Patent Application
20180120328
The present invention relates to a biomarker for a psychiatric/neurological disorder, particularly a biomarker for diagnosing a psychiatric/neurological disorder. The present invention also relates to a test kit for a psychiatric/neurological disorder for detecting the biomarker, and a test method for a psychiatric/neurological disorder using the biomarker.
Diagnosis criteria, such as DMSIV-TR, ICD-10, and PANSS, are currently used as indications in diagnosing psychiatric/neurological disorders, such as schizophrenia, autism, and Alzheimer's disease. However, diagnoses using these criteria are performed by, for example, a medical examination by interview with patients and thus have had the problem of producing variations depending on the evaluator. Thus, there has been a need for a biomarker as an objective biological indication.
Attempts have also been previously made to search for biomarkers for diagnosing psychiatric/neurological disorders. For example, Patent Literature 1 discloses a schizophrenia marker. Patent Literature 2 discloses a biomarker for diagnosing and monitoring mental disorder. However, these markers are not put to practical use in view of accuracy, easiness to practice, cost, and the like.
An object of the present invention is to provide a biomarker for a psychiatric/neurological disorder, particularly a biomarker for diagnosing a psychiatric/neurological disorder, a test kit for a psychiatric/neurological disorder for detecting the biomarker, and a test method using the biomarker.
In view of the above-described problems, the present inventors conducted studies for identifying a biomarker for a psychiatric/neurological disorder. As a result, the present inventors have discovered that the blood concentration of the free κ immunoglobulin chain (containing the constant region) and the free λ immunoglobulin chain (containing the constant region) is significantly increased in patients with psychiatric/neurological disorders compared to that in healthy individuals. Thus, the present invention provides a biomarker for a psychiatric/neurological disorder, comprising at least one selected from the group consisting of the free κ immunoglobulin chain, the free λ immunoglobulin chain, and fragments thereof. The biomarker can be used to readily conduct the test, diagnosis, and the like of a psychiatric/neurological disorder with a blood sample of interest.
In one embodiment, the free κ immunoglobulin chain comprises the amino acid sequence of SEQ ID NO: 1 (constant region) and the free λ immunoglobulin chain comprises the amino acid sequence of SEQ ID NO: 6 (constant region). In one embodiment, the free κ immunoglobulin chain comprises the amino acid sequence of SEQ ID NO: 2 or 3 and the free λ immunoglobulin chain comprises the amino acid sequence of SEQ ID NO: 6.
It is known that the free κ immunoglobulin chain increases also in the serum of patients having multiple myeloma (Non-patent Literature 1). It is known that in this case, an inflammatory cytokine increases simultaneously (Non-patent Literatures 2 and 3). The present inventors have surprisingly discovered that in patients with psychiatric/neurological disorders, the inflammatory cytokine does not increase despite an increase in the free κ immunoglobulin chain. Thus, the present invention provides a biomarker for a psychiatric/neurological disorder, comprising an inflammatory cytokine in addition to the above biomarker. In one embodiment, the inflammatory cytokine is at least one selected from the group consisting of IL-6, IL-1 beta, and TNF-alpha.
The present invention also provides a test kit for a psychiatric/neurological disorder, comprising a substance capable of detecting the biomarker for a psychiatric/neurological disorder. In addition, the present invention provides a test method for a psychiatric/neurological disorder, using the biomarker as an indication. In one embodiment, the test method for a psychiatric/neurological disorder according to the present invention comprises the steps of (a) measuring the amount of the biomarker for a psychiatric/neurological disorder according to the present invention in a blood sample from a subject and (b) comparing the amount with that from a normal subject or with the median value of that from a normal subject.
The biomarker for a psychiatric/neurological disorder according to the present invention can be easily collected and quantified from a blood sample from a subject, and thus the use of the biomarker for a psychiatric/neurological disorder according to the present invention enables the rapid and simple diagnosis and the like of a psychiatric and neurological disorder.
As used herein, the term “psychiatric/neurological disorder” has the meaning generally used in the art, and examples thereof include schizophrenia, autism, Alzheimer's disease, cognitive abnormality, bipolar disorder (manic-depressive psychosis), disorders of cranial nerve development, cognitive impairment ascribed to neuropathy due to infection during pregnancy, mental disorder ascribed to impaired immunity, epilepsy, idiophrenic mental disorder, toxic mental disorder, intellectual disability (mental retardation), psychopathy, and neurosis. In addition, the “psychiatric/neurological disorder” also includes syphilitic mental disorder, senile mental disorder, cerebrovascular mental disorder, mental disorder due to head injury, atypical endogenous psychosis, endocrine mental disorder and exogenous reaction type, and involutional mental disorder. In the present invention, the psychiatric/neurological disorder is preferably schizophrenia, autism, or Alzheimer's disease, most preferably schizophrenia.
As used herein, the term “biomarker for a psychiatric/neurological disorder” refers to a living substance capable of being an indication for determining whether the psychiatric/neurological disorder have been developed or can be potentially developed, or determining prognosis after developing the disorders. For example, the biomarker for a psychiatric/neurological disorder can be used to diagnose the psychiatric/neurological disorder by measuring its amount. The biomarker for a psychiatric/neurological disorder may comprise a plurality of biomarkers. For example, when 2 or more living substances change in quantity in a subject with a psychiatric/neurological disorder, a combination of the 2 or more living substances can be a biomarker for the psychiatric/neurological disorder. For example, when certain 2 living substances simultaneously increase in a subject with another disorder or condition other than a psychiatric/neurological disorder but one of them increases while the other not increasing in a subject with the psychiatric/neurological disorder, a combination of the 2 living substances can also be a biomarker for the psychiatric/neurological disorder. The biomarker for a psychiatric/neurological disorder can be used, by measuring its amount, for the diagnosis and test of the psychiatric/neurological disorder, the screening of subjects having the psychiatric/neurological disorder, the establishment of an efficient therapeutic method, the screening of a candidate agent useful for the psychiatric/neurological disorder, and the development of model animals for the psychiatric/neurological disorder.
