JP3542240B2 - Use as marker for dialysis amyloidosis and immunoreagent for dialysis amyloidosis - Google Patents

Description

【００５５】
この結果から、作成したＣＭ化ヒト血清アルブミンに対する抗体は、ＣＭ化ヒト血清アルブミンのみならず、ＣＭ化ヘモグロビンやＣＭ化β２ＭでもＣＭ化ヒト血清アルブミンとＣＭ化ヒト血清アルブミンに対する抗体の抗原抗体反応が阻害されたことから、ＣＭ化ヘモグロビンおよびＣＭ化β２Ｍとも反応性を示すことが示唆された。
【００５６】
（７） ＤＲＡ患者の血中ＣＭ化ヘモグロビンの測定
ＤＲＡ患者２０人よりＥＤＴＡ−２Ｋを含む真空採血管にて採血した血液５０μｌを、２５０μｌの生理食塩水で１回洗浄し赤血球を得た後、該赤血球に１ｍｌの精製水を加え溶血させたものを被検体とした。
【００５７】
被検体中のＣＭ化ヘモグロビンの測定はドットブロッティング法にて行った。ヘモグロビン濃度をシアンメトヘモグロビン法にて測定した後、該ヘモグロビンが５００ｎｇとなるようにドットブロッティング装置（バイオラッド社製）を用いてＰＶＤＦ膜（バイオラッド社製）に吸着させた。該膜を１０％のスキムミルクを含む２０ｍＭのリン酸緩衝液（ｐＨ７．４）に室温で１時間浸せきし、該膜を取り出し、前記ビオチン標識した１μｇ／ｍｌのＣＭ化ヒト血清アルブミンに対する抗体溶液を５ｍｌ加えた。室温で１時間のインキュベーションの後に、０．０５％のＴｗｅｅｎ２０を含む２０ｍＭのリン酸緩衝液（ｐＨ７．４）５０ｍｌで該膜を３回洗浄した。次いで、該膜にアビジン−ペルオキシダーゼ標識ビオチン複合体溶液（ベクタステインＡＢＣキット：フナコシ社製）を５ｍｌ加え、室温で１時間のインキュベーションした。０．０５％のＴｗｅｅｎ２０を含む２０ｍＭのリン酸緩衝液（ｐＨ７．４）５０ｍｌで該膜を３回洗浄した後、ＥＣＬウエスタンブロッティング検出試薬（アマシャム社製）を２ｍｌ加えた。該膜における発光強度の検出は、バイオラッドＧＳ−３６３モレキュラーイメージャーを用いて行った。
【００５８】
対照として市販のヘモグロビン（シグマ社製）を被検体として上記と同様の方法にてＣＭ化ヘモグロビンの測定を行った。該市販ヘモグロビンの発光強度を１００として、上記ＤＲＡ患者由来のヘモグロビンの発光強度の相対値を表示した。また、発光強度の実測値も併せて表示した。測定結果を表２および図２に示す。
【００５９】
【表２】

【００６０】
比較例１
実施例１でＤＲＡ患者の代わりに、健常者２０人から得た血液を被検体としたこと以外は、実施例１の方法に従って被検体中のＣＭ化ヘモグロビンの測定を行った。実施例１と同様、上記市販ヘモグロビンの発光強度を１００として、上記健常者由来のヘモグロビンの発光強度の相対値を表示した。また、発光強度の実測値も併せて表示した。測定結果を表３および図２に示す。
【００６１】
【表３】

【００６２】
健常者群と実施例１で測定したＤＲＡ患者群の発光強度を統計学的に比較（ｔ検定）したところ、危険率５％未満で、ＤＲＡ患者群の発光強度の方が健常者群の発光強度に比べて有意に高かった。このことは、ＤＲＡ患者由来の血液中には、健常者由来の血液中よりも、有意にＣＭ化ヘモグロビンが多いことを意味する。
【００６３】
上記の結果から、ＣＭ化ヘモグロビンがＤＲＡ用マーカーとしての使用が有効であることが明確になった。
【００６４】
実施例２
実施例１でのＤＲＡ患者の血液の代わりに、該患者血清をフィルター濾過にて分子量３万以上の物質と分子量１万以下の物質を除去した溶液をを被検体としたこと以外は、実施例１の方法に従って被検体中のＣＭ化β２Ｍの測定を行った。発光強度の実測値を表示した。測定結果を表４および図３に示す。
【００６５】
【表４】

【００６６】
比較例２
実施例２でＤＲＡ患者の代わりに、健常者５人から得た血清を被検体としたこと以外は、実施例２の方法に従って被検体中のＣＭ化β２Ｍの測定を行った。発光強度の実測値を表示した。測定結果を表４に示す。
【００６７】
健常者群と実施例２で測定したＤＲＡ患者群の発光強度を比較したところ、健常者血清由来の被検体では検出感度以下であったのに対し、ＤＲＡ患者群の発光強度は有意に高かった。このことは、ＤＲＡ患者由来の血清中には、健常者由来の血清中よりも、有意にＣＭ化β２Ｍが多いことを意味する。
【００６８】
上記の結果から、ＣＭ化β２ＭがＤＲＡ用マーカーとしての使用が有効であることが明確になった。
【図面の簡単な説明】
【図１】本図は、作成したＣＭ化ヒト血清アルブミンに対する抗体の抗原特異性を競合法ＥＬＩＳＡにて調べた結果で、縦軸が４０５ｎｍの吸光度、横軸が各阻害剤の添加量である。
【図２】本図は、健常者とＤＲＡ患者の血液中Ｍ化ヘモグロビンのＣＭ化率との関係を示すグラフである。
【図３】本図は、健常者とＤＲＡ患者の血液中ＣＭ化β２Ｍ濃度との関係を示すグラフである。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to the use of a specific protein or peptide as a marker for dialysis amyloidosis, and to an immunoreagent for dialysis amyloidosis or for evaluating its efficacy.
