JPH0136484B2 - - Google Patents
Info
- Publication number
- JPH0136484B2 JPH0136484B2 JP8174682A JP8174682A JPH0136484B2 JP H0136484 B2 JPH0136484 B2 JP H0136484B2 JP 8174682 A JP8174682 A JP 8174682A JP 8174682 A JP8174682 A JP 8174682A JP H0136484 B2 JPH0136484 B2 JP H0136484B2
- Authority
- JP
- Japan
- Prior art keywords
- latex
- styrene
- emulsifier
- particle size
- reaction
- 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
Links
- 239000004816 latex Substances 0.000 claims description 67
- 229920000126 latex Polymers 0.000 claims description 67
- 239000003995 emulsifying agent Substances 0.000 claims description 19
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims description 18
- 239000003153 chemical reaction reagent Substances 0.000 claims description 18
- 238000010438 heat treatment Methods 0.000 claims description 8
- AGBXYHCHUYARJY-UHFFFAOYSA-N 2-phenylethenesulfonic acid Chemical compound OS(=O)(=O)C=CC1=CC=CC=C1 AGBXYHCHUYARJY-UHFFFAOYSA-N 0.000 claims description 7
- 239000003999 initiator Substances 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 7
- 229910000000 metal hydroxide Inorganic materials 0.000 claims description 6
- 229910044991 metal oxide Inorganic materials 0.000 claims description 6
- 150000004706 metal oxides Chemical class 0.000 claims description 6
- JRKICGRDRMAZLK-UHFFFAOYSA-L peroxydisulfate Chemical compound [O-]S(=O)(=O)OOS([O-])(=O)=O JRKICGRDRMAZLK-UHFFFAOYSA-L 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 239000002245 particle Substances 0.000 description 28
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 22
- 238000006243 chemical reaction Methods 0.000 description 22
- 230000004520 agglutination Effects 0.000 description 14
- 210000002966 serum Anatomy 0.000 description 14
- 239000004471 Glycine Substances 0.000 description 11
- 239000000427 antigen Substances 0.000 description 11
- 102000036639 antigens Human genes 0.000 description 11
- 108091007433 antigens Proteins 0.000 description 11
- 239000004793 Polystyrene Substances 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 229920002223 polystyrene Polymers 0.000 description 8
- 238000007334 copolymerization reaction Methods 0.000 description 5
- 150000004679 hydroxides Chemical class 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 239000006185 dispersion Substances 0.000 description 4
- 108010074605 gamma-Globulins Proteins 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 230000007062 hydrolysis Effects 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 229920000642 polymer Polymers 0.000 description 4
- 238000006116 polymerization reaction Methods 0.000 description 4
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical group N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 3
- 229910001873 dinitrogen Inorganic materials 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- MNCGMVDMOKPCSQ-UHFFFAOYSA-M sodium;2-phenylethenesulfonate Chemical compound [Na+].[O-]S(=O)(=O)C=CC1=CC=CC=C1 MNCGMVDMOKPCSQ-UHFFFAOYSA-M 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-L sulfate group Chemical group S(=O)(=O)([O-])[O-] QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010556 emulsion polymerization method Methods 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000002336 sorption--desorption measurement Methods 0.000 description 2
