-PAGE performed in Example 11. FIG. 10 is an ion peak diagram showing a result of SELDI-TOF-MS performed in Example 11. FIG. 6 is a photograph showing the results of SDS-PAGE performed in Example 12. 10 is an ion peak diagram showing a result of SELDI-TOF-MS performed in Example 12. FIG. 14 is an ion peak diagram showing a result of SELDI-TOF-MS performed in Example 13. FIG.

Claims (15)

Predicting the effect of interferon therapy on the hepatitis C virus persistently infected by using at least one concentration of the following marker substances (A1) to (A4) in the body fluid collected from the hepatitis C virus persistently infected as an index And predicting the effect of interferon therapy.
(A1) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 7780 when subjected to mass spectrometry;
(A2) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 9300 when subjected to mass spectrometry;
(A3) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 51300 when subjected to mass spectrometry;
(A4) A protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 125000 when subjected to mass spectrometry. The method for predicting the effect of interferon therapy according to claim 1, wherein at least one of the following (1) to (3) is satisfied.
(1) The marker substance (A1) is platelet factor 4 or a modified form thereof.
(2) The marker substance (A2) is connective tissue activating peptide 3 or a modified form thereof.
(3) The marker substance (A3) is a vitamin D binding protein or a modified form thereof. Using at least one concentration of a marker substance belonging to any of the following (C1) to (C3) in a body fluid collected from a hepatitis C virus persistently infected patient as an index, the effect of interferon therapy on the hepatitis C virus persistently infected A method for predicting the effect of interferon therapy, characterized by predicting.
(C1) platelet factor 4 or a modified form thereof,
(C2) connective tissue activating peptide 3 or a modified form thereof,
(C3) Vitamin D binding protein or a modified form thereof. The interferon therapy effect prediction method according to any one of claims 1 to 3, wherein at least one concentration of the following marker substances (B1) to (B11) in the body fluid is further used as an index.
(B1) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and generates an ion peak with a mass / charge ratio of about 3360 when subjected to mass spectrometry,
(B2) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 3960 when subjected to mass spectrometry;
(B3) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 4160 when subjected to mass spectrometry;
(B4) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and generates an ion peak with a mass / charge ratio of about 6190 when subjected to mass spectrometry,
(B5) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 6510 when subjected to mass spectrometry;
(B6) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 8610 when subjected to mass spectrometry;
(B7) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 8710 when subjected to mass spectrometry;
(B8) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and generates an ion peak with a mass / charge ratio of about 13900 when subjected to mass spectrometry;
(B9) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 17300 when subjected to mass spectrometry;
(B10) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 17400 when subjected to mass spectrometry;
(B11) A protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M and produces an ion peak with a mass / charge ratio of about 28100 when subjected to mass spectrometry. The method for predicting the effect of interferon therapy according to claim 4, wherein at least one of the following (4) to (13) is satisfied.
(4) The marker substance (B1) is apolipoprotein A1 or a modified form thereof.
(5) The marker substance (B3) is a C1 inhibitor or a modified form thereof.
(6) The marker substance (B4) is apolipoprotein A1 or a modified form thereof.
(7) The marker substance (B5) is apolipoprotein C1 or a modified form thereof.
(8) The marker substance (B6) is complement C4 or a modified form thereof.
(9) The marker substance (B7) is apolipoprotein A2 or a modified form thereof.
(10) The marker substance (B8) is transthyretin or a modified form thereof.
(11) The marker substance (B9) is apolipoprotein A2 or a modified form thereof.
(12) The marker substance (B10) is apolipoprotein A2 or a modified form thereof.
(13) The marker substance (B11) is apolipoprotein A1 or a modified form thereof. The method for predicting the effect of interferon therapy according to any one of claims 1 to 3, wherein at least one concentration belonging to any of the following (D1) to (D6) in the body fluid is further used as an index.
