Patent Description:
Amylospheroids (ASPD; amylospheroids) are each a spherical AB assembly that is formed of about <NUM> numbers of amyloid-β proteins (AB) aggregated together and has a diameter of about <NUM> to <NUM>. ASPD are structures considered to play an important role in neuronal cell death in Alzheimer's disease.

ASPD were isolated as in vitro-synthesized Aβ aggregates (i.e., synthetic ASPD) that are highly neurotoxic (Non-Patent Document <NUM>). Antibodies specific for the synthetic ASPD were produced (Patent Documents <NUM> and <NUM>), and using these antibodies, ASPD formed in vivo (that is, native ASPD) were actually isolated from the brains of human patients with Alzheimer's disease (Non-Patent Document <NUM>).

Both native ASPD and synthetic ASPD induce cell death selectively in mature neuronal cells. The target of ASPD in this neuronal cell death was found to be "α3 subunit of Na+, K+-ATPase pump (hereafter referred to as "NAKα3")", which is a synaptic protein playing a crucial role in neuronal survival and function, and it was revealed that the ion pump function of NAKα3 is impaired owing to binding of ASPD thereto, thereby causing excessive excitation of neuronal cells leading to the death of those neuronal cells (Patent Document <NUM> and Non-Patent Document <NUM>).

The loss of neurons is most highly correlated with clinical symptoms of Alzheimer's disease. It was revealed that, in Alzheimer's disease patients with loss of neurons, the amount of native ASPD in the cerebral cortex increases in correlation with the severity of the Alzheimer's disease, whereas only a trace amount of native ASPD is seen in the cerebellum of Alzheimer's disease patients with little loss of neurons (Non-Patent Document <NUM>). Accordingly, it is considered that ASPD play an important role in the irreversible stage in the development of Alzheimer's disease. Furthermore, native ASPD were also detected in the brains of Lewy body dementia patients (Non-Patent Document <NUM>), and this suggests that ASPD also play an important role in the development of Lewy body dementia.

Synthetic ASPD considered to be equivalent to native ASPD can be produced by slowly stirring a liquid containing AB (Non-Patent Document <NUM> and Patent Document <NUM>).

Also, cell secreted-type ASPD-like structures, which are considered to be equivalent to native ASPD, can be obtained by being secreted from, for example, CHO cells that express any kinds of APP proteins as described below, and can be obtained from culture supernatant of the CHO cells (Patent Document <NUM>). Non-Patent Document <NUM> discloses that Alzheimer Aβ assemblies accumulate in excitatory neurons upon proteasome inhibition and kill nearby NAKα3 neurons by secretion.

As described above, amylospheroids (ASPD) play an important role in Alzheimer's disease and Lewy body dementia. There is a growing need for analyzing ASPD in biospecimens from subjects (patients) for prevention, diagnostics, treatment, and the like of diseases in which ASPD are involved.

In addition, if a therapeutic agent that targets ASPD is developed, ASPD analysis would be necessary for companion diagnostics.

Analysis (including measurement) of a substance generally requires a reference material (standard). The reference material is used for, for example, calibration or preparation of a calibration curve. Native ASPD, synthetic ASPD, and cell secreted-type ASPD-like structures can be used as a reference material for ASPD analysis.

On the other hand, in the case of a reference material to be included in a diagnostic kit, it is desirable that the reference material can be produced stably, has excellent storage stability, and is produced at a low production cost. As a reference material to be included in a kit for ASPD analysis, native ASPD, synthetic ASPD, and cell secreted-type ASPD-like structures have problems in terms of ease of production, storage stability, production cost, and the like.

Thus, viewed from one aspect, the present disclosure provides a substance that can be a substitute for ASPD or a substance that can be a reference material for ASPD in ASPD analysis.

Viewed from another aspect, the present disclosure provides a method capable of analyzing ASPD in a biospecimen such as, for example, blood or cerebrospinal fluid.

Viewed from one aspect, the present disclosure relates to a substance that competes with ASPD against an ASPD-specific antibody,.

Viewed from another aspect, the present disclosure relates to a cross-linked product of Aβ, wherein the AB is cross-linked using a cross-linking agent that has a spacer arm length of between <NUM>Å and <NUM>Å inclusive or a cross-linking agent that has, as a spacer arm, not less than <NUM> and not more than <NUM> groups that are an oxyethylene group and/or an oxypropylene group.

The present invention relates to a method for analyzing ASPD, including the steps of:.

Viewed from one aspect, the present disclosure can provide a substance that can be a substitute for ASPD or a substance that can be a reference material for ASPD in ASPD analysis.

Viewed from another aspect, the present disclosure can provide a method that can analyze ASPD in a biospecimen such as, for example, blood or cerebrospinal fluid.

Viewed from one aspect, the present disclosure is based on the finding that a cross-linked product of Aβ obtained through cross-linking with the use of a predetermined cross-linking agent exhibits reactivity similar to that of ASPD against ASPD-specific antibodies.