- LCPVQAHEFVXVKT-UHFFFAOYSA-N 2-(2,4-difluorophenoxy)pyridin-3-amine Chemical compound NC1=CC=CN=C1OC1=CC=C(F)C=C1F LCPVQAHEFVXVKT-UHFFFAOYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- QPLDLSVMHZLSFG-UHFFFAOYSA-N Copper oxide Chemical class [Cu]=O QPLDLSVMHZLSFG-UHFFFAOYSA-N 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 229910001870 ammonium persulfate Inorganic materials 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- XDIJWRHVEDUFGP-UHFFFAOYSA-N azanium;2-phenylethenesulfonate Chemical compound [NH4+].[O-]S(=O)(=O)C=CC1=CC=CC=C1 XDIJWRHVEDUFGP-UHFFFAOYSA-N 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 239000002612 dispersion medium Substances 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- ZKIAYSOOCAKOJR-UHFFFAOYSA-M lithium;2-phenylethenesulfonate Chemical compound [Li+].[O-]S(=O)(=O)C=CC1=CC=CC=C1 ZKIAYSOOCAKOJR-UHFFFAOYSA-M 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- VTHJTEIRLNZDEV-UHFFFAOYSA-L magnesium dihydroxide Chemical compound [OH-].[OH-].[Mg+2] VTHJTEIRLNZDEV-UHFFFAOYSA-L 0.000 description 1
- 239000000347 magnesium hydroxide Substances 0.000 description 1
- 229910001862 magnesium hydroxide Inorganic materials 0.000 description 1
- 239000000395 magnesium oxide Substances 0.000 description 1
- CPLXHLVBOLITMK-UHFFFAOYSA-N magnesium oxide Inorganic materials [Mg]=O CPLXHLVBOLITMK-UHFFFAOYSA-N 0.000 description 1
- AXZKOIWUVFPNLO-UHFFFAOYSA-N magnesium;oxygen(2-) Chemical compound [O-2].[Mg+2] AXZKOIWUVFPNLO-UHFFFAOYSA-N 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- -1 nonionic Chemical group 0.000 description 1
- 239000012875 nonionic emulsifier Substances 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- QIFCIFLDTQJGHQ-UHFFFAOYSA-M potassium;2-phenylethenesulfonate Chemical compound [K+].[O-]S(=O)(=O)C=CC1=CC=CC=C1 QIFCIFLDTQJGHQ-UHFFFAOYSA-M 0.000 description 1
- 230000001235 sensitizing effect Effects 0.000 description 1
- CHQMHPLRPQMAMX-UHFFFAOYSA-L sodium persulfate Substances [Na+].[Na+].[O-]S(=O)(=O)OOS([O-])(=O)=O CHQMHPLRPQMAMX-UHFFFAOYSA-L 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid group Chemical group S(O)(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Polymerisation Methods In General (AREA)
- Polymerization Catalysts (AREA)
- Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
Description
本発明は免疫血清学的診断試薬用に用いられて
有効なラテツクスの製造方法に関する。
ポリスチレンラテツクスに抗原又は抗体を感作
させ、これを用いて血清中の対応する抗体又は抗
原を、ラテツクスの凝集反応として検出する免疫
血清学的診断法は1956年に血清中のリウマチ因子
の検出に応用されて以来、その簡便性と迅速性の
故に、臨床検査の分野において多くの種類の抗原
又は抗体の検出に拡大適用され今日に至つてい
る。
この目的に用いるポリスチレンラテツクスは、
一般に粒径が0.05ないし１ミクロンであり、粒径
分布が狭く粒径の揃つたものが望ましい。このよ
うなラテツクスは通常公知の乳化重合の方法を用
いて製造できるとされている。その方法とは、例
えば水中にアニオン系、ノニオン系又はカチオン
系の乳化剤の何れか１種又は２種以上を混合した
もの、スチレンモノマー、水溶性ラジカル開始剤
等を共存させて、好ましくは酸素を除いた雰囲気
で、適当な温度に適当な時間保つことである。
このようにして得られるポリスチレンラテツク
スにおいて、その安定性に寄与する乳化剤の存在
形態は重要である。一般には、重合の際に用いた
乳化剤の一部はポリスチレンラテツクス粒子の表
面に吸着されるか化学的に結合されており、他は
ラテツクス中に遊離の状態で存在しており、これ
らの状態の間には乳化剤のポリスチレンラテツク
ス粒子表面に対する吸着脱着平衡が成立してい
る。このように通常の方法で製造されるポリスチ
レンラテツクスにあつては、乳化剤は安定なラテ
ツクスの形成に不可欠である。しかしながら、遊
