JP2000206117A - Method for stabilizing human hemoglobin - Google Patents
Method for stabilizing human hemoglobinInfo
- Publication number
- JP2000206117A JP2000206117A JP11007181A JP718199A JP2000206117A JP 2000206117 A JP2000206117 A JP 2000206117A JP 11007181 A JP11007181 A JP 11007181A JP 718199 A JP718199 A JP 718199A JP 2000206117 A JP2000206117 A JP 2000206117A
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- Prior art keywords
- human hemoglobin
- substance
- solution
- hemoglobin
- guanidino group
- Prior art date
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】 本発明はヒトヘモグロビン
の免疫学的検出における、検体溶液中に含まれるヒトヘ
モグロビンの安定化方法に関するものである。TECHNICAL FIELD The present invention relates to a method for stabilizing human hemoglobin contained in a sample solution in immunological detection of human hemoglobin.
【０００２】[0002]
【従来の技術】 大腸癌などの下部消化器疾患の簡便な
検査法として糞便からのヒトヘモグロビン検出が利用さ
れている。特に抗ヒトヘモグロビン抗体を用いた免疫学
的検出法は、検査前の食事制限を必要とせず高い特異性
が期待できることから、近年広く利用されている検出法
である。2. Description of the Related Art Detection of human hemoglobin from feces is used as a simple test method for lower gastrointestinal diseases such as colorectal cancer. In particular, an immunological detection method using an anti-human hemoglobin antibody is widely used in recent years because high specificity can be expected without requiring dietary restrictions before the test.
【０００３】上記検出法には酵素や放射性元素で標識し
た抗ヒトヘモグロビン抗体を用いる酵素免疫法や放射免
疫法、抗ヒトヘモグロビン抗体を感作した金コロイドや
ラテックス粒子を用いるラテックス凝集法や金コロイド
凝集法、さらに着色ラテックスを標識した抗ヒトヘモグ
ロビン抗体を用いるイムノクロマトグラフ法などがあ
る。The above detection methods include enzyme immunization and radioimmunoassay using an anti-human hemoglobin antibody labeled with an enzyme or a radioactive element, latex agglutination using gold colloid or latex particles sensitized with an anti-human hemoglobin antibody, and gold colloid. There are an agglutination method and an immunochromatography method using an anti-human hemoglobin antibody labeled with a colored latex.
【０００４】これらの検査に使用される糞便検体は、検
査員の不快感や負担を減らすため、糞便を懸濁させた便
懸濁液の形態で提供されることが多い。被験者の自宅等
で採取した糞便を便懸濁用溶液に懸濁し、郵送または病
院に持参する。そのため通常、糞便採取から実際に検査
が行われるまで半日から数日間を要する。[0004] Fecal specimens used for these tests are often provided in the form of a stool suspension in which feces are suspended in order to reduce the discomfort and burden on the examiner. The stool collected at the subject's home or the like is suspended in a stool suspension solution and mailed or brought to a hospital. Therefore, it usually takes from half a day to several days from the collection of feces to the actual test.
【０００５】ところが便懸濁用溶液中に溶解したヒトヘ
モグロビンは、糞便中に含まれる細菌、各種プロテアー
ゼ等の影響や輸送中に高温にさらされたりすることによ
り徐々に変性し、抗原性が低下することが知られてい
る。特に低濃度のヒトヘモグロビンの場合には抗原性の
低下が顕著であり、免疫学的検出法のメリットである高
感度、高精度が維持できず、測定誤差が生じ易い等の問
題が指摘されてきた。However, human hemoglobin dissolved in the stool suspension solution is gradually denatured due to the effects of bacteria and various proteases contained in the stool and exposed to high temperatures during transportation, and the antigenicity is reduced. It is known to Particularly in the case of low-concentration human hemoglobin, the antigenicity is remarkably reduced, and it has been pointed out that the advantages of the immunological detection method, such as high sensitivity and high accuracy, cannot be maintained, and measurement errors easily occur. Was.