The term “κ immunoglobulin chain” and the term “λ immunoglobulin chain” mean the 2 types (κ type and λ type) of an immunoglobulin L chain, respectively. They are herein also referred to as “Ig κ” and “Ig λ”, respectively. The κ immunoglobulin chain (Ig κ) is a polypeptide consisting of a constant region of about 12 kDa called a κ immunoglobulin chain C region (Ig i-C) and a variable region of about 12 kDa (see (a) in
The term “κ immunoglobulin chain C region” is a polypeptide forming the constant region of a κ immunoglobulin chain or a free κ immunoglobulin chain. The polypeptide is herein also referred to as “Ig κ chain C” or “Ig κ-C”. As used herein, the “λ immunoglobulin chain C region” is a polypeptide forming the constant region of a λ immunoglobulin chain or a free λ immunoglobulin chain. The polypeptide is also referred to as “Ig λ chain C” or “Ig λ-C”.
As used herein, the term “free κ immunoglobulin chain” and the term “free λ immunoglobulin chain” mean a κ immunoglobulin chain in free form and a λ immunoglobulin chain in free form, respectively (see (b) in
In the context of the biomarker for a psychiatric/neurological disorder according to the present invention herein, “fragment” refers to a polypeptide containing a partial amino acid sequence of a full-length protein (or a polypeptide). Thus, the “fragment” is a polypeptide which has a length within the full length of the biomarker for a psychiatric/neurological disorder according to the present invention and is at least one amino acid residue shorter, for example, 1 to 100, 1 to 50, 1 to 20, 19, 18, 17, 16, 15, 14, 13, 12, 1, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter, than the full length. Preferably, the fragment of the free κ immunoglobulin chain is the κ immunoglobulin chain C region or a site capable of being an antigen in detection with an antibody. Preferably, the fragment of the free λ immunoglobulin chain is the λ immunoglobulin chain C region or a site capable of being an antigen in detection with an antibody.
As used herein, the term “measurement” includes detection and quantification. The term “quantification” also includes semi-quantification. As used herein, the term “blood sample” refers to blood collected from a subject, and includes serum, plasma, and whole blood.
As used herein, the term “subject” means any mammal, for example, humans, rats, mice, guinea pigs, rabbits, monkeys (for example, marmosets, particularly common marmosets, or rhesus monkeys), dogs, cats, or miniature pigs. The subject is preferably a human. As used herein, the term “normal subject” means a subject not having developed or potentially not developing a psychiatric/neurological disorder, for example, a human, a rat, a mouse, a guinea pig, a rabbit, a monkey (for example, marmosets, particularly common marmosets, or rhesus monkeys), a dog, a cat, or a miniature pig not having developed or potentially not developing a psychiatric/neurological disorder. The normal subject is preferably a human. The “normal subject” and “normal control” can be herein synonymously used. When the normal subject is a human, the normal subject is herein also referred to as a healthy individual.
The biomarker for a psychiatric/neurological disorder according to the present invention comprises at least one selected from the group consisting of a free κ immunoglobulin chain, a free λ immunoglobulin chain, and fragments thereof. Preferably, the biomarker for a psychiatric/neurological disorder according to the present invention consists of at least one selected from the group consisting of a free κ immunoglobulin chain, a free λ immunoglobulin chain, and fragments thereof. More preferably, the biomarker for a psychiatric/neurological disorder according to the present invention comprises a free κ immunoglobulin chain or a fragment thereof. Alternatively, more preferably, the biomarker for a psychiatric/neurological disorder according to the present invention consists of a free κ immunoglobulin chain or a fragment thereof. More preferably, the biomarker for a psychiatric/neurological disorder according to the present invention comprises a free λ immunoglobulin chain or a fragment thereof. Alternatively, more preferably, the biomarker for a psychiatric/neurological disorder according to the present invention consists of a free λ immunoglobulin chain or a fragment thereof. More preferably, the biomarker for a psychiatric/neurological disorder according to the present invention comprises a free κ immunoglobulin chain or a fragment thereof and a free λ immunoglobulin chain or a fragment thereof. Alternatively, more preferably, the biomarker for a psychiatric/neurological disorder according to the present invention consists of a free κ immunoglobulin chain or a fragment thereof and a free λ immunoglobulin chain or a fragment thereof.
In another embodiment, the biomarker for a psychiatric/neurological disorder according to the present invention comprises at least one selected from the group consisting of a free κ immunoglobulin chain, a free λ immunoglobulin chain, and fragments thereof, and an inflammatory cytokine. Preferably, the biomarker for a psychiatric/neurological disorder according to the present invention consists of at least one selected from the group consisting of a free κ immunoglobulin chain, a free λ immunoglobulin chain, and fragments thereof, and an inflammatory cytokine. More preferably, the biomarker for a psychiatric/neurological disorder according to the present invention comprises a free κ immunoglobulin chain or a fragment thereof, and an inflammatory cytokine. Alternatively, more preferably, the biomarker for a psychiatric/neurological disorder according to the present invention consists of a free κ immunoglobulin chain or a fragment thereof, and an inflammatory cytokine. More preferably, the biomarker for a psychiatric/neurological disorder according to the present invention comprises a free λ immunoglobulin chain or a fragment thereof, and an inflammatory cytokine. Alternatively, more preferably, the biomarker for a psychiatric/neurological disorder according to the present invention consists of a free λ immunoglobulin chain or a fragment thereof, and an inflammatory cytokine. More preferably, the biomarker for a psychiatric/neurological disorder according to the present invention comprises a free κ immunoglobulin chain or a fragment thereof, a free λ immunoglobulin chain or a fragment thereof, and an inflammatory cytokine. Alternatively, more preferably, the biomarker for a psychiatric/neurological disorder according to the present invention consists of a free κ immunoglobulin chain or a fragment thereof, a free λ immunoglobulin chain or a fragment thereof, and an inflammatory cytokine.
