WO2011142461A1

WO2011142461A1 - Antibody for diagnosing amyotrophic lateral sclerosis (als)

Info

Publication number: WO2011142461A1
Application number: PCT/JP2011/061069
Authority: WO
Inventors: 秀憲一條; 英起西頭
Original assignee: 国立大学法人東京大学
Priority date: 2010-05-13
Filing date: 2011-05-13
Publication date: 2011-11-17
Also published as: JP5828521B2; JPWO2011142461A1

Abstract

The issues addressed by this invention are the determination of part of the molecular mechanism behind the pathogenesis of amyotrophic lateral sclerosis (ALS) and the use of said molecular mechanism to investigate ALS risks. Said issues are addressed by the provision of a method for detecting ALS risks by detecting mutant SOD1 protein. Namely, said issues are addressed by the provision of an antibody for detecting mutant SOD1 protein, where said antibody binds to mutant SOD1 protein but not to wild-type (i.e., normal) SOD1 protein.

Description

Antibodies for the diagnosis of amyotrophic lateral sclerosis (ALS)

The present invention relates to an antibody that can be used for diagnosis of amyotrophic lateral sclerosis (ALS). The present invention also relates to a method for detecting the risk of developing amyotrophic lateral sclerosis (ALS) using such an antibody.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease in which motor nerves are selectively impaired, with approximately 7,000 patients in Japan and 40,000 patients in the United States. There is. This disease mainly develops in the middle period, muscle atrophy gradually spreads throughout the body, difficult to walk, speech disorder, dysphagia, respiratory disorder, and after the onset 3 It is a very serious disease that will die in 5 years. Since the disease progresses while the patient is conscious, the physical and mental damage to the patient and the caregiver is extremely large.

¡Although ALS has been designated as a “target disease for overcoming intractable diseases,” efforts to develop therapeutic agents have been delayed. The current treatment is only the prevention of muscle weakness through moderate rehabilitation and the temporary delay in the progression of disease due to riluzole (suppressing the release of glutamate to nerve endings), which is the only insurance indication, and is based on the molecular mechanism of ALS pathology. There is no effective treatment.

Breakthrough in elucidating the cause of ALS is a mutation in Cu / Zn superoxide dismutase 1 (SOD1) identified by Rosen et al. In 1993 as a causative gene for familial ALS. About 10% of all ALS patients are familial, of which about 20% (ie, about 2% of all ALS patients) are due to SOD1 gene mutations. In ALS, it is known that mutant SOD1 protein accumulates abnormally.

In the case of familial Alzheimer's disease, the discovery of APP and PS-1 gene mutations as the cause of this greatly contributed to the elucidation of the molecular mechanism of sporadic Alzheimer's disease. The discovery of the causative gene SOD1 was expected to bring about dramatic progress not only in familial ALS but also in elucidating the pathology of sporadic ALS. However, the molecular mechanism that can explain the pathological condition common to ALS has not yet been clarified.

So far, 122 types of point mutations have been reported on the SOD1 gene, and several transgenic mice created for any of these mutations have symptoms similar to ALS, and the function of the SOD1 protein. It has been shown that, depending on the loss, the condition does not develop. Based on these findings, the cause of ALS due to mutations in the SOD1 protein is not due to a decrease in SOD activity (loss of function) based on gene mutations. It is believed to exert neurotoxicity and cause motor neuron cell death and associated neurological symptoms.

To date, more than 100 mutations in the SOD1 gene in familial ALS have been reported, but the mechanism of ALS development that occurs is that the mutant SOD1 protein is expressed in the cell, and the mutant SOD1 protein is expressed in the cell. As a result of accumulation, it is considered that some cytotoxicity is exerted, and the motor nerve is degenerated.

However, at that time, the pathogenesis of ALS, such as what signal the mutant SOD1 protein emits in the cell and what mechanism damages the cell, is still unknown. Did not exist.

We have previously clarified that endoplasmic reticulum stress is deeply involved as a molecular mechanism of neuronal cell death by mutant SOD1 protein in ALS (Japanese Patent Laid-Open No. 2007-151478; Nishitohshitet al. Genes Dev. 2008). That is, as one of the target proteins to which the mutant SOD1 protein specifically binds, the endoplasmic reticulum membrane protein Derlin-1, which plays an important role in the quality control of the endoplasmic reticulum, was identified, and the mutant SOD1 protein and Derlin-1 It was found that the binding causes the breakdown of the endoplasmic reticulum quality control mechanism of nerve cells and induces endoplasmic reticulum stress.

However, as a result of the expression of mutant SOD1 protein in neurons of ALS patients, a series of reasons why it causes binding to Derlin-1 and what molecular mechanism actually damages and denatures cells This mechanism is our original discovery, and research has not yet progressed globally, and the whole picture has not yet been clarified.

JP2007-151478

An object of the present invention is to elucidate a part of the molecular mechanism of the onset of amyotrophic lateral sclerosis (ALS) and to detect the risk of ALS using the elucidated molecular mechanism of the onset of ALS. To do.

The present invention has solved the above problems by providing a method for detecting the risk of ALS by detecting mutant SOD1 protein. That is, in order to detect mutant SOD1 protein, the present invention solves the above problem by providing an antibody that does not bind to wild-type (ie, normal) SOD1 protein but binds to mutant SOD1 protein. did.

The present invention relates to SEQ ID NO: 4 amino acid sequence, SEQ ID NO: 4 amino acid sequence, SEQ 1ID NO: 5, compared to a protein consisting of the amino acid sequence of SEQ ID NO: 2. A three-dimensional structure of a polypeptide region comprising an amino acid sequence, an amino acid sequence in which one amino acid is mutated in SEQ ID NO: 5, an amino acid sequence in SEQ ID NO: 6, or an amino acid sequence in which one amino acid is mutated in SEQ ID NO: 6 Changes have been completed based on the fact that the changes have been shown to be associated with the risk of amyotrophic lateral sclerosis (ALS). Therefore, specifically, in one embodiment of the present invention, based on the above fact, a cell-containing sample collected from a subject is screened with an antibody that binds to a mutant SOD1 protein, and the By detecting the protein that binds to the antibody, except for the protein consisting of the amino acid sequence of SEQ ID NO: アミノ酸 2, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence in which one amino acid is mutated in SEQ ID NO: 4, SEQ ID 含む NO: 5 amino acid sequence, SEQ ID NO: アミノ酸 5 amino acid sequence mutated, SEQ ID NO: アミノ酸 6 amino acid sequence, or SEQ ID NO: ポリ 6 amino acid sequence mutated A method for detecting the risk of ALS is provided by detecting peptides.

