JP7151985B2 - Anti-propanoylated amyloid β protein antibody - Google Patents

Description

The present specification relates to antibodies that specifically bind to propanoylated amyloid beta protein.

Alzheimer's dementia (hereinafter also referred to as AD) is the most common type of dementia and is estimated to account for 40 to 60%. Recent memory impairment is characteristic of AD cognitive impairment, and various psychiatric symptoms such as delusions and depressive symptoms are observed. considered to be rare. Generalized cerebral atrophy, decrease in cerebral cortical neurons, multiple occurrence of senile plaques, neurofibrillary tangles in neurons, etc. have been reported as pathological characteristics of AD. is characterized.

Amyloid β protein (Aβ) has been identified as the major protein that constitutes senile plaques. Therefore, it is considered to be one of the causative molecules involved in the onset of AD. As for Aβ, various molecular species having 38-43 amino acid residues have been produced. Although these Aβs are originally soluble in an aqueous solution, they form senile plaques in a highly aggregated form in AD patient brains. Based on these findings, in the amyloid hypothesis that Aβ is the primary factor in the pathogenesis of AD, this aggregate, and in recent years, the intermediate molecule Aβ oligomer before aggregation and deposition may cause neuronal damage. It is considered.

So far, it has been found that lysine residues in Aβ are propanoyl (hereinafter also referred to as PRL) by peroxidants of ω-3 polyunsaturated fatty acids in the human brain (patent Reference 1). Furthermore, it has also been reported that this PRL-modified Aβ has enhanced aggregation and neuronal toxicity compared to Aβ before PRL modification (Non-Patent Document 1).

JP 2011-202964 A

https://www.sciencedirect.com/journal/free-radical-biology-and-medicine/vol/49/suppl/S?page-size=100&page=3 (17th Annual Meeting of SFRBM and SFRR international Biennal Meeting, Propanoylation of amyloid b is trigger to the aggregateon and enhanced its neurotoxicity)

Since Aβ has two lysine residues, PRL conversion of Aβ is considered to be due to a non-enzymatic reaction. It is presumed that PRL is formed by peroxides of saturated fatty acids. So far, no investigation has been made on the PRL-ylation site, whether both of the two lysine residues in PRL-yated Aβ are modified or only one of them is modified.

The present specification provides antibodies for PRLylation site-specific detection and thus quantification of PRLylated amyloid β.

The present inventors focused on PRL-ylation at two lysine residues in Aβ, and conducted various studies with the aim of detecting Aβ in a PRL-ylation site-specific manner. As a result, one of the two lysine residues (lysine at position 16) is PRL-modified Aβ (16PRL-modified Aβ), and the monoclonal antibody specifically binds to the other lysine residue (lysine at position 28). obtained a monoclonal antibody that specifically binds to PRL-conjugated Aβ (28PRL-conjugated Aβ). Furthermore, in the cerebrospinal fluid collected from AD and non-AD humans, 16PRL-Aβ and 28PRL-Aβ can be detected and quantified using these monoclonal antibodies, respectively. It was found that the distribution of has characteristic tendencies between non-AD patients and AD patients, respectively. This specification provides the following means based on these findings.

(1) an antibody,
The reactivity to propanoylated amyloid β in which the lysine at position 16 of amyloid β42 or the lysine corresponding to the lysine is propanoylated is amyloid β in which the lysine at position 28 of amyloid β42 or the lysine corresponding to the lysine is propanoylated. Antibodies higher than reactive against
(2) The reactivity to the peptide in which the lysine in the amino acid sequence represented by SEQ ID NO: 4 is propanoylated is higher than the reactivity to the peptide in which the lysine in the amino acid sequence represented by SEQ ID NO: 5 is propanoylated, ( The antibody according to 1).
(3) The reactivity to the peptide in which the lysine in the amino acid sequence represented by SEQ ID NO: 6 is propanoylated is higher than the reactivity to the peptide in which the lysine in the amino acid sequence represented by SEQ ID NO: 4 is propanoylated, ( The antibody according to 1) or (2).
(4) Propanoylation in which the lysine at position 16 of amyloid β42 or the lysine corresponding to the lysine is propanoylated. The antibody according to any one of (1) to (3), which has a higher reactivity to amyloid β than the untreated amyloid β.
(5) an antibody,
The reactivity to propanoylated amyloid β in which the lysine at position 28 of amyloid β42 or the lysine corresponding to the lysine is propanoylated is amyloid β in which the lysine corresponding to the lysine at position 16 of amyloid β42 or the lysine corresponding to the lysine is propanoylated. an antibody that is more reactive to
(6) The reactivity to the peptide in which the lysine in the amino acid sequence represented by SEQ ID NO: 5 is propanoylated is higher than the reactivity to the peptide in which the lysine in the amino acid sequence represented by SEQ ID NO: 4 is propanoylated, ( The antibody according to 5).
(7) Propanoylated lysine at position 28 of amyloid β42 or the lysine corresponding to the lysine is propanoylated. The antibody according to (5) or (6), which has higher reactivity to non-propanoylated amyloid β than non-propanoylated amyloid β.
(8) A reagent for characterizing a neurodegenerative disease, comprising the antibody of any one of (1) to (7).
(9) The reagent according to (8), which is for characterization associated with Alzheimer's disease.
(10) A screening method,
contacting one or more test compounds with the antibody according to any one of (1) to (7);
obtaining the reactivity between the one or more test compounds and the antibody;
Evaluating the reactivity of the one or more test compounds with propanoylated amyloid β based on the reactivity;
A method.

FIG. 1 shows a scheme of propanoylation at lysine residues (positions 16 and 28) in the amyloid β sequence. FIG. 1 shows antigen design for generation of antibodies that specifically bind propanoylated amyloid-β. FIG. 4 shows the analysis results of specificity of mAb BST31 by competitive ELISA. FIG. 3 shows results of analysis of specificity by competitive ELISA for mAb 3A11. FIG. 3 shows a standard curve for quantification of PRL16 amyloid β. FIG. 3 shows a standard curve for quantification of PRL28 amyloid β. FIG. 4 is a diagram showing a comparison of quantitative values of PRL16 amyloid β and PRL28 amyloid β in human cerebrospinal fluid. FIG. 4 is a diagram showing a comparison of quantitative values of amyloid β40 and amyloid β42 in human cerebrospinal fluid.

The present disclosure relates to the detection of propanoylated amyloid-β, and more specifically to antibodies for propanoylation site-specific detection of amyloid-β. The scheme of propanoylation of two lysine residues of amyloid β42, which the inventors speculate, is shown in FIG.

According to the antibody disclosed in the present specification, the propanoylation state of two lysine residues in amyloid β, which has not been known at all in the past and has not been paid attention to, was identified. A first propanoylated amyloid β in which lysine and a lysine corresponding to the lysine are propanoylated, a second propanoylated amyloid β in which the lysine at position 28 of amyloid β and a lysine corresponding to the lysine are propanoylated, can be detected independently. According to the present inventors, the cerebrospinal fluid of Alzheimer's disease patients showed different contents of these two propanoylated amyloid-βs. It also differed from the content of primary and secondary propanoylated amyloid β in non-Alzheimer's disease patients. It is considered that the distribution tendency of these two types of propanoylated amyloid β in vivo can be importantly characterized in relation to the onset of Alzheimer's disease and the like. Hereinafter, amyloid β, which the antibody disclosed herein reacts with, will be described, and embodiments of the present disclosure will be described in detail with reference to the drawings as appropriate.