[0002]
[Prior art]
Dialysis-related amyloidosis (hereinafter, also simply referred to as “DRA”) is amyloidosis associated with patients undergoing dialysis treatment, and mainly involves the destruction of bone and joint structures.
[0003]
In recent years, the main component of the deposited amyloid is β2 microglobulin (hereinafter abbreviated as β2M), and amyloid having β2M as a precursor protein is deposited in the synovium of the joint, and carpal tunnel syndrome, polyarthritis, destruction It has been revealed that vertebral spondyloarthritis and the like develop. However, there is no correlation between the onset of dialysis amyloidosis and the blood β2M concentration, suggesting the involvement of factors other than blood β2M concentration. Among them, a modified product of β2M has attracted special attention, and studies on the modified product have been conducted. Recently, Miyata et al. Have revealed that a late product of the Maillard reaction of a protein (hereinafter abbreviated as “AGE”) is present in the deposited amyloid, and the AGE has a pentosidine structure and a carboxymethyl lysine structure. While confirming β2M (hereinafter abbreviated as “AGE-β2M”), it was suggested that AGE-β2M may be involved in the development of bone and joint disorders (J. Clin. Invest. 92: 1243). -1252, 1993 and Clinical Dialysis, Vol. 12, No. 8: 1163-1170, 1996).
[0004]
However, the etiology and pathology of DRA have not been fully elucidated, and no fundamental preventive and therapeutic measures have been established. Therefore, it is important to delay the medical condition by appropriate treatment based on accurate early diagnosis.
[0005]
Thus, it is extremely important to diagnose DRA at an early stage, but the conventional diagnostic methods have problems in terms of accuracy and burden on the subject.
[0006]
That is, conventionally, DRA has been diagnosed by Congo red staining, mainly using a tissue at a site where symptoms have been presented. However, there is a problem that this diagnostic method involves physical pain and damage to a living body. In addition, there is also a problem that the subject may not be stained with Congo Red even if the subject clearly exhibits the symptoms of DRA.
[0007]
Therefore, in recent years, as a more accurate and non-invasive method, a diagnosis has been attempted by labeling β2M in a living body with a radioisotope and obtaining an amyloid deposition site as an image using a scintillation camera or the like. However, this diagnostic method also has problems such as a large effect on the living body such as release and accumulation of the labeled radioisotope, and a complicated operation for responding to the problem.
[0008]
As described above, various studies on the mechanism of the onset of DRA have been carried out, but there is no known example in which the findings obtained in these studies are applied to a new diagnostic method that solves the problems of the above diagnostic methods. For example, the study by Miyata et al. Described above only considers that AGE-β2M is involved in the development of DRA, and correlates AGE-β2M concentration in body fluids such as blood, urine, and lymph with DRA development. At present, it has not been investigated. That is, it was completely unknown whether AGE-β2M could be used effectively as a marker for DRA.
[0009]
[Problems to be solved by the invention]
As described above, a method capable of diagnosing DRA accurately and easily with a light burden on a subject has not yet been developed. Therefore, the object of the present invention
The object is to provide a new DRA diagnostic method that does not have the above-mentioned problems. More specifically, to find a substance present in a body fluid commonly used as a specific sample in a clinical test and exhibiting a certain significant difference between a DRA-onset patient and a healthy person, and furthermore, a reagent for detecting the substance To provide a new diagnostic system capable of accurately and easily diagnosing DRA.