Viewed from another aspect, the present disclosure is based on the finding that ASPD in a biospecimen can be efficiently analyzed using a predetermined combination of antibodies.

In the present disclosure, the term "ASPD" per se may encompass native ASPD, synthetic ASPD, and cell secreted-type ASPD-like structures.

Examples of the ASPD-specific antibody include, in one or more embodiments, polyclonal anti-ASPD antibodies and monoclonal anti-ASPD antibodies disclosed in <CIT> and <CIT>, and in one or more other embodiments, rabbit polyclonal anti-ASPD antibodies (rpASD1, rpASD2, and rpASD3), mouse monoclonal anti-ASPD antibodies (mASD1, mASD2, and mASD3), hamster monoclonal anti-ASPD antibodies (haASD1, haASD2, haASD3, haASD4, and haASD5), and a humanized monoclonal anti-ASPD antibody (huASD2).

AB is a protein that consists of a sequence corresponding to part of an amyloid precursor protein (APP) and has the sequence of a peptide cleaved from APP with β-secretase and γ-secretase in vivo.

In the present disclosure, the term "Aβ" per se may refer to Aβ38 (Aβ1-<NUM>), Aβ39 (AB1-<NUM>), Aβ40 (AB1-<NUM>), Aβ41 (AB1-<NUM>), Aβ42 (AB1-<NUM>), or Aβ43 (AB1-<NUM>), or all of them or only one or some of them.

In the present disclosure, the term "Aβ" per se may encompass human Aβ and nonhuman mammal AB.

In one or more embodiments, examples of human APP include splicing variants such as hAPP770, hAPP751, and hAPP695. Specific examples thereof include, but not limited to, hAPP770 (NCBI Accession No. NP_000475 [VERSION NP_000475. <NUM> GI: <NUM>]).

The N-terminal amino acid of human Aβ (the first amino acid in AB) corresponds to amino acid D (aspartic acid) at position <NUM> in hAPP770.

In the present disclosure, the term "Aβ" per se may encompass wild-type Aβ and mutant AB.

In one or more embodiments, examples of the mutant AB include those having the sequence of a peptide cleaved from mutant APP. The mutant APP may be APP with a mutation linked to familial Alzheimer's disease, and examples of the mutation include the Swedish mutation, the Leuven mutation, the Icelandic mutation, the London mutation, the Iranian mutation, the Austrian mutation, the German mutation, the French mutation, the Florida mutation, the Iberian mutation, the Australian mutation, the Belgian mutation, the Flemish mutation, the Icelandic mutation, the British mutation, the Tottori mutation, the Italian mutation, the Arctic mutation, the Osaka mutation, the Iowa mutation, and the Dutch mutation. Other examples of the mutation include mutations to a modified amino acid, an unnatural amino acid, a D-amino acid, or the like. In one or more embodiments, the modified amino acid may be an amino acid modified on its amino group, carboxyl group, thiol group, or hydroxy group or modified by glycation, PEGylation, or the like.

Viewed from one aspect, the present disclosure relates to a cross-linked Aβ obtained through cross-linking of Aβ using a predetermined cross-linking agent. AB is as described above.

The cross-linking agent used in the cross-linked AB of the present disclosure is, in one or more embodiments, a cross-linking agent that has a spacer arm length of between <NUM>Å and <NUM>Å inclusive. The spacer arm length refers to the molecular span (the distance between bonding molecules) of the cross-linking agent. Since catalogs and instructions for use of commercially available cross-linking agents indicate the spacer arm length of the cross-linking agents, those skilled in the art can select and obtain the cross-linking agent having a desired spacer arm length.

In one or more embodiments, the spacer arm length is <NUM>Å or more, preferably <NUM>Å or more, preferably <NUM>Å or more, more preferably <NUM>Å or more, more preferably <NUM>Å or more, more preferably <NUM>Å or more, still more preferably <NUM>Å or more, still more preferably <NUM>Å or more, still more preferably <NUM>Å or more, still more preferably <NUM>Å or more, still more preferably <NUM>Å or more, and still more preferably <NUM>Å or more, from the viewpoint of allowing the reactivity of the cross-linked AB with ASPD-specific antibodies to be closer to that of ASPD.

In one or more embodiments, the spacer arm length is <NUM>Å or less, preferably <NUM>Å or less, more preferably <NUM>Å or less, still more preferably <NUM>Å or less, yet more preferably <NUM>Å or less, even more preferably <NUM>Å or less, even more preferably <NUM>Å or less, and even more preferably <NUM>Å or less, from the same viewpoint.

In one or more embodiments, the cross-linking agent used in the cross-linked AB according to the present disclosure is a cross-linking agent that has, as a spacer arm, not less than <NUM> and not more than <NUM> groups that are an oxyethylene group(s) (-CH<NUM>CH<NUM>O-, EO group) and/or an oxypropylene group(s) (-CH<NUM>CH<NUM>CH<NUM>O-, PO group). The spacer arm refers to the skeleton or group present between reactive ends of the cross-linking agent.

