WO2014073885A1

WO2014073885A1 - Standard protein complex for quantitative analysis of multimers of multimer-forming polypeptides

Info

Publication number: WO2014073885A1
Application number: PCT/KR2013/010081
Authority: WO
Inventors: 안성수; 강민오; 심규환
Original assignee: 가천대학교 산학협력단
Priority date: 2012-11-07
Filing date: 2013-11-07
Publication date: 2014-05-15
Also published as: KR20140059097A; KR101451797B1

Abstract

The present invention relates to a standard protein complex for a quantitative analysis of multimers of multimer-forming polypeptides and to a kit comprising same. The standard protein complex of the present invention enables signal amplification when applied to MDS, and therefore, the standard protein complex of low concentration (1 ng/㎖) can be detected by MDS. The present invention enables a quantitative analysis of multimers of multimer-forming polypeptides related to various diseases.

Description

【Specification】

[Name of invention]

Standard Protein Complexes for Multimer Quantification of Multimer-forming Polypeptides

Technical Field

The present invention was made by the task number 2011-E53001-00 under the support of the Republic of Korea common infection, the research management professional organization of the project is the disease management headquarters, the research project name is "disease management headquarters R & D", the research project name is "prion Study on the relationship between protein and markers of degenerative brain disease ", the lead organization is Gachon University Industry-Academic Cooperation Group, and the research period is 2011.02.16 ~ 2011.12.15. In addition, the present invention was made by the task number 2012R1A1A2A04 under the support of the Ministry of Education, Science and Technology, the research management specialized organization of the project is the Korea Research Foundation, the research project name is "basic research project- general researcher support project-basic research support project ( Type I) ", the research project titled" Alsheimer's Disease Early Diagnosis-Based Research Using Amyloid-Vitata Complex in Serum ", Gachon University Industry-University Cooperation Group, and the period of study are 2012.09.01 ~ 2013.08.31.

This patent application claims priority to Korean Patent Application No. 10-2012-0125738 filed with the Korean Intellectual Property Office on November 07, 2012, the disclosure of which is incorporated herein by reference.

The present invention relates to standard protein complexes for multimer quantitation of multimer-forming polypeptides, kits comprising the same and multimer quantitation methods.

Background Art

Multimer formation of the polypeptides that make up a protein is generally known to be necessary for the function of the protein. However, the muUimeric form often causes disease in some proteins. In particular, a protein is present as a monomer in a normal state, and then converted into a multimer (or a swarm) when an abnormal state (for example, a misfolded state) is reached. Ms (or accumulation) folded and finally the agglomerated, i.e. proteins that do not have the structure (conformation) that are relevant to the functional are known to ^{^} does not indicate the normal physiological activity. Failure to fold correctly or fail to fold correctly can lead to various forms of physiological dysfunction, resulting in various forms of disease (Massimo Stefani, et al., J. Mol. Med. 81: 678 -699 (2003) and Radford SE, et al., Cell. 97: 291-298 (1999). Many diseases are caused in life due to the presence of protein molecules with incorrect structures, that is, structures that differ from those that function normally.

One of the diseases associated with abnormal aggregation of proteins is Alzheimer's disease. When necropsy of the brain of degenerated Alzheimer's patient was stained by immunohistochemical method, we observed neurofibrillary tangles caused by β-amyloid accumulation and neuronal fibrillation by Tau protein. can do. It is believed that the β-amyloid peptide that constitutes this part is responsible for Alzheimer's disease. β-amyloid exists in the multimer (multimer or aggregated) form in the human body, which is considered to cause cytotoxicity. Several studies have shown that multimer β-amyloid peptides are toxic in the progression of the disease and are involved in the death of brain cells. Therefore, the presence of β-amyloid multimer is an important indicator in the diagnosis of Alzheimer's disease.

Parkinson's disease, a disease associated with abnormal pooling of proteins, is known to cause alpha-synuclein as a result of protein modification and contraction caused by external or internal factors. Therefore, Parkinson's disease is considered to be an accumulation of alpha-synuclein, which is used for the diagnosis of Parkinson's disease.

Prion disease, a disease associated with abnormal aggregation of proteins, changes the structure of normal prion (PrP ^c ) proteins into modified prion protein (PrP ^sc ) structures and accumulates in neurons such as the brain, spleen, and lymphoid organs. Causes neuronal death and is contagious. The normal form of the prion protein _(Pr pG) contains _α -helices and disordered moieties and exists in monomeric form (Zahn, R., et al., Proc. Natl. Acad. Sci. USA 97: 145-150 (2000) ), Scrapie type (PrP ^Sc ) has a highly β-sheet structure Present in the multimer (cold) form or at least dimer form (Caughey B., et al., J. Biol. Chem. 273: 32230-35 (1998)).

