JPH0376425B2 - - Google Patents

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Publication number
JPH0376425B2
JPH0376425B2 JP58066784A JP6678483A JPH0376425B2 JP H0376425 B2 JPH0376425 B2 JP H0376425B2 JP 58066784 A JP58066784 A JP 58066784A JP 6678483 A JP6678483 A JP 6678483A JP H0376425 B2 JPH0376425 B2 JP H0376425B2
Authority
JP
Japan
Prior art keywords
latex
general formula
reagent
collidine
added
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
JP58066784A
Other languages
Japanese (ja)
Other versions
JPS59192962A (en
Inventor
Goro Wakabayashi
Hiroshi Kishioka
Shinichi Mashita
Tsugi Imai
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
KH Neochem Co Ltd
Original Assignee
Kyowa Hakko Kogyo Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kyowa Hakko Kogyo Co Ltd filed Critical Kyowa Hakko Kogyo Co Ltd
Priority to JP6678483A priority Critical patent/JPS59192962A/en
Publication of JPS59192962A publication Critical patent/JPS59192962A/en
Priority to JP28157690A priority patent/JPH03142360A/en
Publication of JPH0376425B2 publication Critical patent/JPH0376425B2/ja
Granted legal-status Critical Current

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Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Compositions Of Macromolecular Compounds (AREA)

Description

【発明の詳細な説明】[Detailed description of the invention]

