JP2015141149A - MEASUREMENT METHOD FOR AMYLOID β PROTEIN AGGLOMERATE - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a measuring method for a marker allowing diagnoses of an Alzheimer disease.SOLUTION: An immunoassay of amyloid β protein aggregate in erythrocytes causes an anti-amyloid β protein aggregate antibody react to an erythrocyte-containing sample. An immunological measuring reagent is for the amyloid β protein aggregate in the erythrocytes containing an anti-amyloid β protein aggregate antibody. An anti-amyloid β protein aggregate antibody specifically combines with an amorphous amyloid β protein aggregate with a particle diameter of 50 nm or larger.

Description

  The present invention relates to an immunoassay and an immunoassay reagent for amyloid β protein aggregates in a red blood cell-containing sample.

  The pathogenesis of Alzheimer's disease is not yet known, and no fundamental treatment has been established. Alzheimer's disease rarely has local neurological symptoms other than cognitive dysfunction or neurological symptoms at an early stage. Although it can be diagnosed by image examination such as CT and MRI after the disease has progressed considerably, there is no fundamental treatment for Alzheimer's disease, so there is only treatment with drugs that suppress progression.

  Therefore, there is a strong demand for the development of a method for early and simple discovery of Alzheimer's disease. Since the main component of senile plaques, which is a major pathological change in Alzheimer's disease, is amyloid β protein (Aβ), the Aβ is deeply involved in the onset and progression of Alzheimer's disease and has been widely studied. ing. For example, means for measuring a decrease in Aβ42 and an increase in tau protein in cerebrospinal fluid have been proposed. However, collecting cerebrospinal fluid is a burden and involves the risk of infection, so it is not a means that can be used in health examinations. Moreover, although image diagnosis by CT or the like is simple, it can be confirmed only after the disease has progressed to some extent.

Recently, it has been reported that Aβ is present in erythrocytes and that Aβ _1-42 and Aβ _{1-40 in} erythrocytes can be measured (Non-patent Document 1).

Kiko et al, PLOSone, 7, Nov. 2012, e49620

However, the amount of Aβ in erythrocytes is reported to be about 10 ng / mL, and the detection lower limit of conventional sandwich ELISA is about 100 ng / mL, and Aβ in erythrocytes cannot be measured by a simple immunoassay.
Therefore, it has been desired to develop a marker measurement method that can diagnose Alzheimer's disease by a simpler means.

  Accordingly, the present inventor has measured the amount in a body fluid sample using an antibody specific for Aβ aggregates instead of Aβ, and found for the first time that high concentrations of Aβ aggregates are present in erythrocytes. As a result of further studies, the concentration of Aβ aggregates in erythrocytes correlates with Alzheimer's disease, and by measuring this, it was found that Alzheimer's disease can be diagnosed easily and early, and the present invention was completed.

  That is, the present invention provides the following [1] to [8].

[1] An immunoassay method for Aβ aggregates in erythrocytes, wherein an anti-Aβ aggregate antibody is reacted with a sample containing erythrocytes.
[2] The immunoassay method according to [1], wherein the anti-Aβ aggregate antibody is an antibody that specifically binds to an amorphous Aβ aggregate having a particle diameter of 50 nm or more.
[3] The immunoassay method according to [1] or [2], wherein the red blood cells are human red blood cells.
[4] The immunoassay method according to any one of [1] to [3], which is measured for the purpose of diagnosing Alzheimer's disease.
[5] A reagent for immunological measurement of Aβ aggregates in erythrocytes containing anti-Aβ aggregate antibodies.
[6] The immunoassay reagent according to [5], wherein the anti-Aβ aggregate antibody is an antibody that specifically binds to an amorphous Aβ aggregate having a particle diameter of 50 nm or more.
[7] The immunoassay reagent according to [5] or [6], wherein the red blood cells are human red blood cells.
[8] The immunoassay reagent according to any one of [5] to [7], which is a diagnostic agent for Alzheimer's disease.

  According to the immunological measurement method of the present invention, Aβ in an erythrocyte-containing sample can be measured by a simple means, and Alzheimer's disease can be diagnosed at an early stage.

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 immunological measurement method for Aβ aggregates in erythrocytes of the present invention is characterized in that an anti-Aβ aggregate antibody is reacted with a sample containing erythrocytes.

  The sample to be measured is a red blood cell-containing sample, and a human red blood cell-containing sample is particularly preferable. The red blood cell-containing sample may be whole blood or a red blood cell fraction in blood, but the red blood cell fraction is more preferable from the viewpoint of measurement accuracy.