[0010]
[Means for Solving the Problems]
The present inventors have generally focused on the fact that AGE has a specific physiological activity, thought that AGE may be a marker for DRA, and variously examined the relationship between in vivo AGE and the development of DRA. I went. As a result, with respect to AGE having a specific substituent (that is, a protein having a specific substituent), the specific protein concentration in a living body of a DRA-onset patient and a healthy subject and the specific protein present in one molecule of the specific protein are considered. A new finding that there is a significant difference from the average number of substituents, and furthermore, the amount of the specific protein in the sample by the reagent using an antibody that specifically reacts with the specific protein, The inventors have found that the average number of specific substituents present in one molecule of the specific protein can be easily measured, and have completed the present invention.
[0011]
That is, the present invention Carboxymethylated hemoglobin in which at least a part of the amino groups of the constituent amino acid units are carboxymethylated As a marker for dialysis amyloidosis.
[0012]
Also, another invention is Using an antibody that specifically reacts with carboxymethylated hemoglobin in which at least a part of the amino groups of the constituent amino acid units are carboxymethylated It is an immunoreagent for dialysis amyloidosis diagnosis or efficacy evaluation.
[0013]
BEST MODE FOR CARRYING OUT THE INVENTION
The protein used as a marker in the present invention is not particularly limited as long as it is a protein in a living body. Examples thereof include albumin, β2M, simple proteins such as histone, collagen, γ globulin, glycoproteins such as erythrocyte membrane protein, low-density lipoprotein and Examples thereof include complex proteins such as lipoproteins such as high-density lipoproteins and metal proteins such as hemoglobin and transferrin, and are preferably proteins derived from body fluids that can easily collect a specimen from a subject. In particular, among proteins derived from body fluids, hemoglobin, which has a long life as a protein and has a high blood concentration, or β2M, which is a main component of deposited amyloid, is more preferable.
[0014]
The peptide used as a marker in the present invention may be an oligopeptide or a polypeptide as long as it is a peptide in a living body, and examples thereof include degradation products of the above proteins.
[0015]
The carboxymethylated protein or carboxymethylated peptide (hereinafter, also simply referred to as “CM product”) in the present invention means that at least a part of the amino groups of the amino acid units constituting the protein or peptide is carboxymethylated (hereinafter, simply referred to as “CM”). (Also called "CM"). Examples of the amino group to be converted into CM include an amino group on a lysine side chain and an amino group at the N-terminus, and do not include -NH- in an amide bond.
[0016]
As shown in the examples described later, the amount of the CM compound present in the body of the DRA patient and the average number of carboxymethyl groups present in one molecule of the CM compound (hereinafter, also simply referred to as “CM conversion rate”) Since was significantly higher than that of healthy subjects (see Tables 1, 2, and 3), the CM compound used in the present invention can be a marker for DRA in the field of clinical examination. That is, by measuring the CM conversion rate and amount of a CM compound in a living body, particularly in blood, it is possible to judge whether or not it is affected by DRA, and to predict the onset and progress degree of DRA. Become. In Example 1 to be described later, CM-modified hemoglobin was used as a CM product, and the DRA patients received artificial dialysis, so that the absolute amount of hemoglobin was smaller than that in healthy subjects. No correlation with the CM compound concentration was investigated. However, as shown in Example 2, in the case of CM2 β2M, a good correlation is observed between the concentration of the CM compound in the sample of the DRA patient and the concentration of the CM compound in the sample of a healthy subject.
[0017]
Although there is no known method for directly measuring the amount and the rate of CM conversion in a living body (for example, in blood or urine), a CM-modified amino acid is obtained by decomposing the CM-modified product, A method for indirectly measuring the amount of a CM compound by measuring the amount is already known. As an example of such an indirect measurement method, a method of hydrolyzing a CM compound and detecting the amino acid converted into a CM by liquid chromatography / mass spectrometry, and a method of hydrolyzing the CM compound and gas chromatography / mass A method for detecting amino acids converted into CM by analysis may be mentioned. However, these indirect measurement methods have problems in that the operation is complicated and the sensitivity is low. Therefore, a direct measurement method that is easy to measure and has high sensitivity has been required. Therefore, the present inventors have developed a method for detecting a CM compound in a living body by an antigen-antibody reaction using an antibody that specifically reacts with the CM product (hereinafter, also simply referred to as “anti-CM antibody”).
[0018]
Such an anti-CM antibody can be obtained by using a CM compound synthesized using an organic synthetic technique as an immunogen (antigen). The protein serving as a raw material for synthesizing such a CM product is not particularly limited. For example, simple proteins such as albumin, β2M, histone, collagen proteins, γ globulin, glycoproteins such as erythrocyte membrane proteins, low density lipoproteins and the like. Examples include complex proteins such as lipoproteins such as high-density lipoproteins and metal proteins such as hemoglobin and transferrin.
[0019]
When a peptide is used as a raw material for synthesizing a commercialized product, the peptide used as the raw material may be an oligopeptide or a polypeptide, for example, a product obtained by artificially synthesizing a degradation product of the protein or a specific region of the protein. Etc. can be used.