In one or more embodiments, the total number of EO groups and PO groups in the spacer arm is <NUM> or more, preferably <NUM> or more, more preferably <NUM> or more, and still more preferably <NUM> or more, from the viewpoint of allowing the reactivity of the cross-linked AB with ASPD-specific antibodies to be closer to that of ASPD.

In one or more embodiments, the total number of EO groups and PO groups in the spacer arm is <NUM> or less, preferably <NUM> or less, more preferably <NUM> or less, still more preferably <NUM> or less, yet more preferably <NUM> or less, and even more preferably <NUM> or less, from the same viewpoint.

In one or more embodiments, the cross-linking target of the cross-linking agent used in the cross-linked AB according to the present disclosure may be, for example, an amino group, a sulfhydryl group, or a carboxyl group, or may be nonselective.

In one or more embodiments, the reactive ends of the cross-linking agent used in the cross-linked AB according to the present disclosure are preferably reactive ends that cross-link between amino groups from the viewpoint of allowing the reactivity of the cross-linked AB with ASPD-specific antibodies to be closer to that of ASPD, and such reactive ends may be NHS esters or imidoesters.

The cross-linking agent used in the cross-linked AB according to the present disclosure may or may not be cleavable. From the viewpoint of stability, it is preferable that the cross-linking agent is not cleavable.

In one or more embodiments, the cross-linking agent used in the cross-linked AB according to the present disclosure is preferably ethylene glycol bis(succinimidyl succinate) (EGS, spacer arm length: <NUM>Å), sulfo-EGS (spacer arm length: <NUM>Å), bis(succinimidyl) penta(ethylene glycol) (BS(PEG)<NUM>, spacer arm length: <NUM>Å), or bis(succinimidyl) nona(ethylene glycol) (BS(PEG)<NUM>, spacer arm length: <NUM>Å), from the viewpoint of allowing the reactivity of the cross-linked AB with ASPD-specific antibodies to be closer to that of ASPD. Of these, BS(PEG)<NUM> and BS(PEG)<NUM> are more preferable, and BS(PEG)<NUM> is still more preferable.

Aβ used in the cross-linked AB according to the present disclosure may be Aβ38, Aβ39, Aβ40, Aβ41, or Aβ42 from the viewpoint of allowing the reactivity of the cross-linked AB with ASPD-specific antibodies to be closer to that of ASPD. Of these, Aβ40 or Aβ42 is preferable, and Aβ42 is more preferable.

In one or more embodiments, the molecular weight of the cross-linked AB according to the present disclosure is more than <NUM> kDa or more than <NUM> kDa, with the molecular weight being a value obtained through analysis by <NUM>% to <NUM>% SDS-PAGE.

In one or more embodiments, the cross-linked AB according to the present disclosure may have cytotoxicity (i.e., the properties of selectively inducing cell death of functionally mature neuronal cells) equivalent to that of ASPD, or may be less toxic than ASPD. Alternatively, the cross-linked AB may be free of cytotoxicity or may be more toxic than ASPD. In one or more embodiments, the cross-linked AB according to the present disclosure may be less toxic than ASPD or is free of cytotoxicity.

In one or more embodiments, the reactivity of the cross-linked Aβ according to the present disclosure with ASPD-specific antibodies is closer to that of ASPD than to those of AB aggregates other than ASPD, and more preferably is equivalent to that of ASPD.

In one or more embodiments, the cross-linked Aβ according to the present disclosure is a substance that competes with ASPD against ASPD-specific antibodies.

In one or more embodiments, the cross-linked Aβ according to the present disclosure can be a substitute for ASPD from the viewpoint of reactivity with ASPD-specific antibodies.

In one or more embodiments, the cross-linked AB according to the present disclosure can be a reference material for ASPD analysis or a reference material to be included in a kit for ASPD analysis.

In the present disclosure, analysis of ASPD may include, in one or more embodiments, detection, measurement, and quantification of ASPD, and screening utilizing them.

In one or more embodiments, ASPD analysis in which the cross-linked Aβ according to the present disclosure is used as a reference material may be analysis utilizing an immunoassay that uses an ASPD-specific antibody(ies). Such analysis may be specifically an enzyme immunoassay that uses an ASPD-specific antibody(ies) or an enzyme immunoassay that is based on chemiluminescence and use an ASPD-specific antibody(ies), and more specifically an enzyme-linked immunosorbent assay (ELISA) or a chemiluminescent enzyme immunoassay (CLEIA), each of which uses an ASPD-specific antibody(ies).

In one or more embodiments, the cross-linked Aβ according to the present disclosure can be used for preparation of a calibration curve in ASPD analysis. In one or more other embodiments, the cross-linked AB according to the present disclosure can be used as a positive control in ASPD analysis.

In one or more embodiments, the cross-linked AB according to the present disclosure can be a reference material for ASPD analysis in diagnostics of a disease involving accumulation of ASPD. In one or more embodiments, examples of the disease involving accumulation of ASPD include Alzheimer's disease and Lewy body dementia.

