JP2627681B2 - Mycoplasma pneumoniae diagnostic reagent - Google Patents
Mycoplasma pneumoniae diagnostic reagentInfo
- Publication number
- JP2627681B2 JP2627681B2 JP2510212A JP51021290A JP2627681B2 JP 2627681 B2 JP2627681 B2 JP 2627681B2 JP 2510212 A JP2510212 A JP 2510212A JP 51021290 A JP51021290 A JP 51021290A JP 2627681 B2 JP2627681 B2 JP 2627681B2
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- Japan
- Prior art keywords
- monoclonal antibody
- antibody
- mycoplasma pneumoniae
- solution
- labeled
- Prior art date
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Description
【発明の詳細な説明】 技術分野 本発明は抗マイコプラズマ・ニューモニエモノクロー
ナル抗体又はその標識体を安定に含有してなるマイコプ
ラズマ・ニューモニエの診断用試薬、並びにハイブリド
ーマの培養上清から抗マイコプラズマ・ニューモニエモ
ノクローナル抗体を大量に、かつ高純度、高収率で簡便
に精製する方法に関する。Description: TECHNICAL FIELD The present invention relates to a diagnostic reagent for Mycoplasma pneumoniae which stably contains an anti-Mycoplasma pneumoniae monoclonal antibody or a labeled form thereof, and an anti-Mycoplasma pneumoniae monoclonal antibody obtained from a culture supernatant of a hybridoma. And a method for easily purifying the compound in a large amount, with high purity and high yield.
背景技術 マイコプラズマ・ニューモニエ感染症はおよそ４年周
期で流行し、小児や若年者では重症化や中枢神経障害等
の重篤な合併症を招くことがある。マイコプラズマ・ニ
ューモニエ感染症はかぜ症候群の中ではウイルスによる
ものに次いで多く、治療にはマクロライド系又はテトラ
サイクリン系の抗生物質が有効であるので、本症の発病
初期に於ける迅速な確定診断が必要とされてきた。しか
し、今日行われている診断方法としては培養法や血清学
的方法等が挙げられるが、いずれも長期間を必要とする
ものであった。特に血清学的診断方法に於ける抗体の検
出は回復期に至って初めて可能になるものであり、非特
異反応も少なくないという欠点があった。本発明者ら
は、かかる欠点を解決すべく研究し、先にマイコプラズ
マ・ニューモニエに特異的な抗マイコプラズマ・ニュー
モニエモノクローナル抗体を得、これがマイコプラズマ
・ニューモニエ感染症の診断薬として有用であることを
見出した（特開昭63-184064号）。BACKGROUND ART Mycoplasma pneumoniae infection is prevalent every four years, and may cause serious complications such as aggravation and central nervous system disorders in children and young people. Mycoplasma pneumoniae infection is the second most common form of cold syndrome following viral infection, and macrolide or tetracycline antibiotics are effective in the treatment, so a prompt definitive diagnosis is needed early in the onset of the disease. And has been. However, the diagnostic methods currently used include a culture method and a serological method, all of which require a long period of time. In particular, detection of antibodies in serologic diagnostic methods is only possible after the recovery period, and has the drawback that non-specific reactions are not infrequent. The present inventors have studied to solve such a drawback, and previously obtained an anti-Mycoplasma pneumoniae monoclonal antibody specific to Mycoplasma pneumoniae, and found that this is useful as a diagnostic agent for Mycoplasma pneumoniae infection. (Japanese Patent Laid-Open No. 63-184064).
しかしながら、当該抗マイコプラズマ・ニューモニエ
モノクローナル抗体の取得手段及びこれを含有する診断
薬にはいくつかの問題点が存することが判明した。すな
わち、抗体の精製は、一般に硫安分画法、イオン交換ク
ロマトグラフィー、ゲル濾過法などの免疫グロブリンの
精製法や、プロテインＡや抗原によるアフィニティーク
ロマトグラフィー法などに基づいて行われている。本発
明者らも、上記特許公開公報の中で、硫安分画法とプロ
テインＡセファロースCL-4B（ファルマシア社製）を組
み合わせて精製された抗マイコプラズマ・ニューモニエ
モクローナル抗体を得ている。しかし大量に抗マイコプ
ラズマ・ニューモニエモノクローナル抗体を得るために
は以下の点で著しく不利であった。However, it has been found that the means for obtaining the anti-Mycoplasma pneumoniae monoclonal antibody and the diagnostic agent containing the same have some problems. That is, antibody purification is generally carried out based on immunoglobulin purification methods such as ammonium sulfate fractionation, ion exchange chromatography, and gel filtration, and affinity chromatography using protein A and antigen. The present inventors have also obtained an anti-mycoplasma pneumoniae moclonal antibody purified by combining the ammonium sulfate fractionation method and Protein A Sepharose CL-4B (Pharmacia) in the above patent publication. However, obtaining an anti-Mycoplasma pneumoniae monoclonal antibody in a large amount was significantly disadvantageous in the following points.