[0020]
The method of carboxymethylating (CM-forming) the amino group of the amino acid constituting the raw material protein or raw material peptide, that is, the method of substituting the hydrogen of each amino group with a -CH2-COOH group, is not limited to any known method. Used without. For example, as in the reductive alkylation method described in “New Chemistry Experiment Course 1, Protein 4” (edited by the Biochemical Society of Japan, pp. 13-16, Tokyo Kagaku Dojin, published on March 20, 1991), CHO- An aldehyde compound represented by COOH and a protein or peptide are dissolved in an aqueous solution such as a borate buffer or a phosphate buffer, and the solution is adjusted to pH 8 to 10 in the presence of a hydride reducing agent such as sodium borohydride or sodium cyanoborohydride. The reaction may be carried out. If the pH is higher than this, proteins and the like may be denatured, and if the pH is lower than this, the hydride reducing agent becomes unstable. In order to allow the reaction to proceed specifically and quantitatively, the reaction is preferably performed at a temperature of 0 to 10 ° C. Alternatively, it can also be obtained by aseptically incubating a reducing sugar and a protein at 37 ° C. for about 60 days in the presence of oxygen.
[0021]
The resulting CM product is generally soluble in water and precipitates upon addition of acetone, alcohol, ammonium sulfate, heavy metal salt, or the like. In addition, the CM compound can be detected by any known method other than the method of detecting an amino group, such as an ultraviolet absorption method, a dye binding method, and a phenol reagent method. In general, in the above method, substances having an amino group, such as proteins, lipids, and carbohydrates, are easily converted to CM. However, in proteins or peptides, the side chain of lysine or the N-terminal amino group is selectively converted to CM. Easy to be.
[0022]
A method for obtaining an anti-CM antibody using the CM compound synthesized in this manner as an antigen is not particularly limited, and a method of immunizing a host animal with the CM compound as an antigen can be arbitrarily adopted.
[0023]
At this time, the antigen is not particularly limited as long as it is a CM compound. However, when a CM peptide is used as the antigen, the ability to induce an immune response may be weak, and therefore, hemocyanin, bovine serum albumin, β-galactosidase, etc. It is preferable to use a known carrier protein with a CM peptide bound thereto as an antigen. For the binding of the CM peptide to the carrier protein, the method usually used for the purpose is used without any limitation.
[0024]
Antibodies produced using such antigens are not particularly limited in their origin. Rabbits, goats, mice, guinea pigs and other host animals can be purified by dialysis, centrifugal concentration, or purified by liquid chromatography or the like. Antiserum obtained by immunization as an antigen, ascites fluid, or the like, or purified by a conventionally known method such as salting out, gel filtration, ion exchange chromatography, affinity chromatography, electrophoresis and the like, and polyclonal antibody Can be used as Alternatively, a monoclonal antibody prepared from a hybridoma obtained by fusing antibody-producing cells such as mammalian spleen cells and lymph node cells sensitized with an antigen with myeloma cells, or a salting-out method known in the art. , Gel filtration, ion exchange chromatography, affinity chromatography, electrophoresis and the like.
[0025]
These antibodies can be used as they are when used as an immunoreagent, but active fragments of antibodies such as Fab, Fab ', and F (ab') 2 obtained by enzymatically treating these antibodies (antigen recognition of antibodies) Portion containing the site) may be used as an anti-CM antibody.
[0026]
The form of the immunoreagent of the present invention is not particularly limited as long as it is a reagent that can detect a CM compound by utilizing an antigen-antibody reaction between an anti-CM antibody and a CM compound. For example, an anti-CM antibody, an antibody against a biological component such as β2M or hemoglobin, and a CM compound are appropriately supported on an insoluble carrier so as to correspond to the non-competitive method, the competitive method, and the sandwich method described below. Can be done. CM products can be measured by detecting an antigen-antibody reaction caused by bringing each of such immunological reagents into contact with an antigen and / or an anti-CM antibody in a sample according to a measurement method. Such an antigen-antibody reaction can be detected by utilizing the agglutination of an insoluble carrier or the like when the immunoreagent of the present invention is a so-called immunoagglutination reagent, and the immunoreagent of the present invention is a so-called labeled immunoreagent. When the reagent is a measurement reagent, it can be detected as a change in a physical quantity such as colorimetry, luminescence, or fluorescence.
[0027]
To illustrate specific embodiments of the immunoreagent of the present invention, qualitative reagents include latex agglutination reagents and microtiter reagents, and quantitative reagents include radioimmunoassay reagents, enzyme immunoassay reagents, and fluorescent immunoassay reagents. , A chemiluminescence immunoassay reagent, a latex quantitative reagent, and the like.