In one or more embodiments, the cross-linked Aβ according to the present disclosure can be a reference material for ASPD analysis in companion diagnostics for a therapeutic agent that targets ASPD, or a reference material to be included in a kit for this ASPD analysis. In one or more embodiments, the cross-linked AB according to the present disclosure can be a component of a companion diagnostic agent for a therapeutic agent that targets ASPD.

Thus, viewed from another aspect, the present disclosure relates to an ASPD analysis kit that includes the cross-linked AB according to the present disclosure as a reference material. In one or more embodiments, the ASPD analysis kit according to the present aspect includes a reagent(s) necessary for an immunoassay that uses an ASPD-specific antibody(ies). In one or more embodiments, examples of the reagent include ASPD-specific antibodies.

In one or more embodiments, the ASPD analysis kit according to the present aspect is an ASPD analysis kit for diagnostics of a disease involving accumulation of ASPD, an ASPD analysis kit for companion diagnostics for a therapeutic agent that targets ASPD, or a companion diagnostic agent for a therapeutic agent that targets ASPD.

In one or more embodiments, examples of the therapeutic agent that targets ASPD include pharmaceutical compositions for prevention, amelioration, and/or treatment of a disease involving accumulation of ASPD.

In one or more embodiments, the cross-linked AB according to the present disclosure is immunogenic and can induce production of an antibody against the cross-linked AB according to the present disclosure.

In one or more embodiments, the cross-linked Aβ according to the present disclosure is immunogenic and can induce production of antibodies against ASPD.

Accordingly, in one or more embodiments, the cross-linked AB according to the present disclosure can be a substitute for ASPD from the viewpoint of capability of inducing antibodies against ASPD.

Using ASPD as an active vaccine for a disease involving accumulation of ASPD has already been disclosed (e.g., <CIT>).

Since the cross-linked AB according to the present disclosure can be a substitute for ASPD, the cross-linked AB according to the present disclosure can also be used as an active vaccine and also can be used in production of a vaccine.

Thus, viewed from one aspect, the present disclosure relates to a pharmaceutical composition containing the cross-linked Aβ according to the present disclosure as an active ingredient. Examples of one or more embodiments of the pharmaceutical composition according to the present disclosure include vaccines containing the cross-linked AB according to the present disclosure. The pharmaceutical composition and vaccine according to the present disclosure may contain a pharmaceutically acceptable excipient and/or adjuvant.

In one or more embodiments, the pharmaceutical composition and vaccine according to the present disclosure can be used for prevention, amelioration, and/or treatment of a disease involving accumulation of ASPD. In one or more embodiments, the pharmaceutical composition and vaccine according to the present disclosure can be used for prevention, amelioration, and/or treatment of Alzheimer's disease and/or Lewy body dementia.

In one or more embodiments, the pharmaceutical composition and vaccine according to the present disclosure can be administered to a subject who is at risk of developing, is suspected of having, or has a disease involving accumulation of ASPD. In one or more embodiments, the pharmaceutical composition according to the present disclosure can be administered to a subject who is at risk of developing, is suspected of having, or has Alzheimer's disease and/or Lewy body dementia. The subject may be a mammal, a human, or a non-human mammal.

One or more embodiments in which the pharmaceutical composition and vaccine according to the present disclosure are used to immunize a subject against ASPD, a disease involving accumulation of ASPD, or Alzheimer's disease and/or Lewy body dementia will be described below. In one or more embodiments, the method for administering the pharmaceutical composition and vaccine according to the present disclosure may be intramuscular, intraperitoneal, intradermal, or subcutaneous injection, or transmucosal administration through the oral tract, gastrointestinal tract, respiratory tract, or genitourinary tract. The dose of the cross-linked AB in the pharmaceutical composition and vaccine according to the present disclosure may be such an amount that a protective immune response is induced without causing severe adverse effects in a subject to which the pharmaceutical composition and vaccine is administered. In one or more embodiments, the dose of the pharmaceutical composition and vaccine according to the present disclosure is estimated to be an amount that contains a cell secreted-type ASPD-like structure in the range from <NUM>µg to <NUM>, <NUM> to <NUM>µg, <NUM> to <NUM>µg, <NUM> to <NUM>µg, or <NUM> to <NUM>µg. Following administration of the initial dose, one or several booster doses may be administered at sufficient intervals.

Thus, viewed from one aspect, the present disclosure relates to a method for preventing, ameliorating, and/or treating a disease involving an accumulation of ASPD (e.g., Alzheimer's disease and/or Lewy body dementia), including administering to a subject the cross-linked AB according to the present disclosure or the pharmaceutical composition or vaccine according to the present disclosure.

Viewed from another aspect, the present disclosure relates to a method for immunizing a subject against ASPD themselves, including administering to the subject the cross-linked AB according to the present disclosure or the pharmaceutical composition or vaccine according to the present disclosure.

Viewed from still another aspect, the present disclosure relates to a method for immunizing a subject against a disease involving an accumulation of ASPD, including administering to the subject the cross-linked AB according to the present disclosure or the pharmaceutical composition or vaccine according to the present disclosure.