【０００６】便懸濁溶液中のヒトヘモグロビンの変性を
抑止し上記問題点を改善するために、便懸濁用溶液にヒ
ト以外の動物ヘモグロビンを添加する方法（特開平２-
２９６１４９号公報）や、プロテアーゼ阻害物質を添加
する方法（特開平３−２７９８５９号公報）、トランス
フェリン及びフェリチンを共存させる方法（特開平８-
２６２０２０号公報）、アスパラギン酸塩、グルタミン
塩酸塩を添加する方法（特開平９−６１４３１号公報）
が提案されている。しかしこれらの方法においてもヒト
ヘモグロビンの変性を完全に防止することはできず、問
題解決に至っていないのが現状である。In order to suppress the denaturation of human hemoglobin in the stool suspension solution and to improve the above-mentioned problems, a method of adding non-human animal hemoglobin to the stool suspension solution is disclosed in
296149), a method of adding a protease inhibitor (JP-A-3-279598), and a method of coexisting transferrin and ferritin (JP-A-8-279).
262020), a method of adding aspartate and glutamine hydrochloride (JP-A-9-61431)
Has been proposed. However, even with these methods, denaturation of human hemoglobin cannot be completely prevented, and at present the problem has not been solved.
【０００７】[0007]
【発明が解決しようとする課題】本発明は免疫学的ヒト
ヘモグロビン検出法において、従来の技術では解決でき
なかった便懸濁液中に溶解したヒトヘモグロビンの変性
を抑制し、高感度、高精度の検出を可能とするヒトヘモ
グロビン安定化方法を提供することを目的とする。DISCLOSURE OF THE INVENTION The present invention relates to an immunological method for detecting human hemoglobin, which suppresses the denaturation of human hemoglobin dissolved in a stool suspension, which cannot be solved by conventional techniques, and provides high sensitivity and high precision. It is an object of the present invention to provide a method for stabilizing human hemoglobin, which enables the detection of human hemoglobin.
【０００８】[0008]
【課題を解決するための手段】本発明者らは、免疫学的
ヒトヘモグロビン検出法においてヒトヘモグロビン含有
溶液中にグアニジノ基を有する物質、所望により蛋白質
を添加することによりヒトヘモグロビンを安定化できる
ことを見出し、本発明を完成するに至った。Means for Solving the Problems The present inventors have found that human hemoglobin can be stabilized by adding a substance having a guanidino group and, if desired, a protein to a solution containing human globin in an immunological method for detecting human hemoglobin. As a result, the present invention has been completed.
【０００９】すなわち本発明はヒトヘモグロビン含有溶
液中にグアニジノ基を有する物質を添加することを特徴
とするヒトヘモグロビンの安定化方法を提供するもので
ある。That is, the present invention provides a method for stabilizing human hemoglobin, which comprises adding a substance having a guanidino group to a solution containing human hemoglobin.
【００１０】また本発明はグアニジノ基を有する物質を
含有することを特徴とする便懸濁用溶液を提供するもの
である。The present invention also provides a stool suspension solution containing a substance having a guanidino group.
【００１１】本発明で用いられるグアニジノ基を有する
物質はグアニジノ基を有する物質であればいずれの物質
を用いてもよいが、アルギニンまたはその塩類、グリコ
シアミンが好ましい。アルギニンまたはその塩類、グリ
コシアミンは市販品を用いることができる。As the substance having a guanidino group used in the present invention, any substance may be used as long as it has a guanidino group, but arginine or salts thereof and glycosiamine are preferred. Arginine or a salt thereof, and glycosiamine may be commercially available products.
【００１２】本発明の便懸濁用溶液に含有させるグアニ
ジノ基を有する物質の濃度としては０．０１〜５Ｍ、好
ましくは０．１〜０．５Ｍである。The concentration of the guanidino group-containing substance contained in the stool suspension solution of the present invention is 0.01 to 5M, preferably 0.1 to 0.5M.
【００１３】本発明では便懸濁用溶液に蛋白質を共存さ
せることによりヒトヘモグロビンの安定性が増すため、
便懸濁用溶液に蛋白質を含有させることが好ましい。蛋
白質としてはアルブミン、カゼイン、スキムミルク、ゼ
ラチン等の免疫学的検出法で通常用いられる蛋白質であ
ればいずれの蛋白質を用いてもよい。蛋白質の濃度とし
ては０．０１〜５％、好ましくは０．１〜２％である。In the present invention, the coexistence of proteins in the stool suspension solution increases the stability of human hemoglobin.