Amyloid-β is generally believed to be produced from the amyloid-β precursor protein by cleavage by β-secretase and γ-secretase. "Amyloid β" as used herein refers to, for example, human amyloid β, which includes amyloid β40 (amyloid β _(1-40) ) having 40 amino acid residues (SEQ ID NO: 1), 42 amino acid residues (SEQ ID NO: 2) amyloid β42 (amyloid β _(1-42) ), 43 (SEQ ID NO: 3) amyloid β43 (amyloid β _(1-43) ), and further amyloid β _(2-42) , Examples include amyloid β _(4-40) and the like, but are not particularly limited, and include amyloid β that may exist in vivo such as humans. In addition, amyloid β in the present specification can include mutants having mutations such as amino acid substitutions and deletions in amyloid β. In this case, the number of mutations in the amino acid sequence represented by SEQ ID NO: 1 is, for example, 1 to 5, or, for example, 1 to 4, or, for example, 1 to 3, or For example, one or two.

Amyloid β with which the antibody disclosed herein reacts is, for example, amyloid β40. It is known that in humans, amyloid β40 is produced in approximately 10 times the amount of amyloid β42. In addition, the amyloid β with which the present antibody reacts is, for example, amyloid β42. It is also known that amyloid β42 is more malignant than amyloid β40 in relation to Alzheimer's disease and the like.

The amyloid β in which the lysine at position 16 of the amino acid sequence represented by SEQ ID NO: 1 is propanoylated means amyloid β40 in which the lysine at position 16 is propanoylated. In addition, the amyloid β in which the lysine corresponding to the lysine at position 16 in the amino acid sequence represented by SEQ ID NO:1 is propanoylated is an amyloid β other than amyloid β40 and has the amino acid sequence represented by SEQ ID NO:1. When aligned, it means amyloid β in which the lysine corresponding to the lysine at position 16 is propanoylated. Therefore, amyloid β42 in which the lysine at position 16 is propanoylated, amyloid β43 in which the same lysine is propanoylated, and the like are included. Alignment can be performed by a person skilled in the art using Protein BLAST or other known databases.

Similarly, amyloid β in which lysine at position 28 in the amino acid sequence represented by SEQ ID NO: 1 is propanoylated means amyloid β40 in which lysine at position 28 is propanoylated. In addition, the amyloid β in which the lysine corresponding to the lysine at position 28 in the amino acid sequence represented by SEQ ID NO:1 is propanoylated is an amyloid β other than amyloid β40 and has the amino acid sequence represented by SEQ ID NO:1. When aligned, it means amyloid β in which the lysine corresponding to the lysine at position 28 is propanoylated. Accordingly, amyloid β42 in which lysine at position 28 is propanoylated, amyloid β43 in which lysine at position 28 is propanoylated, and the like are included.

The structure in which the lysine residue is propanoylated is as shown in FIG.

(antibody)
The antibodies disclosed herein are those whose reactivity to propanoylated amyloid β (first amyloid β) in which the lysine at position 16 of amyloid β or the lysine corresponding to the lysine is propanoylated is It is an antibody (hereinafter also referred to as a first antibody) having higher reactivity with amyloid β (second amyloid β) in which the lysine at the position or the lysine corresponding to the lysine is propanoylated. In addition, other antibodies disclosed herein have reactivity to propanoylated amyloid β (second amyloid β) in which the lysine at position 28 of amyloid β or the lysine corresponding to the lysine is propanoylated, It is an antibody (hereinafter also referred to as a second antibody) having higher reactivity with amyloid β (first amyloid β) in which lysine at position 16 of amyloid β or a lysine corresponding to the lysine is propanoylated. These antibodies are described below in order.

(first antibody)
The first antibody is an antibody that has higher reactivity with the first amyloid β than with the second amyloid β. Here, "reactivity to the first amyloid β" means that the antibody reacts with (typically binds to) amyloid β in which the 16-position lysine of amyloid β is propanoylated. In addition, "reactivity to the second amyloid β" means that the antibody reacts with (typically binds to) amyloid β in which the 28-position lysine of amyloid β is propanoylated. The same applies hereinafter.

Such reactivity possessed by the first antibody can be determined by methods well known to those skilled in the art. Any antibody corresponds to the first antibody if its reactivity to the first amyloid β is higher than its reactivity to the second amyloid β.

The reactivity possessed by the first antibody may have the above reactivity characteristics, but more specifically, for example, the following first aspect can be adopted. That is, the reactivity to the lysine-propanoylated peptide (16PRL peptide) in the amino acid sequence represented by SEQ ID NO: 4 is higher than the reactivity to the lysine-propanoylated peptide (28PRL peptide) in the amino acid sequence represented by SEQ ID NO: 5. Higher than reactive.

Here, the amino acid sequence represented by SEQ ID NO: 4 is the amino acid sequence of positions 11 to 21 of the amino acid sequence of amyloid β40, and contains five amino acid residues before and after the lysine at position 16 of amyloid β. I'm in.

In addition, the amino acid sequence represented by SEQ ID NO: 5 is the amino acid sequence of positions 23 to 33 of the amino acid sequence of amyloid β40, and contains 5 amino acid residues before and after the lysine at position 28 of amyloid β. there is

The reactivity of the first aspect is, for example, that the reactivity of the first antibody to the 16PRL peptide is at least 1.6 times higher than the reactivity to the 28PRL peptide at each concentration of 500 nM (or, for example, 1.7 times or more, for example, 1.8 times or more) higher, and for example, at 167 nM, 1.2 times or more (also, for example, 1.3 times or more, or, for example, 1.4 times or more) higher, and for example, At 55.6 nM, it is 1-fold or more (or, for example, 1.1-fold or more, or, for example, 1.2-fold or more). 1 times or more, or for example 1.2 times or more) higher.

The reactivity possessed by the first antibody can further adopt, for example, the following second aspect. That is, the reactivity to the lysine-propanoylated peptide (16PRL peptide-long) in the amino acid sequence represented by SEQ ID NO: 6 was determined by the lysine-propanoylated peptide (16PRL peptide) in the amino acid sequence represented by SEQ ID NO: 4. ) is higher than the reactivity to

Here, the amino acid sequence represented by SEQ ID NO: 6 is the amino acid sequence of positions 1 to 21 of the amino acid sequence of amyloid β40, and the five amino acids on the C-terminal side of the lysine at position 16 from the N-terminus of amyloid β40. including residues.