Furthermore, viewed from one aspect, the present disclosure relates to a method for producing the pharmaceutical composition or vaccine according to the present disclosure, including combining the cross-linked AB according to the present disclosure with an excipient and/or adjuvant.

Viewed from another aspect, the present disclosure relates to a method for producing a pharmaceutical composition containing the cross-linked AB according to the present disclosure as an active ingredient or for producing a vaccine containing the cross-linked AB according to the present disclosure, including the step of producing the cross-linked AB according to the present disclosure by a production method to be described below.

In one or more embodiments, the cross-linked Aβ according to the present disclosure can be obtained by adding a cross-linking agent to an aqueous AB solution in which Aβ is dissolved in an aqueous solvent to cause a cross-linking reaction.

AB may be prepared through peptide synthesis or may be produced using a recombination technique.

In one or more embodiments, the amount of the cross-linking agent to be added is not less than <NUM> times, not less than <NUM> times, or not less than <NUM> times the amount of Aβ on a molar basis. In one or more embodiments, the amount of the cross-linking agent to be added is not more than <NUM> times, not more than <NUM> times, or not more than <NUM> times the amount of Aβ on a molar basis.

A cross-linked product obtained after the cross-linking reaction may be further subjected to ultrafiltration. The cross-linked AB according to the present disclosure can be obtained in retention liquid or in recovery liquid containing filter residues.

In one or more embodiments, the molecular weight cut-off value (MWCO) of an ultrafiltration filter may be <NUM> kDa, <NUM> kDa, or <NUM> kDa.

In one or more embodiments, the cross-linked Aβ according to the present disclosure can be produced by the method described in examples.

The following listed cross- linked AB [A1] to [A10] may be used in methods of the invention, as described hereinafter.

[A1] A cross-linked Aβ wherein AB is cross-linked using a cross-linking agent that has a spacer arm length of between <NUM>Å and <NUM>Å inclusive.

[A2] A cross-linked Aβ wherein AB is cross-linked using a cross-linking agent that has, as a spacer arm, not less than <NUM> and not more than <NUM> groups that are an EO group and a PO group.

[A3] The cross-linked AB according to [A1] or [A2], wherein Aβ is Aβ38, Aβ39, Aβ40, Aβ41, Aβ42, or Aβ43.

[A4] The cross-linked AB according to any one of [A1] to [A3], wherein the cross-linking agent has reactive ends that cross-link between amino groups.

[A5] The cross-linked AB according to any one of [A1] to [A4], having a molecular weight of more than <NUM> kDa with the molecular weight being a value obtained through analysis by <NUM>% to <NUM>% SDS-PAGE.

[A6] The cross-linked AB according to any one of [A1] to [A5], which is a substance that competes with ASPD against an ASPD-specific antibody.

[A7] The cross-linked AB according to any one of [A1] to [A6], which is a reference material in ASPD analysis.

[A8] The cross-linked AB according to any one of [A1] to [A7], which is a reference material for ASPD analysis in diagnostics of a disease involving accumulation of ASPD.

[A9] The cross-linked AB according to any one of [A1] to [A8], which is a reference material for ASPD analysis in companion diagnostics for a therapeutic agent that targets ASPD.

[A10] The cross-linked AB according to any one of [A1] to [A9], which is a component of a companion diagnostic agent for a therapeutic agent that targets ASPD.

Viewed from one aspect, the present disclosure relates to a method for analyzing ASPD, including the following steps (<NUM>) and (<NUM>) (hereinafter also referred to as "ASPD analysis method according to the present disclosure"):.

In this method, the first antibody is a humanized anti-ASPD monoclonal antibody huASD2 or a monoclonal antibody BAN50a, and
the second antibody is a mouse monoclonal anti-ASPD antibody mASD3 or a labeled antibody that is mASD3 with a label.

The ASPD analysis method according to the present disclosure is a method that utilizes the principle of an immunoassay, and in one or more embodiments, ASPD analysis method is an enzyme immunoassay such as ELISA or a chemiluminescent enzyme immunoassay such as CLEIA.

In one or more embodiments, the first antibody to be bound to the insoluble carrier is preferably huASD2 or BAN50a from the viewpoint of improving the detection sensitivity when blood, plasma components, cerebrospinal fluid, or a diluted solution thereof is used as a sample. huASD2, which is disclosed in <CIT>, is a humanized monoclonal anti-ASPD antibody having heavy chain variable regions whose amino acid sequence is represented by SEQ ID NO: <NUM> in the Sequence Listing and light chain variable regions whose amino acid sequence is represented by SEQ ID NO: <NUM> in the Sequence Listing.

In one or more embodiments, it is possible to use, instead of huASD2 in the present disclosure, an anti-ASPD antibody having heavy chain variable regions whose amino acid sequence is represented by SEQ ID NO: <NUM> in the Sequence Listing and light chain variable regions whose amino acid sequence is represented by SEQ ID NO: <NUM> in the Sequence Listing.