  The anti-Aβ aggregate antibody used for the measurement is preferably an antibody that does not react with Aβ monomer or fibrous Aβ and specifically binds to the Aβ aggregate, and is specific to an amorphous Aβ aggregate having a particle diameter of 50 nm or more. More preferred are antibodies that bind to each other. Such Aβ aggregate-specific anti-Aβ aggregate antibodies include those previously reported by the present inventors (J. Biosci. Bioeng. 115, 216-220 (2013), Adv. Biosci. Biotechnol., 4, 63-66 (2013), JP2013-159596 (antibodies against Aβ aggregates having a particle size of 220 nm or more), and amorphous Aβ aggregates (also referred to as nanoaggregates) having a particle size of 50 nm or more and less than 220 nm. Examples thereof include an antibody having a high reactivity and a relatively low reactivity to an Aβ aggregate having a particle size of 220 nm or more, etc. Here, the relatively low reactivity means that the absorbance by ELISA is ½ or less, preferably Means 1/3 or less.

  The anti-Aβ aggregate antibody used in 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 anti-Aβ aggregate polyclonal antibody used in the present invention may be obtained by immunizing a mammal using an Aβ aggregate having a particle diameter of 50 nm or more as an antigen, collecting the antiserum, and confirming its specificity.

  The anti-Aβ aggregate monoclonal antibody used in the present invention can be prepared by culturing a hybridoma obtained by normal cell fusion. Here, the hybridoma is a fusion of an immune cell of a mammal immunized with an amorphous Aβ aggregate having a particle diameter of 50 nm or more and a myeloma cell. From the obtained fused cell, an amorphous Aβ aggregate of 50 nm or more is obtained. It is obtained by selecting a hybridoma that produces a monoclonal antibody that binds to the aggregate and does not bind to monomeric Aβ and fibrous Aβ aggregates.

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

  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 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 of 50 nm or more by conventional sandwich methods. In this assay, an amorphous Aβ aggregate of 50 nm or more used as an antigen, Aβ _1-42 , Aβ _1-40 , Aβ _16-20 , Aβ fibrous aggregate was used as an antigen for the sandwich method, Specificity can be confirmed.

  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 used in 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 are fixed can be used in combination.

  The antibody used in 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.

  Measurement of Aβ aggregates in erythrocyte-containing samples is performed by immunological measurement means using anti-Aβ aggregate antibodies. More specifically, ELISA, EIA, immunoturbidimetric method, immunoprecipitation method, EMIT method, immunochromatography method and the like can be mentioned. Among these, the ELISA will be explained. The anti-Aβ aggregate antibody is bound to a carrier such as a microtiter plate, beads, nitrocellulose membrane, nylon membrane, etc. Binding to anti-Aβ aggregates on the carrier. After washing the unbound fraction, contact with an anti-Aβ aggregate antibody labeled with a radioisotope, enzyme, fluorescent substance, biotin, etc., and then measure the Aβ aggregate by measuring the label. Can do.

  According to an immunoassay using an anti-Aβ aggregate antibody, the concentration of Aβ aggregate in a 50% solution of normal human erythrocytes was 5.0 μg / mL, which was found to be a sufficiently detectable concentration.

  Further, it was found that the Aβ aggregate concentration in the blood of Alzheimer model mice (tg2576) was significantly lower than the Aβ aggregate concentration in the blood of normal animals, and if the Aβ aggregate concentration in the blood was measured, Early diagnosis of Alzheimer's disease is possible. That is, when the Aβ aggregate concentration in the red blood cell-containing sample in the test red blood cell-containing sample is lower than the Aβ aggregate concentration in the red blood cell-containing sample derived from a healthy subject, the test sample is derived from Alzheimer's disease. Can be determined.

  In order to carry out the immunoassay method of the present invention, it is preferably used as an Aβ immunoassay reagent in erythrocytes containing an anti-Aβ aggregate antibody. The immunological measurement reagent includes an anti-Aβ aggregate antibody, an anti-Aβ aggregate antibody label, a buffer, a standard reagent, a protocol (instruction manual), and the like.

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

Reference 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 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 Inc.) 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 was performed for 5 minutes (37 ° C., 5% CO ₂ ), 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.

Example 1
A. Experimental method (1) Preparation of Aβ _1-42 aggregate 100 μg of commercially available Aβ _1-42 (AnyGen, purity: 95.6% (HPLC, MASS)) was added to a 1.5 mL tube to obtain 1,1,1,3,3. This was dissolved in 200 μl of 3,3-hexafluoro-2-propanol to prepare a 0.5 mg / mL Aβ _1-42 aggregate solution. This solution was incubated at 4 ° C. for 16 hours, and further incubated at 37 ° C. for 3 hours. Thereafter, freeze-drying was performed and the above operation was repeated. This solution was adjusted to 1 mg / mL with ultrapure water to form soluble Aβ _1-42 aggregates (Shimizu T. et al., J. Biosci. Bioeng., 115, 216-220 (2013)). The antigen concentration was calculated by measuring the absorbance at a wavelength of 280 nm and setting the absorbance 1 to 1 mg / mL.