[0028]
The shape of the anti-CM antibody or the antibody against the biological component, or the shape of the insoluble carrier that supports the CM compound in the sample may be appropriately selected depending on the purpose of use, for example, beads, test plates, spheres, and the like. Examples include a disk shape, a tube shape, and a filter shape. In addition, as the material thereof, those used as ordinary immunoassay carriers, for example, glass, polysaccharides or derivatives thereof, silica gel, porous ceramics, metal oxides, red blood cells, propylene, styrene, acrylamide, acrylonitrile, etc. Synthetic resins and those obtained by introducing a reactive functional group such as a sulfone group and an amino group into these by a known method are exemplified.
[0029]
As a method for immobilizing an anti-CM antibody or an antibody against a biological component on an insoluble carrier, or an antigen in a specimen, known methods such as a physical adsorption method, a covalent bonding method, an ionic bonding method, and a crosslinking method can be used without any limitation. .
[0030]
The basic operation of the measurement of the CM compound in the labeled immunoassay reagent can be performed according to a conventional assay method, for example, an enzyme immunoassay (EIA) method such as a radioimmunoassay (RIA) method, an ELISA method, a Western blotting method, a dot blotting method, or the like. it can. The operation, procedure, and the like in each of these assay methods are not particularly different from those generally employed, and can be in accordance with known non-competitive methods, competitive methods, sandwich methods, and the like. In the non-competitive method, the CM compound or the anti-CM antibody in the sample may be supported on an insoluble carrier, and then contacted with the anti-CM antibody or the CM compound. In the competition method, for example, after an artificially produced CM compound is carried on an insoluble carrier, an anti-CM antibody previously reacted with the CM compound in the sample may be brought into contact. In the sandwich method, after an anti-CM antibody or an antibody against a biological component such as β2M or hemoglobin is carried on an insoluble carrier, the antigen in the sample is brought into contact, and an antibody against the biological component or an anti-CM antibody is further brought into contact. Good. By these measurement methods, it is possible to measure the CM rate of the protein or peptide or the amount of the CM product.
[0031]
Examples of the labeling agent in the labeled immunoassay reagent include radioactive substances such as radioactive iodine and radioactive carbon, fluorescent substances such as fluorescein isothiocyanate and tetramethylrhodamine, and enzymes such as alkaline phosphatase and peroxidase. The antigen-antibody reaction product obtained by such a method is detected using radioactivity, colorimetry, fluorescence, luminescence, and the like.
[0032]
For example, an anti-CM antibody or an antigen in a sample is carried on an insoluble carrier at a rate of 0.01 to 1000 μg / cm 2, and 0.001 to 1000 μg of the antigen or the anti-CM antibody in the sample is brought into contact with the carrier for measurement. When an antibody against a biological component is carried on an insoluble carrier, as described above, after the antigen in the sample is brought into contact, an anti-CM antibody is further brought into contact. It is preferable to use an antibody that is not carried on an insoluble carrier and that is labeled with a labeling agent.
[0033]
The basic operation of measuring a CM compound in an immunoagglutination reagent can be performed according to a usual assay method, for example, a hemagglutination method, a passive agglutination method, an immunoassay method, an immunoturbidimetry method, or the like. For example, particles containing 0.001 to 100 mg of anti-CM antibody per gram of particulate insoluble carrier (hereinafter abbreviated as sensitizing particles) in an aqueous medium may be used in an amount of 0.001 to 15% by weight. And used as an active ingredient of an immunoreagent. The particle size of the insoluble carrier carrying the antibody is preferably an insoluble carrier having an average particle size of 0.05 to 10 μm from the viewpoint of the ease of aggregation after the antigen-antibody reaction and the ease of discrimination of aggregation. It is. The sensitized particles prepared by such a method may be brought into contact with an antigen in a sample, and the degree of aggregation of the sensitized particles may be measured. Conventional methods such as visual observation and optical measurement can be used for the degree of particle aggregation without any limitation.
[0034]
When the immunoreagent of the present invention is used as a diagnostic reagent, the concentration of a CM compound in a sample and the CM conversion ratio of the CM compound are measured using an anti-CM antibody. At this time, the biological components serving as samples include, for example, body fluids such as blood, urine, lymph, amniotic fluid, peritoneal fluid, saliva, skin collagen, extracellular matrices such as fibronectin, lens proteins, arteries, and tissues such as kidneys. However, it is more preferable to use a body fluid that is frequently used as a subject in a clinical test.
[0035]
Further, by administering the DRA therapeutic drug using the immunoreagent of the present invention and measuring the amount of a CM compound contained in a specific biological component or the degree of decrease in the CM conversion rate, the efficacy of the therapeutic drug can also be evaluated. .