It is preferable to include the protein in the stool suspension solution. As the protein, any protein may be used as long as it is a protein normally used in immunological detection methods such as albumin, casein, skim milk, gelatin and the like. The concentration of the protein is 0.01 to 5%, preferably 0.1 to 2%.
【００１４】また本発明の便懸濁用溶液にはリン酸塩等
の緩衝用塩類やその他の無機塩類、エチレンジアミン−
N,N,N',N'−四酢酸（ＥＤＴＡ）等のキレート剤、Ｔｗ
ｅｅｎ２０等の界面活性剤を含有させてもよい。ｐHは
６〜９が好ましい。さらに細菌類の増殖を抑制するため
にアジ化ナトリウムを０．０１〜０．５％添加すること
もできる。The stool suspension solution of the present invention may contain a buffering salt such as phosphate, other inorganic salts, ethylenediamine-
Chelating agents such as N, N, N ', N'-tetraacetic acid (EDTA), Tw
A surfactant such as eeen20 may be contained. The pH is preferably from 6 to 9. Further, sodium azide may be added in an amount of 0.01 to 0.5% to suppress the growth of bacteria.
【００１５】本発明では便懸濁用緩衝液にヒトヘモグロ
ビンを溶解するかまたは糞便を懸濁させ試料とする。具
体的には、被験者の糞便一定量を便懸濁用溶液一定量に
加えてまたは混ぜて懸濁することにより試料とする。In the present invention, human hemoglobin is dissolved in a stool suspension buffer or feces are suspended to prepare a sample. Specifically, a sample is prepared by adding or mixing a certain amount of the feces of a subject to a certain amount of a solution for stool suspension or by mixing.
【００１６】本発明を用いてヒトヘモグロビンの検出を
行うには、各種免疫学的ヒトヘモグロビン検出法が利用
可能である。以下に酵素免疫法およびイムノクロマトグ
ラフ法を利用した実施例を挙げ、本発明をさらに詳細に
具体的に説明するが、本発明はこれらの実施例により何
等限定されるものではない。To detect human hemoglobin using the present invention, various immunological methods for detecting human hemoglobin can be used. Hereinafter, the present invention will be described in more detail with reference to examples using enzyme immunoassay and immunochromatography, but the present invention is not limited to these examples.
【００１７】[0017]
【実施例】［実施例１］酵素免疫法によるヒトヘモグロ
ビンの検出 （１）抗ヒトヘモグロビンモノクローナル抗体の作製 ２回結晶ヒトヘモグロビン（Ｓｉｇｍａ社製）を滅菌蒸
留水に溶解し、１０ｍｇ／ｍｌのヒトヘモグロビン溶液
を調製した。次にヒトヘモグロビン溶液とＦｒｅｕｎｄ
のコンプリートアジュバントを当量混合し、オイルエマ
ルジョンとした。これをＢＡＬＢ／ｃＡマウス（ＢＡＬ
Ｂ／ｃＡ ｊｃｌ、７週齢、雌）の背部皮下に０．２ｍ
ｌずつ投与した。初回免疫後、７日目と１４に日目に追
加免疫を行い、さらに細胞融合３日前にヒトヘモグロビ
ン溶液を０．２５ｍｇ／０．２ｍｌずつ腹腔内に投与し
た。最終免疫から３日目のマウス脾細胞とミエローマ細
胞（Ｐ３ｘ６３．Ａｇ８．６５３株）を１０：１の割合
で５０％ポリエチレングリコール４０００を用いて融合
し、ＨＡＴ培地により選択培養した。細胞融合後、１２
日目に培養上清のヒトヘモグロビンに対する抗体活性を
ＥＩＡ法により測定した。測定方法はヒトヘモグロビン
１０μｇ／ｍｌで固相化した９６穴ＥＩＡプレート（コ
ースター社製）を用いて融合細胞の培養液２００μｌを
添加した。その後、３７℃、１時間反応後洗浄を行い、
ぺルオキシダーゼ標識抗マウスＩｇＧ（Ｃａｐｐｅｌ社
製、１：５００）２００μｌを添加した。洗浄後、基質
液（０．１Ｍ Ｏ−フェニレンジアミンと０．０１２％
過酸化水素水）各ウェルに２００μｌずつ添加し、室温
で１５分反応させた。反応後、各ウェルに３．５Ｎ硫酸
を５０μｌずつ添加し酵素反応を停止した。次に４９２
ｎｍにおける吸光値を測定し、ヒトヘモグロビンに反応
するクローンを限界希釈法により２回クローニングを行
った。その結果、クローニング後に腹水として得られた
モノクローナル抗体は１０クローンであった。これらの
得られた各々の腹水１ｍｌをリン酸緩衝生理食塩液（Ｐ
ＢＳ）１ｍｌで２倍に希釈し、飽和硫酸アンモニウム２
ｍｌを滴下して４℃、４時間放置した。その後、３００
０ｒｐｍ、２０分間遠心分離し、沈査をＰＢＳ２ｍｌに
浮遊し透析を行った。この中で反応性の良い２クローン
（Ｋ６、Ｋ８）を選択し、以下の試験に用いた。尚、２
クローンのイムノグロブリンサブクラスはＩｇＧ１、Ｌ