離の乳化剤は前述の抗原又は抗体によるラテツク
スの凝集反応に対しては不都合な影響を与えるの
である。すなわち免疫血清学的診断試薬を製造す
るには、まず前述の如くポリスチレンラテツクス
に抗原又は抗体を感作させる訳であるが、遊離の
乳化剤を含むラテツクスを用いるとこの段階です
でに凝集してしまうことがある。次に、抗原又は
抗体を感作させたラテツクスを用いて、この抗原
又は抗体に対応する抗体又は抗原をラテツクスの
凝集反応によつて検出する際には、検出されるべ
き抗体又は抗原を含む血清（陽性血清）と接触す
れば感作ラテツクスは凝集し、かかる抗体又は抗
原を含まない血清（陰性血清）と接触しても感作
ラテツクスは凝集しないことが必須要件とされる
のであるが、遊離の乳化剤を含む感作ラテツクス
の場合には陰性血清と接触しても凝集してしま
い、いわゆる非特異的凝集反応となることがはな
はだ多いのである。
勿論、ラテツクスに含まれる遊離の乳化剤は、
例えばイオン交換法や透析法の技術を用いて除く
ことは可能である。しかし、遊離の乳化剤をラテ
ツクスから除いてしまつた場合、前述の如く遊離
の乳化剤とラテツクス粒子表面に吸着された乳化
剤との間の吸着脱着平衡の成立によつてラテツク
スが安定化されているために、ラテツクスの安定
性は極端にわるくなり実際上は使用不可能となつ
てしまうのである。
叙上の如く、免疫血清学的診断試薬用ラテツク
スとしては、通常の乳化重合法で製造したポリス
チレンラテツクスは、遊離の乳化剤を含む点にお
いて実用上大きな難点を有しているのである。
本発明は上記の如き欠点のない免疫血清学的診
断試薬として用いられるラテツクスを提供するこ
とを主たる目的として鋭意研究せる結果なされた
ものであり、その要旨はスチレンと該スチレンに
対し10重量％以下のスチレンスルホン酸塩とを乳
化剤の不存在下で、原子価が２価の金属の酸化物
又は水酸化物を含有する水溶液中で過硫酸塩を開
始剤として共重合させ、次いでアルカリ性の条件
下で加熱を行うことを特徴とする、診断試薬用ラ
テツクスの製造方法に存する。
本発明に用いられるスチレンスルホン酸塩とし
ては、スチレンスルホン酸塩ナトリウム、スチレ
ンスルホン酸カリウム、スチレンスルホン酸リチ
ウム、スチレンスルホン酸アンモニウム等があげ
られる。これらのスチレンスルホン酸塩のスチレ
ンに対する使用割合は10重量％以下とされるが、
好ましくは0.0001％から10％、より好ましくは
0.001％から５％の範囲である。
又、本発明において開始剤として用いられる過
硫酸塩としては、過硫酸アンモニウム、過硫酸カ
リウム、過硫酸ナトリウム等があげられる。これ
らの過硫酸塩のモノマー全体に対する割合は0.01
ないし１重量％の範囲が好ましい。
本発明において使用される原子価が２価の金属
の酸化物又は水酸化物としては、鉄、マグネシウ
ム、カルシウム、銅の酸化物又は水酸化物等があ
げられる。これらの２価の金属の酸化物又は水酸
化物の使用量は、スチレンモノマーに対し0.003
〜3.0重量％の範囲とされるのが好ましい。
本発明方法によりラテツクス製造のための共重
合を行うには水が仕込まれた反応器内にスチレ
ン、スチレンスルホン酸塩、原子価が２価の金属
の酸化物又は水酸化物及び開始剤を加えて撹拌し
ながら加熱すればよく、その際の重合反応温度は
通常50〜100℃、好ましくは60〜85℃の範囲とす
るのがよい。又、重合反応に要する時間はモノマ
ー組成、モノマー濃度、開始剤濃度等の条件によ
り変るが、通常５〜50時間の範囲である。
本発明において、原子価が２価の金属の酸化物
又は水酸化物が使用されるのは次の理由による。
すなわち乳化剤の不存在下でスチレンとスチレン
スルホン酸塩を共重合させて得られるラテツクス
で粒子径のよく揃つたものを得ようとすれば、ス
チレンモノマーに対する触媒量を増加するだけで
もよい。しかしこの場合、得られたラテツクスを
用いて試薬化した際に感度が悪く、又非特異的凝
集反応を示す確率が多く、安定性にすぐれたラテ
ツクス試薬が得られない。かりに非特異的凝集反
応の少ない良好なラテツクス試薬を得ようとすれ
ば、非常に純度の高い精製された抗体又は抗原を
用いなければならず、試薬製造に時間と手間を要
するため、高価な試薬となる。
これに対し原子価が２価の金属の酸化物又は水
酸化物を使用する場合には、これらがラテツクス
粒子の核として働き、この核のまわりをスチレン
とスチレンスルホン酸塩の共重合体が取巻いて均
一な粒子のラテツクスを形成する。
この場合においてラテツクス粒子が均一に分散
状態を保持し、分散媒に分散された際に沈降した
り浮き上つてしまつたりしないことが必要であ
る。原子価が２価の金属の酸化物又は水酸化物が
ラテツクス粒子の核として働く場合は、ラテツク
ス粒子を均一に分散できる程良い比重を有するも
のとなり、ラテツクス粒子の沈降、浮き上りを生
じないものとすることができる。
本発明方法により平均粒径が0.1ないし1.5ミク
ロンで、粒径のばらつきが変動係数（粒径の標準
偏差／平均粒径）で表わして0.02以下である粒径
が非常によく揃つた単分散ラテツクスを得ること
が出来る。
該ラテツクスが、乳化剤を全く含まないにも拘
らず極めて安定な理由は次のように説明できる。
即ち開始剤として過硫酸塩を用いるからポリマー
鎖の両端に硫酸基（SO4 -）が存在することにな
り、ポリマー鎖同志の間にはこの硫酸基による電
気的反発力が作用してラテツクスが安定化され
る。しかし、ポリマー鎖末端の硫酸基による電気
的反発力のみではラテツクスの安定化には不十分
であり、これに加えてスチレンスルホン酸塩を共
重合させてポリマー鎖中にスルホン基を導入する
ことにより、それによる電気的反発力をも加えて
始めて十分に安定なラテツクスが得られるのであ
る。ここにおいて言及すべきことは、前述のポリ
マー鎖末端の硫酸基は比較的不安定であり、加水
分解を受け易く水酸基を経てカルボン酸になる傾
向があることである。この場合、加水分解が不完
全で水酸基の段階で留つていれば、水酸基はイオ
ン化しにくいためラテツクスの安定化に寄与しな
い。
従つて加水分解を進めて大部分がカルボキシル
基である状態にしておくことが、ラテツクスの安
定化にとつて重要であり、そしてカルボキシル基
の解離を促進するためには加水分解をアルカリ性
で行うことが必要である。
そこで本発明においては上記により得られたラ
テツクスをアルカリ性の条件下で加熱するのであ
る。この際の加熱温度は通常50〜90℃、好ましく
は60〜80℃とするのがよく、又加熱時間は10〜
100時間とするのがよい。
そして上記加熱は、反応系をアルカリ性にして
行われるのであるが、その際の反応系のPH値が
7.5〜12.5とくに８〜11.5の範囲の保たれるのがよ
い。
本発明のラテツクスの製造方法は上述の通りの
構成であるので、乳化剤を全く含まないにもかゝ
わらず極めて安定にしてしかも粒径がよく揃つた
ラテツクスを製造することが出来るのであり、そ
して該ラテツクスは乳化剤を全く含まないために
免疫血清学的診断試薬として用いられた場合にい
わゆる非特異的凝集反応を起すことがなく、該診
断試薬としてすぐれた性能を有するものである。
次に本発明の実施例について説明する。
実施例 １
スチレンモノマー90ｇ、スチレンスルホン酸ナ