The reactivity of the second aspect is, for example, that the reactivity of the first antibody to 16PRL peptide-long is 5 times or more (or, for example, 5.2 times or more) than the reactivity to 16PRL peptide at each concentration of 500 nM. , or, for example, 5.3 times or more, also, for example, 5.4 times or more) higher, and for example, 2.5 times or more (or, for example, 2.6 times or more, also, for example, 2.7 times) at the same 167 nM times or more, for example, 2.8 times or more, or for example, 2.9 times or more) higher, and for example, 1.3 times or more at the same 55.6 nM (also, for example, 1.4 times or more, also for example, 1.5 times or more, for example, 1.6 times or more) higher, and for example, 1 times or more (or, for example, 1.1 times or more, or for example, 1.2 times or more) higher at 18.5 nM.

The first antibody further has, for example, a higher reactivity to the first amyloid β than an antibody to amyloid β in which the lysine at position 16 of amyloid β or the lysine corresponding to the lysine is not propanoylated. is.

The first antibody may have such reaction characteristics, but more specifically, the following aspects can be adopted. That is, the reactivity to the 16PRL peptide or 16PRL-long is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 4 in which the lysine residue is not PRL (hereinafter also referred to as 16 peptide) or the lysine residue is PRL This embodiment has a higher reactivity than the peptide consisting of the amino acid sequence represented by SEQ ID NO: 6 (hereinafter also referred to as 16 peptide-long).

The reactivity of this aspect is, for example, that the reactivity of the first antibody to the 16PRL peptide is 1.3 times or more (or, for example, 1.4 times or more, or For example, 1.5 times or more) higher, for example, 1.2 times or more at the same 167 nM (also, for example, 1.3 times or more, also, for example, 1.4 times or more, or for example, at the same 55.6 nM) at 1 times or more (also, for example, 1.1 times or more, also, for example, 1.2 times or more), and for example, at 18.5 nM at 1 times or more (also, for example, 1.1 times or more, also for example, 1.2 times or more) high.

Further, for example, the reactivity of the first antibody to 16PRL peptide-long is 7 times or more (or, for example, 7.2 times or more, or, for example, 7.2 times or more) than the reactivity to 16 peptide-long at each concentration of 500 nM. 4 times or more, or, for example, 7.7 times or more) higher, or, for example, 3.5 times or more (or, for example, 3.6 times or more, or, for example, 3.8 times or more) higher at 167 nM, and For example, at 55.6 nM, it is 1.5 times or more (also, for example, 1.6 times or more, or, for example, 1.7 times or more), and for example, at 18.5 nM, it is 1 time or more (also, for example, 1.1 times or more, or for example, 1.2 times or more) higher.

(Second antibody)
The second antibody is an antibody that has higher reactivity with the second amyloid β than with the first amyloid β. Here, "reactivity to the second amyloid β" and "reactivity to the first amyloid β" are as already explained.

Such reactivity possessed by the second antibody can be determined by methods well known to those skilled in the art. Any antibody corresponds to a second antibody if its reactivity to the second amyloid β is higher than the reactivity to the first amyloid β.

The reactivity possessed by the second antibody may have the reactivity characteristics as described above, but more specifically, for example, the following first aspect can be adopted. That is, the reactivity to the lysine-propanoylated peptide (28PRL peptide) in the amino acid sequence represented by SEQ ID NO: 5 is the same as the reactivity to the lysine-propanoylated peptide (16PRL peptide) in the amino acid sequence represented by SEQ ID NO: 4. Higher than reactive.

The reactivity of the first aspect is, for example, that the reactivity of the second antibody to the 28PRL peptide is 4.5 times or more (or, for example, 4.6 times or more) at each concentration of 50 nM than the reactivity to the 16PRL peptide. , or, for example, 4.7 times or more) higher, or, for example, 2.5 times or more at the same 16.7 nM (also, for example, 2.6 times or more, also, for example, 2.7 times or more, or for example, 2.8 times or more) ) higher, for example, 1.7 times or more (also, for example, 1.8 times or more, also, for example, 1.9 times or more) at the same 5.56 nM, and for example, 1.5 at the same 1.85 nM times or more (also such as 1.6 times or more, also such as 1.7 times or more).

The second antibody further has, for example, a higher reactivity to the second amyloid β than an antibody to amyloid β in which the lysine at position 28 of amyloid β or the lysine corresponding to the lysine is not propanoylated. is.

The second antibody may have such reaction characteristics, but more specifically, it can adopt the following aspects. That is, in this embodiment, the reactivity to the 28PRL peptide is higher than the reactivity to the peptide consisting of the amino acid sequence represented by SEQ ID NO: 5 in which the lysine residue is not PRL-modified (hereinafter also referred to as the 28peptide).

The above-described comparison of the reactivity of the first and second antibodies is performed by determining the antigen to which the antibody originally binds for each antibody using competitive ELISA using a substance to be compared as a competitor. can be done.

Such antibodies may be polyclonal antibodies and monoclonal antibodies, but are preferably monoclonal antibodies. Moreover, such an antibody may be biotinylated so that it can be complexed with streptavidin or the like. Moreover, in addition to human antibodies, if the origin is not human, it may be a humanized antibody obtained by a method well known to those skilled in the art.

(Preparation of antibody)
A person skilled in the art can prepare an antibody having the above reactivity by appropriately adopting a method well known to those skilled in the art. For example, the antibody can be prepared by the following method.

(Preparation of antigen)
An overview of antigen production is shown in FIG. The antigen for producing the antibody that specifically binds to the first propanoylated amyloid β (hereinafter simply referred to as the first antibody) is lysine at position 16 in the amino acid sequence represented by SEQ ID NO: 1 (hereinafter , 16Lys) is prepared in advance, and using such propanoylated lysine, an amino acid containing, for example, about 3 to 8 amino acid residues before and after, typically 5 amino acid residues, is prepared. Synthesize a peptide of sequence. In addition, propanoylation of lysine can be carried out, for example, as follows.

57 μl of propionic anhydride was added to 105 mg of Nα-[(9-fluorenylmethoxy)carbonyl]-lysine (Nα-Fmoc-Lys) dissolved in 500 μl of 83% methanol and allowed to react at room temperature for 4 hours. After that, 57 μl of propionic anhydride is further added and reacted at room temperature. After the addition, the reaction solution was further reacted for 4 hours, and the propanoylated Fmoc-Lys was purified by high performance liquid chromatography. Purification conditions are separation conditions using a single solvent, and the solvent used is H ₂ O containing 0.1% trifluoroacetic acid/55% acetonitrile. The column used for separation was C30-UG-5 (10φ×250 mm), and the flow rate was 2 ml/min. The propanoylated Fmoc-lysine fractionated and purified in this manner is powdered by a rotary evaporator and freeze-dried, and then used as a material for peptide synthesis.

This 16Lys-propanoylated peptide was conjugated with a known carrier protein KLH (keyhole limpet hemocyanin) or BSA (bovine serum albumin) (PRL16-KLH or PRL16-BSA) via a Cys residue attached to the N-terminus. can be used as an antigen for producing antibodies. The carrier protein is not particularly limited, and a protein such as OVA (ovalbumin) or a protein bound or polymerized with a polymer can be used as appropriate, but a carrier is not necessarily required.

