JP2014122179A - MONOCLONAL ANTIBODY SPECIFIC TO NANO-AGGREGATE OF β-AMYLOID PRECURSOR PROTEIN - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a material for specifically examining only amorphous Aβ aggregate of a nano size.SOLUTION: An antibody having relatively low reactivity with an amyloid-β protein aggregate whose particle diameter is not less than 220 nm binds specifically to an aggregate of an amorphous amyloid-β protein having a particle diameter from not less than 50 nm to less than 220 nm, and does not bind to an aggregate of a monomer amyloid-β protein and a fibrous amyloid-β protein.

Description

  The present invention relates to an antibody that specifically binds to an aggregate having a nano-size particle size among aggregates of amyloid β protein (Aβ) and use thereof.

The major pathological changes in Alzheimer's disease include senile plaques and neurofibrillary tangles. These senile plaques are recognized from the early stage of onset, and the main component is amyloid β protein (Aβ). The Aβ has attracted attention and has been extensively studied because it is deeply involved in the onset and progression of Alzheimer's disease.
Aβ is a peptide consisting of 40-42 amino acids that are produced by β-secretase and γ-secretase excised from β-amyloid precursor, and two types of Aβ _1-40 and Aβ _1-42 Has been identified (Non-Patent Document 1).

  Since the theory that the onset of Alzheimer's disease occurs when Aβ aggregates and deposits in brain tissue is prevalent, Aβ vaccine therapy and anti-Aβ monoclonal antibody therapy have been developed as therapeutic agents (Patent Literature) 1, 2).

  On the other hand, it is not monomeric Aβ that deposits in the brain, but fibrous or amorphous Aβ aggregates, and small amorphous Aβ aggregates of less than 32 mer are highly neurotoxic. Therefore, research is being carried out (Non-Patent Documents 2 and 3). Antibodies against these small amorphous Aβ aggregates (Non-patent Document 3) and fibrous Aβ aggregates (Non-patent Document 4) have also been produced.

International Publication No. 2008/156622 JP 2010-215651 A

Human Molecular Genetics, 13, 159-170 (2004) Neurol Sci. 2009 December; 30 (6): 471-477 J. Biol. Chem. (2011) Vol. 286, No.13, pp11555-11562 J. Biol. Chem. (2009) Vol. 284, No. 47, pp32895-32905

However, among the Aβ aggregates, nano-sized and large-sized amorphous aggregates have been confirmed to be in the brain tissue, but their actions have hardly been elucidated. The nano-sized aggregate is actually detected using an anti-Aβ antibody or the like, and its detection method and function elucidation have not been studied at all.
Accordingly, an object of the present invention is to provide a material for specifically detecting only nano-sized amorphous Aβ aggregates.

  Accordingly, the present inventor has focused on nano-sized amorphous Aβ aggregates, which have not been noticed until now, and decided to produce antibodies specific to the nano-sized amorphous Aβ aggregates. Therefore, by using an amorphous nanoaggregate that is easily dissolved in water as an antigen, an antibody that specifically binds to an amorphous Aβ aggregate having a particle size of 50 nm or more and less than 220 nm, which is nanosized, is obtained. Successful.

That is, the present invention relates to the following [1] to [6].
[1] Amyloid β that specifically binds to an aggregate of amorphous amyloid β protein having a particle size of 50 nm or more and less than 220 nm, does not bind to monomeric amyloid β protein or fibrous amyloid, and has a particle size of 220 nm or more Antibodies with relatively low reactivity with protein aggregates.
[2] The antibody according to [1], which is a monoclonal antibody.
[3] The antibody according to [2], which is produced by a hybridoma deposited as NITE P-1489.
[4] Detection of an amorphous amyloid β protein aggregate having a particle diameter of 50 nm or more and less than 220 nm in the sample, wherein the sample is reacted with the antibody according to any one of [1] to [3] Law.
[5] It is obtained by a cell fusion method using an amorphous nanoaggregate easily soluble in water of amyloid β protein as an immunogen, and specifically binds to an aggregate of amyloid β protein having a particle size of 50 nm or more and less than 220 nm. A hybridoma that produces an antibody that does not bind to monomeric amyloid β protein and fibrous amyloid β protein aggregates and has a relatively low reactivity with amyloid β protein aggregates having a particle size of 220 nm or more.
[6] The hybridoma according to [5], deposited as NITE P-1489.