トリウム0.63ｇ、水酸化マグネシウム0.4ｇ、過
硫酸カリウム0.5ｇ、イオン交換水450ｇを反応容
器に仕込み、容器を窒素ガスで置換し反応温度70
℃で24時間共重合した。
共重合終了後、反応容器の内部を空気で置換
し、ラテツクスのPHを8.5に調節し70℃で24時間
加熱を続けた。このようにして得られたラテツク
スの平均粒径は0.70ミクロン、粒径のばら付きは
変動係数で表わして0.018であつた。PH8.5のグリ
シン緩衝液に分散したラテツクス分散液１容に対
し、グリシン緩衝液で0.1％に希釈したヒトガン
マグロブリン溶液１容を混合し、37℃に60分保つ
た後、26000×Ｇで遠心分離して未吸着のヒトガ
ンマグロブリンを除き、沈降したラテツクス粒子
をグリシン緩衝液に再分散して均一な感作ラテツ
クス分散液とした。この１滴とグリシン緩衝液で
種々の倍率に希釈したリウマチ因子を含む血清１
滴とをガラス板上で混合し、３分間ガラス板をゆ
るやかに前後左右に傾けて凝集反応の強さを観察
し次表の結果を得た。
The present invention relates to a method for producing a latex effective for use in immunoserological diagnostic reagents. The immunoserological diagnostic method, which involves sensitizing polystyrene latex with an antigen or antibody and using this to detect the corresponding antibody or antigen in serum as an agglutination reaction of the latex, was developed in 1956 for the detection of rheumatoid factor in serum. Due to its simplicity and rapidity, it has been widely applied to the detection of many types of antigens or antibodies in the field of clinical testing, and continues to this day. The polystyrene latex used for this purpose is
Generally, the particle size is 0.05 to 1 micron, and particles with a narrow particle size distribution and uniform particle size are desirable. It is said that such a latex can be produced using a commonly known emulsion polymerization method. The method includes, for example, coexisting in water with one or more of anionic, nonionic, or cationic emulsifiers, styrene monomer, water-soluble radical initiator, etc., and preferably oxygen. The method is to maintain the temperature at an appropriate temperature for an appropriate amount of time in an atmosphere that is free of heat. In the polystyrene latex thus obtained, the form in which the emulsifier exists, which contributes to its stability, is important. Generally, some of the emulsifiers used during polymerization are adsorbed or chemically bonded to the surface of polystyrene latex particles, while others are present in the latex in a free state, and these states During this period, an adsorption/desorption equilibrium of the emulsifier on the surface of the polystyrene latex particles is established. In polystyrene latex manufactured by conventional methods, emulsifiers are essential for forming a stable latex. However, free emulsifiers have an unfavorable effect on the agglutination reaction of the latex by the aforementioned antigens or antibodies. That is, in order to produce an immunoserological diagnostic reagent, polystyrene latex is first sensitized with an antigen or antibody as described above, but if a latex containing a free emulsifier is used, it may have already aggregated at this stage. Sometimes I put it away. Next, when detecting an antibody or antigen corresponding to the antigen or antibody by a latex agglutination reaction using latex sensitized with an antigen or antibody, serum containing the antibody or antigen to be detected is used. It is essential that the sensitized latex does not agglutinate when it comes into contact with (positive serum) and does not agglutinate even when it comes into contact with serum that does not contain such antibodies or antigens (negative serum). In the case of a sensitized latex containing an emulsifier, it often aggregates even when it comes into contact with negative serum, resulting in a so-called non-specific agglutination reaction. Of course, the free emulsifier contained in latex is