鎖はカッパー型であった。EXAMPLES Example 1 Detection of Human Hemoglobin by Enzyme Immunoassay (1) Preparation of Anti-Human Hemoglobin Monoclonal Antibody Twice-crystal human hemoglobin (manufactured by Sigma) was dissolved in sterile distilled water and 10 mg / ml of human A hemoglobin solution was prepared. Next, human hemoglobin solution and Freund
An equivalent amount of the complete adjuvant was mixed to obtain an oil emulsion. This was transferred to a BALB / cA mouse (BAL
B / cA jcl, 7 weeks old, female)
Each dose was administered. After the first immunization, booster immunizations were performed on the 7th and 14th days, and a human hemoglobin solution was administered intraperitoneally 0.25 mg / 0.2 ml 3 days before the cell fusion. Three days after the final immunization, mouse spleen cells and myeloma cells (P3x63.Ag8.653 strain) were fused at a ratio of 10: 1 using 50% polyethylene glycol 4000, and selectively cultured in a HAT medium. After cell fusion, 12
On the day, the antibody activity of the culture supernatant against human hemoglobin was measured by the EIA method. As a measuring method, 200 μl of a culture solution of the fused cells was added using a 96-well EIA plate (Coaster) immobilized with human hemoglobin at 10 μg / ml. After that, washing was performed after the reaction at 37 ° C. for 1 hour.
200 μl of peroxidase-labeled anti-mouse IgG (Cappel, 1: 500) was added. After washing, the substrate solution (0.1 M O-phenylenediamine and 0.012%
(Hydrogen peroxide solution) 200 μl was added to each well and reacted at room temperature for 15 minutes. After the reaction, 50 μl of 3.5N sulfuric acid was added to each well to stop the enzyme reaction. Then 492
The absorbance at nm was measured, and clones reacting with human hemoglobin were cloned twice by the limiting dilution method. As a result, the monoclonal antibody obtained as ascites after cloning was 10 clones. 1 ml of each of the obtained ascites was added to a phosphate buffered saline (P
BS) Dilute 2 times with 1 ml and add saturated ammonium sulfate 2
ml was added dropwise and left at 4 ° C. for 4 hours. Then 300
After centrifugation at 0 rpm for 20 minutes, the precipitate was suspended in 2 ml of PBS and dialyzed. Among them, two clones (K6 and K8) having good reactivity were selected and used for the following tests. 2
The immunoglobulin subclasses of the clones are IgG 1 , L
The chains were copper-shaped.
【００１８】（２）便懸濁用溶液の調製 ＰＢＳにエチレンジアミン−N,N,N',N'−四酢酸（ＥＤ
ＴＡ）２ｍＭ（和光純薬社製）、アジ化ナトリウム（和
光純薬社製）０．１％、Ｔｗｅｅｎ２０（和光純薬社
製）０．０５％となるように加えベース緩衝液とした。
このベース緩衝液に以下のように各試薬を組み合わせて
添加し、便懸濁用溶液とした。 ：ベース緩衝液 ＋ Ｌ型アルギニン塩酸塩 ０．５
Ｍ ：ベース緩衝液 ＋ Ｄ型アルギニン塩酸塩 ０．５
Ｍ ：ベース緩衝液 ＋ グリコシアミン ０．０１Ｍ ： ＋ ウシ血清アルブミン １％ ： ＋ ウシ血清アルブミン １％ ： ＋ ウシ血清アルブミン １％ ：ベース緩衝液のみ(2) Preparation of a stool suspension solution Ethylenediamine-N, N, N ', N'-tetraacetic acid (ED
TA) 2 mM (manufactured by Wako Pure Chemical Industries, Ltd.), sodium azide (manufactured by Wako Pure Chemical Industries, Ltd.) 0.1%, Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.) 0.05%, and a base buffer solution was prepared.