Similarly, the antigen for producing an antibody that specifically binds to the second propanoylated amyloid β (hereinafter, simply referred to as the second antibody) is also the 28th position in the amino acid sequence represented by SEQ ID NO: 1. A propanoylated lysine for use in lysine (hereinafter also simply referred to as 28Lys) is prepared in advance, and the propanoylated lysine is used to prepare a peptide consisting of an appropriate number of amino acid residues containing 28-position lysine. to synthesize. This 28Lys propanoylated peptide is conjugated with a known carrier protein KLH or BSA (PRL28-KLH or PRL28-BSA) via the Cys residue attached to the N-terminus, and used as an antigen for antibody production. do.

In addition, as an antigen used for screening to improve the binding specificity of the first antibody, the above 16Lys neighborhood sequence includes an amino acid sequence portion closer to the N-terminus, and a long sequence of about 16Lys + 5 residues C A long-type antigen (long PRL16-BSA) can also be prepared by introducing a Cys residue into the terminal side and complexing the C-terminal side with a carrier protein. Furthermore, for the same purpose, a native type antigen (Native PRL16-BSA) consisting of a natural amino acid sequence in which 16Lys is not propanoylated in the long sequence and conjugated with a carrier protein at the C-terminus can also be prepared. can.

(Immunity)
An immunizing antigen (KLH complex) for producing the first antibody and the second antibody is administered, optionally together with a complete adjuvant, to a known antibody-producing animal such as a mouse by intraperitoneal administration or the like. , primary immunization, followed by booster immunizations with incomplete adjuvant at regular intervals. Blood is collected from the animal in a timely manner, the serum is used as the primary antibody, and the antibody titer is evaluated by the ELISA method using various antigens that are BSA complexes, and after confirming a sufficient increase in the antibody titer. , the final immunization is performed about 3 days before the slaughter, the animals are sacrificed, and the spleen is removed. The animals used for immunization are not particularly limited, but rabbits, goats, sheep, hamsters, mice, rats, guinea pigs, chickens, non-human animals that produce human antibodies, and the like can all be used.

(Establishment of hybridoma cells)
Spleen cells obtained from the spleens of sacrificed animals are prepared, and the spleen cells are fused with myeloma cells selectably lacking a specific gene by a known method using polyethylene glycol 1500 or the like. As the immortalization method, a known method other than cell fusion can also be used. For example, transformation using Epstein-Barr virus (D. Kozbor et al., Eur J Immunol, 14:23 (1984)) can also be used.

A selective medium is then used to select only the hybridoma cells. Hybridoma cells having an antibody titer against PRL16-BSA can be screened and cloned using PRL16-BSA and, if necessary, long PRL16-BSA to obtain a single clone that produces the first antibody. can. In addition, screening and cloning using PRL28-BSA for hybridoma cells having an antibody titer against PRL28-BSA can yield a single clone that produces the second antibody.

(Obtaining antibodies)
The first antibody can be obtained from the culture supernatant of the first antibody-producing hybridoma cells. Any suitable protein purification method can be used, but it is convenient to use affinity chromatography. Similarly, the second antibody can be obtained from the culture supernatant of the second antibody-producing hybridoma cells.

The specificity of the first antibody and the second antibody can be evaluated by indirect ELISA, competitive ELISA, or the like. Also, the characteristic reactivity of the first antibody and the second antibody can be determined using the competitive ELISA disclosed in the Examples.

The first antibody specifically detects 16Lys, and the second antibody specifically detects 28Lys. , will be bound by both the first antibody and the second antibody.

Humanized antibodies are non-human (mouse, rat, hamster, rabbit, etc.) antibodies that are humanized (hereinafter referred to as humanized antibodies) are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab', F(ab') ₂ or other antigen-binding subsequence of an antibody) that contains minimal sequence derived from non-human immunoglobulin. Humanized antibodies, particularly antibodies constructed partially or wholly from amino acid sequences derived from human antibody germline by altering the sequence of the antibody with the complementarity determining regions (CDRs) of non-human antibodies is preferred. Such alterations can be accomplished by replacing non-human antibody constant regions with human antibody constant regions to create human/non-human chimeras with acceptable low immunogenicity for pharmaceutical use. is. More preferably, the variable regions and even the CDRs of the antibodies are also humanized by techniques that are by now well known in the art. The framework regions of the variable region are replaced by the corresponding human framework regions and the non-human CDRs are either substantially unchanged or may be replaced with sequences from the human genome.

A humanized antibody further refers to one comprising a human framework and at least one CDR from a non-human antibody, wherein any constant regions present are substantially identical to human immunoglobulin constant regions. Substantially identical refers to at least 85-100%, preferably 95-100%, amino acid sequence identity. That is, the humanized antibody will be identical in all portions, except for the CDR portions, to portions that correspond to one or more naturally occurring human immunoglobulin sequences.

Methods for humanizing non-human antibodies are well known in the art. For example, Winter et al. (Patent No. 2912618), Jones et al. (Nature, 321: 522 (1986)), Riechmann et al. (Nature, 332: 323 (1988)), Verhoeyen et al. , 239: 1534 (1988)), the method of Queen et al. (Proc. Natl. Acad. Sci. USA 88: 2869 (1991)), and the like. In the process of obtaining humanized antibodies, it is desirable to perform silent mutation of codons for the purpose of optimizing expression in host cells expressing humanized antibodies (for example, the method of Nakamura et al.: Nuclecic Acid Res 29: 292 (2000) ). An antibody thus obtained is characterized by having an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the variable region, as long as it has the specificity described in the present application. Also included in the present invention is a humanized antibody.

(Method for detecting propanoylated amyloid β)
By using the first antibody and/or the second antibody, propanoylated amyloid β can be detected and quantified in a propanoylated modification site-specific manner. For example, the method for detecting propanoylated amyloid β disclosed in the present specification (hereinafter also simply referred to as the present detection method) is amyloid β in which two lysine residues of amyloid β are each modified with a propanoyl group. can be provided.

(subject)
In this detection method, a biological sample in which amyloid β may be present can be used as a test subject without any particular limitation. Examples include body fluids such as cerebrospinal fluid containing cerebrospinal fluid, blood, urine, and tears, as well as tissues (slices) in which various nerve cells are present, such as the brain and eyes. In particular, it is preferable that the subject is an individual suffering from a neurodegenerative disease such as Alzheimer's disease, which may accumulate, produce, or be involved in amyloid β, or the neurodegenerative disease model animal. In addition, nasal discharge and nasal mucosa can also be tested.

Such neurodegenerative diseases include Alzheimer's disease, vascular amyloid lesions, amyloidosis, and the like.

In this detection method, for example, such antibodies can be used to detect the first propanoylated amyloid β and the second propanoylated amyloid β, respectively. For example, ELISA, enzyme immunoassay such as competitive ELISA, dot blotting, western blotting, affinity chromatography, tissue staining, and various detection methods using antibodies can be used to detect amyloid β in various test subjects. can be done.

In this detection method, each of these propanoylated amyloid βs is detected, and not only detection but also relative or absolute amounts can be quantified.

In this detection method, only the first propanoylated amyloid β may be detected, only the second propanoylated amyloid β may be detected, or both may be detected. Alternatively, these may be detected simultaneously.