[0036]
【The invention's effect】
The present inventors obtained an anti-CM antibody by immunizing rabbits with a CM protein in which the amino group of human serum albumin was converted to CM, and purifying the same. The antigen recognized by the antibody was confirmed to be a carboxymethyl group added to the protein without depending on the type of the protein. Furthermore, it was confirmed that the anti-CM antibody reacts significantly more strongly with blood and serum of a DRA patient than with blood and serum of a healthy subject. This indicates that the CM compound can be effectively used as a novel clinical marker for diagnosing the onset or progression of DRA, and also indicates the utility of the immunoreagent of the present invention. That is, the present invention provides a new diagnostic system using the above-mentioned novel marker in the diagnosis of DRA, and its significance is large.
[0037]
【Example】
EXAMPLES Examples will be shown below for more specifically describing the present invention, but the present invention is not limited to these examples.
[0038]
Example 1
(1) Production of CM protein
To convert the amino group of the protein into CM, 1 ml of 1 mg / ml human serum albumin (Fraction V, manufactured by Sigma) adjusted to pH 9 and 1 ml of 0.25 M glyoxylic acid (manufactured by Sigma) adjusted to pH 9 were added. Mix and leave at 0 ° C. for 12 hours. Thereafter, 1 mg of sodium cyanoborohydride was added, and the mixture was further left for 12 hours.
[0039]
As a control, human serum albumin was treated in the same manner except that glyoxylic acid was not added.
[0040]
The CM conversion rate of human serum albumin after each of the above treatments was determined by measuring unreacted amino groups using trinitrobenzenesulfonic acid (hereinafter abbreviated as TNBS) by the following method.
[0041]
That is, 0.5 ml of each sample was added to 0.5 ml of a 0.1 M aqueous sodium hydroxide solution containing 0.1 M sodium tetraborate. Next, 20 μl of 1.1 M TNBS which had been recrystallized and washed with diluted hydrochloric acid was added thereto, followed by stirring. Thirty minutes later, 2 ml of 98.5 mM sodium dihydrogen phosphate containing 1.5 mM sodium sulfite was added to stop the reaction. After diluting 10 times with distilled water, the absorbance at 420 nm was measured. The absorbance of albumin was 0.03, and the absorbance of human serum albumin (control) not treated with glyoxylic acid was 1.25. When the same measurement was carried out in a system not containing any of the above human serum albumins, the absorbance was 0.03, and it was found that the CM conversion rate of the CM human human albumin was 100%.
[0042]
CM-modified human serum albumin obtained by such a method is dialyzed against a 20 mM phosphate buffer (pH 7.4) at 4 ° C. for 2 days to remove unreacted glyoxylic acid and sodium cyanoborohydride, and then CM Served as an immunogen for the production of antibodies against the conjugated protein.
[0043]
(2) Preparation of antibody against CM protein
Rabbits weighing 2 kg or more were immunized as follows using the CM human human albumin prepared in Example 1 as an antigen.
[0044]
0.5 ml of the antigen solution adjusted to 2 mg / ml and 0.5 ml of Freund's complete adjuvant were added to the ear vein of the rabbit. Thereafter, every two weeks, 0.25 ml of the 2 mg / ml antigen and 0.25 ml of Freund's incomplete adjuvant were boosted. During this period, rabbits were partially bled from the peripheral ear vein once every two weeks to confirm whether antibodies against CM-serum albumin were produced. Six weeks later, it was confirmed by an enzyme-linked immunosorbent assay (ELISA) that production of antibodies against CM serum human albumin was performed, and the whole blood was collected.
[0045]
(3) Preparation of affinity purification column
After washing 25 ml of Affigel 15 (manufactured by Bio-Rad) with 75 ml of 10 mM acetate buffer (pH 4.5), 62.5 ml of a 10 mg / ml human serum albumin solution was added, followed by gentle stirring at room temperature for 1 hour. Next, unreacted human serum albumin was removed by filtration, 30 ml of 1 M ethanolamine was added, and the mixture was gently stirred at room temperature to block unreacted N-hydroxysuccinimide ester. The support on which the human serum albumin was immobilized was packed in a column, and washed with ion-exchanged water until the absorbance at 280 nm became zero. Further, the column was equilibrated with a 20 mM phosphate buffer (pH 7.4) containing 0.15 M sodium chloride.
[0046]
(4) Affinity purification of antibody against CM-serum albumin
The prepared antibody to CM serum albumin diluted with a 20 mM phosphate buffer (pH 7.4) containing 0.15 M sodium chloride so as to have a concentration of 1 mg / ml was adjusted to about 100 mg with the affinity. It was applied to a purification column. Then, the phosphate buffer was flowed at a flow rate of 0.5 ml / min until the absorbance at 280 nm became 0. The antibody that did not bind to the column was recovered as an antibody against CM-converted human serum albumin. When the absorbance at 280 nm became 0, the phosphate buffer was changed to 0.1 M glycine buffer (pH 3.0), and the antibody bound to the column was eluted. The column was equilibrated with a phosphate buffer (pH 7.4), the recovered antibody was applied to the column again, and the antibody not bound to the column was recovered. This operation was repeated once more, and the resultant was used as an antibody for biotin labeling.