本発明はラテツクス試薬およびラテツクスの安
定化法に関する。さらに詳しく本発明はγ−コリ
ジン; 一般式() (式中、R1、R2、R3およびR4は同一もしくは異
なつて水素原子又は炭素数1−5のアルキル基を
表わす。)で表わされるイミダゾール又はその誘
導体;および 一般式() (式中、R1は水素原子又は炭素数1−5のアル
キル基を表わす。)で表わされるプロパン1,3
−ジオール誘導体からなる群から選ばれる化合物
の少なくとも1種を含有する抗原もしくは抗体感
作ラテツクス試薬に関し、さらに抗原又は抗体感
作ラテツクスにγ−コリジン;一般式()で表
わされるイミダゾール又はその誘導体;および一
般式()で表わされるプロパン1,3−ジオー
ル誘導体からなる群から選ばれる化合物の少なく
とも1つを共存させることを特徴とする抗原又は
抗体感作ラテツクスの安定化法に関する。 本発明のラテツクス試薬は例えばα−フエトプ
ロテイン、C−反応性タンパク、フイプリノーゲ
ン、リウマチ因子、イムノグロプリンなどの測定
用試薬として有用である。 従来のラテツクス試薬は保存期間が長くなると
ラテツクス粒子の自然凝集が起り、抗原又は抗体
を精度良く測定することができない。 上記の欠点を改良すべく種々検討した結果、抗
原又は抗体感作ラテツクスを水性溶媒に懸濁した
後、γ−コリジン;一般式()で表わされるイ
ミダゾール又はその誘導体;および一般式()
で表わされるプロパン1,3−ジオール誘導体か
らなる群から選ばれる化合物の少なくとも1つを
共存せしめることにより得たラテツクス試薬が長
期間保存してもラテツクス粒子の自然凝集が非常
に少なく、かつ長期間保存した後でも抗原又は抗
体を精度良く測定できることが見い出された。 以下に本発明を詳細に説明する。 本発明に使用する抗原又は抗体としては、ヒト
イムノグロブリンG、ヒトイムノグロブリンA、
ヒトイムノグロブリンM、ヒト−γ−グロブリ
ン、ヒト血清アルブミン、α−フエトプロテイ
ン、β2−ミクログロブリン、ミオグロビン、C−
反応性蛋白、フイブリノーゲン、人絨毛性ゴナド
トロピンなどがあげられる。 抗原又は抗体を感作するラテツクスとしては、
ポリスチレン、カルボキシル化ポリスチレン、ポ
リビニルトルエン、スチレン−ブタジエン共重合
体、カルボキシル化スチレン−ブタジエン共重合
体、アクリル酸エステル重合体、メタクリル酸エ
ステル重合体などがあげられる。 水性溶媒としては、生理食塩水、緩衝液(グリ
シン緩衝液、リ酸緩衝液、ホウ酸緩衝液など)お
よびそれらの組み合わせが用いられる。一般式
()および()中の炭素数1−5のアルキル
基とはメチル基、エチル基、プロピル基、ブチル
基等である。 ラテツクス試薬を製造する際には、例えば吸着
法、共有結合法が使用できる。吸着法による場合
には抗原又は抗体を溶解した水性溶媒中にラテツ
クスを添加し、撹拌又は振盪し、必要に応じて加
熱し、抗原又は抗体をラテツクスに感作する。次
にこの感作ラテツクスを遠心分離によつて分離
し、上清を除去する。得られた感作ラテツクスを
再び水性溶媒に再分散し、しかる後この分散液に
γ−コリジン;一般式()で表わされるイミダ
ゾール又はその誘導体;および一般式()で表
わされるプロパン1,3−ジオール誘導体からな
る群から選ばれる化合物の少なくとも1つを添加
し、混合することによりラテツクス試薬を得る。 又、共有結合法による場合には、カルボキシル
化ラテツクスの懸濁液にカツプリング剤として、
例えば、水溶性カルボジイミドなどを室温〜5℃
で撹拌しながら混合する。しかる後に、この液に
水性溶媒に溶解した抗原又は抗体を加え、撹拌混
合した後、遠心分離する。得られた抗原又は抗体
感作ラテツクスを再び水性溶媒に再分散する。つ
いでこの分散液にγ−コリジン;一般式()で
表わされるイミダゾール又はその誘導体;および
一般式()で表わされるプロパン1,3−ジオ
ール誘導体からなる群から選ばれる化合物の少な
くとも1つを添加し、混合することによりラテツ
クス試薬を得る。 上記の如くして得られるラテツクス試薬中の感
作ラテツクス濃度としては0.01〜5.0w/v%(以
下、%はw/v%を意味する。)、好ましくは0.05
〜2.0%の範囲であり、γ−コリジン、一般式
()で表わされるイミダゾール又はその誘導体
又は一般式()で表わされるプロパン1,3−
ジオールの濃度としては0.05〜2.0%、好ましく
は0.1〜1.0%の範囲である。 以下に実施例を示す。 実施例 1 粒子径0.2μmのポリスチレンラテツクスを
0.1Mリン酸緩衝液(PH8)で2.0%になるように
分散した懸濁液5mlにヒトγ−グロブリンを上記
緩衝液(PH8.0)に溶解して0.5%とした液5mlを
添加し、ついで混合し56℃で1時間ゆつくり撹拌
しながら感作する。遠心分離後0.1%牛血清アル
ブミンを含有する0.1Mグリシン緩衝液(PH8.0)
に前記で得られた感作ラテツクス濃度が0.5%に
なるように再分散する。この分散した懸濁液10ml
にγ−コリジン40μをゆつくり撹拌しながら混
合し、更にアジ化ナトリウム0.01gを添加してリ
ウマチ因子側定用試薬を得る。 比較のため上記配合でγ−コリジンを添加しな
い試薬を調整する。 これら2つの試薬を約4℃で長期間保存した場
合の懸濁液粒子の自然凝集の度合を分光光度計に
よる吸光度測定から判定する。測定は波長800n
m、光路長5mm、試薬を上記0.1Mリン酸緩衝液
(PH8)で5倍に希釈して試料とする。その結果
を第1表に示す。
The present invention relates to latex reagents and methods for stabilizing latex. More specifically, the present invention relates to γ-collidine; general formula () (In the formula, R 1 , R 2 , R 3 and R 4 are the same or different and represent a hydrogen atom or an alkyl group having 1 to 5 carbon atoms.) Imidazole or a derivative thereof; and the general formula () Propane 1,3 represented by (wherein, R 1 represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms)
- An antigen or antibody sensitized latex reagent containing at least one compound selected from the group consisting of diol derivatives, further including γ-collidine; imidazole or a derivative thereof represented by the general formula (); The present invention relates to a method for stabilizing an antigen or antibody sensitized latex, which comprises coexisting at least one compound selected from the group consisting of propane 1,3-diol derivatives represented by the general formula (). The latex reagent of the present invention is useful as a reagent for measuring α-fetoprotein, C-reactive protein, fibrinogen, rheumatoid factor, immunoglobulin, and the like. When conventional latex reagents are stored for a long time, natural aggregation of latex particles occurs, making it impossible to accurately measure antigens or antibodies. As a result of various studies to improve the above drawbacks, after suspending an antigen or antibody sensitized latex in an aqueous solvent, γ-collidine; imidazole or its derivative represented by the general formula (); and the general formula ()
A latex reagent obtained by coexisting with at least one compound selected from the group consisting of propane 1,3-diol derivatives represented by It has been found that antigens or antibodies can be measured with high accuracy even after storage. The present invention will be explained in detail below. The antigen or antibody used in the present invention includes human immunoglobulin G, human immunoglobulin A,
Human immunoglobulin M, human γ-globulin, human serum albumin, α-fetoprotein, β 2 -microglobulin, myoglobin, C-
Examples include reactive protein, fibrinogen, and human chorionic gonadotropin. Latexes that sensitize antigens or antibodies include:
Examples include polystyrene, carboxylated polystyrene, polyvinyltoluene, styrene-butadiene copolymer, carboxylated styrene-butadiene copolymer, acrylic ester polymer, and methacrylic ester polymer. As the aqueous solvent, physiological saline, buffer solutions (glycine buffer, phosphate buffer, borate buffer, etc.), and combinations thereof are used. The alkyl group having 1 to 5 carbon atoms in the general formulas () and () includes a methyl group, an ethyl group, a propyl group, a butyl group, and the like. When producing latex reagents, for example, adsorption methods and covalent bonding methods can be used. When using the adsorption method, latex is added to an aqueous solvent in which the antigen or antibody is dissolved, stirred or shaken, and heated if necessary to sensitize the latex with the antigen or antibody. This sensitized latex is then separated by centrifugation and the supernatant is removed. The obtained sensitized latex was redispersed in an aqueous solvent, and then this dispersion was mixed with γ-collidine; imidazole or its derivative represented by the general formula (); and propane 1,3- represented by the general formula (). A latex reagent is obtained by adding and mixing at least one compound selected from the group consisting of diol derivatives. In addition, when using a covalent bonding method, a coupling agent is added to the suspension of carboxylated latex.
For example, water-soluble carbodiimide etc. at room temperature to 5°C.
Mix while stirring. Thereafter, an antigen or antibody dissolved in an aqueous solvent is added to this solution, mixed by stirring, and then centrifuged. The obtained antigen or antibody sensitized latex is redispersed in an aqueous solvent. Then, to this dispersion, at least one compound selected from the group consisting of γ-collidine; imidazole or its derivative represented by the general formula (); and a propane 1,3-diol derivative represented by the general formula () is added. , a latex reagent is obtained by mixing. The concentration of the sensitizing latex in the latex reagent obtained as described above is 0.01 to 5.0 w/v% (hereinafter, % means w/v%), preferably 0.05
~2.0%, γ-collidine, imidazole represented by the general formula () or its derivatives, or propane 1,3- represented by the general formula ()
The concentration of diol is in the range of 0.05 to 2.0%, preferably 0.1 to 1.0%. Examples are shown below. Example 1 Polystyrene latex with a particle size of 0.2 μm
To 5 ml of a suspension dispersed in 0.1M phosphate buffer (PH 8) to a concentration of 2.0%, 5 ml of a solution obtained by dissolving human γ-globulin in the above buffer (PH 8.0) to a concentration of 0.5% was added. Then, mix and sensitize by slowly stirring at 56°C for 1 hour. 0.1M glycine buffer (PH8.0) containing 0.1% bovine serum albumin after centrifugation
Then, the sensitized latex obtained above is redispersed so that the concentration is 0.5%. 10ml of this dispersed suspension
40μ of γ-collidine was slowly mixed with stirring, and 0.01g of sodium azide was added to obtain a reagent for determining rheumatoid factor. For comparison, a reagent with the above formulation without the addition of γ-collidine was prepared. When these two reagents are stored at about 4° C. for a long period of time, the degree of spontaneous aggregation of the suspension particles is determined from absorbance measurement using a spectrophotometer. Measurement is at wavelength 800n
m, optical path length 5 mm, and dilute the reagent 5 times with the above 0.1 M phosphate buffer (PH8) to use as a sample. The results are shown in Table 1.