In one embodiment, the inflammatory cytokine is at least one selected from the group consisting of IL-6, IL-1 beta, and TNF-alpha. Preferably, the inflammatory cytokine is at least two selected from the group consisting of L-6, IL-1 beta, and TNF-alpha. More preferably, the inflammatory cytokine is IL-6, IL-1 beta, and TNF-alpha. In one embodiment, the biomarker for a psychiatric/neurological disorder according to the present invention is present in a blood sample collected from a subject.
The amino acid sequence of a free c immunoglobulin chain is well-known to those of ordinary skill in the art. The human-derived free κ immunoglobulin chain comprises a constant region (κ immunoglobulin chain C region). For example, the free κ immunoglobulin chain comprises the amino acid sequence of SEQ ID NO: 1 (C region). Preferably, the free κ immunoglobulin chain comprises the amino acid sequence of SEQ ID NO: 2 or 3. Those of ordinary skill in the art can detect and quantify the free κ immunoglobulin chain using a substance capable of recognizing the constant region (κ immunoglobulin chain C region) or an antigen site thereof. Examples of the substance include Anti-Ig κ light chain antibody (MAR-C8) (Abcam Co., Ltd.) and FREELITE κ chain (MBL Co., Ltd., code number: BS-LK016BD).
The constant region (κ immunoglobulin chain C region) preferably comprises the amino acid sequence of SEQ ID NO: 1. Alternatively, the constant region (κ immunoglobulin chain C region) preferably consists of the amino acid sequence of SEQ ID NO: 1. More preferably, the constant region (κ immunoglobulin chain C region) comprises the amino acid sequence of SEQ ID NO: 2 or 3. The antigen site of the constant region (κ immunoglobulin chain C region) preferably comprises the amino acid sequence of SEQ ID NO: 2 or 3. Alternatively, the antigen site of the constant region (κ immunoglobulin chain C region) preferably consists of the amino acid sequence of SEQ ID NO: 2 or 3. The fragment of the free κ immunoglobulin chain preferably comprises the constant region (κ immunoglobulin chain C region) or an antigen site thereof. Alternatively, the fragment of the free κ immunoglobulin chain preferably consists of the constant region (κ immunoglobulin chain C region) or an antigen site thereof.
A free κ immunoglobulin chain comprising the amino acid sequence of SEQ ID NO: 4 (rat-derived) or SEQ ID NO: 5 (rat-derived) can be used to test, diagnose, and/or determine whether psychiatric/neurological disorder model animals (particularly, rats) have developed or can potentially develop the psychiatric/neurological disorder. It is useful, particularly for the preparation, quality control, or the like of psychiatric/neurological disorder model animals. The test method can be similarly performed by the method shown in the section “Test Method for Psychiatric/Neurological Disorder” below. Thus, in another embodiment, the free κ immunoglobulin chain comprises the amino acid sequence of SEQ ID NO: 4 (C region). Preferably, the free κ immunoglobulin chain comprises the amino acid sequence of SEQ ID NO: 5. The constant region (κ immunoglobulin chain C region) preferably comprises the amino acid sequence of SEQ ID NO: 4. Alternatively, the constant region (κ immunoglobulin chain C region) preferably consists of the amino acid sequence of SEQ ID NO: 4. The antigen site of the constant region (κ immunoglobulin chain C region) preferably comprises the amino acid sequence of SEQ ID NO: 5. Alternatively, the antigen site of the constant region (κ immunoglobulin chain C region) preferably consists of the amino acid sequence of SEQ ID NO: 5. The fragment of the free κ immunoglobulin chain preferably comprises the constant region (κ immunoglobulin chain C region) or an antigen site thereof. Alternatively, the fragment of the free κ immunoglobulin chain preferably consists of the constant region (κ immunoglobulin chain C region) or an antigen site thereof.
The amino acid sequence of a free λ immunoglobulin chain is also well-known to those of ordinary skill in the art. The free λ immunoglobulin chain comprises a constant region (λ immunoglobulin chain C region). For example, the free λ immunoglobulin chain comprises the amino acid sequence of SEQ ID NO: 6. Preferably, the free λ immunoglobulin chain comprises the antigen site of the constant region (λ immunoglobulin chain C region). Those of ordinary skill in the art can readily detect and quantify the free λ immunoglobulin chain using a substance capable of recognizing the constant region (λ immunoglobulin chain C region) or an antigen site thereof. Examples of the substance include Anti-Ig λ light chain (human) pAb-FITC (MBL Co., Ltd.) and FREELITE λ chain (MBL Co., Ltd., code number: BS-LK018BD).