For example, it is possible to remove it using ion exchange or dialysis techniques. However, when the free emulsifier is removed from the latex, the latex is stabilized due to the establishment of adsorption-desorption equilibrium between the free emulsifier and the emulsifier adsorbed on the surface of the latex particles, as described above. The stability of the latex becomes extremely poor, making it practically unusable. As mentioned above, as latex for immunoserological diagnostic reagents, polystyrene latex produced by the usual emulsion polymerization method has a major practical drawback in that it contains free emulsifier. The present invention was made as a result of intensive research with the main purpose of providing a latex to be used as an immunoserological diagnostic reagent free from the above-mentioned drawbacks, and the gist of the invention is to provide a latex for use as an immunoserological diagnostic reagent that does not have the above-mentioned drawbacks. styrene sulfonate in the absence of an emulsifier in an aqueous solution containing a divalent metal oxide or hydroxide using a persulfate as an initiator, and then under alkaline conditions. The present invention relates to a method for producing a latex for diagnostic reagents, the method comprising heating a latex for diagnostic reagents. Examples of the styrene sulfonate used in the present invention include sodium styrene sulfonate, potassium styrene sulfonate, lithium styrene sulfonate, and ammonium styrene sulfonate. The ratio of these styrene sulfonates to styrene is said to be 10% by weight or less, but
Preferably from 0.0001% to 10%, more preferably
It ranges from 0.001% to 5%. Further, examples of the persulfate used as an initiator in the present invention include ammonium persulfate, potassium persulfate, sodium persulfate, and the like. The proportion of these persulfates to the total monomer is 0.01
A range of from 1% to 1% by weight is preferred. Examples of the divalent metal oxides or hydroxides used in the present invention include iron, magnesium, calcium, and copper oxides or hydroxides. The amount of these divalent metal oxides or hydroxides used is 0.003 to styrene monomer.
The range is preferably 3.0% by weight. To perform copolymerization for latex production according to the method of the present invention, styrene, styrene sulfonate, an oxide or hydroxide of a divalent metal, and an initiator are added to a reactor charged with water. The polymerization reaction temperature at this time is usually in the range of 50 to 100°C, preferably 60 to 85°C. The time required for the polymerization reaction varies depending on conditions such as monomer composition, monomer concentration, and initiator concentration, but is usually in the range of 5 to 50 hours. The reason why divalent metal oxides or hydroxides are used in the present invention is as follows.