【表】 実施例で得られた試薬では吸光度変化がほとん
ど認められないことから、感作ラテツクス粒子の
自然凝集が起つていないことがわかる。一方、比
較例のものでは吸光度変化が大きく、自然凝集が
生じている。 実施例 2 カルボキシル化ポリスチレンラテツクス(粒子
径0.25μm)を0.1Mリン酸緩衝液−生理食塩水
(以下PBSと称す。)(PH7.0)で1%の濃度に調整
した懸濁液6mlに0.6mgの1−シクロヘキシル−
3−〔2−モルホリニル−(4)−エチル〕カルボジ
イミド−メト−P−トルエンスルホネートを20ml
のPBSに溶解した液280μを4℃恒温下で撹拌
しながら添加する。5分後にウサギ抗C反応性蛋
白抗血清を0.1%含有してなるPBS2.2mlをゆるや
かに撹拌しながら添加し、4℃で24時間撹拌を続
けて共有結合によつて感作する。ついで、10000
回転で遠心分離を行ない上清を捨て得られた沈殿
物をPBSに再分散する。再度、遠心分離を行な
い、0.1%牛血清アルブミンを含有したPBSに感
作ラテツクス濃度が0.5%になるように再分散す
る。ついでこの懸濁液6mlにγ−コリジン12μ
をゆるやかに撹拌しながら添加し、更にアジ化ナ
トリウム6mgを添加混合してC反応清蛋白測定用
試薬を得る。 比較のためにγ−コリジン無添加の試薬も同時
に調整する。 これら2つの試薬を4℃〜7℃で12カ月保存
し、自然凝集の有無を肉眼で観察する。γ−コリ
ジン添加の場合はほとんど凝集を認めなかつた。
又ヒト血清25μをスライドガラス板上に滴下
し、次に保存後の上記試薬25μを加えて2.5分間
ゆり動かし、凝集反応状態を肉眼で判定した結果
を第2表に示す。
[Table] Almost no change in absorbance was observed in the reagents obtained in the examples, which indicates that spontaneous aggregation of the sensitized latex particles did not occur. On the other hand, in the comparative example, the absorbance change was large and spontaneous aggregation occurred. Example 2 Carboxylated polystyrene latex (particle size 0.25 μm) was added to 6 ml of a suspension adjusted to a concentration of 1% with 0.1 M phosphate buffer-physiological saline (hereinafter referred to as PBS) (PH 7.0). 0.6 mg of 1-cyclohexyl
20 ml of 3-[2-morpholinyl-(4)-ethyl]carbodiimide-meth-P-toluenesulfonate
280μ of a solution dissolved in PBS is added while stirring at a constant temperature of 4°C. After 5 minutes, 2.2 ml of PBS containing 0.1% rabbit anti-C-reactive protein antiserum is added with gentle stirring, and stirring is continued at 4° C. for 24 hours to sensitize by covalent bonding. Then 10000
Perform centrifugation by rotating, discard the supernatant, and redisperse the resulting precipitate in PBS. Centrifuge again and redistribute the sensitized latex in PBS containing 0.1% bovine serum albumin to a concentration of 0.5%. Next, add 12μ of γ-collidine to 6ml of this suspension.
was added with gentle stirring, and 6 mg of sodium azide was further added and mixed to obtain a reagent for measuring C reaction protein. For comparison, a reagent without γ-collidine was also prepared at the same time. These two reagents are stored at 4°C to 7°C for 12 months, and the presence or absence of spontaneous aggregation is visually observed. When γ-collidine was added, almost no aggregation was observed.
Table 2 shows the results of dropping 25μ of human serum onto a glass slide plate, adding 25μ of the above-mentioned reagent after storage, and shaking for 2.5 minutes.The state of the agglutination reaction was determined visually.Table 2 shows the results.