Each of the reagents was combined and added to the base buffer as follows to prepare a stool suspension solution. : Base buffer + L-arginine hydrochloride 0.5
M: base buffer + D-type arginine hydrochloride 0.5
M: base buffer + glycosiamine 0.01M: + bovine serum albumin 1%: + bovine serum albumin 1%: + bovine serum albumin 1%: base buffer only
【００１９】（３）試料液の調製 市販の２回結晶化ヘモグロビン（シグマ社製）を、上記
７種類の便懸濁用溶液にて１００ｎｇ／ｍｌとなるよう
希釈した。(3) Preparation of sample solution A commercially available twice-crystallized hemoglobin (manufactured by Sigma) was diluted to 100 ng / ml with the above seven types of stool suspension solutions.
【００２０】（４）サンドイッチＥＬＩＳＡ法によるヘ
モグロビンの測定 抗ヒトヘモグロビン抗体溶液（Ｋ６）を２μｇ／ｍｌで
固相化した９６穴ＥＩＡプレート（コースター社製）に
試料液２００μｌを添加した。その後、３７℃、１時間
反応後洗浄を行い、ぺルオキシダーゼ標識抗ヒトヘモグ
ロビン抗体（１：５０００）２００μｌを添加した。洗
浄後、基質液（０．１Ｍ Ｏ−フェニレンジアミンと
０．０１２％過酸化水素水）各ウェルに２００μｌずつ
添加し、室温で１５分反応させた。反応後、各ウェルに
３．５Ｎ硫酸を５０μｌずつ添加し酵素反応を停止し、
４９２ｎｍにおける吸光値を測定した。その後、各試料
液を４０℃にて保存し３日、７日、１４日後に同様操作
で結果を判定し、０日の吸光値を１００％とした保存率
を算出した。表１に示したように、ベース緩衝液と比べ
てグアニジノ基を有する物質を含有する便懸濁用溶液中
ではヒトヘモグロビンの安定性が著しく高まることがわ
かった。(4) F by sandwich ELISA
Measurement of Moglobin 200 μl of a sample solution was added to a 96-well EIA plate (manufactured by Coaster) on which an anti-human hemoglobin antibody solution (K6) was immobilized at 2 μg / ml. Thereafter, washing was performed after reaction at 37 ° C. for 1 hour, and 200 μl of peroxidase-labeled anti-human hemoglobin antibody (1: 5000) was added. After washing, 200 μl of the substrate solution (0.1 M O-phenylenediamine and 0.012% aqueous hydrogen peroxide) was added to each well, and reacted at room temperature for 15 minutes. After the reaction, 50 μl of 3.5N sulfuric acid was added to each well to stop the enzyme reaction,
The absorbance at 492 nm was measured. Thereafter, each sample solution was stored at 40 ° C., and after 3, 7, and 14 days, the results were determined in the same manner, and the preservation rate was calculated with the absorbance value on day 0 as 100%. As shown in Table 1, the stability of human hemoglobin was found to be significantly higher in the stool suspension solution containing the substance having a guanidino group than in the base buffer.