  The antibody of the present invention is specific to nano-sized amorphous Aβ aggregates of 50 nm or more and less than 220 nm that have not been focused on and their function has not been known so far, and they are monomeric Aβ and fibrous Aβ aggregates. It does not react with aggregates and is relatively low in reactivity with amyloid β protein aggregates having a particle size of 220 nm or more. Therefore, using the antibody of the present invention, nanosized amorphous Aβ aggregates that have been known to be deposited in the brain but whose function has not been elucidated can be specifically detected. The antibody of the present invention is useful for elucidating the function of nano-sized amorphous Aβ aggregates, investigating the cause of Alzheimer's disease progression, clinical diagnostic agents, and the like.

It is a figure which shows the AFM image of an aggregate and a monomer. (A) Nanoaggregates: Large aggregates having a diameter of 200 nm or more are observed when mixed with elliptical nanoaggregates having a diameter of about 50 nm. (B) Microaggregate (LOA) (C) Fibrous Aβ _1-42 (D) Monomer It is a figure which shows the reactivity of each monoclonal antibody with respect to each aggregate. It is a figure which shows the reactivity of each monoclonal antibody with respect to the nano-aggregate fraction which carried out the size fractionation.

  The antibody of the present invention specifically binds to an amorphous Aβ aggregate (also referred to as nanoaggregate) having a particle size of 50 nm or more and less than 220 nm, does not bind to monomer Aβ and fibrous Aβ aggregate, Reactivity with amyloid β protein aggregates having a particle size of 220 nm or more is relatively low. Here, the amorphous Aβ aggregate having a particle diameter of 50 nm or more and less than 220 nm is different from the monomer Aβ, the fibrous Aβ aggregate and the micro-sized Aβ aggregate (LOA) which have been attracting attention. Moreover, the relatively low reactivity means that the absorbance by ELISA is ½ or less, preferably ３ or less.

  The antibody of the present invention may be either a monoclonal antibody or a polyclonal antibody as long as it has the above-mentioned characteristics, but a monoclonal antibody is preferable in that a highly specific antibody can be easily obtained.

  The polyclonal antibody of the present invention may be obtained by immunizing a mammal using an amorphous Aβ aggregate having a particle size of 50 nm or more and less than 220 nm as an antigen, collecting its antiserum, and confirming its specificity.

  The monoclonal antibody of the present invention can be prepared by culturing a hybridoma obtained by normal cell fusion. Here, the hybridoma is obtained by fusing a mammalian immune cell immunized with an amorphous Aβ aggregate having a particle size of 50 nm or more and less than 220 nm, and a myeloma cell, and from the obtained fused cell, an amorphous material having a size of 50 nm or more and less than 220 nm It is obtained by selecting a hybridoma that produces a monoclonal antibody that binds to a high quality Aβ aggregate, does not bind to monomeric Aβ and fibrous Aβ aggregates, and has a relatively low reactivity with microaggregates.

  As an antigen for producing a hybridoma, one of amyloid β protein aggregates, which is an amorphous nanoaggregate easily dissolved in water, that is, an amorphous Aβ aggregate having a particle diameter of 50 nm or more and less than 220 nm, is used. Use.

  Examples of animals used for the production of hybridomas include mice and rabbits, but it is preferable to use the most widely used mice for the production of hybridomas. Immunization of mice is performed by injecting the above-mentioned amorphous Aβ aggregates of 50 nm or more and less than 220 nm into mice with Freund's adjuvant as necessary. Next, spleen cells are collected from the immunized mouse, and cell fusion with a mouse myeloma cell line such as P3U1 is performed according to a conventional method.

The culture supernatant of the hybridoma grown in HAT medium is tested for the presence of monoclonal antibodies that bind to amorphous Aβ aggregates between 50 nm and less than 220 nm by conventional sandwich methods. In this assay, the amorphous Aβ aggregates, Aβ _1-42 , Aβ _1-40 , Aβ _16-20 , Aβ fibrous aggregates, and _{microaggregates} used as the antigen were used in the sandwich method. Use as an antigen to confirm specificity.