In the above embodiment, the mutant SOD1 protein is detected by detecting only the mutant SOD1 protein using an antibody that does not bind to the wild-type (ie, normal) SOD1 protein but binds to the mutant SOD1 protein. Can be realized. Examples of such antibodies include the amino acid sequence of Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly (SEQ ID NO: 4), the amino acid sequence in which one amino acid is mutated in SEQ ID NO: 4, Lys Val Trp Gly Ser The amino acid sequence of Ile Lys Gly Leu Thr Glu (SEQ ID NO: 5), the amino acid sequence in which one amino acid is mutated in SEQ ID NO: 5, the amino acid of Arg Leu Ala Cys Gly Val Ile Gly Ile (SEQ ID NO: 6) Mention may be made of antibodies that bind to a sequence or a polypeptide comprising an amino acid sequence in which one amino acid has been mutated in SEQ ID NO: 6. This detection method did not detect the wild-type SOD1 protein consisting of the amino acid sequence of SEQ ID NO: 2, but one amino acid was mutated in SEQ ID NO: 4 in the mutant SOD1 protein, SEQ 、 ID NO: 4. Amino acid sequence, SEQ ID NO: アミノ酸 5 amino acid sequence, SEQ ID NO: アミノ酸 5 amino acid sequence mutated, SEQ ID NO: 6 amino acid sequence, or SEQ ID NO: 6 amino acid sequence If a conformational change of the polypeptide region containing the sequence is detected, it is determined that the individual who is the source of the sample is at risk of developing ALS.

In another embodiment of the present invention, an amino acid sequence of Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly (SEQ ID NO: 4), an amino acid sequence in which one amino acid is mutated in SEQ ID NO: 4, Lys Val Trp Gly Ser Ile Lys Gly Leu Thr Glu (SEQ ID NO: 5) amino acid sequence, SEQ ID NO: 5 amino acid sequence mutated, Arg アミノ酸 Leu Ala Cys Gly Val Ile Gly Ile (SEQ ID NO: 6) Provided are antibodies themselves that bind to an amino acid sequence or a polypeptide comprising an amino acid sequence in which one amino acid is mutated in SEQ ID NO: 6.

In these embodiments, the substance to which the antibody binds is the amino acid sequence of SEQ ID NO: 4, the amino acid sequence in which one amino acid is mutated in SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, SEQ ID ID NO: Mutant SOD1 protein with a conformational change in a polypeptide region comprising an amino acid sequence in which one amino acid is mutated in SEQ ID NO: 6, or an amino acid sequence in which one amino acid is mutated in SEQ ID NO: 6 That is, the antibody of the present invention can detect a mutant SOD1 protein. Specifically, the antibody of the present invention has

amino acid numbers

3, 4, 5, 6, 8, 10, 12, 14, 16, 19, 20, 22, 29, 37 in the amino acid sequence of SEQ ID NO: 2. , 38,40,41,43,45,46,47,48,49,57,59,65,66,72,76,80,84,85,86,87,89,90,93,95,97 99, 100, 101, 104, 105, 106, 108, 111, 112, 113, 114, 115, 116, 117, 118, 124, 125, 126, 127, 132, 133, 134, 139, 140, 141 , 144, 145, 146, 147, 148, 149, and 151, any one amino acid residue selected from the group consisting of 1 amino acid substitution, deletion or addition can be detected. In addition, for SOD1 gene mutations that have not been discovered yet, the mutant SOD1 protein produced by the mutation is SEQ ID NO: アミノ酸 4 amino acid sequence, SEQ ID NO: アミノ酸 4 amino acid sequence, ID 含む NO: 5 amino acid sequence, SEQ ID NO: アミノ酸 5 amino acid sequence mutated, SEQ ID NO: アミノ酸 6 amino acid sequence, or SEQ ID NO: ポリ 6 amino acid sequence mutated It is possible to detect whether or not it is accompanied by a change in the three-dimensional structure of the peptide region.

The amino acid sequence of SEQ ID NO: 結合 4 to which the above antibody binds, the amino acid sequence in which one amino acid was mutated in SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, and one amino acid in SEQ ID NO: 5 was mutated Conformational changes in the polypeptide region containing the amino acid sequence, the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence in which 1 amino acid is mutated in SEQ ID NO: X6 is associated with amyotrophic lateral sclerosis (ALS) It was shown that it has the feature of.

The antibody produced and used in the present invention may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody. In the present invention, # 406, # 659, or # 785 (Deposit numbers NITE P-941, NITE BP-942, and NITE BP-943 (the deposit date is May 12, 2010)) or # 27 (deposit number NITE BP-1100) (the deposit date is 2011) The above-mentioned antibody produced by any of the hybridomas of May 12, 2011) can be provided.

In this specification, when an amino acid is “mutated” in a specific sequence, it means that the amino acid is deleted, the amino acid is substituted, or the amino acid is added. To do.

In the present invention, “antibody” also includes antibody fragments or modified antibodies that bind to mutant SOD1 protein. To such an antibody fragment, for example, Fab, F (ab ′) ₂ , Fv of the above-described monoclonal antibody, or single chain Fv derived from the above-described antibody (H chain Fv and L chain Fv are bound by a linker) Stuff, scFv). In addition, such an antibody fragment is treated with an enzyme such as papain or pepsin, or a gene encoding the antibody fragment is constructed, incorporated into an expression vector, and the vector in an appropriate host cell. It can also be prepared recombinantly by expressing. As the modified antibody, an antibody bound to various molecules such as polyethylene glycol (PEG) can be used.

According to the present invention, a method for detecting the risk of ALS can be newly provided by detecting a mutant SOD1 protein. The present invention further provides an antibody that does not bind to wild-type (ie, normal) SOD1 protein but binds to mutant SOD1 protein, which can be used to detect mutant SOD1 protein in the above method. You can also. With these effects, it is possible to reliably detect ALS or the risk of ALS that has been difficult to differentiate clinically until now.

Figure 1 shows the amino acid sequence of wild-type SOD1 protein (identical to SEQ ID NO: 2) and its specific mutations that are known to be involved in familial amyotrophic lateral sclerosis (ALS) It is a figure which shows the variation | mutation of SOD1 protein. FIG. 2 shows that among the antibodies of the present invention, five antibodies derived from hybridomas # 242, # 406, # 659, # 785, and # 1175 were able to detect Flag-SOD1 (G93A). FIG. FIG. 3 shows that among the antibodies of the present invention, three types of antibodies derived from hybridomas # 406, # 659, and # 785 can detect Flag-SOD1 (G85R) in addition to Flag-SOD1 (G93A). It is a figure which shows having been made. FIG. 4-1 shows that various mutant SOD1 proteins can be precipitated (ie, detected) by an antibody obtained from a # 785 hybridoma selected as one of the antibodies of the present invention. It is. FIG. 4-2 shows that various mutant SOD1s can be precipitated (ie, detected) by the antibody obtained from the # 785 hybridoma selected as one of the antibodies of the present invention. is there. FIG. 4-3 is a diagram showing that various mutant SOD1s can be precipitated (ie, detected) by an antibody obtained from the # 785 hybridoma selected as one of the antibodies of the present invention. is there. FIG. 5 shows that antibodies derived from the # 27 hybridoma of the present invention were able to detect Flag-SOD1 (A4V), Flag-SOD1 (G93A) and Flag-SOD1 (G85R) (FIG. 5). 5A), and in addition to these, various mutant SOD1 proteins having mutations in the region corresponding to SEQ ID NO: 4 in the amino acid sequence of SEQ ID NO: 2 (peptide region consisting of the 6th to 16th amino acid sequences) FIG. 5 shows that immunoprecipitation is possible (FIG. 5B). FIG. 6 shows that the antibody obtained from the # 406 hybridoma selected as one of the antibodies of the present invention also shows the conformational change (exposed to the Derlin-1 binding site) associated with the mutation of SOD1 protein in cell immunostaining. It is a figure which shows that it can detect. FIG. 7 shows that an antibody obtained from the # 785 hybridoma selected as one of the antibodies of the present invention is a mutant SOD1 protein in the cytoplasm of B cells in the blood of an amyotrophic lateral sclerosis (ALS) individual. It is a figure which shows that an immunoprecipitation is possible.