The constant region (λ immunoglobulin chain C region) preferably comprises the amino acid sequence of SEQ ID NO: 6. Alternatively, the constant region (λ immunoglobulin chain C region) preferably consists of the amino acid sequence of SEQ ID NO: 6. The fragment of the free λ immunoglobulin chain preferably comprises the λ immunoglobulin chain C region or an antigen site thereof. Alternatively, the fragment of the free λ immunoglobulin chain preferably consists of the λ immunoglobulin chain C region or an antigen site thereof.
The relation between SEQ ID NO and the κ immunoglobulin chain C region or the λ immunoglobulin chain C region is as follows.
SEQ ID NO: 1—κ immunoglobulin chain C region (human-derived)
SEQ ID NO: 2—antigen site (human-derived)
SEQ ID NO: 3—antigen site (human-derived)
SEQ ID NO: 4—κ immunoglobulin chain C region (rat-derived)
SEQ ID NO: 5—antigen site (rat-derived)
SEQ ID NO: 6—λ immunoglobulin chain C region (human-derived)
The free κ immunoglobulin chain is a polypeptide consisting of a constant region (λ immunoglobulin chain C region) of about 12 kDa and a variable region of about 12 kDa (see
In another embodiment, the biomarker for a psychiatric/neurological disorder according to the present invention comprises a polypeptide containing the antigen site of a free κ immunoglobulin chain, or a fragment thereof. Alternatively, the biomarker for a psychiatric/neurological disorder according to the present invention consists of a polypeptide containing the antigen site of a free κ immunoglobulin chain, or a fragment thereof. In still another embodiment, the biomarker for a psychiatric/neurological disorder according to the present invention comprises a polypeptide containing a free κ immunoglobulin chain C region, or a fragment thereof. Alternatively, the biomarker for a psychiatric/neurological disorder according to the present invention consists of a polypeptide containing a free κ immunoglobulin chain C region, or a fragment thereof. In another embodiment, the biomarker for a psychiatric/neurological disorder according to the present invention comprises a polypeptide containing the antigen site of a free λ immunoglobulin chain, or a fragment thereof. Alternatively, the biomarker for a psychiatric/neurological disorder according to the present invention consists of a polypeptide containing the antigen site of a free λ immunoglobulin chain, or a fragment thereof. In still another embodiment, the biomarker for a psychiatric/neurological disorder according to the present invention comprises a polypeptide containing a free λ immunoglobulin chain C region, or a fragment thereof. Alternatively, the biomarker for a psychiatric/neurological disorder according to the present invention consists of a polypeptide containing a free λ immunoglobulin chain C region, or a fragment thereof.
Those of ordinary skill in the art will understand that deletion, addition, variation, or substitution in the amino acid sequence of each of the polypeptides having the above amino acid sequences can be permitted provided that the uniqueness of the polypeptide, i.e., a specific amino acid sequence characterizing the polypeptide (e.g., a motif, a domain, a box sequence, or the like), or the like, is retained. The number of such deletions, additions, variations, or substitutions is not limited (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) provided that the uniqueness of the biomarker for a psychiatric/neurological disorder according to the present invention is retained. In general, variation or substitution between amino acids having the same properties has no or little effect on the function of the polypeptide; thus, a polypeptide containing the variation or substitution can be equated with the original polypeptide (for example, a polypeptide specified by any of the above SEQ ID NOS). The properties of such amino acids can be categorized, for example, as follows: acidic amino acids (aspartic acid and glutamic acid), basic amino acids (lysine, histidine, and arginine), hydrophobic (aliphatic) amino acids (glycine, alanine, valine, leucine, and isoleucine), sulfur-containing amino acids (methionine and cysteine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), acidic amino acid amides (asparagine and glutamine), or neutral amino acids (glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, proline, serine, threonine, cysteine, methionine, asparagine, and glutamine).
For example, an amino acid sequence in which Glu at the 105th position and Cys at the 106th position are deleted in the amino acid sequence of SEQ ID NO: 1 is also included within the scope of the present invention.
Those of ordinary skill in the art will understand that the biomarker for a psychiatric/neurological disorder according to the present invention can also include one modified, for example, with phosphorylation, sugar chain addition, amidation, ubiquitination, or acetylation.
The biomarker for a psychiatric/neurological disorder according to the present invention can be present in a blood, urine, or spinal fluid sample from a subject. The sample is preferably a blood sample (serum, plasma, or whole blood). The blood sample is more preferably serum. The collection of a blood sample from a subject can be performed by a common method well-known to those of ordinary skill in the art. The blood sample can be collected, for example, using a syringe.
All of a free κ immunoglobulin chain, a free λ immunoglobulin chain, and fragments thereof in the blood sample of the subject increase significantly. Thus, their change can be measured to determine the subject as having developed or capable of potentially developing the psychiatric/neurological disorder.
The free κ immunoglobulin chain may also increase, for example, in patients with multiple myeloma. However, it is known that an inflammatory cytokine increases simultaneously in this case (Non-patent Literatures 1 to 3). On the other hand, the present inventors have found that in a subject with psychiatric/neurological disorder, the free κ immunoglobulin chain increases while an increase in an inflammatory cytokine is not observed. Thus, a psychiatric/neurological disorder can be more clearly determined by the combination of the free κ immunoglobulin chain and the inflammatory cytokine, i.e., by using the amount of the free κ immunoglobulin chain or a fragment thereof and the amount of the inflammatory cytokine as indications. The amount of the free λ immunoglobulin chain or a fragment thereof and the amount of the inflammatory cytokine may also be used as indications. Most preferably, the three of the free κ immunoglobulin chain or a fragment thereof, the free λ immunoglobulin chain or a fragment thereof, and the inflammatory cytokine are used as indications.