Various reclamation-related diseases can be diagnosed by discriminating and detecting the packed form of protein (multimer form), which is known to cause various diseases as described above, with the monomeric form. As a technology for this, the present inventors have developed a multi timer detection system (MDS) and received a patent registration (Korean Patent No. 10-0987639). The multimer detection method is a new EUSA technology that can fractionally detect multimeric forms of multimer-forming polypeptides from monomeric forms. The general ELISA cannot distinguish between the multimer type and the monomer type, but according to the above method, it is possible to effectively detect this, thereby ultimately diagnosing a bulb-related disease. In the immunoassay using the multimer detection method, a standard protein is used for quantitative analysis of the detected protein. Existing multimer-type standard proteins are prepared by multimer-forming monomers of multimer-forming polypeptides using Bradford protein assay, HPLC, SDS-phage, etc. It is carried out by measuring the concentration of. However, the prior art is not uniform because the synthesis of the multimer is not constant and is synthesized by various forms of multimers such as dimers, trimers, and fibrils, and thus the exact mole number of the peptide molecules of the multimer is determined. There is a problem that is difficult to measure. In addition, after the synthesis of the multimer, the characteristics of the multimer are denatured through purification, quantification, and the like, and the quantitative concentration varies greatly, so that the control of the experimental conditions is very difficult. Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

[Content of invention]

[Problem to solve]

The present inventors improve the problems of the conventional standard protein as above, Research efforts have been made to develop a standard protein applicable to ELISA (MDS) for the fractional detection of multimer forms from the monomeric forms of multimer-forming polypeptides associated with the pathogenesis of herb-related diseases. As a result, the present invention was completed by developing a standard protein complex having a novel structure in which a plurality of monomer-type peptides of mulmeromer-forming polypeptides are bonded to a carrier protein.

It is therefore an object of the present invention to provide a standard protein complex for multimer quantitation of multimer-forming polypeptides.

Another object of the present invention is to provide a kit for the differential detection of a multimeric form from the monomeric form of a multimer-forming polypeptide.

It is another object of the present invention to provide a multimer quantification method of a multimer-forming polypeptide. Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

[Measures of problem]

According to one aspect of the invention, the invention provides a standard protein complex for multimer quantitation of multimer-forming polypeptides comprising:

(a) a carrier protein having a linker; And

(b) a polypeptide wherein the linker is covalently bonded to the N- or C-terminus, the polypeptide is a monomeric form of a multimer-forming polypeptide or a fragment thereof and has a semicoupling group covalently bonded to the linker at the N- or C-terminus, 3-12 each bind to each linker of carrier protein through the reactor, and

The carrier protein is a protein that does not bind an antibody to the polypeptide. The inventors have improved the problems of such conventional standard proteins, and the monomers of multimer-forming polypeptides associated with the pathogenesis of aggregation-related diseases. Research efforts have been made to develop standard proteins applicable to ELISA (MDS) for the detection of multimer types from fractions. As a result, the present invention has been completed by developing a standard protein complex of a novel structure in which a large number of monomer-type peptides of a multimer-forming polypeptide are bound to a carrier protein.

As used herein, the term "multimer-forming polypeptide" refers to a polypeptide capable of forming a globule. In particular, the following structural changes cause various diseases. For example, Alzheimer's disease Creutzfeldt-Jakob disease Spongiform encephalopathies, Parkinson's disease, Huntington's disease, Amyotrophic lateral sclerosis, Serpin deficiency, emphysema, cirrhosis Type II diabetes, primary systemic amyloidosis, secondary systemic amyloidosis, Fronto-temporal dementias, systemic amyloidosis in the elderly, familial amyloid polyneuropathy, hereditary cerebral amyloid angiopathy ) And hemodialysis-related amyloidosis. Thus, the term "multimer-forming polypeptide" can be used interchangeably with the term "column-forming polypeptide".

In general, the monomeric (ie non-aggregated) type of the multimer-forming polypeptide is normal and the multimeric (ie aggregated) type is responsible for diseases, particularly neurodegenerative diseases such as Alzheimer's disease and Creutzfeldt-Jakob disease. cause. Thus, the multimer-formation of the multimer-forming polypeptide can be fractionally detected from the monomeric form or the quantitative analysis of the multimer form contained in the sample can be used to diagnose a bowel-related disease. For the quantitative analysis of these multimer types, a standard protein of a multimer type having a precise concentration is required. Accordingly, the present inventors have developed a standard protein (Miligomer Mimicking Standard Protein) that mimics a multimeric peptide as a standard protein for multimer quantification of multimer-forming polypeptides.