By detecting the first propanoylated amyloid β and / or the second propanoylated amyloid β in various test subjects, it is detected according to the oxidation state of amyloid β, and its distribution in nerve tissue and cerebrospinal cord By evaluating the concentration in body fluids such as fluid and blood, we will investigate the relationship between amyloid-β acylation (propanoylation is oxidation caused by lipid peroxides) and neurodegenerative diseases such as Alzheimer's disease. Characterization is possible.

For example, 1 selected from the group consisting of the amount of the first propanoylated amyloid β, the amount of the second propanoylated amyloid β, and the amount of the second propanoylated amyloid β/the amount of the first propanoylated amyloid β A species or two or more can be associated with any of the incidence, severity, prognosis and complications of neurodegenerative diseases such as Alzheimer's disease. Also, using such an association, one or more of these numerical values can be used as a marker for diagnosing such neurodegenerative diseases.

(Method for detecting amyloid β associated with diagnosis of Alzheimer's disease)
The method for detecting amyloid β related to the diagnosis of Alzheimer's disease disclosed in the present specification includes lysine at position 16 of amyloid β42 in spinal fluid, blood, nasal discharge and nasal mucosa of Alzheimer's disease patients or a lysine corresponding to the lysine. Either or both of the first propanoylated amyloid β in which is propanoylated and the second propanoylated amyloid β in which the lysine at position 28 of amyloid β42 or the lysine corresponding to the lysine is propanoylated, independently and detecting. According to this method, the diagnosis of Alzheimer's disease can be aided by propanoylation site-specific detection of amyloid β in association with the diagnosis of Alzheimer's disease.

In view of the above, the first antibody and/or the second antibody can be used as reagents for characterization associated with neurodegenerative diseases. Also, the first antibody and/or the second antibody can be used as reagents for characterization associated with neurodegenerative diseases such as Alzheimer's disease.

(Screening method for compounds reactive with propanoylated amyloid β)
The screening method disclosed herein comprises a step of contacting one or more test compounds with a first antibody and/or a second antibody, and the one or more test compounds and the antibody and evaluating the reactivity of the one or more test compounds with propanoylated amyloid β based on the reactivity. Since the first antibody and the second antibody specifically bind to the propanoylated amyloid β and the propanoylated modification site, this reactivity is used to obtain the first propanoylated amyloid β and the second propanoylated amyloid Compounds with binding properties to β can be screened. binding of the test compound to the first propanoylated amyloid-β and/or the second propanoylated amyloid-β by competing the first antibody and/or the second antibody with the potential test compound Reactivity can be evaluated. Using this binding reactivity as an index, a compound that specifically binds to the first propanoylated amyloid β and/or the second propanoylated amyloid β can be selected.

Such screened compounds, like the first antibody and the second antibody, bind to the first propanoylated amyloid β and/or the second propanoylated amyloid β, and are therefore used for the treatment, prevention, and diagnosis of Alzheimer's disease. It is effective for characterization such as

The test compound in this screening method is not particularly limited, but examples include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. These compounds may be novel compounds or known compounds. Reactivity with the first and second propanoylated amyloid β is evaluated by ELISA, competitive ELISA, affinity chromatography, dot blotting, etc., in the same manner as the reaction specificity evaluation method for the first and second antibodies. can. The test compound may be labeled with a known labeling substance or the like.

Examples are given below as specific examples in order to more specifically describe the disclosure of the present specification. The following examples are intended to illustrate the disclosure herein, but not to limit its scope.

(Preparation of Antigen for Antibody Preparation)
Since amyloid β has two (16th (Lys16) and 28th (Lys28) from the N-terminus) lysine residues that can be PRL-conjugated, an antibody that can individually recognize these two PRL-conjugation sites. (PRL conversion of 16th lysine residue: mAb BST31, PRL conversion of 28th lysine residue: mAb3A11) were prepared. First, as shown in FIG. 2, as shown in the upper two types (PRL16-antigen, PRL28-antigen), lysine residues targeted to carrier proteins (keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA)) An antigen (PRL16-antigen (KLH or BSA)) for producing an antibody that recognizes the PRL conversion of Lys16 and an antibody that recognizes the PRL conversion of Lys28 by binding a peptide having five amino acid residues around the group. An antigen (PRL28-antigen (KLH or BSA)) for

With respect to PRL16-antigen, LongPRL16-antigen (containing the 21st amino acid sequence from the N-terminus of the Aβ sequence) having a long peptide chain and Native16-antigen, which is an unmodified form, have a long peptide chain for the purpose of producing more specific antibodies. was made. Peptides used for all antigens were also prepared in a free form without being bound to a carrier protein.

(Obtaining antibodies)
Among the antigens prepared in Example 1, two antigens, PRL16-antigen (KLH) or PRL28-antigen (KLH), were added to female BALB/c mice (Freund's complete adjuvant, FCA) together with 6-week-old) were administered intraperitoneally for initial immunization. Thereafter, booster immunizations were given biweekly with Freund's incomplete adjuvant (FIA). Specifically, it was implemented as follows.

(1) Immunization by mouse intraperitoneal administration For the initial immunization, an emulsion was formed with 150 μl of PRL16-KLH (1 mg/ml) or PRL28-KLH (1 mg/ml) + 350 μl of phosphate buffered saline (PBS) + 500 μl of FCA, and 200 μl of the emulsion was formed. /mouse was administered intraperitoneally. A booster immunization was performed two weeks after the first immunization. An emulsion was formed with 150 μl of PRL16-KLH (1 mg/ml) or PRL28-KLH (1 mg/ml)+350 μl of PBS+500 μl of FIA, and 200 μl/animal was intraperitoneally administered every other week.

Blood was collected from the tail vein, and antibody titers against various antigens (native BSA, PRL-BSA (an antigen obtained by directly converting BSA to PRL), Native16-BSA, PRL16-BSA, LongPRL16-BSA, PRL28-BSA) were determined by ELISA. was confirmed over time. That is, one week after each booster immunization, blood was collected from the tail vein of the mouse, and the antibody titer against various antigens was examined by ELISA using the serum obtained by centrifugation as the primary antibody. In addition, the ELISA method was implemented as follows.

(1) Coating (solid phase of antigen on 96-well plate):
Native BSA (1 mg/ml), PRL-BSA (1 mg/ml), Native16-BSA (1 mg/ml), LongPRL16-BSA (1 mg/ml), PRL16-BSA (1 mg/ml), PRL28-BSA (1 mg/ml) ml) was diluted with PBS to 0.5 μg/ml, and 100 μl/well was spread on a 96-well plate. After that, it was allowed to stand overnight at 4°C.
(2) Washing: Washing was performed three times with 200 μl/well of PBS containing 0.05% Tween (TPBS).
(3) Blocking:
A 1% solution of powdered block ace was prepared with ultrapure water and sprayed at 200 μl/well. After that, the mixture was kept at 37°C for 1 hour.
(4) Washing: Washed 3 times with 200 μl/well of TPBS.
(5) Primary antibody:
Serum, hybridoma culture supernatant, and purified antibody to be measured for antibody titer were diluted 1000-fold with TPBS, serially diluted 3-fold, and each diluted solution was sprayed at 100 μl/well. After that, the mixture was kept at 37°C for 1 hour.
(6) Washing: Washed 3 times with 200 μl/well of TPBS.
(7) secondary antibody:
A horseradish peroxidase (HRP)-labeled anti-mouse IgG antibody was diluted 5000-fold with TPBS and sprayed at 100 μl/well. After that, the mixture was kept at 37°C for 1 hour.
(8) Washing: Washed 3 times with 200 μl/well of TPBS.
(9) Color development and measurement:
3,3′,5,5′-Tetramethylbenzidine (TMB) coloring solution [1% TMB: 40 mM citrate phosphate buffer (pH 5.0): 30% H ₂ O ₂ = 50:5000:2 mixed 100 μl/well of the mixed solution] was applied to develop color. After developing the color for 10 minutes at room temperature, the plate was stopped with 1N phosphoric acid (100 μl/well) and the absorbance at 450 nm was measured with a plate reader.