１）硫安分画での収率が著しく悪い。50％飽和硫安濃度
でも充分な抗体の沈澱物が得られない。1) The yield in the ammonium sulfate fractionation is extremely poor. Even at a 50% saturated ammonium sulfate concentration, no sufficient antibody precipitate can be obtained.
２）中性の緩衝液に対して溶解性が悪く、硫安沈澱物を
少量の緩衝液に高濃度に溶解させることが困難である。
低塩濃度の緩衝液に対しては更に溶解性が悪く、透析す
ると多量の沈澱物が析出して失活する。2) Poor solubility in neutral buffers, making it difficult to dissolve ammonium sulfate precipitates in a small amount of buffer at a high concentration.
It has poor solubility in low-salt buffer solutions, and upon dialysis, a large amount of precipitate precipitates and is inactivated.
３）プロテインＡセファロース4Bは高価である。3) Protein A Sepharose 4B is expensive.
これらの問題点により、本発明者らの見出した抗マイ
コプラズマ・ニューモニエモノクローナル抗体は、一般
的なIgGに属する抗体の精製法によって精製することが
困難であった。Due to these problems, it has been difficult to purify the anti-Mycoplasma pneumoniae monoclonal antibody discovered by the present inventors by a general method for purifying antibodies belonging to IgG.
また、得られた抗マイコプラズマ・ニューモニエモノ
クローナル抗体は、通常の保存条件では不安定であり診
断薬として開発するには問題があった。In addition, the obtained anti-Mycoplasma pneumoniae monoclonal antibody is unstable under ordinary storage conditions, and has a problem in developing as a diagnostic agent.
従って、大量生産に適した抗マイコプラズマ・ニュー
モニエモノクローナル抗体の精製法及び当該モノクロー
ナル抗体を安定に維持した診断薬の開発が望まれてい
た。Therefore, there has been a demand for a method for purifying an anti-Mycoplasma pneumoniae monoclonal antibody suitable for mass production and development of a diagnostic agent stably maintaining the monoclonal antibody.
本発明者らは、かかる現状に鑑み鋭意研究した結果、
抗マイコプラズマ・ニューモニエモノクローナル抗体が
産生するハイブリドーマの培養上清を濃縮した後塩析
し、pHを一定の範囲に調整すれば、精製が容易に行い得
ること、及び得られたモノクローナル抗体をアルブミン
とともに一定のpHを有する緩衝液に溶解し、又はこれを
更に凍結乾燥すれば安定な診断薬が得られることを見出
し、本発明を完成するに到った。The present inventors have conducted intensive research in view of the current situation,
After concentrating the culture supernatant of the hybridoma produced by the anti-Mycoplasma pneumoniae monoclonal antibody and salting it out and adjusting the pH to a certain range, purification can be easily performed, and the obtained monoclonal antibody can be fixed together with albumin. It has been found that a stable diagnostic agent can be obtained by dissolving it in a buffer solution having a pH of or by freeze-drying the solution, and completed the present invention.
発明の開示 本発明は抗マイコプラズマ・ニューモニエモノクロー
ナル抗体を産生するハイブリドーマの培養上清を２〜20
倍に濃縮し、次いで塩析して得られた画分をpH6.0〜7.5
の緩衝液で洗浄後pH8〜10の緩衝液に溶解することを特
徴とする、抗マイコプラズマ・ニューモニエモノクロー
ナル抗体の精製法； 抗マイコプラズマ・ニューモニエモノクローナル抗体
又はその標識体を、当該抗体又は標識体に対して12.5〜
250重量倍のアルブミンを含有し糖類を含有しないpH5.5
〜8.5の水性溶液に溶解するか、62.5〜625重量倍のアル
ブミンを含有し糖類を含有しないpH5.5〜8.5の水性溶液
に溶解後凍結乾燥することを特徴とする抗マイコプラズ
マ・ニューモニエモノクローナル抗体の安定化法；並び
に 抗マイコプラズマ・ニューモニエモノクローナル抗体
（以下、「本モノクローナル抗体」と略す）又はその標
識体を、当該抗体又は標識体に対して12.5〜250重量倍
のアルブミンを含有し糖類を含有しないpH5.5〜8.5の水
性溶液に溶解するか、62.5〜625重量倍のアルブミンを
含有し糖類を含有しないpH5.5〜8.5の水性溶液に溶解後
凍結乾燥することにより得られるマイコプラズマ・ニュ
ーモニエ（以下、「M.p」と略す）診断用試薬を提供す
るものである。DISCLOSURE OF THE INVENTION The present invention relates to a method for preparing a culture supernatant of a hybridoma producing an anti-Mycoplasma pneumoniae monoclonal antibody from 2 to 20.