According to a preferred embodiment of the present invention, the multimer-forming polypeptide is an Aβ peptide (β-amyloid) involved in Alzheimer's disease, tau protein, Creutzfeldt-Jakob disease, and sponges involved in brain disease, Parkinson's. Α-synuclein, involved in disease, primary systemic Ig gradient involved in amyloidosis, serum amyloid A involved in secondary systemic amyloidosis, tau protein involved in frontal-temporal dementia, transthyretin involved in elderly systemic amyloidosis, transthyretin involved in familial amyloid polyneuropathy, Cystatin involved in hereditary cerebral amyloid vasculitis (: β ₂ -microglobulin involved in hemodialysis-related amyloidosis, huntingtin involved in Huntington's disease, superoxide dismutase, surfine involved in amyotrophic lateral sclerosis) Deficiencies, emphysema, and surfines involved in cirrhosis, amylin involved in type II diabetes and phosphorylated forms of the peptides.

Preferably, the multimer forming polypeptide is an Αβ peptide, α-synuclein, prion protein or phosphorylated form of the peptide. When the method of the present invention is applied to prion protein (PrP), the monomer type is PrP ^c (cell or normal form of a prion) and the multimer type is PrP ^Sc (schematic or infectious type of prion).

Carrier proteins of the present invention have a plurality of linkers so that they can bind to the monomeric forms of the multimer-forming polypeptide or fragments thereof to form a complex.

According to a preferred embodiment of the present invention, the carrier protein of the present invention has a surface modified with a functional group, the functional group serves as a linker to bind with one end of the monomeric form or fragment thereof of the multimer-forming polypeptide. do. Preferably, the functional group is selected from the group consisting of maleimide group, amino group, carboxyl group, aldehyde group, propionaldehyde group, butyl aldehyde group succinimidyl propionate, succinimidyl carboxymethyl, hydroxy succinimidyl and succinimidyl carbonate. More preferably, it is a maleimide group.

The number of functional groups as the linker is preferably 3 to 12, more preferably 3 to 10, even more preferably 4 to 7.

According to a preferred embodiment of the present invention, the carrier protein is a globular protein. The term “sphere protein” refers to a protein whose shape is spherical or near spherical, and is understood as a glossary of fibrous protein. According to a more preferred embodiment of the present invention, the carrier protein is albumin, ovalbumin, lactoalbumin, serum albumin, leucosin (leucosin), legumelin, keyhole-lymphpet hemocyanin (keyholelimpet hemocyanin), globin And globulin. Preferably, the carrier protein is albumin, ovalbumin or keyhole-limpet hemocyanin, more preferably ovalbumin.

The monomeric type or fragment thereof of the multimer-forming polypeptide of the present invention has a reactor covalently bonded to the linker of the carrier protein at the N- or C-terminal, and binds to the linker of the carrier protein through the reaction to form a standard protein complex. Form. The monomer type of the multimer-forming polypeptide refers to a monomer molecule of a multimer-forming polypeptide such as Aβ peptide, tau protein, prion protein.

According to a preferred embodiment of the present invention, monomeric forms or fragments of 3 to 12 multimer-forming polypeptides bind each linker of the carrier protein via a reactor. Preferably, monomeric forms of 3 to 10 multimer-forming polypeptides or fragments thereof bind to the linker of the carrier protein, respectively, more preferably monomeric forms of 4 to 7 multimer-forming polypeptides or fragments thereof. Each of these carrier proteins binds to a linker.

According to a preferred embodiment of the present invention, the reaction group of the polytide covalently bonded to the linker is a thiol group, a hydroxyl group or an amino group. Preferably, the reactor is a thiol group, more preferably a thiol group of a cysteine residue. When there is no cysteine residue at the N- or C-terminal end of the monomeric form or fragment thereof of the multimer-forming polypeptide, the cysteine residue may be introduced at the terminal to have a thiol group.

According to a preferred embodiment of the invention the monomeric form or fragment thereof of the multimer-forming polypeptide comprises 10 to 30 amino acids.