Amyotrophic lateral sclerosis (ALS) is known as a conformational disease, and the causative protein of familial ALS among these diseases is SOD1 protein. The amino acid sequence of the wild-type human SOD1 (superoxide dismutase 1) protein and the nucleotide sequence of the gene encoding it are available as GenBank Accession No. NM_000454. In this specification, the protein The amino acid sequence is described as SEQ ID NO: 2 and the nucleotide sequence encoding it is described as SEQ ID NO: 1. Regarding the amino acid sequence of this SEQ ID NO: 2, it is common in the art to exclude the first methionine of the wild-type SOD1 protein when counting the amino acid number of the SOD1 protein. The amino acid sequence of SEQ ID NO: 2 is described as the amino acid sequence disclosed in GenBank Accession No. NM_000454 excluding the first methionine.

To date, 122 types of point mutations have been reported in the SOD1 protein, and of these, it is known that more than 100 types of mutant SOD1 proteins are actually involved in familial ALS at this stage ( (See Figure 1 for specific SOD1 protein mutations known to be involved in familial ALS). In FIG. 1, the row indicated by “WT” indicates the amino acid sequence of the wild-type SOD1 protein, and the amino acids described above indicate the mutant amino acids of the respective mutant SOD1 proteins. Among the mutant amino acid designations, Δ indicates a deletion, + indicates an insertion, and * indicates a stop codon.

From previous studies, four of the mutant SOD1 proteins known to be involved in these familial ALS proteins (SEQ AIDV, based on the amino acid sequence number of SEQ ID NO: X 2) G85R, G93A, and L126stop), specifically with Derlin-1 (CT4) (Phe Leu Tyr Arg Trp Leu Pro Ser Arg Grg Gly (SEQ ID NO: 7)) It has been shown to combine (Nishitoh et al. Genes Dev. 2008).

Based on the binding between various mutant SOD1 proteins involved in familial ALS and Derlin-1 (CT4), the mutant SOD1 protein can be combined with Derlin-1, depending on the mutant SOD1 protein, each mutant amino acid site. It was unlikely that this was due to the formation of a Derlin-1 binding site in the vicinity, and it was thought that a Derlin-1 binding region originally existed in the amino acid sequence of the wild-type SOD1 protein.

Next, when the interaction site with Derlin-1 on the SOD1 protein side was examined, it was defined by the amino acid sequence shown by SEQ ID NO: 2 (consisting of the amino acid sequence excluding the first methionine). Of the wild-type SOD1 protein consisting of amino acids 5 to 18 on the N-terminal side (Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly Ile Ile (SEQ ID NO: 3)) binds to Derlin-1 It became clear that it was a region (single underlined portion in FIG. 1).

From this result, in the wild type, the peptide region consisting of the 5th to 18th amino acid sequence (SEQ ID NO: 3) hidden in the 3D structure is 3D structurally exposed in the mutant SOD1 protein involved in familial ALS. As a result, it was expected to cause familial ALS through binding to Derlin-1. Therefore, in the present invention, an antibody capable of binding to a peptide region consisting of amino acids 5 to 18 (SEQ ID NO: 3) in the amino acid sequence of the mutant SOD1 protein (SEQ ID NO: 2) is prepared. It was decided to.

Of the amino acid sequences of SEQ ID NO: 3, the N-terminal Val and the C-terminal two Ile are hydrophobic, which may reduce the water solubility of the immunogen. The peptide region consisting of the amino acid sequence (Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly (SEQ ID NO: 4)) was used as an immunogen.

The resulting antibody binds to the mutant SOD1 protein actually produced in the cell (eg, G85R and G93A), but does not bind to the wild-type SOD1 protein (SEQ ID NO: 2) Using these as indicators, screening was performed and candidate antibodies were selected. At that time, as described above, it was suggested that binding between Derlin-1 and mutant SOD1 protein may result from a change in conformation based on mutation in SOD1 protein. Screening was performed below (ie, without destroying the SOD1 protein conformation). As a result, three hybridoma cell lines producing antibodies with the desired characteristics, # 406, # 659, and # 785 (respectively 2-5-8, Kazusa Kamashichi, Kisarazu, Chiba Prefecture, product evaluation Deposited as NITE P-941, NITE BP-942, and NITE 技術 BP-943 at the National Institute of Technology (NITE) and Patent Microorganisms Depository (NPMD); Could also get on May 12, 2010).

Based on such findings, in one embodiment of the present invention, an amino acid sequence of Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly (SEQ ID NO: 4) or an amino acid in which one amino acid is mutated in SEQ ID NO: 4 Antibodies that bind to a polypeptide comprising the sequence are provided.

In one embodiment of the present invention, an antibody having the characteristics of binding to a mutant SOD1 protein (eg, G85R and G93A) but not to a wild type SOD1 protein (SEQ ID NO: 2) is further searched. did.

When the three-dimensional structure of the wild-type SOD1 protein was analyzed, the 5-18th amino acid sequence (SEQ ID NO: 3) of the wild-type SOD1 protein (SEQ ID NO: 2) formed a β-strand structure. Β-strand structure formed by peptide region (Lys Val Trp Gly Ser Ile Lys Gly Leu Thr Glu (SEQ ID NO: 5)) consisting of amino acids 30-40 amino acids on the N-terminal side or C-terminal side 143- It is clear that the β-strand structure formed by the peptide region consisting of the 151st amino acid (Arg Leu Ala Cys Gly Val Ile Gly Ile (SEQ ID NO: して 6)) is located in close proximity to each other became. And by having such a structure, all of the three β-strand structures described above are concealed in the three-dimensional structure in the wild-type SOD1 protein, but are three-dimensionally exposed in the mutant SOD1 protein, It was expected to be detectable by the antibody.

Therefore, in the wild-type SOD1 protein, the three-dimensional structure is hidden in the wild-type SOD1 protein, but as a candidate polypeptide region that is three-dimensionally exposed in the mutant-type SOD1 protein, among the wild-type SOD1 protein (SEQ ID NO: 2), Two peptide regions that are three-dimensionally close to the 5th to 18th amino acid sequence that changes its structure due to mutation, a peptide region consisting of amino acids 30 to 40 on the N-terminal side (Lys Val Trp Gly Ser Ile Lys Gly Leu Thr Glu (SEQ ID NO: 5)) or a peptide region consisting of amino acids 143 to 151 on the C-terminal side (Arg Leu Ala Cys Gly Val Ile Gly Ile (SEQ ID NO: 6)) was selected and used as an immunogen ( (Double underlined part in Fig. 1)

The resulting antibody binds to the mutant SOD1 protein actually produced in the cell (eg, G85R and G93A), but does not bind to the wild-type SOD1 protein (SEQ ID NO: 2) Using these as indicators, screening was performed under non-denaturing conditions (ie, without destroying the three-dimensional structure of the SOD1 protein), and candidate antibodies were selected. As a result, it was possible to obtain one type of hybridoma cell line, # 27 (NITE BP-1100; the date of entrustment was May 12, 2011) that produced an antibody having the desired characteristics.