[0047]
(5) Biotin labeling of antibody against CM serum human albumin
Biotin labeling of the purified antibody was performed using a protein biotilation system (manufactured by Gibco).
[0048]
A solution prepared by diluting or concentrating the purified antibody against CM human serum albumin with a 20 mM phosphate buffer (pH 7.4) containing 0.15 M sodium chloride to a concentration of 1.5 mg / ml was added to 0.05 M. Sodium carbonate buffer (pH 9.0) was added. Next, 26 μl of a 50 mg / ml CAB-NHS ester solution was added to 6.7 ml of the antibody solution, and the mixture was gently stirred at room temperature for 1 hour, and ammonium chloride was added to 0.11 M to terminate the reaction. Thereafter, the antibody solution was desalted using a column attached to this kit. Furthermore, when the number of moles of biotin introduced by Avidin / HABA included in the kit was calculated, 14 moles of biotin was bound to 1 mole of the antibody to CM-enriched human serum albumin.
[0049]
(6) Antigen specificity of antibody against CM human serum albumin
Antigen specificity of the antibody against CM-human human albumin was confirmed by competition ELISA.
[0050]
An antibody against CM human serum albumin diluted with 10 mM phosphate buffer (pH 7.4) containing 0.15 M sodium chloride (hereinafter abbreviated as PBS) so as to be 1 μg / ml was added to the prepared CM human serum. Albumin was added at 0.1, 1, 10, 100 μg / ml, respectively. This solution was left at 37 ° C. for 1 hour, and used as an antibody solution inhibited by CM-modified human serum albumin.
[0051]
In the same manner as in the preparation of CM human serum albumin, CM hemoglobin prepared from hemoglobin (manufactured by Sigma) was prepared. The CM-modified hemoglobin was added to a 1 μg / ml antibody solution against CM-modified human serum albumin at a concentration of 0.1, 1, 10, 100 μg / ml, respectively. This solution was left at 37 ° C. for 1 hour and used as an antibody solution inhibited by CM-modified hemoglobin.
[0052]
CM-modified β2M prepared from β2-microglobulin (manufactured by Seikagaku Corporation) was prepared in the same manner as in the preparation of CM-modified human serum albumin. The CM2 β2M was added to a 1 μg / ml antibody solution against CM human serum albumin at a concentration of 0.1, 1, 10, 100 μg / ml, respectively. This solution was left at 37 ° C. for 1 hour and used as an antibody solution inhibited by CM2 β2M.
[0053]
In performing the competition ELISA, the prepared CM-converted human serum albumin was diluted with PBS to 1 μg / ml. Next, 100 μl of the diluted CM human serum albumin solution was applied per well to a 96-well immunoplate (manufactured by NUNC) and allowed to stand at 37 ° C. for 1 hour to immobilize the CM human human albumin on the immunoplate. After one hour, CM-human albumin not bound to the immunoplate was removed, 100 μl of PBS containing 0.5% gelatin was applied per well, and the plate was allowed to stand at 37 ° C. for one hour, and the CM-human serum albumin was removed. Blocked the unbound portion. After 1 hour, the gelatin solution is removed, and the plate is washed three times with PBS. Then, the antibody solution inhibited by the above-mentioned concentration of CM-human serum albumin, or the antibody inhibited by the above-mentioned concentration of CM-containing hemoglobin or CM-β2M 100 μl of the solution was applied per well and left at 37 ° C. for 1 hour. Thereafter, the plate was washed three times with PBS, 100 µl of an anti-rabbit IgG antibody solution (manufactured by Cosmo Bio) labeled with 1 µg / ml of alkaline phosphatase was applied per well, and left at 37 ° C for 1 hour. Further, the plate was washed three times with PBS, and 100 μl of a substrate solution prepared per well using an alkaline phosphatase substrate kit (manufactured by Bio-Rad) according to the instructions was applied. After standing at room temperature for 5 minutes, a 0.4 M sodium hydroxide solution was added at 100 μl per well to stop the alkaline phosphatase reaction, and the absorbance at 405 nm was measured. The results are shown in Table 1 and FIG.
[0054]
[Table 1]

[0055]
From these results, the antibody against CM human serum albumin prepared showed that the antigen-antibody reaction of the CM human serum albumin and the antibody against CM human serum albumin not only with CM human hemoglobin but also with CM hemoglobin and CM β2M. The inhibition suggested that CM-activated hemoglobin and CM-activated β2M also showed reactivity.
[0056]
(7) Measurement of CM hemoglobin in blood of DRA patients
50 μl of blood collected from 20 DRA patients using a vacuum blood collection tube containing EDTA-2K was washed once with 250 μl of physiological saline to obtain red blood cells, and 1 ml of purified water was added to the red blood cells to cause hemolysis. Was used as a subject.