【表】 〓
[Table] 〓

Claims (1)

【特許請求の範囲】 1 一般式 (式中、R1は水素原子又は炭素数1−5のアル
キル基を表わす。)で表わされるプロパン1,3
−ジオール誘導体を含有する抗原もしくは抗体感
作ラテツクス試薬。
[Claims] 1. General formula Propane 1,3 represented by (wherein, R 1 represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms)
- Antigen or antibody sensitizing latex reagents containing diol derivatives.
JP6678483A 1983-04-15 1983-04-15 Latex reagent Granted JPS59192962A (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP6678483A JPS59192962A (en) 1983-04-15 1983-04-15 Latex reagent
JP28157690A JPH03142360A (en) 1983-04-15 1990-10-19 Latex reagent

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP6678483A JPS59192962A (en) 1983-04-15 1983-04-15 Latex reagent

Related Child Applications (1)

Application Number Title Priority Date Filing Date
JP28157690A Division JPH03142360A (en) 1983-04-15 1990-10-19 Latex reagent

Publications (2)

Publication Number Publication Date
JPS59192962A JPS59192962A (en) 1984-11-01
JPH0376425B2 true JPH0376425B2 (en) 1991-12-05

Family

ID=13325830

Family Applications (1)

Application Number Title Priority Date Filing Date
JP6678483A Granted JPS59192962A (en) 1983-04-15 1983-04-15 Latex reagent

Country Status (1)

Country Link
JP (1) JPS59192962A (en)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62218866A (en) * 1986-03-20 1987-09-26 Hitachi Chem Co Ltd Reagent for quantitative analysis of human c-reactive protein
JPS62218864A (en) * 1986-03-20 1987-09-26 Hitachi Chem Co Ltd Quantitative analysis of human c reactive protein
JP2534067B2 (en) * 1987-07-09 1996-09-11 日水製薬株式会社 Quantitative method for C-reactive protein
US5948820A (en) 1994-08-22 1999-09-07 Yoshitomi Pharmaceutical Industries, Ltd. Benzene compound and pharmaceutical use thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS49133092A (en) * 1971-11-22 1974-12-20

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS49133092A (en) * 1971-11-22 1974-12-20

Also Published As

Publication number Publication date
JPS59192962A (en) 1984-11-01

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