The biomarker for a psychiatric/neurological disorder according to the present invention is preferably a biomarker for schizophrenia, autism, disorders of cranial nerve development, cognitive impairment ascribed to neuropathy due to infection during pregnancy, or mental disorder ascribed to impaired immunity. Most preferably it is a biomarker for schizophrenia.
The details of the determination of the development or potential development of a psychiatric/neurological disorder will be described in “Test Method for Psychiatric/Neurological Disorder” below.
The present invention provides a test kit for a psychiatric/neurological disorder. The test kit can be used to measure the amount of the biomarker for a psychiatric/neurological disorder according to the present invention in a blood sample from a subject to determine whether the subject has developed or is at risk of developing the psychiatric/neurological disorder.
The test kit of the present invention contains a substance capable of detecting the biomarker for a psychiatric/neurological disorder according to the present invention. When the biomarker for a psychiatric/neurological disorder comprises a plurality of biomarkers, the kit can contain a plurality of substances capable of detecting the plurality of biomarkers respectively. The substance is not particularly limited; however, it can be any molecule having affinity to the biomarker for a psychiatric/neurological disorder according to the present invention. The substance can be a substance capable of detecting a free cK immunoglobulin chain C region or an antigen site thereof. The substance can be a substance capable of detecting a free λ immunoglobulin chain C region or an antigen site thereof. The substance includes, for example, an antibody, a low molecular compound, a high molecular compound, a protein, a peptide, and a nucleic acid. The substance is preferably labeled with any marker, such as a dye or fluorescent dye, a radioactive isotope, and an enzyme so that the biomarker for a psychiatric/neurological disorder according to the present invention can be detected. The substance is more preferably an antibody. Preferably, the antibody is an anti-Ig κ antibody, an anti-Ig κ-C antibody, an anti-Ig λ antibody, or an anti-Ig λ-C antibody. The antibody may be a monoclonal antibody or a polyclonal antibody.
Examples of the substance capable of detecting the biomarker for a psychiatric/neurological disorder according to the present invention include Anti-Ig κ light chain antibody (MAR-C8) (Abcam Co., Ltd.), FREELITE κ chain (MBL Co., Ltd., code number. BS-LK016BD), Anti-Ig λ light chain (human) pAb-FITC (MBL Co., Ltd.), and FREELITE λ chain (MBL Co., Ltd., code number: BS-LK018BD).
The antibody may be prepared by a method well-known to those of ordinary skill in the art. For example, the biomarker for a psychiatric/neurological disorder according to the present invention can be administered to a mammal, such as a rabbit, for immunization to prepare an antibody from its serum. The anti-Ig κ antibody can be prepared by administering the polypeptide of the κ immunoglobulin chain C region or an antigen site thereof to a mammal, such as a rabbit. For example, the anti-Ig κ antibody is prepared by administering a polypeptide comprising the amino acid sequence of any one of SEQ ID NOS: 1 to 5 or a polypeptide consisting of the amino acid sequence of any one of SEQ ID NOS: 1 to 5. The anti-Ig λ antibody can be prepared by administering the polypeptide of the λ immunoglobulin chain C region or an antigen site thereof to a mammal, such as a rabbit. For example, the anti-Ig λ antibody is prepared by administering a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6. The antibody may be labeled with any marker, such as a dye (e.g., a fluorescent dye), a radioactive isotope, and an enzyme.
The test kit for a psychiatric/neurological disorder according to the present invention may further contain a labeled secondary antibody. The labeling can be performed using any marker, such as a dye (e.g., a fluorescent dye), a radioactive isotope, and an enzyme.
In a particular embodiment, the substance capable of detecting the biomarker for a psychiatric/neurological disorder according to the present invention, for example, an antibody, is immobilized on a solid-phase support. The solid-phase support may be made of any appropriate material, such as a plastic (e.g., polystyrene resin, polycarbonate resin, polyethylene resin, or polypropylene resin) or glass. The solid-phase support may be a substrate of any shape, such as a dish, a well, a strip, or a chip. The substrate surface may be optionally coated and modified.
The test kit for a psychiatric/neurological disorder according to the present invention may contain instructions showing a method for measuring the amount of the biomarker for a psychiatric/neurological disorder according to the present invention.
In addition, the test kit for a psychiatric/neurological disorder according to the present invention may contain, for example, reagents (for example, a buffer solution, a blocking reagent, an enzyme substrate, and a fluorescent reagent) and/or devices or instruments (for example, a container, a reaction device, a fluorescence measuring instrument) used in the test method for a psychiatric/neurological disorder.
The test kit for a psychiatric/neurological disorder according to the present invention is preferably a test kit for schizophrenia, autism, disorders of cranial nerve development, cognitive impairment ascribed to neuropathy due to infection during pregnancy, or mental disorder ascribed to impaired immunity. Most preferably, it is a test kit for schizophrenia.
The test kit for a psychiatric/neurological disorder according to the present invention can be used for determining the development or potential development of a psychiatric/neurological disorder and prognosis after developing the disorder. The details of the determination will be described in the item of “Test Method for Psychiatric/Neurological Disorder” below. The test kit for a psychiatric/neurological disorder according to the present invention can be used for diagnosing a psychiatric/neurological disorder. The test kit for a psychiatric/neurological disorder according to the present invention can be used in a test method for a psychiatric/neurological disorder shown below.