Based on such findings, in one embodiment of the present invention, the amino acid sequence of Lys Val Trp Gly Ser Ile Lys Gly Leu Thr Glu (SEQ ID NO: 5) or Arg Leu Ala Cys Gly Val Ile Gly Ile (SEQ ID NOle) An antibody that binds to a polypeptide comprising: 6) is provided.

We selected some of the 122 mutant SOD1 proteins that are actually known to be related to familial ALS and detected whether they could bind to these antibodies. All have been shown to bind to all mutant SOD1 proteins tested under non-denaturing conditions. Therefore, the antibody of the present invention does not bind to a protein consisting of the amino acid sequence of SEQ ID NO: 2 (ie, wild type SOD1 protein), but the other amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 4 1 amino acid sequence mutated in SEQ ID NO: 5 amino acid sequence, SEQ ID 配列 NO: amino acid sequence mutated in 1 amino acid in SEQ ID NO: アミノ酸 6 amino acid sequence, or in SEQ ID NO: 6 A polypeptide comprising an amino acid sequence in which one amino acid is mutated can be bound.

Here, the amino acid sequence of SEQ ID NO: 4 to which these antibodies are bound, the amino acid sequence in which one amino acid is mutated in SEQ ID NO: 4, the amino acid sequence of SEQ ID アミノ酸 NO: 5, SEQ ID NO: 5 A polypeptide comprising an amino acid sequence in which one amino acid is mutated in SEQ ID NO: 6, or an amino acid sequence in which one amino acid is mutated in SEQ ID NO: 6 includes a mutant SOD1 protein, and more Specifically, among the amino acid sequences of SEQ ID NO: 2, for example, amino acid numbers 3, 4, 5, 6, 8, 10, 12, 14, 16, 19, 20, 22, 29, 37, 38, 40, 41, 43, 45, 46, 47, 48, 49, 57, 59, 65, 66, 72, 76, 80, 84, 85, 86, 87, 89, 90, 93, 95, 97, 99, 100, 101, 104, 105, 106, 108, 111, 112, 113, 114, 115, 116, 117, 118, 124, 125, 126, 127, 132, 133, 134, 139, 140, 141, 144, 145, One of 146, 147, 148, 149, or 151 One amino acid residue includes those having one amino acid substitution, deletion or addition.

From these results, except for the protein consisting of the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID 、 NO: 4, the amino acid sequence in which one amino acid was mutated in SEQ ID NO: 4, the amino acid of SEQ ID NO: 5 3D structural change of a polypeptide region comprising the sequence, an amino acid sequence mutated by 1 amino acid in SEQ ID NO: 5, an amino acid sequence of SEQ ID NO: 6, or an amino acid sequence mutated by 1 amino acid in SEQ ID NO: 6 Has been shown to be a risk factor associated with the development of amyotrophic lateral sclerosis (ALS).

Based on such findings, in another embodiment of the present invention, it further binds to mutant SOD1 protein (for example, but not limited to, for example, G85R and G93A), but wild-type SOD1 protein ( By using an antibody that does not bind to SEQ ID NO: 2), screening for a cell-containing sample taken from a subject and detecting the protein that binds to that antibody, the sequence of SEQ ID NO: 2 Other than proteins consisting of amino acid sequences, SEQ ID NO: 4 amino acid sequence, SEQ ID NO: アミノ酸 4 amino acid sequence mutated, SEQ ID NO: 5 amino acid sequence, SEQ ID NO: 5 A method for detecting a polypeptide comprising a mutated amino acid sequence, the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence in which one amino acid is mutated in SEQ ID NO: 6, and Through this method, a method for detecting a risk associated with the development of familial ALS is provided.

Example 1: Production of antibody against mutant SOD1 protein (1)
In this example, the purpose was to produce an antibody that does not bind to the wild-type SOD1 protein but can bind to the mutant SOD1 protein.

(1) Immunity of rats Hydrophobic amino acids present at the N-terminus and C-terminus of SOD1 (5-18 aa) (Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly Ile Ile (SEQ ID NO: 3)) (V and I) may reduce water solubility, so the peptide consisting of amino acids 6 to 16 of SOD1 protein (Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly (SEQ ID NO: 4)) Peptides were made as immunogens.

The peptide consisting of amino acids 6 to 16 of this SOD1 protein was complexed with keyhole limpet hemocyanin (KLH; Operon) and prepared to 1 mg / mL. 400 μL of a peptide solution of a peptide consisting of amino acids 6 to 16 of SOD1 protein and 800 μL of Freund's complete adjuvant (Sigma) were stirred and emulsified with a Luer syringe to obtain an antigen. The prepared antigen was intradermally injected into 3 rats (WKY / Izm female, 8 weeks old at the time of immunization; Japan Claire) in an amount of 100 μL per leg from the hindlimb paws.

(2) Preparation of myeloma cells, preparation of lymph node cells, cell fusion Mouse myeloma cell line SP2 / 0-Ag14 (SP2) (DS Pharma Medical), GIT medium containing 10% fetal bovine serum (biowest) (Wako Jun) In the drug), the cells were cultured under the conditions of 37 ° C. and 5% CO ₂ .

This mouse myeloma cell line arose from the stock 4 days before the scheduled fusion date with cells collected from the immunized mouse, and on the day of cell fusion, 6 10 cm dishes per rat were confluent with myeloma cells. Sowed. On the day of cell fusion, the GIT medium was removed by aspiration, and 4 μmL of DMEM high glucose (this culture medium is simply referred to as “DMEM”; Sigma Aldrich) was added to each dish to remove SP2.

The suspension was collected in a 50-mL Falcon tube, centrifuged at 1000-rpm for 3 minutes, and the supernatant was removed by suction. Further, the cells were suspended in 20 mL of DMEM and centrifuged again at 1000 rpm for 3 minutes, and the supernatant was removed by suction. Finally, the cells were suspended in 5 mL DMEM to prepare SP2 cell solution used for fusion.

The rat immunized in (1) was deeply anesthetized with ether, and blood was collected from the heart. Then, after cervical dislocation and lethal death, the abdomen was opened and the iliac lymph nodes were removed. Using a cell strainer (Becton, “Dickinson” and “Company”), lymph node tissue cells containing antibody-producing B cells were recovered from 2 lymph nodes in 2 mL of DMEM.

The lymph node cell solution was added to the SP2 cell solution, suspended in DMEM to a total volume of 40 mL, and then centrifuged at 1200 rpm for 10 minutes. The supernatant was removed by aspiration, and the precipitate was loosened by tapping. Then, it was held by hand and incubated at body temperature for 2 minutes. 1 mL of polyethylene glycol (PEG) prepared in advance was slowly added dropwise over 1 minute while shaking the falcon tube. The Falcon tube was shaken and rotated to perform a cell fusion reaction for 2 minutes.