[0057]
The measurement of CM hemoglobin in the subject was performed by the dot blotting method. After measuring the hemoglobin concentration by the cyanmethemoglobin method, the hemoglobin was adsorbed to a PVDF membrane (manufactured by Bio-Rad) using a dot blotting apparatus (manufactured by Bio-Rad) so that the hemoglobin became 500 ng. The membrane was immersed in a 20 mM phosphate buffer (pH 7.4) containing 10% skim milk for 1 hour at room temperature. The membrane was taken out, and the biotin-labeled 1 μg / ml antibody solution against CM human serum albumin was removed. 5 ml was added. After 1 hour incubation at room temperature, the membrane was washed three times with 50 ml of 20 mM phosphate buffer (pH 7.4) containing 0.05% Tween20. Next, 5 ml of an avidin-peroxidase-labeled biotin complex solution (Vectastain ABC kit: manufactured by Funakoshi) was added to the membrane, and the mixture was incubated at room temperature for 1 hour. After washing the membrane three times with 50 ml of a 20 mM phosphate buffer (pH 7.4) containing 0.05% Tween 20, 2 ml of an ECL western blotting detection reagent (manufactured by Amersham) was added. The emission intensity of the film was detected using a Bio-Rad GS-363 molecular imager.
[0058]
As a control, commercially available hemoglobin (manufactured by Sigma) was used as a subject, and the measurement of commercialized hemoglobin was performed in the same manner as above. With the luminescence intensity of the commercially available hemoglobin as 100, the relative value of the luminescence intensity of the hemoglobin derived from the DRA patient was displayed. The measured values of the emission intensity are also shown. The measurement results are shown in Table 2 and FIG.
[0059]
[Table 2]

[0060]
Comparative Example 1
CM hemoglobin in the subject was measured according to the method of Example 1 except that blood obtained from 20 healthy subjects was used as the subject instead of the DRA patient in Example 1. In the same manner as in Example 1, the relative value of the luminescence intensity of the hemoglobin derived from the healthy subject was displayed, with the luminescence intensity of the commercially available hemoglobin being 100. The measured values of the emission intensity are also shown. The measurement results are shown in Table 3 and FIG.
[0061]
[Table 3]

[0062]
Statistical comparison (t-test) of the luminescence intensity between the healthy group and the DRA patient group measured in Example 1 showed that the luminescence intensity of the DRA patient group was lower than that of the healthy group at a risk factor of less than 5%. It was significantly higher than the strength. This means that there is significantly more CM-modified hemoglobin in blood from DRA patients than in blood from healthy individuals.
[0063]
From the above results, it has been clarified that the use of CM-modified hemoglobin as a marker for DRA is effective.
[0064]
Example 2
The procedure of Example 1 was repeated, except that a solution obtained by removing a substance having a molecular weight of 30,000 or more and a substance having a molecular weight of 10,000 or less from the serum of the patient by filtration with a filter was used instead of the blood of the DRA patient in Example 1. According to the method 1, CM2 β2M in the subject was measured. The measured value of the emission intensity was displayed. The measurement results are shown in Table 4 and FIG.
[0065]
[Table 4]

[0066]
Comparative Example 2
In Example 2, CM2 β2M in a subject was measured according to the method of Example 2 except that serum obtained from five healthy subjects was used as the subject instead of the DRA patient. The measured value of the emission intensity was displayed. Table 4 shows the measurement results.
[0067]
When the luminescence intensities of the healthy subject group and the DRA patient group measured in Example 2 were compared, the luminescence intensity of the DRA patient group was significantly higher while the sensitivity of the subject derived from the serum of the healthy subject was lower than the detection sensitivity. . This means that the serum from DRA patients has significantly more CM2 β2M than the serum from healthy individuals.
[0068]
From the above results, it has been clarified that the use of CM2 β2M as a marker for DRA is effective.
[Brief description of the drawings]
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the results of examining the antigen specificity of a prepared antibody to CM-human serum albumin by a competitive ELISA. The vertical axis indicates the absorbance at 405 nm, and the horizontal axis indicates the amount of each inhibitor added. .
FIG. 2 is a graph showing the relationship between the rate of CM hemoglobin in blood of healthy subjects and DRA patients.
FIG. 3 is a graph showing the relationship between the concentration of CM2 β2M in blood of healthy subjects and DRA patients.

Claims (2)

Use of carboxymethylated hemoglobin in which at least a part of the amino group of the constituent amino acid units is carboxymethylated as a marker for dialysis amyloidosis. An immunoreagent for dialysis amyloidosis or efficacy evaluation using an antibody that specifically reacts with carboxymethylated hemoglobin in which at least a part of the amino groups of the constituent amino acid units is carboxymethylated .

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