  Among these, monoclonal antibodies that bind to amorphous Aβ aggregates of 50 nm or more and less than 220 nm, do not bind to monomeric Aβ and fibrous Aβ aggregates, and have low reactivity with microaggregates. Nine clones were found that produced. Among these, one clone was named Anti-LFI 79-3, and was deposited as NITE P-1489 at the National Institute of Technology and Evaluation Microbiological Research Center.

  In order to produce the monoclonal antibody of the present invention, for example, any of the above hybridomas may be cultured in a conventional medium, and the target monoclonal antibody may be collected from the culture supernatant. Alternatively, any one of the hybridomas may be inoculated into the abdominal cavity of a mouse, ascites collected from the mouse, and the target monoclonal antibody collected from the ascites. Collection of monoclonal antibodies from the culture supernatant or ascites, that is, isolation and purification can be performed according to a conventional method. For example, salting out with ammonium sulfate, affinity chromatography using a support on which Aβ aggregates of 50 nm or more and less than 220 nm are fixed can be used in combination.

  The antibody of the present invention can be a chimeric antibody or a humanized antibody depending on the purpose. Furthermore, it can also be set as label | markers, such as fluorescence, light emission, a radioisotope, and a metal colloid.

  The obtained antibody of the present invention binds to amorphous Aβ aggregates of 50 nm or more and less than 220 nm, does not bind to monomer Aβ and fibrous Aβ aggregates, and is relatively reactive with microaggregates. Since it is low, it is useful for specifically detecting amorphous Aβ aggregates of 50 nm or more and less than 220 nm.

In order to detect an amorphous Aβ aggregate of 50 nm or more and less than 220 nm using the antibody of the present invention, the sample is reacted with the antibody of the present invention, and the amorphous particle having a particle diameter of 50 nm or more and less than 220 nm in the sample is detected. Amyloid β protein aggregates can be detected.
Examples of the detection means include ELISA, immunostaining, western blotting, immunohistochemical staining, fluorescent immunostaining, immunoprecipitation, and the like.
Examples of specimens include excised tissues, tissue sections such as brain sections, cultured cells, and the like.

  EXAMPLES Next, an Example is given and this invention is demonstrated in detail.

Example 1
[Method for preparing monoclonal antibody]
(1) Production of antigen

(Nano-aggregate)
Aβ _1-42 (AnyGen Co. Ltd, Korea) was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol to a concentration of 0.22 mM, and allowed to stand at 4 ° C. for 16 hours. After leaving still at 37 degreeC for 3 hours, it was made to dry under reduced pressure. This dissolution and drying under reduced pressure were repeated twice to produce amorphous nanoaggregates that were easily dissolved in water. Since this fraction contains aggregates of various sizes, size fractionation was performed by filtering with 0.22 μm, 300 kDa (about 50 nm) and 100 kDa filters.

(Micro agglomerates)
Add 0.22 mM Aβ _1-42 (AnyGen Co. Ltd, Korea) and 2.2 mM Aβ _16-20 (KLVFF) to suppress the formation of fibrous aggregates in phosphate buffered saline (Dulbecco's PBS) 37 The mixture was stirred at 3 ° C. for 16 hours. The residue obtained by filtering this solution through a 0.22 μm filter was used as a microaggregate (LOA).

(Fibrous Aβ aggregate)
Preparation of _{microaggregates} was carried out without adding Aβ _16-20, and then allowed to stand at 37 ° C. for 16 hours to produce fibrous Aβ aggregates.

(monomer)
Aβ _1-42 is added to 1,1,1,3,3,3-hexafluoro-2-propanol to a _{concentration} of 50 μM, suspended by sonication, and allowed to stand at 30 ° C. for 16 hours for complete dissolution. It was. What removed the solvent by drying under reduced pressure was used as a monomer.

The nano-aggregates, micro-aggregates (LOA), fibrous Aβ aggregates, and monomers formed as described above were observed using an atomic force microscope (AFM, JSPM-5200, JEOEL). Each aggregate or monomer of Aβ _1-42 was adjusted to 1 μg / ml, and 10 μl was dropped on the mica piece and dried under reduced pressure. The measurement was performed in the AC mode at a resonance frequency of 190 kHz. FIG. 1 shows an AFM image.