After completion of the reaction, 4.5 μmL of DMEM was added dropwise over 3 minutes while shaking the falcon tube. Subsequently, 4.5 μmL of DMEM was similarly added dropwise over 2 minutes and centrifuged at 900 rpm for 5 minutes. The supernatant was removed by aspiration, and BM-Condimed-H1 (Roche) 4 mL and HAT medium 36 mL were added to suspend the cells. For HAT medium, dissolve 6 ml of thymidine (Wako Pure Chemical Industries) and hypoxanthine (Wako Pure Chemical Industries) solution at 60 ℃, stir with aminopterin (Wako Pure Chemical Industries) 0.6 ml, and filter with 0.22μm filter (Millipore). Of these, 6 ml is prepared by dissolving in 555 ml of GIT medium (Wako Pure Chemicals) containing 10% FBS.

The cell solution of the lymph node tissue cells described above was seeded at 100 μL / well in four 96-well plates and cultured in HAT medium for about 7 to 10 days to obtain hybridoma cells.

(3) Primary screening by ELISA As a primary screening, ELISA using a peptide consisting of amino acids 6 to 16 of SOD1 protein (SEQ ID NO: 4) was performed.

In a 96-well Maxisorp ELISA plate (Nunc), dispense 3 μg / mL KLH-conjugated SOD1 protein peptide consisting of amino acids 6 to 16 or PBS containing KLH at 100 μL / well and incubate overnight at 4 ° C. Then, the antigen was adsorbed to the surface of the plate well. After discarding the antigen solution and dispensing PBS containing 1% bovine serum albumin at 100 μL / well and incubating at 37 ° C. for 1 hour for blocking, the hybridoma culture supernatant was dispensed at 40 μL / well and 1 at 37 ° C. Incubated for hours.

Discard the culture supernatant, wash the wells twice with PBS, dispense PBS containing 0.1% bovine serum albumin diluted 1000-fold with an HRP-labeled anti-rat IgG antibody (GE Healthcare) stock solution at 100 μL / well at 37 ° C Incubated for 1 hour. Discard the secondary antibody solution and wash the wells 3 times with PBS, and add 30% H ₂ O ₂ at a rate of 0.4 μL / mL to PBS containing 0.4 mg / mL o-phenylenediamine (OPD) just before color development. The chromogenic substrate solution was dispensed at 100 μL / well and allowed to stand at room temperature for 1 hour for color development.

As a result, 266 positive clones were obtained (data not shown).

(4) Limiting dilution Hybridomas cultured in 24-well plates were suspended by pipetting, and the cells were diluted in an appropriate amount of culture solution (HAT medium) and the number of cells was counted. This cell solution was added to 30 mL of HAT medium supplemented with 10% BM condimed H1 so as to be 0.5 cells / 150 μL / well and 1.5 cells / 150 μL / well, and seeded on two 96-well plates at 200 μL / well.

After limiting dilution in this manner, each of the obtained hybridomas was further grown in a culture solution (HAT medium), and further screening was performed using the culture supernatant of the well in which colonies were formed. .

(5) Secondary screening by immunoprecipitation As a secondary screening, the hybridoma supernatant was evaluated by immunoprecipitation (IP) in order to evaluate whether or not SOD1 protein was recognized in a non-denaturing state.

First, to overexpress mutant SOD1 in HEK293A cells cultured at 37 ° C in 5% CO ₂ in DMEM high glucose containing 10% fetal bovine serum and 100 unit / mL penicillin G. A gene encoding -SOD1 (G93A) was introduced using a pcDNA3-Flag-SOD1 (G93A) vector. The nucleotide sequence of the gene encoding Flag-SOD1 (G93A) was formed in the pcDNA3-Flag-SOD1 (G93A) vector as follows. First, a circular vector pcDNA3 treated with a BamHI-EcoRI restriction enzyme from a nucleotide sequence (ggatccaccg ccatggacta caaggacgat gatgacaagg gcgaattc; SEQ ID NO: 8) encoding Flag (Asp Tyr Lys Asp Asp Asp Asp Lys; SEQ ID NO: 9). It was introduced into the multicloning site of 0 vector (Invitrogen) (pcDNA3-Flag vector). Next, nucleotides encoding SOD1 (G93A) (SEQ ID NO: 11) are obtained by performing PCR using primers (gaattcgcga cgaaggccg (SEQ ID NO: 14) and ggatccttgg gcgatcccaa tt (SEQ ID NO: 15)). The sequence (SEQ ID NO: 10) was amplified while introducing EcoRI and XhoI restriction enzyme sites at both ends. This PCR product is treated with EcoRI-XhoI, and then introduced into the cloning site of pcDNA3-Flag vector treated with EcoRI-XhoI, so that the nucleotide sequence encoding SEQ ID NO: 8 and SOD1 (G93A) A pcDNA3-Flag-SOD1 (G93A) vector containing Flag-SOD1 (G93A) fused with the encoding nucleotide sequence (SEQ ID NO: 10) was prepared. The pcDNA3-Flag-SOD1 (G93A) vector having this nucleotide sequence was introduced into HEK293A cells using FuGENE6 (Roche).

After culturing Flag-SOD1 (G93A) transfected cells seeded in a 6-well plate for 48 hours in DMEM containing 10% FBS, 1 μmL of IP lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM) Cells by incubating the cells for 30 minutes at 4 ° C with NaCl, 10 mM EDTA pH 8.0, 1% sodium deoxycholate, 1% Triton-

X

100, 1 mM mM phenylmethylsulfonyl fluoride, 5 μg / mL leupeptin) After dissolution, the supernatant was collected by centrifugation at 4 ° C., 15000 rpm for 10 minutes.

Next, the cell lysate prepared as described above was immunoprecipitated with the hybridoma culture supernatant. 500 μL of culture supernatant containing the antibody of the present invention prepared in (1) to (4) for immunoprecipitation, and anti-SOD1 antibody (Stressgen) or anti-Flag antibody (Sigma; M2) that recognizes wild-type SOD1 protein as a control antibody 1 μg / 500 μL of IP lysis buffer (as described above) was added to 500 μL of the cell lysate, and the mixture was mixed by inversion overnight at 4 ° C. for reaction.

After the reaction, 20 μL of protein G sepharose and M2 affinity agarose gel (Sigma) were added in a liquid amount, and the mixture was further mixed by inversion at 4 ° C. for 2 hours to react. After the reaction, the supernatant is removed and washed 4 times with 1 mL of IP lysis buffer.Finally, 50 μL of 2 × SDS sample buffer containing 20 μM dithiothreitol is added and boiled at 98 ° C. for 3 minutes. -Used for PAGE.

After electrophoresis, transfer the protein from the gel to polyvinylidene difluoride membrane (0.45μm, Pall), and TBS-T containing 1% skim milk (150 mM NaCl, 50 mM Tris-HCl pH 8.0, 0.05% Tween 20) After blocking at room temperature for 2 hours, the mixture was reacted at 4 ° C. overnight with a primary antibody diluted with TBS-T containing 5% bovine serum albumin and 0.1% NaN ₃ . After washing twice with TBS-T, the mixture was reacted with a secondary antibody diluted with TBS-T containing 1% skim milk at room temperature for 1 hour and washed with TBS-T for 2 hours.

Detection was performed by a highly sensitive chemiluminescence method (enhanced chemiluminescence; ECL) (GE Healthcare). In Western blotting, primary antibodies include anti-Flag antibody (M2; Sigma), anti-SOD1 antibody (SOD-100; Stressgen), secondary antibodies include anti-mouse IgG antibody (GE Healthcare), and anti-rat IgG antibody (Cell signaling). , Anti-rabbit IgG antibody (Cell signaling), both HRP-labeled were used.