After confirmation of an increase in antibody titer, mice were sacrificed, and PRL16-KLH antigen + sterilized PBS suspension or PRL28-KLH antigen + sterilized PBS suspension was administered to the tail vein 3 days before removal of the spleen for final immunization. were sacrificed and the spleen was removed.

(2) Establishment of hybridoma cells Next, in the serum of mice immunized with each antigen, PRL16-amyloid β antibody from mice with antibody titers against PRL16-BSA and LongPRL16-BSA, and mice with antibody titers against PRL28-BSA. established a PRL28-amyloid β antibody. That is, by fusing myeloma cells that can be semipermanently maintained in culture with spleen cells obtained from each mouse, hybridoma cells that can continue to produce antibodies semipermanently were established. Specifically, it was implemented as follows.

Splenocytes were prepared from the spleens of sacrificed mice and fused with myeloma cells (P3U1) via polyethylene glycol 1500. Then, by culturing the fused cells using HAT medium, only cells that became hybridomas were selected. In HAT medium, aminopterin inhibits the DNA synthesis pathway via the de novo pathway and promotes DNA synthesis in the salvage pathway. It cannot synthesize pathway-dependent DNA, and cannot proliferate or survive. However, since spleen cells themselves have this HGPRT, myeloma cells fused with spleen cells, that is, hybridoma cells can synthesize DNA and survive in HAT medium. After selection in HAT medium in this manner, ELISA was performed using the culture supernatant to select only hybridomas producing the antibody of interest. ELISA was performed according to the ELISA protocol described above.

By repeating selection (screening and cloning) of these cells, a single clone was established as the antibody-producing strain. The specific antibody-producing clone against PRL16-Aβ was named "BST31", and the specific antibody-producing clone against PRL28-Aβ was named "3A11". Hereinafter, the antibodies are referred to as mAb BST31 and mAb3A11.

Regarding the establishment of this clone, not only PRL16-BSA but also LongPRL16-BSA were screened in parallel during screening in ELISA after cell fusion. A selection of non-specific antibodies that would also have cross-reactivity to PRL28-BSA was eliminated. In addition, since it can recognize PRL conversion of lysine residues in peptides having more amino acid sequences of amyloid β, it is more specific for "PRL conversion of the 16th lysine residue in the amyloid β sequence". It can be said that this led to the establishment of antibodies with high

(3) Purification of Each Antibody Using a Protein G Column Antibodies were purified from the culture supernatant of antibody-producing hybridoma cells by affinity purification using a protein G column. After purifying each antibody, the protein was quantified, prepared at a concentration of 1 mg/ml as the antibody concentration, and stored at -30°C or -80°C. Affinity chromatography was performed using a peristaltic pump or manually as follows.

(1) A Protein G column was connected and equilibrated while running ultrapure water through the line.
(2) Substitution with 0.02 M phosphate buffer (pH 7.0) (PB) and equilibration for 20 minutes.
(3) After equilibration with 0.02 M PB, the column was replaced with the culture supernatant, and the entire culture supernatant was passed through the column.
(4) After flowing the culture supernatant through the column, the column was washed with 0.02M PB for 20 minutes.
(5) 75 µl of 1 M Tris aqueous solution was added to five 1.5 ml tubes during washing.
(6) After washing with 0.02M PB, it was eluted with 0.1M Glysine/HCl (pH 2.5). 1 ml of the eluate was collected in each 1.5 ml tube containing the 1 M Tris aqueous solution (collected as fractions for a total of 5 tubes).
(7) The protein content of each fraction was measured by BCA assay, and antibody-eluted fractions were identified.
(8) The antibody-eluted fraction was collected, dialyzed against PBS at 4°C for 2 days, adjusted to a concentration of 1 mg/ml, and stored at -30°C or -80°C.

(Specificity analysis of established monoclonal antibodies mAb BSA31 and mAb3A11)
(1) Examination by Indirect ELISA Using purified monoclonal antibodies, cross-reactivity to each antigen was verified. For mAb BST31, use native BSA, PRL-BSA, Native16-BSA, PRL16-BSA, LongPRL16-BSA, PRL28-BSA, and for mAb3A11, use Native BSA, PRL-BSA, PRL16-BSA, PRL28-BSA Then, it was performed according to the ELISA method protocol described above. As a result, mAb BST31 was found to cross only two species, PRL16-BSA and LongPRL16-BSA, and was found to react more strongly to LongPRL16-BSA. Therefore, it was decided to use LongPRL16-BSA (0.5 μg/ml, 100 μl/well) containing longer amino acid residues and containing 21 residues, which is about half of amyloid β, as an antigen for competitive ELISA coating. Antibody concentrations of mAb BST31 were set to 0.2 μg/ml and 50 μl/well. In mAb 3A11 cross-reactivity was observed only to PRL28-BSA. Therefore, PRL28-BSA (0.5 μg/ml, 100 μl/well) was used as the antigen for coating, and the mAb3A11 antibody concentration was 0.1 μg/ml, 50 μl/well for competitive ELISA. did.

(2) Examination by Competitive ELISA Method Competitive ELISA is an experiment for verifying the specificity of the prepared antibodies. Competitive ELISA is performed by coexisting an antibody, an antigen on a plate, and a competing substance to determine whether or not the competitor inhibits the antibody from crossing with the antigen on the plate. It is an experiment that can examine the sex. The conditions for each antigen and antibody used were as described above, and the ELISA method was performed according to the following protocol. The results are shown in FIGS. 3 and 4. FIG.