(2) Preparation of antibody The nanoaggregate prepared as described above (0.1 mg) was mixed with complete or incomplete Freund's adjuvant, and mice (BALB / C, 8 weeks, female) were immunized. Immunization was performed 3 times every 2 weeks. The spleen of the immunized mouse was removed, 20% of the number of mouse myeloma cells (P3U1) were mixed, and 1 ml of PEG (Roche Diagnostics) was stirred into this mixed cell for 1 minute. Slowly added. After stirring for 1 minute, 3 ml of serum-free medium FCS was slowly added with stirring over 3 minutes. Subsequently, 10 ml of serum-free medium was slowly added with stirring over 3 minutes. Thereafter, incubation (37 ° C., 5% CO ₂ ) was performed for 5 minutes, and the supernatant was discarded by centrifugation (1000 rpm, 5 min). Cells were denatured and cultured by diluting with HAT medium.

  Using this culture supernatant, cells that produce antibodies that react with nanoaggregates were screened by ELISA. Cells that obtained positivity were subjected to limiting dilution to unify hybridomas.

  In the ELISA method, each antigen was immobilized non-covalently by incubation with Nunc immunoplate maxi soap (Thermo scientific Inc.) at 4 ° C. for 16 hours. After washing with a solution (PBS-T) in which 0.05% Tween 20 was added to Dulbecco's PBS, a blocking buffer (ImmunoBlock, DS Pharma Biomedical Co., Ltd.) was added and incubated at 37 ° C. for 2 hours. After washing with PBS-T, HRP-labeled anti-mouse IgG goat (Sigma-Aldrich) as a secondary antibody was added and incubated at 37 ° C. for 2 hours. After washing with PBS-T, color was developed according to the description of SIGMAFAST OPD (Sigma-Aldrich) and measured at 492 nm.

ELISA was performed using the antibodies of the present invention using _{nanoaggregates at} various concentrations, fibrous Aβ _1-42 , monomeric Aβ _1-42 and _{microaggregates} as antigens.
2 and 3 show the results of ELISA.

As shown in FIG. 2, the monoclonal antibody of the present invention showed high reactivity to the nanoaggregate fraction. Although it was weak, reactivity was also observed in LOA, and it hardly reacted with fibrous and monomeric Aβ _1-42 and showed high reactivity only with nanoaggregates. On the other hand, the antibody 31-2 against LOA (Patent Document 3) showed equivalent reactivity to the nanoaggregate fraction and LOA.
Of the nine hybridomas obtained, one having relatively high reactivity with the nanoaggregate fraction and low reactivity with the fibrous and monomer was named Anti-LFI 79-3.

  As shown in FIG. 3, the monoclonal antibody of the present invention reacts best with the aggregate that passes through the 220 nm filter but does not pass through the 300 kDa filter in the nanoaggregate fraction. It reacts slightly with aggregates that do not pass through a 220 nm filter larger than this aggregate, but hardly reacts with aggregates of 300 kDa or less.

Claims (6)

  It specifically binds to an amorphous amyloid β protein aggregate having a particle size of 50 nm or more and less than 220 nm, does not bind to monomeric amyloid β protein and fibrous amyloid β protein aggregate, and has an particle size of 220 nm or more. Antibodies with relatively low reactivity with β protein aggregates.   The antibody according to claim 1, which is a monoclonal antibody.   The antibody according to claim 2, which is produced by a hybridoma deposited as NITE P-1489.   A method for detecting an amorphous amyloid β protein aggregate having a particle diameter of 50 nm or more and less than 220 nm in the sample, wherein the antibody according to any one of claims 1 to 3 is reacted with the sample.   One of the aggregates of amyloid β protein, which is obtained by a cell fusion method using an amorphous nanoaggregate that is easily dissolved in water as an immunogen, and aggregates of amorphous amyloid β protein having a particle size of 50 nm or more and less than 220 nm A hybridoma that produces an antibody that specifically binds to an aggregate, does not bind to amyloid β protein and fibrous amyloid β protein aggregates, and has relatively low reactivity with amyloid β protein aggregates having a particle size of 220 nm or more.   The hybridoma according to claim 5, which has been deposited as NITE P-1489.