As a result, Flag-SOD1 (G93A) could be detected in the hybridoma supernatants # 242, # 406, # 659, # 785, and # 1175 (FIG. 2).

(6) Further examination of antibodies In addition, in order to evaluate whether other mutant SOD1 proteins can also be detected, Flag-SOD1 (G85R) transgenic cells are also cultured in the same manner, and Flag-SOD1 (G85R) is The cells were overexpressed, the cell lysate was immunoprecipitated with the above five hybridoma supernatants, and immunoprecipitated Flag-SOD1 (G85R) was further examined by Western blotting.

The nucleotide sequence of the gene encoding Flag-SOD1 (G85R) was introduced into the pcDNA3-Flag vector prepared in (5) and formed in the pcDNA3-Flag-SOD1 (G85R) vector as follows. . PCR using primers (gaattcgcga cgaaggccg (SEQ ID NO: 14) and ggatccttgg gcgatcccaa tt (SEQ ID NO: 15)) allows nucleotide sequence encoding SOD1 (G85R) (SEQ ID NO: 13) ID NO: 12) was amplified while introducing EcoRI restriction enzyme sites and XhoI restriction enzyme sites at both ends. This PCR product is treated with EcoRI-XhoI, and then introduced into the cloning site of pcDNA3-Flag vector treated with EcoRI-XhoI, so that the nucleotide sequence coding for Flag (SEQ ID NO: 8) and SOD1 (G85R) A pcDNA3-Flag-SOD1 (G85R) vector containing Flag-SOD1 (G85R) fused with a nucleotide sequence encoding SEQ ID NO: SEQ ID NO: 12 was prepared. The pcDNA3-Flag-SOD1 (G85R) vector having this nucleotide sequence was introduced into HEK293A cells using FuGENE6 (Roche) in the same manner as in (5). Further, immunoprecipitation and western blotting for examination were performed as described above in (5).

As a result, Flag-SOD1 (G85R) was detected in the hybridoma supernatants of # 406, # 659, and # 785 (FIG. 3).

From these results, in the present invention, three types of hybridomas # 406, # 659, and # 785 that were able to bind to both SOD1 (G93A) and SOD1 (G85R) were selected as preferred, and these Cell lines were deposited with the National Institute of Technology and Evaluation (NITE) and the Patent Microorganism Depositary Center (NPMD) (the deposit numbers of # 406, # 659, and # 785 were NITE P-941 and NITE BP-, respectively) 942 and NITE BP-943; the date of entrustment is May 12, 2010).

Example 2: Examination of antibody antigen recognizability The culture supernatants of the three hybridoma cells # 406, # 659, and # 785 obtained in Example 1 did not recognize wild-type SOD1 protein, and SOD1 (G85R ) And SOD1 (G93A), in this example, it was examined whether or not other various mutant SOD1 proteins are also commonly recognized.

In the experiment, using the cell supernatant obtained from the # 785 hybridoma, cells expressing various mutant SOD1 proteins were prepared in the same manner as in Example 1 (5) and (6). Immunoprecipitation is performed by mixing the cell supernatant obtained from # 785 hybridoma, and each of the obtained Flag-mutant SOD1 proteins is used as a primary antibody for anti-Flag antibody (M2; Sigma), secondary antibody. As these, anti-mouse IgG antibodies labeled with HRP (GE Healthcare), anti-rat IgG antibodies (Cell signaling), and anti-rabbit IgG antibodies (Cell signaling) were used.

As a result, SOD1 (K3E), SOD1 (A4S), SOD1 (A4T), SOD1 (A4V), SOD1 (V5L), SOD1 (C6F), SOD1 (C6G), SOD1 (C6Y), SOD1 ( G16A), SOD1 (G16S), SOD1 (F20C), SOD1 (V29A), SOD1 (G37R), SOD1 (L38R), SOD1 (L38V), SOD1 (E40G), SOD1 (G41D), SOD1 (G41S), SOD1 ( H43R), SOD1 (F45C), SOD1 (H46R), SOD1 (V47A), SOD1 (V47F), SOD1 (H48Q), SOD1 (H48R), SOD1 (C57R), SOD1 (S59I), SOD1 (N65S), SOD1 ( P66A), SOD1 (G72C), SOD1 (G72S), SOD1 (D76V), SOD1 (D76Y), SOD1 (H80R), SOD1 (L84F), SOD1 (L84V), SOD1 (G85R), SOD1 (G85S), SOD1 ( N86D), SOD1 (N86I), SOD1 (N86K), SOD1 (N86S), SOD1 (V87A), SOD1 (V87M), SOD1 (T88Δ) (meaning a deletion of three amino acids T88, A89 and D90), SOD1 ( A89T), SOD1 (A89V), SOD1 (D90A), SOD1 (D90V), SOD1 (G93A), SOD1 (G93C), SOD1 (G93D), SOD1 (G93R) , SOD1 (G93S), SOD1 (G93V), SOD1 (A95T), SOD1 (A95V), SOD1 (V97L), SOD1 (V97M), SOD1 (I99V), SOD1 (E100G), SOD1 (E100K), SOD1 (D101G) , SOD1 (D101H), SOD1 (D101N), SOD1 (D101Y), SOD1 (I104F), SOD1 (S105L), SOD1 (S105Δ), SOD1 (L106V), SOD1 (G108V), SOD1 (C111Y), SOD1 (I112M) , SOD1 (I112T), SOD1 (I113F), SOD1 (I113T), SOD1 (G114A), SOD1 (R115G), SOD1 (T116R), SOD1 (L117V), SOD1 (V118L), SOD1 (V118 +), SOD1 (D124G) , SOD1 (D124V), SOD1 (D125H), SOD1 (L126S), SOD1 (L126 *), SOD1 (L126Δ), SOD1 (E132 +), SOD1 (E133V), SOD1 (E133Δ), SOD1 (S134N), SOD1 (N139D ), SOD1 (N139H), SOD1 (N139K), SOD1 (A140G), SOD1 (G141E), SOD1 (G141 *), SOD1 (L144F1), SOD1 (L144F2), SOD1 (L144S), SOD1 (A145G), SOD1 ( A145T), SOD1 (C146R), SOD1 (C146 *), SOD1 (G147D), SOD1 (G147R), SOD1 (V148G), SOD1 (I1 49T), SOD1 (I151S), and SOD1 (I151T), all 107 types can be immunoprecipitated by the antibody of the present invention, that is, although there is a difference in degree, the antibody of the present invention is a mutant SOD1 protein. It was revealed that the mutant SOD1 protein can be recognized regardless of the mutation site (Fig. 4a) to i)). In FIG. 4, “del” is described in place of the notation “Δ” in FIG. 1 to indicate deletion, “ins” is described in place of the notation “+” in FIG. 1 to indicate insertion, and In order to indicate a stop codon, “Z” or “stop” may be used instead of “*” in FIG.

Example 3: Production of antibody against mutant SOD1 protein (2)
In this example, the aim was to produce additional antibodies that did not bind to the wild-type SOD1 protein but could bind to the mutant SOD1 protein.