That is, in order to obtain a latex with well-uniformed particle sizes obtained by copolymerizing styrene and styrene sulfonate in the absence of an emulsifier, it is sufficient to simply increase the amount of catalyst relative to the styrene monomer. However, in this case, when the obtained latex is used to form a reagent, the sensitivity is poor and there is a high probability of non-specific agglutination reactions occurring, making it impossible to obtain a latex reagent with excellent stability. In order to obtain a good latex reagent with low non-specific agglutination reactions, it is necessary to use purified antibodies or antigens of extremely high purity, which requires time and effort to manufacture, resulting in expensive reagents. becomes. On the other hand, when divalent metal oxides or hydroxides are used, these act as the core of the latex particles, and the copolymer of styrene and styrene sulfonate surrounds this core. Roll to form a uniform particle latex. In this case, it is necessary that the latex particles maintain a uniformly dispersed state and do not settle or float when dispersed in the dispersion medium. When the oxide or hydroxide of a divalent metal acts as the nucleus of the latex particles, it has a specific gravity that is sufficient to uniformly disperse the latex particles, and does not cause settling or floating of the latex particles. It can be done. By the method of the present invention, a monodisperse latex with an average particle size of 0.1 to 1.5 microns and a variation in particle size of 0.02 or less expressed as a coefficient of variation (standard deviation of particle size/average particle size) with very uniform particle sizes can be produced. can be obtained. The reason why this latex is extremely stable despite not containing any emulsifier can be explained as follows.
That is, since persulfate is used as an initiator, sulfate groups (SO 4 - ) are present at both ends of the polymer chains, and the electrical repulsion caused by these sulfate groups acts between polymer chains, forming a latex. stabilized. However, the electrical repulsion caused by the sulfate group at the end of the polymer chain is not sufficient to stabilize the latex. A sufficiently stable latex can only be obtained by adding the electrical repulsion caused by this. It should be mentioned here that the sulfuric acid groups at the ends of the polymer chains mentioned above are relatively unstable and tend to be susceptible to hydrolysis and become carboxylic acids via hydroxyl groups. In this case, if the hydrolysis is incomplete and remains at the hydroxyl group stage, the hydroxyl group is difficult to ionize and therefore does not contribute to stabilizing the latex. Therefore, it is important for the stabilization of the latex to advance the hydrolysis to a state where most of the carboxyl groups are present, and to promote the dissociation of the carboxyl groups, the hydrolysis should be carried out in an alkaline environment. is necessary. Therefore, in the present invention, the latex obtained as described above is heated under alkaline conditions. The heating temperature at this time is usually 50-90℃, preferably 60-80℃, and the heating time is 10-90℃.
It is better to set it to 100 hours. The above heating is performed by making the reaction system alkaline, but the PH value of the reaction system at that time is
It is best to keep it within the range of 7.5 to 12.5, especially 8 to 11.5. Since the method for producing latex of the present invention has the above-mentioned structure, it is possible to produce latex which is extremely stable and has a well-uniformed particle size even though it does not contain any emulsifier, and Since the latex does not contain any emulsifier, it does not cause so-called non-specific agglutination reactions when used as an immunoserological diagnostic reagent, and has excellent performance as a diagnostic reagent. Next, examples of the present invention will be described. Example 1 90 g of styrene monomer, 0.63 g of sodium styrene sulfonate, 0.4 g of magnesium hydroxide, 0.5 g of potassium persulfate, and 450 g of ion-exchanged water were charged into a reaction vessel, the vessel was replaced with nitrogen gas, and the reaction temperature was set at 70°C.
Copolymerization was carried out at ℃ for 24 hours. After the copolymerization was completed, the inside of the reaction vessel was replaced with air, the pH of the latex was adjusted to 8.5, and heating was continued at 70°C for 24 hours. The average particle size of the latex thus obtained was 0.70 microns, and the variation in particle size was 0.018 expressed as a coefficient of variation. 1 volume of a human gamma globulin solution diluted to 0.1% with a glycine buffer was mixed with 1 volume of a latex dispersion in a pH 8.5 glycine buffer, kept at 37°C for 60 minutes, and then heated at 26,000×G. Unadsorbed human gamma globulin was removed by centrifugation, and the precipitated latex particles were redispersed in a glycine buffer to obtain a uniform sensitized latex dispersion. This drop and serum 1 containing rheumatoid factor diluted to various ratios with glycine buffer
The strength of the aggregation reaction was observed by mixing the droplets on a glass plate and gently tilting the glass plate back and forth for 3 minutes to obtain the results shown in the following table.