(D1) Apolipoprotein A1 or a modified product thereof,
(D2) C1 inhibitor or a modified form thereof,
(D3) Apolipoprotein C1 or a modified product thereof,
(D4) complement C4 or a modified product thereof,
(D5) Apolipoprotein A2 or a modified product thereof,
(D6) Transthyretin or a modified product thereof. Predicting the effect of interferon therapy on the hepatitis C virus persistently infected with at least one concentration of the following marker substances (B1) to (B11) in body fluid collected from the hepatitis C virus persistently infected And predicting the effect of interferon therapy.
(B1) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and generates an ion peak with a mass / charge ratio of about 3360 when subjected to mass spectrometry,
(B2) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 3960 when subjected to mass spectrometry;
(B3) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 4160 when subjected to mass spectrometry;
(B4) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and generates an ion peak with a mass / charge ratio of about 6190 when subjected to mass spectrometry,
(B5) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 6510 when subjected to mass spectrometry;
(B6) a protein that binds to a weak cation exchanger at pH 4.0 and produces an ion peak with a mass / charge ratio of about 8610 when subjected to mass spectrometry;
(B7) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 8710 when subjected to mass spectrometry;
(B8) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and generates an ion peak with a mass / charge ratio of about 13900 when subjected to mass spectrometry;
(B9) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 17300 when subjected to mass spectrometry;
(B10) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 17400 when subjected to mass spectrometry;
(B11) A protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M and produces an ion peak with a mass / charge ratio of about 28100 when subjected to mass spectrometry. The method for predicting the effect of interferon therapy according to claim 7, wherein at least one of the following (4) to (13) is satisfied.
(4) The marker substance (B1) is apolipoprotein A1 or a modified form thereof.
(5) The marker substance (B3) is a C1 inhibitor or a modified form thereof.
(6) The marker substance (B4) is apolipoprotein A1 or a modified form thereof.
(7) The marker substance (B5) is apolipoprotein C1 or a modified form thereof.
(8) The marker substance (B6) is complement C4 or a modified form thereof.
(9) The marker substance (B7) is apolipoprotein A2 or a modified form thereof.
(10) The marker substance (B8) is transthyretin or a modified form thereof.
(11) The marker substance (B9) is apolipoprotein A2 or a modified form thereof.
(12) The marker substance (B10) is apolipoprotein A2 or a modified form thereof.
(13) The marker substance (B11) is apolipoprotein A1 or a modified form thereof. Using at least one concentration of a marker substance belonging to any of the following (D1) to (D6) in a body fluid collected from a hepatitis C virus persistently infected patient as an index, the effect of interferon therapy on the hepatitis C virus persistently infected A method for predicting the effect of interferon therapy, characterized by predicting.
(D1) Apolipoprotein A1 or a modified product thereof,
(D2) C1 inhibitor or a modified form thereof,
(D3) Apolipoprotein C1 or a modified product thereof,
(D4) complement C4 or a modified product thereof,
(D5) Apolipoprotein A2 or a modified product thereof,
(D6) Transthyretin or a modified product thereof.   The method for predicting the effect of interferon therapy according to any one of claims 1 to 9, wherein the body fluid is blood.   The bodily fluid or bodily fluid component is brought into contact with a carrier on which a substance having affinity for the marker substance is immobilized, and the marker substance in the bodily fluid is captured on the carrier, and based on the amount of the captured marker substance The method for predicting the effect of interferon therapy according to any one of claims 1 to 10, wherein the concentration of the marker substance in a body fluid is calculated.   The method for predicting the effect of interferon therapy according to claim 11, wherein the carrier has a planar portion, and the substance having affinity for the marker substance is immobilized on a part of the planar portion.   The method for predicting the effect of interferon therapy according to claim 11 or 12, wherein the substance having affinity for the marker substance is an ion exchanger, a metal chelate or an antibody.   14. A kit for use in the method for predicting the effect of interferon therapy according to any one of claims 1 to 13, comprising a carrier on which a substance having affinity for the marker substance is immobilized. Kit for predicting the effect of therapy.   The interferon therapy effect prediction kit according to claim 14, wherein the substance having affinity for the marker substance is an ion exchanger, a metal chelate or an antibody.