Peptide consisting of amino acids 30-40 of wild-type SOD1 protein (Lys Val Trp Gly Ser Ile Lys Gly Leu Thr Glu (SEQ ID NO: 5)) peptide for the purpose of producing an antibody having such characteristics Were used as immunogens to generate antibodies.

Peptide consisting of amino acids 30-40 of wild type SOD1 protein is prepared to 1 mg / mL, and 400 µL of peptide solution and Freund's complete adjuvant (Sigma) 800 µL are emulsified by stirring with a Luer syringe, and the antigen It was. The prepared antigen was intradermally injected into 3 rats (WKY / Izm female, 8 weeks old at the time of immunization; Japan Claire) in an amount of 100 μL per leg from the hindlimb paws.

The subsequent antibody production step and antibody screening step were carried out as described in Example 1.

As a result, the hybridoma supernatant of # 27 did not detect the wild type SOD1 protein, but detected the mutant SOD1 protein of Flag-SOD1 (A4V), Flag-SOD1 (G85R) and Flag-SOD1 (G93A) (Fig. 5A). The cell line was deposited with the National Institute for Product Evaluation and Technology (NITE) and the Patent Microorganism Depositary Center (NPMD) with the obtained # 27 hybridoma as preferred (the deposit number of # 27 is NITE BP -1100; contract date is May 12, 2011).

In this example, the culture supernatant of the obtained # 27 hybridoma cells was further transformed into various mutant SOD1 proteins other than SOD1 (A4V), SOD1 (G85R), and SOD (G93A), particularly SEQ ID Whether or not to recognize a mutant SOD1 protein having a mutation in the part corresponding to SEQ ID NO: NO4 in the amino acid sequence of NO: 2 is determined using an antibody obtained from the hybridoma of Example 27. As described in.

As a result, the antibodies obtained from the # 27 hybridoma were SOD1 (A4V), SOD1 (L8Q), SOD1 (L8V), SOD1 (G10R), SOD1 (G10V), SOD1 (G12R), SOD1 (V14G), SOD1 (V14M), SOD1 (G85R), and SOD1 (G93A) are capable of immunoprecipitation of all 10 mutant SOD1 proteins, that is, the antibody obtained from the # 27 hybridoma is an immunogen It was revealed that the mutant SOD1 protein can be recognized regardless of the mutation site of the mutant SOD1 protein other than the peptide region consisting of amino acids 30 to 40 of the wild-type SOD1 protein used as (FIG. 5B).

Example 4: Cellular immunostaining with mutant SOD1 antibody In this example, it was examined whether the mutant SOD1 antibody of the present invention can detect mutant SOD1 protein even in cell immunostaining.

12 to 16 hours after seeding HEK293A cells at 0.4 × 10 ⁵ cells / well in a 24-well plate containing a collagen-coated φ12 mm cover glass, Flag-SOD1 (WT) (Flag-encoding nucleotide sequence (SEQ ID NO: 8) and nucleotide sequence encoding wild-type SOD1 protein (SEQ ID NO: 1) or nucleotide sequence encoding Flag-SOD1 (G93A) (SEQ ID NO: 10), 0.5 Gene transfer was performed at μg / well, and Flag-SOD1 (WT) and Flag-SOD1 (G93A) were overexpressed.

24 hours after the gene transfer, the cover glass was washed by immersing it in about 5 ml of PBS three times, and the cells were fixed with 4% paraformaldehyde. After fixation, the cover glass was subjected to 3 times with about 5 mL of PBS and subjected to cell membrane permeabilization with 0.2% Triton® X-100. After the Triton® X-100 treatment, the cover glass was treated with about 5 μmL of PBS three times, and the cover glass was placed on 500 μL of sodium borohydride and incubated at room temperature for 1 minute to prevent discoloration. Thereafter, the cover glass was washed 3 times with about 5 ml of PBS, blocked with 5% FBS, and cell immunostaining was performed using # 406 hybridoma culture supernatant as a primary antibody, followed by anti-Flag antibody (M2 antibody). Recognized Flag. Secondary antibodies were incubated with Alexa 488 anti-rat IgG and Alexa 633 anti-mouse IgG.

When the fluorescence intensity of the mutant SOD1 antibody was compared in cells with approximately the same fluorescence intensity of the anti-Flag antibody, the fluorescence intensity of Flag-SOD1 (G93A) was stronger than that of Flag-SOD1 (WT) (FIG. 5). From this, it is considered that # 406 can detect a mutation-specific three-dimensional structural change (exposing the Derlin-1 binding site) of the SOD1 protein even in cell immunostaining.

Example 5: Detection of mutant SOD1 protein present in cytoplasm of human blood-derived B cells by mutant SOD1 antibody In this example, the antibody of the present invention was used to detect human amyotrophic lateral sclerosis (ALS). We examined whether mutant SOD1 protein present in the cytoplasm of B cells in the blood of individuals can be detected. This example was approved by the “Human Genome / Gene Analysis Research Ethics Review Committee” at the University of Tokyo Graduate School of Medicine and the “Research Ethics Review Committee for Humans” at the University of Tokyo Graduate School of Pharmaceutical Sciences. Based on.

From a human patient with ALS, 10 ml of venous blood was collected and 2 x 10 ⁷ cells per tube were used for each sample B cell using B cells immortalized by Epstein-Barr virus infection at SRL. Aliquot and 500 μL IP lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM EDTA pH 8.0, 1% sodium deoxycholate, 1% Triton-

X

100, 1 mM per tube The cells were lysed by incubating the cells with phenylmethylsulfonyl fluoride (5 μg / mL leupeptin) at 4 ° C. for 30 minutes, and centrifuged at 4 ° C., 15000 rpm for 10 minutes to collect the supernatant.

The cell lysate prepared as described above was then immunoprecipitated with the culture supernatant obtained from the # 785 hybridoma as described below, and the immunoprecipitated antibody-SOD1 complex was then purified using an anti-SOD1 antibody. Immunoblotting was performed.

Add 500 μL of the culture supernatant containing the antibody obtained from the # 785 hybridoma that undergoes immunoprecipitation, add 1 μg / 500 μL of IP lysis buffer (as described above) to 500 μL of the cell lysate, and mix by inverting at 4 ° C overnight. I let you.

After the reaction, 20 μL of protein G Sepharose agarose gel (Sigma) was added in a liquid amount, and the mixture was further mixed by inversion at 4 ° C. for 2 hours to react. After the reaction, the supernatant is removed and washed 4 times with 1 mL of IP lysis buffer.Finally, 50 μL of 2 × SDS sample buffer containing 20 μM dithiothreitol is added and boiled at 98 ° C. for 3 minutes. -Used for PAGE.

Detection was performed by a highly sensitive chemiluminescence method (enhanced chemiluminescence; ECL) (GE Healthcare). In Western blotting, an anti-SOD1 antibody (SOD-100; Stressgen) was used as the primary antibody, and an HRP-labeled anti-rabbit IgG antibody (Cell signaling) was used as the secondary antibody.

As a result, it was shown that various SOD1 mutations collected from human ALS patients can be detected by using an antibody obtained from a hybridoma of the antibody of the present invention (for example, # 785) (FIG. 7). ).