【表】
また、リウマチ因子を含む血積のかわりにグリ
シン緩衝液で20倍に希釈したリウマチ因子を含ま
ない血清を用いて同じ試験をした場合、凝集は全
く観察されなかつた。
これらの結果から明らかなように、本発明の方
法によつて得られたラテツクスを用いて調製した
免疫血清学的診断試薬は感度が高く、かつ非特異
的な凝集反応を起こさないものである。
実施例 ２
スチレンモノマー90ｇ、スチレンスルホン酸ナ
トリウム0.63ｇ、酸化マグネシウム0.4ｇ、過硫
酸カリウム0.5ｇ、イオン交換水450ｇを反応容器
に仕込み、容器を窒素ガスで置換し反応温度70℃
で30時間共重合した。
共重合終了後、反応容器の内部を空気で置換
し、ラテツクスのPHを8.5に調節し70℃で24時間
加熱を続けた。このようにして得られたラテツク
スの平均粒径は0.96ミクロン、粒径のばら付きは
変動係数で表わして0.013であつた。PH8.5のグリ
シン緩衝液に分散したラテツクス分散液１容に対
し、グリシン緩衝液で0.1％に希釈したヒトガン
マグロブリン溶液１容を混合し、37℃に60分保つ
た後、26000×Ｇで遠心分離して未吸着のヒトガ
ンマグロブリンを除き、沈降したラテツクス粒子
をグリシン緩衝液に再分散して均一な感作ラテツ
クス分散液とした。実施例１におけると同様にし
てこの１滴とグリシン緩衝液で種々の倍率に希釈
したリウマチ因子を含む血清１滴とをガラス板上
で混合し、３分間ガラス板をゆるやかに前後左右
に傾けて凝集反応の強さを観察し次表の結果を得
た。[Table] Furthermore, when the same test was performed using rheumatoid factor-free serum diluted 20 times with glycine buffer instead of blood volume containing rheumatoid factor, no agglutination was observed. As is clear from these results, the immunoserological diagnostic reagent prepared using the latex obtained by the method of the present invention has high sensitivity and does not cause non-specific agglutination reactions. Example 2 90 g of styrene monomer, 0.63 g of sodium styrene sulfonate, 0.4 g of magnesium oxide, 0.5 g of potassium persulfate, and 450 g of ion-exchanged water were charged into a reaction container, the container was replaced with nitrogen gas, and the reaction temperature was 70°C.
Copolymerization was carried out for 30 hours. After the copolymerization was completed, the inside of the reaction vessel was replaced with air, the pH of the latex was adjusted to 8.5, and heating was continued at 70°C for 24 hours. The average particle size of the latex thus obtained was 0.96 microns, and the variation in particle size was 0.013 expressed as a coefficient of variation. 1 volume of a human gamma globulin solution diluted to 0.1% with a glycine buffer was mixed with 1 volume of a latex dispersion in a pH 8.5 glycine buffer, kept at 37°C for 60 minutes, and then heated at 26,000×G. Unadsorbed human gamma globulin was removed by centrifugation, and the precipitated latex particles were redispersed in a glycine buffer to obtain a uniform sensitized latex dispersion. In the same manner as in Example 1, one drop of this and one drop of serum containing rheumatoid factor diluted to various ratios with glycine buffer were mixed on a glass plate, and the glass plate was gently tilted back and forth and left and right for 3 minutes. The strength of the agglutination reaction was observed and the results shown in the following table were obtained.
【表】
また、リウマチ因子を含む血清のかわりにグリ
シン緩衝液で20倍に希釈したリウマチ因子を含ま
ない血清を用いて同じ試験をした場合、凝集は全
く観察されなかつた。
これらの結果から明らかなように、本発明の方
法によつて得られたラテツクスを用いて調製した
免疫血清学的診断試薬は感度が高く、かつ非特異
的な凝集反応を起こさないものである。
比較例
スチレンモノマー91ｇ、ノニオン乳化剤（第一
工業製薬社製、商品名エマルジツド49）２ｇ、過
硫酸カリウム0.3ｇ、イオン交換水440ｇを反応容
器に仕込み、容器を窒素ガスで置換し反応温度70
℃で24時間重合した。得られたラテツクスの平均
粒径は0.48ミクロン、粒径のばらつきは変動係数
で表わして0.15であつた。
このラテツクスを用い実施例１と全く同じ方法
で免疫血清学的診断試薬を調製し、リウマチ因子
を含む血清による凝集反応の強さを観察し、次表
の結果を得た。[Table] Furthermore, when the same test was performed using rheumatoid factor-free serum diluted 20 times with glycine buffer instead of serum containing rheumatoid factor, no agglutination was observed. As is clear from these results, the immunoserological diagnostic reagent prepared using the latex obtained by the method of the present invention has high sensitivity and does not cause non-specific agglutination reactions. Comparative example: 91 g of styrene monomer, 2 g of nonionic emulsifier (manufactured by Dai-ichi Kogyo Seiyaku Co., Ltd., trade name: Emulgide 49), 0.3 g of potassium persulfate, and 440 g of ion-exchanged water were charged into a reaction container, and the container was replaced with nitrogen gas, and the reaction temperature was 70
Polymerization was carried out at ℃ for 24 hours. The average particle size of the obtained latex was 0.48 microns, and the variation in particle size was 0.15 expressed as a coefficient of variation. Using this latex, an immunoserological diagnostic reagent was prepared in exactly the same manner as in Example 1, and the strength of the agglutination reaction with serum containing rheumatoid factor was observed, and the results shown in the following table were obtained.