(1) Coating (solid phase of antigen on 96-well plate):
For competitive ELISA using mAb BST31: LongPRL16-BSA (0.5 μg/ml, 100 μl/well),
For competitive ELISA using mAb 3A11: PRL28-BSA (0.5 μg/ml, 100 μl/well) was spread on a 96-well plate. After that, it was allowed to stand overnight at 4°C.
(2) Washing: Washed 3 times with 200 μl/well of TPBS.
(3) Blocking:
A 1% solution of powder block ace was prepared with ultrapure water and sprayed at 200 μl/well. After that, the mixture was kept at 37°C for 1 hour.
(4) Washing: Washed 3 times with 200 μl/well of TPBS.
(5) Primary antibody: The peptides listed below were added simultaneously with each antibody.
mAb BST31: PRL16-peptide, PRL28-peptide, Long16-peptide, Native16-peptide (free peptides to which the carrier protein of each antigen is not bound) were diluted in three stages with TPBS with an upper limit of 500 nM as competitors. was added at 50 µl/well.
mAb 3A11: PRL16-peptide and PRL28-peptide were diluted in three stages with TPBS with an upper limit of 50 nM and added at 50 μl/well as competitors. Both were then left overnight at 4°C.
(6) Washing: Washed 3 times with 200 μl/well of TPBS.
(7) secondary antibody:
A horseradish peroxidase (HRP)-labeled anti-mouse IgG antibody was diluted 5000-fold with TPBS and sprayed at 100 μl/well. After that, the mixture was kept at 37°C for 1 hour.
(8) Washing: Washed 3 times with 200 μl/well of TPBS.
(9) Color development and measurement:
3,3',5,5'-Tetramethylbenzidine (TMB) coloring solution [1% TMB: 40 mM citrate phosphate buffer (pH 5.0): 30% H ₂ O ₂ = 50: 5000: 2 mixed 100 μl/well of the mixed solution] was applied to develop color. After developing the color for 20 minutes at room temperature, the plate was stopped with 1N phosphoric acid (100 μl/well) and the absorbance at 450 nm was measured with a plate reader.

As shown in FIG. 3, mAb BST31 does not cross Native16-peptide and PRL28-peptide, but crosses only PRL16-peptide and LongPRL16-peptide, and among these antibodies crosses LongPRL16-peptide more strongly. was found to be In addition, as shown in FIG. 4, mAb 3A11 was found to be an antibody that crosses only to PRL28-peptide, but not to PRL16-peptide. From the above, it was confirmed that these antibodies are capable of site-specifically detecting PRL conversion in the amyloid β sequence.

(Biotinization for higher sensitivity of each antibody)
Biotin is a low-molecular weight compound found in egg yolk and has the property of forming a strong non-covalent bond with avidin in egg white. Therefore, by binding a plurality of biotins to an antibody, a large amount of avidin can be bound to the biotinylated antibody, and detection sensitivity can be increased via avidin. Therefore, biotin labeling of mAb BST31 and mAb3A11 was performed. The biotin labeling was confirmed by using Streptavidin-HRP (BD Biosciences) as a probe for antibody detection.

(1) 0.0023 g of succinimidyl-6-(biotinamide) hexanoate (EZ-link NHS-LC-Biotin, Thermo Fisher) was dissolved in 500 μl N,N-Dimethylformamide (DMF) (biotinylation reagent).
(2) 27 μl of the above biotin reagent was added to 2 mg each of mAb BST31 and mAb 3A11 (in PBS solution).
(3) Leave on ice for 2 hours.
(4) 1 L of PBS was dialyzed for 2 days using a cellophane membrane (Spectra/Por 7 Dialysis Membrane MWCO 10000, SPECTRUM LABORATORIES).
(5) Protein concentration was measured by BCA assay, mAb BST31 was adjusted to 0.8 mg/ml and mAb3A11 to 0.5 mg/ml, and stored at -30°C.

(Construction of competitive ELISA quantification method using mAb BST31)
A method for quantification of PRL16-Aβ was constructed by a competitive ELISA based on Biotin-labeled mAb 3A11. As a result of examining various conditions, the following protocol was developed. A calibration curve thus created is shown in FIG.

(Competitive ELISA-PRL16-method for quantifying amyloid β)
なお、本定量法においては、９６Ｆ ＭＡＸＩＳＯＲＰ ＢＬＡＣＫ ＭＩＣＲＯＷＥＬＬ、ＳｔａｒｔｉｎｇＢｌｏｃｋ ^TM （ＰＢＳ）Ｂｌｏｃｋｉｎｇ Ｂｕｆｆｅｒ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ）及びＰｉｅｒｃｅ ^TM Ｈｉｇｈ Ｓｅｎｓｉｔｉｖｉｔｙ Ｓｔｒｅｐｔａｖｉｄｉｎ－ＨＲＰ（Ｐｉｅｒｃｅ）、ＳｕｐｅｒＳｉｇｎａｌ（登録商標）ＥＬＩＳＡ Ｆｅｍｔｏ Ｍａｘｉｍｕｍ Ｓｅｎｓｉｔｉｖｉｔｙ Ｓｕｂｓｔｒａｔｅ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Scientific) was used.

(procedure)
(1) Coating (solid phase of antigen on 96-well plate):
LongPRL16-BSA (1 mg/ml) was diluted 500,000 times with PBS, and 100 μl of the diluted solution was sprayed on each well of 96F MAXI SORP BLACK MICROWELL. After that, it was allowed to stand overnight at 4°C.
(2) Washing: Washed 5 times with 200 μl/well of TPBS solution.
(3) Blocking:
StartingBlock ^™ (PBS) Blocking Buffer was sprinkled at 200 μl/well and allowed to stand at room temperature for 30 minutes.
(4) Washing: Washed 5 times with 200 μl/well of TPBS solution.
(5) Competitive reaction:
・Competitor preparation (for standard curve creation)
Long PRL16-Peptide
It was adjusted to 1000 nM with TPBS and serially diluted 10-fold with this as the upper limit. 50 μl/well was added to 96 wells.
- Undiluted human cerebrospinal fluid was directly added at 50 μl/well.
- Preparation of primary antibody mAb BST31-biotin (0.8 mg/mL) was diluted 50,000 times with TPBS and used.
When adding the above prepared solutions to the 96-well plate, the competitor (LongPRL16-peptide) or human cerebrospinal fluid was added first, and then the primary antibody was added.
After the above addition, the mixture was allowed to stand overnight at 4°C.
(6) Washing: Washed 5 times with 200 μl/well of TPBS solution.
(7) secondary antibody:
Pierce ^™ High Sensitivity Streptavidin-HRP was diluted 100,000 times with TPBS and sprayed at 100 μl/well.
After that, the mixture was kept at 37°C for 1 hour.
(8) Washing: Washed 5 times with 200 μl/well of TPBS solution.
(9) Chemiluminescence:
SuperSignal (registered trademark) ELISA Femto Maximum Sensitivity Substrate was added at 100 µl/well, chemiluminescence was caused, and the luminescence intensity was measured with a plate reader 1 minute after the addition.

(Construction of competitive ELISA quantification method using mAb3A11)
A method for quantification of PRL28-Aβ was constructed by a competitive ELISA based on Biotin-labeled mAb 3A11. As a result of examining various conditions, the following protocol was developed. FIG. 6 shows the calibration curve thus created.

(Competitive ELISA-PRL28 amyloid β quantification method)
In this quantification method, F96 CERT MAXISORP NUNC-IMMUNO PLATE, Block Ace Powder (DS Pharma Biomedical Co., Ltd.), and Pierce ^™ High Sensitivity Streptavidin-HRP (Pierce) were used.