【００２１】[0021]
【表１】 [Table 1]
【００２２】［実施例２］イムノクロマトグラフ法によ
る糞便中ヒトヘモグロビンの検出 （１）抗体固相化支持体の作製 ニトロセルロースシート（ＳＲＨＦ ミリポア社製）を
６ｍｍ×２０ｍｍに裁断し、その下端より９ｍｍの位置
に抗ヒトヘモグロビン抗体溶液０．５ｍｇ／ｍｌ（Ｋ
８）、１４ｍｍの位置に抗ウサギＩｇＧポリクローナル
抗体０．２５ｍｇ／ｍｌ（Ｃａｐｐｅｌ社製）を各々エ
アーブラシ（バイオドット社製）を用いて塗布し、抗ヒ
トヘモグロビン抗体および抗ウサギＩｇＧ抗体のライン
を作製した。室温で２時間乾燥後、１％スキムミルク
（ＤＩＦＣＯ社製）−０．１％Ｔｗｅｅｎ２０を含むＰ
ＢＳに３７℃１時間浸漬しブロッキングを行った。その
後、充分に乾燥し抗体固相化支持体とした。Example 2 Detection of Human Hemoglobin in Feces by Immunochromatography (1) Preparation of Antibody- Immobilized Support Nitrocellulose sheet (manufactured by SRHF Millipore) was cut into 6 mm × 20 mm, and 9 mm from the lower end. At the position of 0.5 mg / ml anti-human hemoglobin antibody solution (K
8) Apply 0.25 mg / ml of anti-rabbit IgG polyclonal antibody (manufactured by Cappel) to each 14 mm position using an airbrush (manufactured by Biodot), and line the lines of the anti-human hemoglobin antibody and anti-rabbit IgG antibody. Produced. After drying at room temperature for 2 hours, P containing 1% skim milk (manufactured by DIFCO) -0.1% Tween 20
Blocking was performed by immersing in BS at 37 ° C. for 1 hour. Thereafter, the support was sufficiently dried to obtain an antibody-immobilized support.
【００２３】（２）着色ラテックス粒子標識物の調製 ａ．レッドラテックス粒子標識抗ヒトヘモグロビンモノ
クローナル抗体 レッドラテックス粒子分散液（ＰＬ−Ｌａｔｅｘ，１０
％，４５０ｎｍ、Ｐｏｌｙｍｅｒ Ｌａｂｏｒａｔｏｒ
ｉｅｓ社製）３００μｌにＰＢＳ１．２ｍｌを加え、１
００００ｒｐｍ、５分間遠心分離を行った。沈渣に抗ヒ
トヘモグロビンモノクローナル抗体溶液０．５ｍｇ／ｍ
ｌ（Ｋ６）１ｍｌを加え、充分に混和して室温で１時間
反応を行った。未反応の抗ヒトヘモグロビンモノクロー
ナル抗体を除去するため１００００ｒｐｍ、５分間遠心
分離を行い、沈渣をＰＢＳ１．５ｍｌに懸濁し、再度遠
心分離を行った。１％スキムミルク１ｍｌを加え室温で
１時間反応させブロッキングを行った。その後、１００
００ｒｐｍ、５分間遠心分離を行い、沈渣を１％のスキ
ムミルクおよび０．０１％のアジ化ナトリウムを含むＰ
ＢＳ１ｍｌに懸濁し、冷蔵保存した。 ｂ．着色ラテックス粒子標識物凍結乾燥担体 レッドラテックス粒子標識抗ヒトヘモグロビンモノクロ
ーナル抗体をベンリーゼ（商標登録）不織布（旭化成社
製）６ｍｍ×１０ｍｍに１５μｌ含浸させ凍結乾燥をし
て調製した。(2) Preparation of a labeled colored latex particle a. Red latex particle labeled anti-human hemoglobin mono
Clonal antibody red latex particle dispersion (PL-Latex, 10
%, 450 nm, Polymer Laborator
ises) (1.2 μl PBS)
Centrifugation was performed at 0000 rpm for 5 minutes. 0.5 mg / m of anti-human hemoglobin monoclonal antibody solution
1 (K6) was added, mixed well, and reacted at room temperature for 1 hour. To remove unreacted anti-human hemoglobin monoclonal antibody, centrifugation was performed at 10,000 rpm for 5 minutes, and the precipitate was suspended in 1.5 ml of PBS and centrifuged again. 1 ml of 1% skim milk was added and reacted at room temperature for 1 hour to perform blocking. Then 100
After centrifugation at 00 rpm for 5 minutes, the sediment was washed with 1% skim milk and 0.01%
The cells were suspended in 1 ml of BS and stored refrigerated. b. Colored latex particle labeled freeze-dried carrier Red latex particle labeled anti-human hemoglobin monoclonal antibody was impregnated with 15 μl of 6 mm × 10 mm nonwoven fabric (manufactured by Asahi Kasei Corporation) and freeze-dried.