【表】
また、リウマチ因子を含まない血清をグリシン
緩衝液で20倍に希釈したものを用いて同じ試験を
した場合、明らかな凝集がみとめられた。[Table] Furthermore, when the same test was conducted using rheumatoid factor-free serum diluted 20 times with glycine buffer, clear agglutination was observed.
Claims (1)
スチレンスルホン酸塩とを乳化剤の不存在下で、
原子価が２価の金属の酸化物又は水酸化物を含有
する水溶液中で過硫酸塩を開始剤として共重合さ
せ、次いでアルカリ性の条件下で加熱を行なうこ
とを特徴とする、診断試薬用ラテツクスの製造方
法。 ２ アルカリ性の条件下での加熱を、50〜90℃で
10〜100時間行なうことを特徴とする、特許請求
の範囲第１項記載の診断試薬用ラテツクスの製造
方法。[Claims] 1. Styrene and styrene sulfonate in an amount of 10% by weight or less based on the styrene in the absence of an emulsifier,
A latex for diagnostic reagents characterized by copolymerizing in an aqueous solution containing a divalent metal oxide or hydroxide using a persulfate as an initiator, and then heating under alkaline conditions. manufacturing method. 2 Heating under alkaline conditions at 50-90℃
2. The method for producing a latex for diagnostic reagents according to claim 1, which is carried out for 10 to 100 hours.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8174682A JPS58198508A (en) | 1982-05-14 | 1982-05-14 | Production of latex for diagnostic agent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8174682A JPS58198508A (en) | 1982-05-14 | 1982-05-14 | Production of latex for diagnostic agent |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS58198508A JPS58198508A (en) | 1983-11-18 |
| JPH0136484B2 true JPH0136484B2 (en) | 1989-08-01 |
Family
ID=13754996
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP8174682A Granted JPS58198508A (en) | 1982-05-14 | 1982-05-14 | Production of latex for diagnostic agent |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS58198508A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004325416A (en) * | 2003-04-28 | 2004-11-18 | Sekisui Chem Co Ltd | Carrier particle latex for measurement reagent, and measurement reagent |
| EP2023141A2 (en) | 2001-07-02 | 2009-02-11 | Sekisui Medical Co., Ltd. | Carrier particle latex for assay reagent and assay reagent |
| JP2024052454A (en) * | 2022-09-30 | 2024-04-11 | 積水メディカル株式会社 | Method for producing latex particle and method for producing measurement reagent |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5989301A (en) * | 1982-11-12 | 1984-05-23 | Sekisui Chem Co Ltd | Production of latex for reagent of diagnosis |
| JP6207198B2 (en) * | 2012-03-28 | 2017-10-04 | 積水メディカル株式会社 | Latex particles for diagnostic reagent and method for producing the same |
-
1982
- 1982-05-14 JP JP8174682A patent/JPS58198508A/en active Granted
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2023141A2 (en) | 2001-07-02 | 2009-02-11 | Sekisui Medical Co., Ltd. | Carrier particle latex for assay reagent and assay reagent |
| US7867785B2 (en) | 2001-07-02 | 2011-01-11 | Sekisui Medical Co., Ltd. | Carrier particle latex for assay reagent and assay reagent |
| JP2004325416A (en) * | 2003-04-28 | 2004-11-18 | Sekisui Chem Co Ltd | Carrier particle latex for measurement reagent, and measurement reagent |
| JP2024052454A (en) * | 2022-09-30 | 2024-04-11 | 積水メディカル株式会社 | Method for producing latex particle and method for producing measurement reagent |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS58198508A (en) | 1983-11-18 |
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