According to a preferred embodiment of the present invention, when the monomeric form or fragment thereof of the multimer-forming polypeptide is Aβ peptide, it is β-amyloid peptide, preferably β-amyloid peptide. N-terminal region peptide, more preferably a peptide having amino acid sequence 1-15 of the β-amyloid _W2 peptide. In addition, when the monomer type or fragment thereof of the multimer-forming polypeptide is α-synuclein, peptides 122-136 of α-synuclein are preferred.

According to a preferred embodiment of the present invention, the standard protein complex of the present invention can be applied to a multimer detection system (MDS). The multimer detection method is disclosed in Korean Patent No. 10-0987639 as a method for fractionally detecting a multimer type from a monomer type of a multimer-forming polypeptide in a raw sample developed by the present inventors. This document is incorporated herein by reference. Thus, the standard protein complex of the present invention does not react (bind) the carrier protein with the antibody to the monomeric form or fragment thereof of the multimer-forming polypeptide for application to MDS. Accordingly, the present invention can use as a carrier protein any protein that does not react with the antibody against the monomeric form of the multimer-forming polypeptide or fragment thereof. Preferably, the antibody is a capture antibody that recognizes an epitope on the monomeric form of the multimer-forming polypeptide or a fragment thereof and a detection antibody that recognizes an epitope that overlaps with the epitope. As used herein, the term "capture antibody" refers to an antibody capable of binding to a multimer-forming polypeptide to be detected in a raw sample, and the term "detection antibody" refers to a multimer-formation captured by the capture antibody to It means an antibody capable of binding to a polypeptide. "Antibody" means an immunoglobulin protein capable of binding an antigen. Antibodies as used herein include antibody fragments (eg F (ab ') 2, Fab', Fab, Fv) as well as whole antibodies capable of binding epitopes, antigens or antigen fragments to be detected.

The MDS is characterized by using a set of capture antibodies and detection antibodies that specifically recognize epitopes on a multimer-forming polypeptide, wherein the epitopes that the capture antibodies and detection antibodies specifically recognize are identical or overlapping each other. It is. The term "overlapped with," used while referring to epitopes for capture and detection antibodies, encompasses epitopes comprising amino acid sequences that have been fully or partially overlapped. Preferably, the capture antibody and the detection antibody Specifically recognized epitopes are sequences that are not repeated in multimer-forming polypeptides.

Once dug into MDS, the multimer-forming polypeptide bound to the capture antibody can no longer bind to the detection antibody because there is no additional epitope recognized by the detection antibody. Thus, as shown in FIG. 1, when applying the standard protein complex of the present invention to MDS, the capture antibody binds to and captures the monomeric form of a plurality of multimer-forming polypeptides on the standard protein complex or part of fragmentation thereof. The detection antibody binds to the remaining monomeric types or fragments thereof in which the antibody is not bound. ^As' shown in Figure 2, the conventional dimer (dimer) in the form of peptides to obtain a signal amplification effect is structurally eoryeowoona, standard protein complex of the present invention, it is possible to obtain a signal amplification effect it detects an antibody can catch a lot. As demonstrated in the examples below, the standard protein complexes of the present invention are excellent in detection sensitivity such that they can be detected by MDS even at low concentrations of 1 ng / ^.

The detection of "multimer type-detecting antibody complex" and "standard protein complex-detecting antibody complex" can be carried out by various methods known in the art. The formation of the complex indicates the presence of a multimer form in the raw sample. The step is carried out according to conventional methods, for example, Enzyme I unoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980 and Harlow and Lane, eds. Antibodies: A Laboratory Manua K1988) Can be performed quantitatively or qualitatively using various detectable label / substrate pairs as described in Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. According to another aspect of the present invention, the present invention provides a kit for the differential detection of a multimer form from the monomeric form of a multimer-forming polypeptide in a Mosample comprising a standard protein complex of the invention.

Since the kit of the present invention includes the standard protein complex described above, the common content between the two is omitted in order to avoid excessive complexity of the present specification.

As used herein, the term "biosample" refers to the organism-derived sample to be analyzed. The raw sample may be a cell, tissue, or Biological fluid, or other medium that can be analyzed in accordance with the present invention, which includes samples taken from humans, samples taken from animals, samples taken from food for humans or animals. Preferably, the raw sample is blood, serum, plasma, lymph, milk, urine, feces, tears, saliva, semen, brain extract (eg brain homogenate), spinal fluid (SCF), layered water, spleen and tonsil tissue extract A fluid sample containing the body. More preferably, the raw sample is brain homogenate or plasma.

According to a preferred embodiment of the present invention, the kit of the present invention is a kit for quantitative analysis of the multimer type.