The present invention provides a test method for a psychiatric/neurological disorder. The test method uses the amount of the biomarker for a psychiatric/neurological disorder according to the present invention in a blood sample from a subject as an indication. The test method of the present invention is useful for determining the development or potential development of a psychiatric/neurological disorder and prognosis after developing the disorder. The test method of the present invention can also be used for diagnosing a psychiatric/neurological disorder. The test method of the present invention can also provide data necessary for the treatment, prevention, diagnosis, differentiation, or prognosis estimation of a psychiatric/neurological disorder. The test method of the present invention can also provide information useful for determining the development or potential development of a psychiatric/neurological disorder and prognosis after developing the disorder. In addition, a treatment method and a treatment regimen for a subject can be decided from the results obtained using the test method of the present invention. Further, a primary screening of a psychiatric/neurological disorder in a medical checkup and the like can be performed using the test method of the present invention. A screening of a psychiatric/neurological disorder can also be carried out in the first contact of patients in psychiatry.
The test method for a psychiatric/neurological disorder according to the present invention is preferably a test method for schizophrenia, autism, disorders of cranial nerve development, cognitive impairment ascribed to neuropathy due to infection during pregnancy, or mental disorder ascribed to impaired immunity. Most preferably, it is a test kit for schizophrenia.
The test method of the present invention can comprise a step of measuring the amount of the biomarker for a psychiatric/neurological disorder according to the present invention in a subject and a step of comparing the measured amount of the biomarker for a psychiatric/neurological disorder according to the present invention with that in a normal subject. The test method of the present invention can comprise the steps of (a) measuring the amount of at least one selected from the group consisting of a free κ immunoglobulin chain, a free λ immunoglobulin chain, and fragments thereof in a blood sample from a subject and (b) comparing the amount with that from a normal subject. Preferably, the test method of the present invention can comprise the steps of (a) measuring the amount of a free κ immunoglobulin chain or a fragment thereof and a free λ immunoglobulin chain or a fragment thereof in a blood sample from a subject and (b) comparing the amount with that from a normal subject. The comparing step comprises a step of comparison with the median value in the normal control group. The amount is preferably an amount by combining, i.e., the total amounts of a free c immunoglobulin chain or a fragment thereof and a free λ immunoglobulin chain or a fragment thereof in a blood sample from a subject.
In another embodiment, the test method of the present invention can comprise the steps of (a) measuring the amount of at least one selected from the group consisting of a free κ immunoglobulin chain, a free λ immunoglobulin chain, and fragments thereof and the amount of an inflammatory cytokine in a blood sample from a subject and (b) comparing the amount with that from a normal subject. More preferably, the test method of the present invention comprises the steps of (a) measuring the amount of a free κ immunoglobulin chain or a fragment thereof and a free λ immunoglobulin chain or a fragment thereof and the amount of an inflammatory cytokine in a blood sample from a subject and (b) comparing these amounts with those from a normal subject. The comparing step comprises a step of comparison with the median value in the normal control group. The amount is preferably an amount by combining, i.e., the total amounts of a free κ immunoglobulin chain or a fragment thereof and a free λ immunoglobulin chain or a fragment thereof in a blood sample from a subject.
The details of a psychiatric/neurological disorder, a free κ immunoglobulin chain, a free λ immunoglobulin chain, and fragments thereof, and an inflammatory cytokine have been described in the section “Biomarker for Psychiatric/Neurological Disorder According to Present Invention” and will not be again described.
In the step (a) of the test method of the present invention, the measurement includes detection and quantification. The level of the quantification can be a level that enables the determination of the development or potential development of a psychiatric/neurological disorder or the determination of prognosis after developing the disorders. The quantification may be semi-quantification. The measurement of a free κ immunoglobulin chain may be the detection or quantification of a free κ immunoglobulin chain C region and/or an antigen site thereof. The measurement of a free λ immunoglobulin chain may be the detection of a free λ immunoglobulin chain C region and/or an antigen site thereof. The measurement may be the measurement of the amount of mRNA encoding the biomarker for a psychiatric/neurological disorder according to the present invention.
In the step (a) of the test method of the present invention, the measurement of the amount of the biomarker for a psychiatric/neurological disorder according to the present invention can be carried out by a common method well-known to those of ordinary skill in the art. Examples thereof include a one- or two-dimensional electrophoresis method, a Western blotting method, an enzyme linked immunosorbent assay (ELISA), a sandwich ELISA method, an immunoelectrophoresis method, single radial immunodiffusion (SRID), a radioimmunoassay (RIA), an enzyme immunoassay (EIA), a latex immunoassay (LIA), and a fluorescence immunoassay (FIA). The amount of the biomarker for a psychiatric/neurological disorder according to the present invention can also be measured using the detection kit for a psychiatric/neurological disorder according to the present invention. The amount of the biomarker for a psychiatric/neurological disorder according to the present invention can also be measured using, for example, a device (e.g., a radiation counter, an optical measurement device, or a fluorescence intensity measuring device) and an image analysis software which are well-known to those of ordinary skill in the art. The amount of the biomarker for a psychiatric/neurological disorder according to the present invention may be measured once or a plurality of times.
In the step (a) of the test method of the present invention, the amount of the biomarker for a psychiatric/neurological disorder according to the present invention may be a concentration (e.g., mg/l, μg/l, or pg/ml), a radiation intensity, or a fluorescence intensity. The unit of the amount of the biomarker for a psychiatric/neurological disorder according to the present invention will depend on the measurement method. In a certain embodiment, the amount of the biomarker for a psychiatric/neurological disorder according to the present invention may be a relative ratio to that in a normal subject.
The blood sample may be serum, plasma, or whole blood. The blood sample is preferably serum. The method for collecting the blood sample may be a common method, such as collection using a syringe or the like from a subject, and is not particularly limited. The blood sample collected from a subject may be denatured and/or stored by a method well-known to those of ordinary skill in the art.