(1) Coating (solid phase of antigen on 96-well plate):
PRL28-BSA (1 mg/ml) was diluted 8000-fold with PBS, and 100 μl/well was sprayed on a 96-well plate. After that, it was allowed to stand overnight at 4°C.
(2) Washing: Washed 3 times with 200 μl/well of TPBS solution.
(3) Blocking:
A 1% solution of Block Ace powder was prepared with ultrapure water and sprayed at 200 μl/well. After that, the mixture was kept at 37°C for 1 hour.
(4) Washing: Washed 3 times with 200 μl/well of TPBS solution.
(5) Competitive reaction:
・Competitor preparation (for standard curve creation)
PRL28-Peptide
It was adjusted to 280 nM with TPBS, and serially diluted 3-fold with this as the upper limit. 50 μl/well was added to 96 wells.
- Undiluted human cerebrospinal fluid was directly added at 50 μl/well.
- Preparation of primary antibody mAb 3A11-biotin (0.5 mg/mL) was diluted 40,000 times with TPBS and used.
When the above prepared solutions were added to the 96-well plate, the competitor (PRL28-peptide) or human cerebrospinal fluid was added first, followed by the primary antibody.
After the above addition, the mixture was allowed to stand overnight at 4°C.
(6) Washing: Washed 3 times with 200 μl/well of TPBS solution.
(7) secondary antibody:
Pierce ^™ High Sensitivity Streptavidin-HRP was diluted 20,000 times with TPBS and sprayed at 100 μl/well. After that, the mixture was kept at 37°C for 1 hour.
(8) Washing: Washed 3 times with 200 μl/well of TPBS solution.
(9) Color development and measurement:
By spraying 100 μl/well of a TMB coloring solution [1% TMB:40 mM citrate phosphate buffer (pH 5.0):30% H ₂ O ₂ =50:5000:2 mixed solution] colored. After developing the color at room temperature for 20 minutes, it was stopped with 1N phosphoric acid (100 μl/well), and the absorbance at 450 nm was measured with a plate reader.

In addition, in FIG. 6, the cross-reactivity of the antibody to the competitor is expressed as B/B ₀ .
B: Absorbance in the presence of competitors at various concentrations B ₀ : Absorbance of mAb3A11 in the absence of competitors (maximum absorbance during color development)

(Quantification of PRL16-Aβ and PRL28-Aβ from human cerebrospinal fluid)
As described in Example 5, a quantifiable ELISA method was developed using each modified site-specific antibody. Using the calibration curve prepared from these two protocols, each PRL-modified site of Aβ contained in human cerebrospinal fluid was quantified. In addition, a comparison was made between non AD patients (patients who are not AD) and AD patients. Quantitative values of PRL16-Aβ and PRL28-Aβ are shown in FIG. FIG. 8 also shows a comparison of the quantitative values of Aβ40 and 42 in the same cerebrospinal fluid.

As shown in FIG. 7, when each PRL-conjugated site of Aβ in human cerebrospinal fluid was quantified by the developed ELISA method, the 16th PRL-conjugated Aβ was found to be unmodified Aβ (40 and 42) "decrease" compared to the control group (non AD) as in the quantified value (see FIG. 8), whereas the 28th PRL-conjugated Aβ is rather "increased" in AD patient cerebrospinal fluid showed the value. Based on these changes, it is thought that by proposing two new quantification methods as biomarkers for AD, it may be possible to elucidate the onset mechanism of AD and become a biochemical biomarker for diagnosis. The present invention, which is a quantitative method, was considered highly novel.

Regarding the abundance of PRL-Aβ in the cerebrospinal fluid, PRL28-Aβ is 200-600 pM, while PRL16-Aβ is 10 pM or less. It became clear that the abundance of

In AD patients, Aβ42 in the cerebrospinal fluid is said to decrease and tau in the cerebrospinal fluid increases, and by combining these, it is possible to diagnose AD with sensitivity and specificity exceeding 80% (Knopman, DS; DeKosky, ST; Cummings, JL; et al, 2001), and the ratio of toxic oligomers with a specific structure among Aβ oligomers to Aβ42 is significantly increased in human cerebrospinal fluid [Kazuhiro Irie, Kazuma Murakami , Experimental Medicine (Yodosha) Vol.35 No.12 p52-59 (2017). ] have been reported. According to the present disclosure, AD patients had increased PRL28-Aβ in cerebrospinal fluid, which is analogous to elevated toxic Aβ oligomers/Aβ42 ratios in AD patients. From this common point as well, it was speculated that the PRL conversion of the 28th lysine residue in Aβ is accelerated in AD patients during the process of AD onset, and may be a factor in the onset of AD.

Claims (7)

An antibody that specifically binds to propanoylated amyloid β in which the lysine at position 16 of amyloid β42 or the lysine corresponding to the lysine is propanoylated (however, an anti-propanoyl lysine that binds to propanoyl lysine alone) excluding noyllysine antibodies.)
specifically binds to a peptide in which lysine in the amino acid sequence represented by SEQ ID NO: 4 is propanoylated, or specific to a peptide in which lysine in the amino acid sequence represented by SEQ ID NO: 6 is propanoylated an antibody that binds to an antibody,
The antibody is an antibody that specifically binds to propanoylated amyloid β in which the lysine at position 28 of amyloid β42 or the lysine corresponding to the lysine is propanoylated (however, anti-propanoyl lysine that binds to propanoyl lysine alone) excluding antibodies.)
An antibody that specifically binds to a peptide in which a lysine in the amino acid sequence represented by SEQ ID NO:5 is propanoylated. An agent for detecting propanoylation modification sites of propanoylated amyloid β,
A detection agent comprising one or more antibodies selected from the antibodies according to claim 1 or 2 . A reagent for characterization associated with neurodegenerative diseases, comprising one or more antibodies selected from the antibodies of claims 1 or 2 . 5. The reagent of claim 4 , which is a reagent for characterization associated with Alzheimer's disease. A method for detecting propanoylated amyloid β, comprising:
A step of contacting one or more test subjects containing body fluids and tissues that may contain amyloid β with one or more antibodies selected from the antibodies according to claim 1 or 2 ;
The antibody, 16-propanoylated amyloid β in which lysine at position 16 of amyloid β42 or a lysine corresponding to the lysine is propanoylated and/or lysine at position 28 of amyloid β42 or a lysine corresponding to the lysine is propanoylated obtaining reactivity with 28-propanoylated amyloid β;
detecting the 16-propanoylated amyloid β and/or the 28-propanoylated amyloid β based on the reactivity;
A method. A method of screening for a compound reactive with propanoylated amyloid β, comprising:
1 or 2 or more test compounds, 1 or 2 or more antibodies selected from the antibodies according to claim 1 or 2 , and lysine at position 16 of amyloid β42 or a lysine corresponding to the lysine is propanoylated a step of contacting the 16-propanoylated amyloid β and/or the 28-propanoylated amyloid β in which the lysine at position 28 of amyloid β42 or the lysine corresponding to the lysine is propanoylated;
obtaining reactivity between the one or more antibodies and the 16-propanoylated amyloid β and/or the 28-propanoylated amyloid β;
evaluating the reactivity of the one or more test compounds with the 16-propanoylated amyloid β and/or the 28-propanoylated amyloid β based on the reactivity;
A method.

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