【００２４】（３）試験片の作製 抗体固相化支持体の下端から２ｍｍの位置まで着色ラテ
ックス粒子標識物凍乾担体を重ねた。さらに、着色ラテ
ックス粒子標識物凍乾担体上に被験液浸漬用担体を下端
から２ｍｍの位置まで重ねた。また、抗体固相化支持体
の上端から２ｍｍの位置まで吸水性担体６ｍｍ×３０ｍ
ｍ（３ＭＭ Ｃｈｒ、ワットマン社製）を重ね、最後に
透明なテープを上部に貼り固定して試験片とした。(3) Preparation of Test Piece A freeze-dried carrier of colored latex particle-labeled substance was superimposed to a position 2 mm from the lower end of the support on which the antibody was immobilized. Further, the carrier for immersion in the test liquid was placed on the freeze-dried carrier of the labeled colored latex particle to a position 2 mm from the lower end. Further, the water-absorbing carrier 6 mm × 30 m from the upper end of the antibody-immobilized support to a position 2 mm from the upper end.
m (3MM Chr, manufactured by Whatman), and finally, a transparent tape was stuck on the upper part and fixed to obtain a test piece.
【００２５】（４）便懸濁用溶液の調製 実施例１と同様に以下の７種類の便懸濁用溶液を調製し
た。 ：ベース緩衝液 ＋ Ｌ型アルギニン塩酸塩 ０．５
Ｍ ：ベース緩衝液 ＋ Ｄ型アルギニン塩酸塩 ０．５
Ｍ ：ベース緩衝液 ＋ グリコシアミン ０．０１Ｍ ： ＋ ウシ血清アルブミン １％ ： ＋ ウシ血清アルブミン １％ ： ＋ ウシ血清アルブミン １％ ：ベース緩衝液のみ(4) Preparation of Fecal Suspension Solutions In the same manner as in Example 1, the following seven types of fecal suspension solutions were prepared. : Base buffer + L-arginine hydrochloride 0.5
M: base buffer + D-type arginine hydrochloride 0.5
M: base buffer + glycosiamine 0.01M: + bovine serum albumin 1%: + bovine serum albumin 1%: + bovine serum albumin 1%: base buffer only
【００２６】（５）試料液の調製 市販のヘモグロビン検出試薬ＯＣ−ヘモキャッチ‘栄
研’（栄研化学社製）を用いて測定した結果、ヘモグロ
ビン陽性と判定された便検体２５例について、上記７種
類の便懸濁用緩衝液を充填した採便容器（ニッショー社
製）を用いて糞便を採取し、よく混和して懸濁させ試料
液とした。(5) Preparation of Sample Solution As a result of measurement using a commercially available hemoglobin detection reagent OC-Hemocatch 'Eiken' (manufactured by Eiken Chemical Co., Ltd.), 25 stool samples determined to be hemoglobin-positive were subjected to the above 7 tests. Stool was collected using a stool collection container (manufactured by Nissha) filled with various stool suspension buffers, mixed well, and suspended to prepare a sample solution.
【００２７】（６）ヘモグロビンの検出 各試料液１００μｌを試験片の被験液浸漬用担体に滴加
して展開し、１０分後に抗ヒトヘモグロビン抗体固相化
位置の赤いラインの発色の有無を肉眼で確認することで
結果を判定した。その後、各試料液を３０℃にて保存
し、３日、７日後に同様操作で結果を判定し、陽性率を
算出した。表２に示したように、本発明グアニジノ基を
含有する便懸濁用溶液中ヒトヘモグロビンの安定性は著
しく高まった。(6) Detection of hemoglobin 100 μl of each sample solution was added dropwise to the carrier for immersion of the test piece in the test solution and developed. After 10 minutes, the presence or absence of color development of the red line at the position where the anti-human hemoglobin antibody was immobilized was visually checked. The result was determined by confirming with. Thereafter, each sample solution was stored at 30 ° C., and after 3 days and 7 days, the results were determined by the same operation, and the positive rate was calculated. As shown in Table 2, the stability of human hemoglobin in the stool suspension solution containing a guanidino group of the present invention was significantly increased.
【００２８】[0028]
【表２】 [Table 2]
【００２９】[0029]
【発明の効果】以上のように、本発明によりヘモグロビ
ンの安定性が著しく改善され、採便後から検査に供され
るまでに通常要する期間にわたりヘモグロビンの抗原性
の低下を抑制することが可能である。As described above, according to the present invention, the stability of hemoglobin is remarkably improved, and it is possible to suppress a decrease in the antigenicity of hemoglobin for a period normally required after stool collection and before being used for examination. is there.