(In SEQ ID NO: <NUM>, X at position <NUM> is G or A, X at position <NUM> is L or V, and X at position <NUM> is T or R. In SEQ ID NO: <NUM>, X at position <NUM> is S or A, X at position <NUM> is S or A, X at position <NUM> is Y or F, X at position <NUM> is L or F, X at position <NUM> is K, F, or R, X at position <NUM> is A or T, and X at position <NUM> is G or A.

In the present disclosure, BAN50a is a known monoclonal antibody that specifically recognizes the N-terminal region ofAB and is disclosed in <CIT>. Commercially available products can be used as BAN50a, or BAN50a can be produced by a known method using Hybridoma BAN50 (Accession number: FERM BP4163) deposited in NITE Patent Microorganisms Depositary.

In one or more embodiments, the second antibody may be a labeled antibody that is mASD3 with a label from the viewpoint of improving the detection sensitivity. In one or more embodiments, labeling may be achieved through biotinylation.

In one or more embodiments, examples of the insoluble carrier used in the ASPD analysis method according to the present disclosure include solid phase materials such as beads and multi-well plates.

In one or more embodiments, examples of the biological sample that may contain ASPD include blood, plasma, and cerebrospinal fluid, as well as dilutions thereof.

In one or more embodiments, the ASPD analysis method according to the present disclosure may include a step (<NUM>) of preparing the biological sample prior to the step (<NUM>). In one or more embodiments, this sample preparation step (<NUM>) includes diluting a biospecimen such as blood, plasma, or cerebrospinal fluid with a suitable solvent or a suitable buffer. In one or more embodiments, the solvent or buffer used in this sample preparation step (<NUM>) may contain a surfactant and/or any other drug to the extent that the analysis is not hindered.

In one or more embodiments, the ASPD analysis method according to the present disclosure may further includes the following step (<NUM>):.

In one or more embodiments, the reporter may be a substance that utilizes chemiluminescence or bioluminescence, a fluorescent protein, or the like. From the viewpoint of improving the detection sensitivity, the reporter is preferably a substance that utilizes chemiluminescence or bioluminescence.

In one or more embodiments, the substance that utilizes chemiluminescence or bioluminescence may be an enzyme such as a bioluminescent enzyme or a variant thereof.

In one or more embodiments, examples of the enzyme include HRP (horseradish peroxidase) and alkaline phosphatase.

In one or more embodiments, the binding partner may be streptavidin or the like when the label is biotin.

In one or more embodiments, measurement of luminescence can be performed using a luminometer or a multi-plate reader.

In one or more embodiments, the ASPD analysis method according to the present disclosure may use the cross-linked AB according to the present disclosure as a reference material.

As disclosed hereinbefore, the invention relates to a method for analyzing ASPD, including the following steps (<NUM>) and (<NUM>):.

In a preferred aspect, this analysis method is a method utilizing the principle of an immunoassay.

In a further preferred aspect of this analysis method, the method utilizes the principle of an immunoassay is ELISA or CLEIA.

In a preferred aspect of any of the above analysis methods, the biological sample is blood, plasma components, cerebral spinal fluid, or a diluted solution thereof.

In a further preferred aspect of any of the above analysis methods, the method further includes a step (<NUM>) of preparing the biological sample prior to the step (<NUM>).

In a preferred aspect of the above analysis method, the sample preparation step (<NUM>) includes diluting a biospecimen selected from the group consisting of blood, plasma, and cerebrospinal fluid with a solvent or a buffer.

In a further preferred aspect of any of the above analysis methods , the method further includes the following step (<NUM>):.

In a preferred aspect of any of the above analysis methods, a substance according to [<NUM>] described below is used as a reference material for ASPD or the method includes using the cross-linked AB according to any one of [A1] to [A10] as described hereinbefore as a reference material.

Substances and cross-linked products that may be used in methods of the invention as described hereinbefore and hereinafter are set out below:.

The method of the invention for analyzing ASPD, includes the steps of:.

In a preferred aspect the above analysis method further comprises the step of:.

In a yet further preferred aspect, the analysis method described above uses the substance according to [<NUM>] described hereinbefore or the cross-linked product according to [<NUM>] as described herein before as a reference material for ASPD.

In the following, one or more embodiments of the present invention will be further described with reference to examples.

In the following examples, the concentration of a cross-linked AB and the concentration of ASPD are AB monomer-equivalent concentrations, unless otherwise stated.

The cross-linking agents used were as follows.

The cross-linked Aβ1, cross-linked Aβ2, and cross-linked Aβ3 obtained were subjected to SDS-PAGE (<NUM>% to <NUM>% Gel) to check whether they were cross-linked. <FIG> shows an example of the results obtained.

As can be seen from <FIG>, the cross-linked AB <NUM> to <NUM> were each found to be polymerized and to have a molecular weight of <NUM> kDa or more.

Reactivities of the obtained cross-linked Aβ1, cross-linked Aβ2, and cross-linked Aβ3 in ASPD CLEIA were examined.