Before measuring the blood sample, the blood sample collected from a subject can be prepared using a reagent and a means which are well-known to those of ordinary skill in the art. Examples of the reagent include buffers, such as acetic acid-acetate, phosphoric acid-phosphate, citric acid-citrate, or tris(hydroxymethyl)aminomethane (tris); denaturating agents, such as urea and/or thiourea; reducing agents, such as dithiothreitol (DTT); and surfactants, such as sodium lauryl sulfate (SDS). Examples of the means include filtration, ultrafiltration, centrifugation, homogenization, incubation, shaking, and lyophilization.
In the test method of the present invention, when the free κ immunoglobulin chain, the free λ immunoglobulin chain, and fragments thereof in a blood sample from a subject are measured, denaturation (cleavage of S—S bonds) by DTT may not be conducted. Urea and/or thiourea may also not be used. For example, the blood sample may be denatured only with SDS. The reason therefor is to eliminate the κ immunoglobulin chain and the λ immunoglobulin chain expressed in IgG, IgA, IgM and the like and efficiently measure only the free κ immunoglobulin chain, the free λ immunoglobulin chain, and fragments thereof.
In the step (a) of the test method of the present invention, the subject is not particularly limited. The subject is preferably a human. The subject is, for example, a subject for whom it is necessary to be determined whether or not the subject has developed a psychiatric/neurological disorder. The subject may be a subject determined as having developed a psychiatric/neurological disorder using conventional diagnosis criteria, such as DMSIV-TR, ICD-10, and PANSS. The subject may be a subject not determined as having developed a psychiatric/neurological disorder using conventional diagnosis criteria, such as DMSIV-TR, ICD-10, and PANSS. In addition, the subject may be a subject for whom no information about a psychiatric/neurological disorder is obtained. It may be confirmed in advance whether the subject has a cancer and/or an inflammatory disorder.
In preparing a blood sample, the blood sample is preferably incubated at about 60° C., about 70° C., about 80° C., about 90° C., about 100° C., about 110° C., or about 120° C. for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, or about 10 minutes. Most preferably, it may be incubated at about 100° C. for about 5 minutes.
In the step (b), the normal subject is a subject not having developed or potentially not developing a psychiatric/neurological disorder. For example, the subject is a subject determined as not having developed a psychiatric/neurological disorder using conventional diagnosis criteria, such as DMSIV-TR, ICD-10, and PANSS. The method for the comparison in the step (b) may be a method well-known to those of ordinary skill in the art and is not particularly limited. For example, the measured value obtained in the step (a) can be compared with the measured value of the biomarker for a psychiatric/neurological disorder according to the present invention in a normal subject obtained in advance. Preferably, the results obtained by a two-dimensional electrophoresis method or an enzyme linked immunosorbent assay (ELISA) are compared. When the biomarker for a psychiatric/neurological disorder according to the present invention comprises a plurality of biomarkers, the values measured therefor may be added together for comparison. The measurement of the biomarker for a psychiatric/neurological disorder according to the present invention in a normal subject may be the same as the measurement method described in the step (a).
In most normal subjects, an inflammatory cytokine is below the limit of detection. Thus, the comparison of the amount of the inflammatory cytokine with that from a normal subject may be simply whether or not the inflammatory cytokine is detected. No detection of the inflammatory cytokine can be determined as indicating no change in the cytokine amount as compared with that from a normal subject.
Using the test method of the present invention, whether or not a subject has developed or can potentially develop a psychiatric/neurological disorder can be determined as follows. When the amount of a free κ immunoglobulin chain or a fragment thereof in a blood sample from a subject increases compared to that from a normal subject or is larger than the median value of that from a normal control group, the subject can be determined as having developed or potentially developing a psychiatric/neurological disorder. Alternatively, when the amount of a free λ immunoglobulin chain or a fragment thereof in a blood sample from a subject increases compared to that from a normal subject, the subject can be determined as having developed or potentially developing a psychiatric/neurological disorder. Alternatively, when the amount of a free κ immunoglobulin chain or a fragment thereof and a free λ immunoglobulin chain or a fragment thereof in a blood sample from a subject increases compared to that from a normal subject or is larger than the median value of that from a normal control group, the subject can be determined as having developed or potentially developing a psychiatric/neurological disorder.
Using the test method of the present invention, whether or not a subject has developed or can potentially develop a psychiatric/neurological disorder can also be determined as follows. When the amount of a free κ immunoglobulin chain or a fragment thereof in a blood sample from a subject increases compared to that from a normal subject or is larger than the median value of that from a normal control group, and the amount of an inflammatory cytokine does not change compared to that from a normal subject or is not detected, the subject can be determined as having developed or potentially developing a psychiatric/neurological disorder. Alternatively, when the amount of a free λ immunoglobulin chain or a fragment thereof in a blood sample from a subject increases compared to that from a normal subject or is larger than the median value of that from a normal control group, and the amount of an inflammatory cytokine does not change or is not detected compared to that from a normal subject, the subject can be determined as having developed or potentially developing a psychiatric/neurological disorder. Alternatively, when the amount of a free κ immunoglobulin chain or a fragment thereof and a free λ immunoglobulin chain or a fragment thereof in a blood sample from a subject increases compared to that from a normal subject and the amount of an inflammatory cytokine does not change or is not detected compared to that from a normal subject, the subject can be determined as having developed or potentially developing a psychiatric/neurological disorder.