According to a preferred embodiment of the present invention, the kit of the present invention further comprises the detection antibody and / or capture antibody. According to another aspect of the invention, the invention provides a multimer quantitative method for multimer-forming polypeptides using a standard protein complex, comprising the following steps:

(a) contacting the raw sample with a capture antibody capable of capturing the monomeric and multimeric forms of the multimer-forming polypeptide by recognizing an epitope on the multimer-forming polypeptide;

(b) contacting the raw sample with a detection antibody that recognizes an epitope overlapped with the epitope of step (a) to form a capture antibody-multimer type-detection antibody complex, wherein the detection antibody generates a detectable signal. Has a marker;

(c) removing a substance other than the capture antibody-multimer-type-detecting antibody complex formed in step (b) and measuring the signal value generated by the detection antibody in the complex; And

(d) comparing the signal value obtained in step (c) with a calibration curve obtained using the standard protein complex to quantify the multimer of the multimer-forming polypeptide, wherein the standard protein complex comprises a linker Having a carrier protein; And a polypeptide wherein the linker is covalently bonded to the N- or C- terminus, wherein the polypeptide is a monomeric form of a multimer-forming polypeptide or a fragment thereof at the N- or C- terminus. Having a semicoupling group covalently bonded to the linker, wherein each of 3-12 polypeptides binds to each linker of a carrier protein through the semicoupling group, the carrier protein is a protein which does not bind to an antibody to the polypeptide,

The calibration curve comprises (i) quantifying a polypeptide of the standard protein complex; (Π) contacting the standard protein complex with a capture antibody capable of recognizing an epitope on the polypeptide and capturing the standard protein complex; (iii) contacting the standard protein complex with a detection antibody that recognizes the epitope overlapped with the epitope of step (Π) to form a capture antibody-standard protein complex-detection antibody complex, wherein the detection antibody generates a detectable signal To have a label; (iv) removing substances other than the capture antibody-standard protein complex-detecting antibody complex formed in step (iii) and measuring signal values generated by the detection antibody in the complex; And (V) implementing a calibration curve using the polypeptide quantitative value of the standard protein complex obtained in step (i) and the signal value obtained in step (iv).

Steps (a) to (c) of the method are described in detail in Korean Patent No. 10—0987639, which is incorporated herein by reference.

The detection antibody has a label that produces a detectable signal. Such labels include compound labels (eg biotin), enzyme labels (eg alkaline phosphatase, peroxidase, β-galactosidase and β-glucosidase), radioactive labels (eg I ¹²⁵ and C). ¹⁴ ), including but not limited to fluorescent labels (such as ᅳ fluorescein), luminescent labels, chemiluminescent labels, and FRET (fluorescence resonance energy transfer) labels. Various labels and methods for labeling antibodies are known in the art (Harlow and Lane, eds. Antibodies: A Laboratory Manual (1988) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

For quantitative analysis of the multimer type, data obtained by applying a standard protein complex to MDS, specifically showing the relationship (or aspect) between the amount of standard protein (e.g. concentration, mole number) and the MDS detent ion signal Data, preferably of a multimer-forming polypeptide bound to a carrier protein Data showing the relationship (or aspect) of the amount of monomeric form or fragment thereof to the MDS detection signal is available. The data can be implemented in the form of a calibration curve. To obtain these data, first, the quantification of standard protein complexes is required. Quantification of monomeric forms or fragments thereof in standard protein complexes can be performed by measuring the total protein molecular weight that increases as the monomeric form or fragment thereof attaches to a carrier protein using methods such as SDS-PAGE, MALDI-TOFF, and the like. have. The present invention synthesizes a standard protein through a process of binding a plurality of the monomer types or fragments thereof to a carrier protein having a molecular weight (or easily measured), and the molecular weight of the generated standard protein can be easily experimented based on Lab. Because it can be easily measured through the quantitation of the standard protein complex can be performed quickly and accurately. Finally, the amount of multimers detected in the experimental group is quantified by comparison with data values of standard protein complexes. 【Effects of the Invention】

The features and advantages of the present invention are summarized as follows:

(0 The present invention provides standard protein complexes and kits comprising the same for multimer quantitation of multimer-forming polypeptides.

(Π) The standard protein complex of the present invention is capable of signal amplification when applied to MDS so that even small concentrations of standard protein complex (1 ng /) can be detected by MDS.