ASPD CLEIA used was the system shown in <FIG>, in which an anti-ASPD polyclonal antibody (first antibody) immobilized on a solid phase, a mouse anti-ASPD monoclonal antibody mASD3 (second antibody) as a detection-side antibody, and an anti-mouse IgG-HRP were used. mASD3 is a monoclonal antibody described in <CIT> and <CIT>, and a hybridoma for mASD3 has been deposited in NITE Patent Microorganisms Depositary (Accession number: FERM BP-<NUM>). HRP stands for "horseradish peroxidase".

The reactivity in ASPD CLEIA [anti-ASPD polyclonal antibody/mASD3] when each of the cross-linked Aβ1, cross-linked Aβ2, and cross-linked Aβ3 was used as an analyte was compared with the reactivity in ASPD CLEIA when ASPD were used as an analyte, and <FIG> shows an example of the results obtained.

As can be seen from <FIG>, the reactivities of the cross-linked Aβ1 and cross-linked Aβ2 were closer to the reactivity of ASPD than the reactivity of the cross-linked Aβ3 was. In particular, the reactivity of the cross-linked Aβ1 was equivalent to the reactivity of ASPD.

Subsequently, the reactivity of a cross-linked AB was compared using a commercially available Aβ oligomer measurement kit that uses a monoclonal antibody 82E <NUM>, which specifically recognizes the N-terminus of Aβ.

The cross-linked Aβ1, ASPD, and an Aβ1-<NUM> dimer were used as analytes.

Furthermore, reactivities of the cross-linked Aβ1, ASPD, and the Aβ1-<NUM> dimer in ASPD CLEIA [anti-ASPD polyclonal antibody/mASD3] were also compared.

<FIG> shows an example of the results obtained.

As can be seen from <FIG>, when the commercially available Aβ oligomer measurement kit was used, the cross-linked Aβ1 also exhibited the reactivity equivalent to that of ASPD, similarly to the case of ASPD CLEIA.

In the case where the commercially available Aβ oligomer measurement kit using 82E1 was used, the reactivities of the cross-linked Aβ1 and ASPD were much lower than the reactivity of the Aβ1-<NUM> dimer.

In contrast, in ASPD CLEIA, the reactivity of the Aβ1-<NUM> dimer was much lower than the reactivities of the cross-linked Aβ1 and ASPD.

Moreover, it was also found that ASPD CLEIA exhibits high detection sensitivity for the cross-linked Aβ1 and ASPD.

Rabbits (New Zealand White) were immunized six times with the cross-linked Aβ1 (<NUM>µg/single immunization) together with Freund's adjuvant (FCA, first time) and incomplete Freund's adjuvant (FIA, five times), followed by exsanguination. Then, the reactivities of antibodies contained in serum with the cross-linked AB <NUM> and a synthetic ASPD were evaluated. <FIG> shows an example of the results obtained.

In <FIG>, "pre-serum" indicates the reactivity of pre-immune serum with a plate having the cross-linked Aβ1 immobilized thereon, "immobilized cross-linked Aβ" indicates the reactivity of the obtained serum with a plate having the cross-linked Aβ1 immobilized thereon, "immobilized synthetic ASPD" indicates the reactivity of the obtained serum with a plate having synthetic ASPD immobilized thereon, "control" indicates the reactivity of the obtained serum with a plate having a F12 protein immobilized thereon, and "Empty" indicates the reactivity of the obtained serum with an empty plate.

The results shown in <FIG> demonstrate that the cross-linked AB <NUM> is immunogenic and can induce antibodies reactive with ASPD.

ASPD in human CSF specimens were measured using an ASPD CLEIA system [anti-ASPD polyclonal antibody/mASD3]. The flow of the measurement was specifically as follows.

As a standard for measurement, the cross-linked Aβ1 or a cell secreted-type ASPD-like structure (<CIT>) was used.

As a specimen diluent, <NUM>% BSAin PBS (-) with <NUM>% antiseptic (product name: ProClin, Sigma-Aldrich) was used.

PBS (-) with <NUM>% Tween <NUM> was used as a washing solution.

Table <NUM> below shows one example of the results obtained when known concentrations of ASPD (high concentration H: <NUM> pM, medium concentration M: <NUM> pM, low concentration L: <NUM> pM, and none N: <NUM> pM) were added to human CSF (Age <NUM>/Caucasian/M), the resultant solutions were diluted <NUM>-fold, and the thus-obtained diluted solutions were subjected to quantification.

As can be seen from Table <NUM>, a recovery rate of approximately <NUM>% was achieved. Similar results were obtained when human CSF (Age <NUM>/Caucasian/M) and human CSF (Age <NUM>/Caucasian/M) were used.

Next, <FIG> shows the results obtained when human CSF was spiked with ASPD to prepare a sample, the sample was then diluted <NUM>-fold, <NUM>-fold, and <NUM>-fold with a specimen diluent, and the samples with the dilution ratios of <NUM>, <NUM>/<NUM>, <NUM>/<NUM>, and <NUM>/<NUM> were subjected to measurement.