Claims (6)
ノ基を有する物質を添加することを特徴とするヒトヘモ
グロビンの安定化方法。1. A method for stabilizing human hemoglobin, comprising adding a substance having a guanidino group to a solution containing human hemoglobin.
ノ基を有する物質および蛋白質を共存させることを特徴
とするヒトヘモグロビンの安定化方法。2. A method for stabilizing human hemoglobin, comprising coexisting a substance having a guanidino group and a protein in a solution containing human hemoglobin.
またはその塩類、グリコシアミンから選ばれる少なくと
も一種類である請求項１または２記載のヒトヘモグロビ
ン安定化方法。3. The method for stabilizing human hemoglobin according to claim 1, wherein the substance having a guanidino group is at least one selected from arginine or salts thereof and glycosamine.
とを特徴とする便懸濁用溶液。4. A stool suspension solution containing a substance having a guanidino group.
を含有することを特徴とする便懸濁用溶液。5. A stool suspension solution containing a substance having a guanidino group and a protein.
またはその塩類、グリコシアミンから選ばれる少なくと
も一種類である請求項４または５記載の便懸濁用溶液。6. The stool suspension solution according to claim 4, wherein the substance having a guanidino group is at least one selected from arginine or salts thereof and glycosamine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11007181A JP2000206117A (en) | 1999-01-14 | 1999-01-14 | Method for stabilizing human hemoglobin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11007181A JP2000206117A (en) | 1999-01-14 | 1999-01-14 | Method for stabilizing human hemoglobin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2000206117A true JP2000206117A (en) | 2000-07-28 |
Family
ID=11658907
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP11007181A Pending JP2000206117A (en) | 1999-01-14 | 1999-01-14 | Method for stabilizing human hemoglobin |
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| Country | Link |
|---|---|
| JP (1) | JP2000206117A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102253225A (en) * | 2011-04-20 | 2011-11-23 | 珠海经济特区海泰生物制药有限公司 | Kit for detecting Southeast Asia type alpha-thalassemia |
| JP2018072045A (en) * | 2016-10-25 | 2018-05-10 | 福井県 | Method for inspection of human blood and human blood inspection kit |
| WO2019168109A1 (en) * | 2018-03-02 | 2019-09-06 | 栄研化学株式会社 | Method for stabilizing protein contained in sample and solution for stabilizing protein contained in sample |
| WO2023286821A1 (en) * | 2021-07-15 | 2023-01-19 | 積水メディカル株式会社 | Composition containing tarc, diluted solution, method for preventing carry over of tarc, adsorption-preventing agent, and continuous analysis method |
-
1999
- 1999-01-14 JP JP11007181A patent/JP2000206117A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102253225A (en) * | 2011-04-20 | 2011-11-23 | 珠海经济特区海泰生物制药有限公司 | Kit for detecting Southeast Asia type alpha-thalassemia |
| CN102253225B (en) * | 2011-04-20 | 2013-07-10 | 珠海经济特区海泰生物制药有限公司 | Kit for detecting Southeast Asia type alpha-thalassemia |
| JP2018072045A (en) * | 2016-10-25 | 2018-05-10 | 福井県 | Method for inspection of human blood and human blood inspection kit |
| WO2019168109A1 (en) * | 2018-03-02 | 2019-09-06 | 栄研化学株式会社 | Method for stabilizing protein contained in sample and solution for stabilizing protein contained in sample |
| JPWO2019168109A1 (en) * | 2018-03-02 | 2021-02-25 | 栄研化学株式会社 | A method for stabilizing the protein contained in the sample, a solution for stabilizing the protein contained in the sample |
| JP7237924B2 (en) | 2018-03-02 | 2023-03-13 | 栄研化学株式会社 | Method for stabilizing protein contained in specimen, solution for stabilizing protein contained in specimen |
| WO2023286821A1 (en) * | 2021-07-15 | 2023-01-19 | 積水メディカル株式会社 | Composition containing tarc, diluted solution, method for preventing carry over of tarc, adsorption-preventing agent, and continuous analysis method |
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