(iii) The present invention enables quantitative analysis of multimers of multimer-forming polypeptides associated with various diseases. [Brief Description of Drawings]

Figure 1 is a schematic diagram showing the detection process of the standard protein complex of the present invention applied to MDS.

Figure 2 is a schematic diagram showing a problem that occurs when the conventional dimer standard protein is applied to MDS.

3 to 5 are graphs showing the results of ELISA experiments of β-amyloid standard protein complexes. 6 is a graph showing the results of ELISA experiments of α-synuclein standard protein complex.

Figure 7 is a graph showing the results of ELISA experiments of phosphorylated α-synuclein standard protein complex.

8 is a photograph showing the molecular weight change of the standard protein complex (0MSP) of the present invention. MA-0VA represents ovalbumin modified with maleimide group, and Aβ 1-OVA represents 0MSP in which Aβ 1-15 (^ AEFRHDSGYEVHHQ ^ C) peptide is covalently bound to MA-0VA via a linker. The temperature is room temperature. 4 ° CAP1VA represents 0MSP obtained by performing covalent linkage between the Aβ1-15 peptide and MA-0VA through an overnight at 4 ^° C., and α-S—P represents 0MSP to which peptide 1 is bound. AS represents 0MSP to which peptide 3 is bound.

[Specific contents to carry out invention]

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention more specifically, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. . Example

Example 1. Preparation and ELISA experiment of β-amyloid standard protein complex

Orimalbumin (Thermo Scientific, # 77125) (maleimide-activated ovalbumin, MA-OVA) modified with maleimide group was dissolved in tertiary distilled water to a final concentration of 10 mg / ^. PBS + 10 mM EDTA (pH 7.2) binding buffer and MA-0VA (10 mg / ni «were diluted to 1 mg / ^. Synthesis of Ν-terminal 1-15 peptide (Αβΐ) of β-amyloid 卜₄₂ protein After the preparation of the ^ AEFRHDSGYEVHHQ ¹ ^ peptide (SEQ ID NO: 1) with cysteine at the C-terminus, 1 mg of the prepared 1-15 peptide and 0.5 mg of MA-0VA were mixed with the binding buffer (pH 7.2) (concentration). 5 mg / ^ 1-15 peptide 100 μΑ, concentration 10 mg / iui MA-OVA 50 ≠, binding buffer 350 ≠ ovalbumin based final concentration of 0.5 mg / 500! Λ = 1 mg / ^) MA Cysteine to block maleimide residues that may remain in the OVA 1 ^ of a solution (solution of Sigma C-1276 dissolved in PBS pH 4.5 and adjusted to 120 mg / m £) was added and incubated at room temperature for 1 hour. Put the solution in a dialysis membrane (Spectra / Por Dialysis membrane 4, MWCO 12-14,000, Norm la flat width: 10mm, diameter: 6.4mm, Vol / Length: 0.32 mi / cm, Reorder No: 132 697), and immerse in PBS Dialysis gave a standard protein complex. The completed standard protein complex was named Oligomer Mimicking Standard Protein (OMSP). The completed standard protein complex was stored at -20 ° C.

TBST was prepared by diluting Î ± -OVA Oligomer Miking Protein, A ^ OMSP, 1 mg /) with β-amyloid to 15, 100 and 10 ng / i. It was dispensed on plates coated with 2 ^ g / i of anti-β-amyloid _W2 antibody (1E11, Covance) by 100 ^ and then incubated at 37 ^° C for 1 hour. After 3 washes with TBST, 200 ng / m £ solution of detection antibody anti-β-amyloid (HV) -attached detection antibody anti-β-amyloid, Covance) was added to the same plate in 100 ^ increments, 37 ^° Incubate at C for 1 hour. After washing 3 times with TBST, the ECL solution (100 ^) was dispensed and the luminescence intensity was measured. Β-amyloid used as a control was dissolved in DMS0 peptide (Abeta 1-42, Bachem), immediately diluted in PBS, incubated at 37 ^° C and used to form a multimer. Some monomers and multimers coexist in the control group. Experimental results are shown in FIGS. 3 to 5.