As can be seen from <FIG>, dilution linearity was observed as a result of plotting the results obtained for the diluted samples.

ASPD in human plasma specimens were measured using ASPD CLEIA [anti-ASPD polyclonal antibody/mASD3]. The flow of the measurement was specifically as follows.

PBS (-) with <NUM>% Tween20 was used as a washing solution.

Table <NUM> below shows one example of the results obtained when known concentrations of ASPD were added to human plasma (Age <NUM>/Caucasian/F), the resultant solutions were diluted <NUM>-fold with <NUM>% BSA PBS (-), and the obtained diluted solutions were subjected to quantification.

As can be seen from Table <NUM>, ASPD could be measured when human plasma was used instead of human CSF. However, the recovery rate was reduced approximately by half. It is considered that this reduction was caused by the influence of plasma components.

Next, <FIG> shows the results obtained when human plasma was spiked with ASPD to prepare a sample, the sample was then diluted <NUM>-fold, <NUM>-fold, <NUM>-fold, and <NUM>-fold with a specimen diluent, and the samples with the dilution ratios of <NUM>, <NUM>/<NUM>, <NUM>/<NUM>, <NUM>/<NUM>, and <NUM>/<NUM> were subjected to measurement.

As can be seen from <FIG>, dilution linearity was observed as a result of plotting the results obtained for the diluted samples. However, the inclination was reduced to <NUM>/<NUM> as compared with the inclination obtained when the buffer was used. It is considered that this was caused by the influence of plasma components.

The influence on the recovery rate in the human plasma samples could be eliminated by setting the sample dilution ratio to <NUM>/<NUM>.

ASPD were measured in ASPD CLEIA in which humanized anti-ASPD monoclonal antibody huASD2 was used as an antibody immobilized on a solid phase and mouse anti-ASPD monoclonal antibody mASD3 and anti-mouse IgG-HRP were used (<FIG>).

<FIG> shows an example of the results obtained. As can be seen from <FIG>, as a result of four independent ASPD measurements, substantially the same luminescence values were obtained.

In order to improve the ASPD detection sensitivity, a system was constructed in which a biotinylated mASD3 antibody and HRP with streptavidin (SA) bound thereto were used (<FIG>).

As a result of measuring ASPD in the huASD2/biotinylated mASD3 system, the detection sensitivity was improved as compared with the case where the measurement was performed using the huASD2/mASD3 system (<FIG>).

Moreover, the huASD2/biotinylated mASD3 system was highly specific for ASPD and hardly detected a multiple antigenic peptide having <NUM> molecules of Aβ1-<NUM> bound thereto (MAP <NUM>, FUJIFIIM Wako Pure Chemical Corporation) (<FIG>). On the other hand, as a control, measurement was also performed using a BAN50/biotinylated BAN50 system (High Molecular Amyloid β Oligomer ELISA Kit, FUJIFILM Wako Pure Chemical Corporation). As a result, this system could hardly detected ASPD whereas it detected MAP <NUM> with high sensitivity (<FIG>).

Out of various combinations of a first antibody to be immobilized and a detection-side second antibody, combinations capable of reducing the influence of plasma were found.

ASPD CEILA systems were constructed with the following combinations of [first antibody/second antibody] and used to measure ASPD in plasma samples.

The combinations of [first antibody/second antibody] employed were [huASD2/biotinylated mASD3], [mASD3/biotinylated mASD3], [82E <NUM>/biotinylated mASD3], [BAN50a/biotinylated mASD3], and [6E <NUM>/biotinylated mASD3]. As samples, plasma samples containing ASPD and diluted at a dilution ratio of <NUM>/<NUM> were prepared and compared with a buffer sample.

As can be seen from <FIG>, it was found that the influence of plasma components can be avoided most effectively when employing the combination of [BAN50a/biotinylated mASD3].

For ten plasma samples from patients diagnosed as having Alzheimer's disease (AD) (open circle) and ten plasma samples from healthy subjects (open triangle), measurement was performed in ASPD CLEIA employing the combination of [huASD2/biotinylated mASD3]. As a result, ASPD were detected from four samples from the AD patients. <FIG> shows the results obtained.

Claim 1:
A method for analyzing amylospheroids (ASPD), comprising the steps of:
(<NUM>) bringing a biological sample that may contain ASPD into contact with an antibody (first antibody) that is capable of binding to ASPD and is bound to an insoluble carrier, thereby causing, when the biological sample contains ASPD, the ASPD to bind to the first antibody; and
(<NUM>) reacting an anti-ASPD monoclonal antibody (second antibody) with the ASPD that has bound to the first antibody in the step (<NUM>),
wherein the first antibody is a humanized anti-ASPD monoclonal antibody huASD2 or a monoclonal antibody BAN50a, and
the second antibody is a mouse monoclonal anti-ASPD antibody mASD3 or a labeled antibody that is mASD3 with a label.