“Increase” can be increasing about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 50-fold, about 100-fold, or more. “Not change” refers to that the amount of the biomarker for a psychiatric/neurological disorder according to the present invention is the same or is not significantly different between the amount from a subject in the step (a) and that from a normal subject in the step (b). “Not significantly different” refers to, for example, that the p value is less than 0.05, or the difference is about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 1%0 about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, or about 30%.
“Larger than the median value” means, for example, larger than the median value in the normal control group. The median value in the normal control group may be a known median value as described, for example, in Non-patent Literature 1, or a value calculated from the actual measurement in the normal control group.
Examples of the present invention will be described below. The following Examples are described for a better understanding of the scope of claims of the present invention, and not intended to limit the scope of claims of the present invention.
Poly IC (polyriboinosinic-polyribocytidilic acid) (Sigma Co., Ltd.) dissolved in saline was intraperitoneally administered in an amount of 4 mg/kg to a Wistar rat at day 15 of gestation before normal delivery to prepare maternal immune activation model rats (MIA rats) which were schizophrenic and autistic model animals. The MIA rat is one of psychiatric/neurological disorder model animals (see Limin Shi et al, J. Neurosci. 2003 Jan. 1; 23(1): 297-302 and Arata Oh-Nishi et al, Brain Research, Volume 1363, 6 Dec. 2010, Pages 170-179). Only an equal volume of saline was intraperitoneally administered to another Wistar rat at day 15 of gestation before normal delivery to prepare control rats (hereinafter, referred to as “control” rats) as a control group. The MIA rats had significantly attenuated PPI compared to the control rats and were confirmed to be capable of being used as psychiatric/neurological disorder model animals.
The sera of the MIA rats (12-week old) and the control rats (12-week old) were collected and denatured with urea/thiourea, DTT, and SDS, followed by separating protein by a 2-dimensional electrophoresis method. Electrophoresis gel images are shown in
The free κ immunoglobulin chain consists of a constant region (κ immunoglobulin chain C region) of about 12 kDa and a variable region of about 12 kDa. The variable region frequently changes in the amino acid sequence and thus is not detected by a mass spectrometer (MS). The spot in which the significant difference was seen was observed around about 25 kDa and thus it may be actually a free κ immunoglobulin chain.
A rat-derived κ immunoglobulin chain C region (SEQ ID NO: 4) was used as an antigen and administered to a rabbit for immunization to prepare an antibody (anti-Ig κ-C antibody). Using the same method as that in Example 1, MIA rats and control rats were prepared. The sera of the MIA rats (12-week old, N=7) and the control rats (12-week old, N=7) were denatured only with SDS (100° C., 5 minutes) and subjected to electrophoresis with SDS-polyacrylamide gel to separate protein. Thereafter, Western blotting was carried out (gel: 10 to 20%, primary antibody (1:1,000), secondary antibody (1:5,000)) (
The intensity of a band of about 25 kDa significantly increased in the MIA rats (“Poly IC” in
The amounts of inflammatory cytokines in the serum were measured in the MIA rats (“Poly IC” in the table, 12-week old, N=7) and the control rats (“Saline” in the table, 12-week old, N=7) of Example 2. The measurement of the amount of inflammatory cytokines used Quantikine ELASA Rat IL-1β, IL-6, and TNF-α (from R&D Co., Ltd.).
No increase of the inflammatory cytokines in sera was observed in the MIA rats (“Poly IC” in the table) and the control rats (“Saline” in the table).
The usefulness of the biomarker for a psychiatric/neurological disorder according to the present invention was tested in a group of schizophrenia patients (SZ) and a group of healthy individuals (Normal).
Serum samples from the SZ and Normal groups, collected according to an IRB-approved protocol, which was given sufficient ethical consideration, were purchased from PrecisionMed Inc. (USA) (acting import agent: Wako Pure Chemical Industries, Ltd.).
The amounts of the free κ immunoglobulin chain and the free λ immunoglobulin chain in the sera were measured using FREELITE κ Chain (MBL Co., Ltd., code number. BS-LK016BD) and FREELITE λ chain (MBL Co., Ltd., code number: BS-LK018BD) according to the instructions of the provider. The inflammatory cytokines were measured using AlphaLASA Human IL-1 beta, IL-6, and TNF-alpha Kits (from PerkinElmer Inc., code numbers: AL223 C/F, AL220 C/F, and AL208 C/F).
Basic data and the data summarizing average values in the group of schizophrenia patients (SZ) and the normal group are shown in Tables 2 and 3 below (psychopathological score: PANSS score).
There was no statistically significant difference in age, and the inflammatory cytokines (IL-6, IL-1 beta, and TNF-alpha) were not detected in both of the SZ and normal groups.
In addition, the concentration of free Ig κ in the sera from the 11 patients of the SZ group had higher values (minimum: 9.11 mg/I, maximum: 23.8 mg/l) compared with the median value (7.5 mg/l) of the concentration of free Igh in the sera from healthy individuals aged 40 to 49 years described in Non-patent Literature 1. On the other hand, the median value of the concentration of free Ig λ in the sera from the healthy individuals aged 40 to 49 years is 12.8 mg/l, and 8 of 11 patients in the SZ group had a higher value than the median value (minimum: 9.05 mg/l, maximum: 20.8 mg/l).
The biomarker of the present invention in a blood sample can be measured to perform the primary screening of a psychiatric/neurological disorder in a medical checkup and the like. The screening of a psychiatric/neurological disorder can also be carried out in the first contact of patients in psychiatry.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2013104124 | May 2013 | JP | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14891301 | Nov 2015 | US |
| Child | 15645907 | US |