As shown in Figures 3-5, it was confirmed that the amyloid standard protein complex can be detected by ELISA (MDS) even at a low concentration of 1 ng / m. This is a significant difference used as a control. These results indicate that a large number of detection antibodies can be attached to the standard protein complex of the present invention, which is effective in amplifying signals. Example 2. Preparation and ELISA experiment of α-synuclein standard protein complex

Ovalbumin (MA-0VA) modified with maleimide group was dissolved in tertiary distilled water to a final concentration of 10 nig / ^. Peptide 3 (peptides 122-136 of α-synuclein was synthesized using a binding buffer and cysteine was attached to the Ν-terminal. CNEAYEMPSEEGYQDY as a peptide; SEQ ID NO: 2)) was 50 and OVA was 100 zg / ii ^ (865 μ binding buffer + OVA 10 ^ + peptide 125 id; 0VA = 0.1 mg, Pep = 0.05 mg). And then incubated at room temperature for 2 hours. Cysteine solution (120 mg /) was added in 1 ^ peptide peptides and left at room temperature for 1 hour. Peptide solution was placed in a membrane tube, sealed, immersed in PBS, and dialyzed (change every three hours, then incubate overnight at 4 ^° C). The solution from the membrane tube was transferred to an appendorf tube (1 ^ g / \\ ii). The finished standard protein complex was stored at -20 ^° C.

PBST was used to dilute the peptide solution at 1600 ng / m, 800, 400,

Stepped to 200, 100, 50 and 25 ng / m £. After incubation for 3 hours at 37 ^° C. and washed 5 times with PBST. Biotinylated p-sl29 antibody (Santa Cruz) 1 g / i was dispensed into 211 Ab (Santa Cruz) coated plates, incubated at 37 ^° C for 3 hours and washed 5 times with PBST. Biotin-bound strept-avidin-poly HRP (Strept-avidin-poly HRP) was 5000 times divided into 100 each well and placed for 30 minutes at room temperature. After washing with PBST, the ECL solution was dispensed and the fluorescence intensity was measured (read signal at Lumin 5times-2s). Experimental results are shown in FIG. 6.

As shown in Figure 6, it was confirmed that a small concentration of standard protein complex can also be detected by ELISA, Example 3. Preparation of phosphorylated α-synuclein standard protein complex and ELISA experiment

Ovalbumin (MA-0VA) modified with maleimide group was dissolved at 10 mg / ^ in tertiary distilled water. 50 zg / i of Peptide 1 (CNEAYEMPpSEEGYQDY; SEQ ID NO: 2) as a peptide having cysteine at the Ν-terminus after synthesizing peptide No. 122-136 of α-synuclein phosphorylated serine 129 using a binding buffer And OVA diluted to 100 (865 μΐ binding buffer + OVA 10 + peptide 125 ≠ OVA = 0.1 mg, Pep = 0.05 mg). And incubated at room temperature for 2 hours. Cysteine solution (120 rag /) was added in 1 ^ peptide peptides and placed in silver for 1 hour. Peptide Solution Membrane Placed in a tube, sealed, soaked in PBS and dialyzed (change every 3 hours, then incubate overnight at 4 ^° C). The solution of the membrane tube was transferred to an appendorf tube (l / g /) and the finished standard protein complex was stored ^at -20 ^° C.

PBST was used to dilute the peptide 3 solution at 640 ng / ra £, 320, 160

Stepped to 80, 40, 20 and 10 ng / ^. After incubation at 37 ^° C. for 2 hours, it was washed 5 times with PBST. Biotinylated 211 antibody (Santa Cruz) 1 / ^ was dispensed on 211 Ab-coated plates, incubated at 37 ° C for 2 hours and washed 5 times with PBST. 100 ^ ul was dispensed into each well with Strap-avidin-poly HRP at 5000 times and allowed to stand at room temperature for 30 minutes. After washing with PBST, the ECL solution was dispensed and the fluorescence intensity was measured (signal reading at Lumin 5times-2s). The experimental results are shown in FIG.

As shown in Figure 7, it was confirmed that a small concentration of standard protein complex can also be detected by EUSA. Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims

[Patent Claims]

[Claim 1]

Standard protein complex for multimer quantitation of multimer-forming polypeptides comprising:

(a) a carrier protein having a linker; And

(b) a polypeptide wherein the linker is covalently bonded to the N- or C-terminus, the polypeptide is a monomeric form or fragment thereof of a multimer-forming polypeptide having a reactor covalently bonded to the linker at the N- or C-terminus Each of 3-12 polypeptides binds to each linker of a carrier protein through the reaction step, and

The carrier protein is a protein that does not bind an antibody to the polypeptide.

[Claim 2]

The method of claim 1, wherein the multimer-forming polypeptide is β-amyloid, tau protein, prion protein α-synuclein, Ig light chain, serum amyloid A, transthyretin, cystatin C, β 2-microglobulin , Standard protein complex, characterized in that selected from the group consisting of huntingtin, superoxide dismutase, surfine and amylin.