According to the present invention, a method for detecting the risk of ALS can be newly provided by detecting a mutant SOD1 protein. The present invention further provides an antibody that does not bind to wild-type (ie, normal) SOD1 protein but binds to mutant SOD1 protein, which can be used in the above method to detect mutant SOD1 protein. You can also With these effects, it is possible to reliably detect ALS or the risk of ALS that has been difficult to differentiate clinically until now.

SEQ ID NO: 1: Nucleotide sequence of DNA encoding human wild-type SOD1 protein
SEQ ID NO: 2: Amino acid sequence of human wild-type SOD1 protein
SEQ ID NO: 3: Amino acid sequence of a polypeptide consisting of amino acids 5 to 18 of SEQ ID NO: 2
SEQ ID NO: 4: Amino acid sequence of a polypeptide consisting of amino acids 6 to 16 of SEQ ID NO: 2.
SEQ ID NO: 5: Amino acid sequence of a polypeptide consisting of amino acids 30 to 40 of SEQ ID NO: 2
SEQ ID NO: 6: Amino acid sequence of a polypeptide consisting of amino acids 143 to 151 of SEQ ID NO: 2
SEQ ID NO: 7: Amino acid sequence of Derlin-1 (CT4)
SEQ ID NO: 8: Nucleotide sequence encoding the Flag tag
SEQ ID NO: 9: Amino acid sequence of Flag tag
SEQ ID NO: 10: nucleotide sequence encoding SOD1 (G93A) protein
SEQ ID NO: 11: SOD1 (G93A) protein amino acid sequence
SEQ ID NO: 12: Nucleotide sequence encoding SOD1 (G85R) protein
SEQ ID NO: 13: Amino acid sequence of SOD1 (G85R) protein
SEQ ID NO: 14: SOD1 amplification primer
SEQ ID NO: 15: SOD1 amplification primer

Claims

Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly (SEQ ID NO: 4), SEQ ID NO: アミノ酸 4 amino acid sequence mutated, Lys Val Trp Gly Ser Ile Lys Gly Leu Thr Glu ID NO: 5) amino acid sequence, SEQ ID NO: 5 amino acid sequence mutated, Arg Leu Ala Cys Gly Val Ile Gly Ile (SEQ ID NO: 6) amino acid sequence, or SEQ ID NO: 6 An antibody that binds to a polypeptide comprising an amino acid sequence in which one amino acid has been mutated.
SEQ ID NO: アミノ酸 4 amino acid sequence, SEQ ID アミノ酸 NO: アミノ酸 4 amino acid sequence mutated, SEQ ID NO: アミノ酸 5 amino acid sequence, SEQ ID NO: 5 amino acid sequence mutated, SEQ ID 2. The antibody according to claim 1, wherein the polypeptide comprising an amino acid sequence of NO: 6 or an amino acid sequence in which one amino acid is mutated in SEQ ID NO: 6 is a mutant SOD1 protein.
SEQ ID NO: アミノ酸 4 amino acid sequence, SEQ ID アミノ酸 NO: アミノ酸 4 amino acid sequence mutated, SEQ ID NO: アミノ酸 5 amino acid sequence, SEQ ID NO: 5 amino acid sequence mutated, SEQ ID A polypeptide comprising an amino acid sequence of NO: 6 or an amino acid sequence in which one amino acid is mutated in SEQ ID NO: 6 is amino acid number 3, 4, 5, 6, 8 of the amino acid sequence of SEQ ID NO: 2. , 10, 12, 14, 16, 19, 20, 22, 29, 37, 38, 40, 41, 43, 45, 46, 47, 48, 49, 57, 59, 65, 66, 72, 76, 80 , 84, 85, 86, 87, 89, 90, 93, 95, 97, 99, 100, 101, 104, 105, 106, 108, 111, 112, 113, 114, 115, 116, 117, 118, 124 , 125, 126, 127, 132, 133, 134, 139, 140, 141, 144, 145, 146, 147, 148, 149, and 151 in any one amino acid residue, 1 Have amino acid substitutions, deletions or additions The antibody of claim 1 or 2.
SEQ ID NO: アミノ酸 4 amino acid sequence, SEQ ID アミノ酸 NO: アミノ酸 4 amino acid sequence mutated, SEQ ID NO: アミノ酸 5 amino acid sequence, SEQ ID NO: 5 amino acid sequence mutated, SEQ ID The amino acid sequence of NO: 6 or a polypeptide comprising an amino acid sequence in which one amino acid is mutated in SEQ ID NO: 6 is associated with amyotrophic lateral sclerosis (ALS) The antibody according to item 1.
The antibody according to any one of claims 1 to 4, which does not bind to a protein consisting of the amino acid sequence of SEQ ID NO: 2.
The antibody according to any one of claims 1 to 5, which is a monoclonal antibody.
7. The antibody according to claim 6, which is produced by any one of NITE® P-941, NITE® BP-942, NITE® BP-943, and NITE® BP-1100 hybridomas.
A cell-containing sample collected from a subject is screened using the antibody of any one of claims 1 to 7 and detecting a protein that binds to the antibody, SEQ ID NO: 2 SEQ ID NO: アミノ酸 4 amino acid sequence, SEQ ID NO: amino acid sequence mutated in 4, SEQ ID NO: 配列 5 amino acid sequence, SEQ ID NO: アミノ酸 5 amino acid sequence A method for detecting a polypeptide comprising an amino acid sequence mutated in SEQ ID NO: 6, or an amino acid sequence in which one amino acid is mutated in SEQ ID NO: 6.
SEQ ID NO: アミノ酸 4 amino acid sequence, SEQ ID NO: 4 amino acid sequence, SEQ ID NO: アミノ酸 5 amino acid sequence, SEQ ID NO, SEQ ID NO : Amino acid sequence mutated by 1 amino acid in 5, SEQ ID NO: アミノ酸 6 amino acid sequence, or a polypeptide comprising an amino acid sequence mutated by 1 amino acid in SEQ ID NO: 6, SEQ ID NO: アミノ酸 2 amino acid sequence Of these, amino acid numbers 3, 4, 5, 6, 8, 10, 12, 14, 16, 19, 20, 22, 29, 37, 38, 40, 41, 43, 45, 46, 47, 48, 49 57, 59, 65, 66, 72, 76, 80, 84, 85, 86, 87, 89, 90, 93, 95, 97, 99, 100, 101, 104, 105, 106, 108, 111, 112 , 113, 114, 115, 116, 117, 118, 124, 125, 126, 127, 132, 133, 134, 139, 140, 141, 144, 145, 146, 147, 148, 149, and 151 Any one amino acid residue selected from 9. The detection method according to claim 8, wherein the detection method has one amino acid substitution, deletion or addition.
SEQ ID NO: アミノ酸 4 amino acid sequence, SEQ ID NO: 4 amino acid sequence, SEQ ID NO: アミノ酸 5 amino acid sequence, SEQ ID NO, SEQ ID NO : Amino acid sequence mutated by 1 amino acid in 5, SEQ ID NO: アミノ酸 6 amino acid sequence, or the presence of a polypeptide comprising an amino acid sequence mutated by 1 amino acid in SEQ ID NO: 6 is atrophic lateral sclerosis 10. The detection method according to claim 8 or 9, wherein the detection method is associated with a risk of illness (ALS).