(2) Preparation of calibration curves for Aβ _1-42 aggregates by sandwich ELISA Nine types of monoclonal antibodies that recognize only Aβ _1-42 aggregates without reacting with fibrous Aβ _1-42 and Aβ _1-42 monomers (Shimizu T et al., J. Biosci. Bioeng., 115, 216-220 (2013) and Shimizu T. et al., Adv. Biosci. Biotechnol., 4, 63-66 (2013)) Aβ _1-42 aggregates were detected by sandwich ELISA using two antibodies as capture antibody and HRP-labeled detection antibody, and the combination with the highest detection sensitivity was selected. 100 μl of anti-Aβ _1-42 aggregate monoclonal antibody 2 μg / mL as a primary antibody was added to each well of a 96-well plate (Nunc Immunoplate Maxi Soap, Thermo Fisher Scientific Inc) and left at 37 ° C. for 1 hour. Thereafter, the solution was removed and washed with PBS-T in which 0.05 V / v% of Tween 20 was added to Dulbecco's PBS (Wako Pure Chemical Industries), 200 μl of immunoblock was added as a blocking buffer, and the mixture was allowed to stand at 37 ° C. for 1 hour. After removing the solution, the _plate was washed with PBS-T, and 100 μl of Aβ _1-42 aggregate was added as an antigen. After standing at 37 ° C. for 1 hour, the solution was removed and washed with PBS-T. 100 μl of HRP-labeled antibody was added as a detection antibody, allowed to stand at 37 ° C. for 1 hour, removed from the solution, and washed with PBS-T. Color development was performed with o-phenylenediamine dihydrochloride (SIGMA), and the absorbance at a wavelength of 492 nm was measured using a microplate reader.

(3) Detection of Aβ _1-42 aggregates in erythrocytes An equal amount of distilled water was added to human erythrocytes (Lee BioSolutions, Inc) and mixed by shaking to lyse. The protein concentration in this sample was measured by a BCA protein measurement kit (Pierce), and the Aβ _1-42 aggregate concentration was measured by a sandwich ELISA.
As an Alzheimer's disease model mouse, a genetically modified mouse called Tg2576 is widely used. Tg2576 the recombinant mouse models so as to produce a large amount of A [beta] _1-42 by introducing mutations into the precursor protein gene of A [beta] _1-42, senile plaques are formed on the brain rearing 12 months. Blood samples were collected from the tails of 3 each of Tg2576 and wild type 17-month-old male mice, and the concentration of Aβ _1-42 aggregates contained in the sample mixed with the same amount of distilled water and shaken was measured.

B. Results When the antigen Aβ _1-42 aggregate 1 μg / mL was measured, the antibody combination that gave the highest absorbance was when 77-3 and 37-11 were used as the capture antibody and the detection antibody, respectively. Thereafter, sandwich ELISA was performed using this antibody combination. At this time, when the detection standard value was 3.3 times the standard deviation of the absorbance of the negative control to which no antigen was added, the detection minimum value was 0.01 μm / mL.
The concentration of Aβ _1-42 aggregate in a sample obtained by treating human erythrocytes with an equal amount of distilled water was 1.7 μg / mL, which was a measurable value in consideration of the lower limit of detection. The protein concentration in this sample was 23 mg / mL, and erythrocytes were found to contain 226 ng (50 pmol) of Aβ _1-42 aggregate per mg of protein.
Table 1 shows the amount of Aβ _1-42 aggregate per mg of protein contained in the blood of Tg2576 and wild type mice. When eight mouse blood samples were examined, the average amount of Aβ _1-42 of Tg2576 mice was 8.1 ng, while that of wild-type mice was 10.1 ng, which was low for model mice. It was. This result is consistent with the result that the Aβ _1-42 concentration in the cerebrospinal fluid of Alzheimer's disease patients is about 50% lower than that of healthy individuals (Mikio Tokai Hayashi, Cognition and Dimentia, 8, 2790-275 (2009) ). The measurement of the Aβ _1-42 aggregate concentration in erythrocytes by this method is a more simple method.

Claims (8)

  An immunological assay for amyloid β protein aggregates in erythrocytes, wherein an anti-amyloid β protein aggregate antibody is reacted with an erythrocyte-containing sample.   The immunoassay method according to claim 1, wherein the anti-amyloid β protein aggregate antibody is an antibody that specifically binds to an amorphous amyloid β protein aggregate having a particle diameter of 50 nm or more.   The immunological assay according to claim 1 or 2, wherein the erythrocytes are human erythrocytes.   The immunological measurement method according to any one of claims 1 to 3, which is measured for the purpose of diagnosing Alzheimer's disease.   A reagent for immunological measurement of amyloid β protein aggregates in erythrocytes containing anti-amyloid β protein aggregate antibodies.   The immunoassay reagent according to claim 5, wherein the anti-amyloid β protein aggregate antibody is an antibody that specifically binds to an amorphous amyloid β protein aggregate having a particle diameter of 50 nm or more.   The immunoassay reagent according to claim 5 or 6, wherein the erythrocytes are human erythrocytes.   The immunological measurement reagent according to any one of claims 5 to 7, which is a diagnostic agent for Alzheimer's disease.