[Claim 3]

The method of claim 1, wherein the linker of the carrier protein is a maleimide group, amino group, carboxyl group, aldehyde group, propionaldehyde group, butyl aldehyde group, succinimidyl propionate, succinimidyl carboxymethyl, hydroxy succinimidyl and succinimidyl Standard protein complex, characterized in that the functional group selected from the group consisting of carbonate.

[Claim 4]

The standard protein complex of claim 1, wherein the carrier protein is a globular protein.

[Claim 5]

According to claim 1, wherein the carrier protein is composed of albumin, ovalbumin, lactoalbumin, serum albumin, leucosin, legumelin, keyholelimpet hemocyanin, globin and globulin Standard protein complex, characterized in that selected from the group.

[Claim 6]

The standard protein complex of claim 1, wherein the polypeptide consists of 10 to 30 amino acids.

[Claim 7]

The standard protein complex according to claim 1, wherein the reactor of the polypeptide covalently bonded with the linker is a thiol group, a hydroxy group or an amino group.

[Port 8].

The standard protein complex of claim 1, wherein the antibody to the polypeptide is a capture antibody that recognizes an epitope on the polypeptide and a detection antibody that recognizes an epitope overlapped with the epitope.

[Claim 9]

A kit for the fractional detection of a multimer form from the monomeric form of a multimer-forming polypeptide in a biosample, comprising the standard protein complex of any one of claims 1 to 8.

[Claim 10]

Multimer quantification method of multimer-forming polypeptides using a standard protein complex, comprising the following steps:

(a) Recognize epitopes on the multimer-forming polypeptide to capture monomeric and multimer forms of the multimerizing polypeptide. Contacting the raw sample with a possible capture antibody;

(c) removing substances other than the capture antibody-multimer-type-detecting antibody complex formed in step (b) and measuring signal values generated by the detection antibody in the complex; And

(d) comparing the signal value obtained in step (c) with a calibration curve obtained using the standard protein complex to quantify the multimer of the multimer-forming pulley peptide, wherein the standard protein complex comprises a linker Having a carrier protein; And a polypeptide wherein the linker is covalently bonded to the N- or C-terminus, wherein the polypeptide is a monomeric form of a multimer-forming polypeptide or a fragment thereof and a semicoupling group covalently bonded to the linker at the N- or C-terminus. Wherein each of 3-12 polypeptides binds to each linker of a carrier protein through the reaction period, the carrier protein is a protein that does not bind to an antibody to the polypeptide,

The calibration curve comprises (i) quantifying a polypeptide of the standard protein complex; (ii) contacting said standard protein complex with a capture antibody capable of recognizing an epitope on said polypeptide and capturing said standard protein complex; (iii) contacting the standard protein complex with a detection antibody that recognizes the epitope overlapped with the epitope of step (ii) to form a capture antibody-standard protein complex-detection antibody complex, wherein the detection antibody generates a detectable signal To have a label; (iv) removing substances other than the capture antibody-standard protein complex-detecting antibody complex formed in step (iii) and measuring signal values generated by the detection antibody in the complex; And (V) implementing a calibration curve using the polypeptide quantitative value of the standard protein complex obtained in step (i) and the signal value obtained in step (iv).

[Claim 11]

The method of claim 10, wherein the multimer-forming polypeptide is β-amyloid, tau protein, prion protein, α-synuclein, Ig light chain, serum amyloid A, transthyretin, cystatin C, β2-microglobulin ， Bangbab, characterized in that selected from the group consisting of huntingtin, superoxide dismutase, surfine and amylin.

[Claim 12]

The linker of the carrier protein according to claim 10, wherein the linker of the carrier protein is a maleimide group, an amino group, a carboxyl group, an aldehyde group, a progoon aldehyde group, a butyl aldehyde group, succinimidyl propionate, succinimidyl carboxymethyl, hydroxy succinimidyl and succinimi And a functional group selected from the group consisting of dill carbonate.

[Claim 13]

The method of claim 10, wherein the carrier protein is a globular protein.

[Claim 14]

The method of claim 10, wherein the carrier protein is composed of albumin, ovalbumin, lactoalbumin, serum albumin, leucosin, legumelin, keyholelimpet hemocyanin, globin and globulin Selected from

[Claim 15]

The method of claim 10, wherein the polypeptide consists of 10 to 30 amino acids.

[Claim 16]

The method of claim 10, wherein the reactor of the polypeptide covalently bonded with the linker is a thiol group, a hydroxyl group or an amino group.