Concentration and then salting out, the resulting fraction was pH 6.0-7.5.
Washing with a buffer solution of pH 8 to 10, and dissolving in a buffer solution of pH 8 to 10, a method for purifying an anti-Mycoplasma pneumoniae monoclonal antibody; an anti-Mycoplasma pneumoniae monoclonal antibody or a label thereof, 12.5 ~
PH 5.5 containing 250 weight times albumin and no saccharides
Dissolving in an aqueous solution of pH 8.5 to 8.5, or dissolving in an aqueous solution containing 62.5 to 625 times by weight of albumin and containing no saccharides and having a pH of 5.5 to 8.5, followed by freeze-drying, the anti-Mycoplasma pneumoniae monoclonal antibody Stabilization method; and anti-Mycoplasma pneumoniae monoclonal antibody (hereinafter abbreviated as "the present monoclonal antibody") or a labeled product thereof, containing albumin 12.5 to 250 times the weight of the antibody or the labeled product and containing no saccharides Mycoplasma pneumoniae obtained by dissolving in an aqueous solution of pH 5.5 to 8.5 or by dissolving in an aqueous solution of pH 5.5 to 8.5 containing 62.5 to 625 times by weight of albumin and containing no saccharide and freeze-drying (hereinafter referred to as "mycoplasma pneumoniae") , Abbreviated as “Mp”).
図面の簡単な説明 図−１は、実施例３において中性緩衝液で洗浄した硫
安分画のゲル濾過パターンを示し、図−２は実施例３に
おける未洗浄硫安分画のゲル濾過パターンを示す図面で
ある。BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the gel filtration pattern of the ammonium sulfate fraction washed with a neutral buffer in Example 3, and FIG. 2 shows the gel filtration pattern of the unwashed ammonium sulfate fraction in Example 3. It is a drawing.
発明を実施するための最良の形態 本モノクローナル抗体の精製に用いられるハイブリド
ーマの培養上清は、例えば特開昭63-184064号に記載の
方法によって得られる。すなわち、M.pで免疫した哺乳
動物の免疫担当細胞と骨髄種細胞とを常法により融合さ
せて得られたハイブリドーマ（例えばC2-G3,G1-B8,G1-E
6）を適当な培地で培養するか、該ハイブリドーマを動
物の腹腔内に投与した後腹水を採取することによって得
られる。BEST MODE FOR CARRYING OUT THE INVENTION The culture supernatant of a hybridoma used for purification of the present monoclonal antibody can be obtained, for example, by the method described in JP-A-63-184064. That is, a hybridoma (eg, C2-G3, G1-B8, G1-E) obtained by fusing an immunocompetent cell and a myeloid cell of a mammal immunized with Mp by a conventional method.
6) is cultured in an appropriate medium, or the hybridoma is obtained by intraperitoneally administering the hybridoma and collecting ascites.
本発明精製法を実施するには、まず上記の如くして得
た培養上清をpH8〜９に調整した後、２〜20倍、好まし
くは５〜10倍に濃縮する。濃縮方法は沈澱物が生じない
方法であれば特に限定されないが、限外濾過法又は透析
法が好ましい。次いで、濃縮した培養上清を塩析して沈
澱物を採取する。塩析は、例えば硫安分画法によって行
うことができる。すなわち、濃縮液にpHを７に調整した
等量の飽和硫安溶液を加えて（50％飽和）一夜静置し、
生じた沈澱物を0.5N炭酸塩緩衝液（pH9.5）に溶解し、
２分の１量の飽和硫安溶液を加えて（33％飽和）生じた
沈澱を採取する。To carry out the purification method of the present invention, the culture supernatant obtained as described above is first adjusted to pH 8 to 9, and then concentrated 2 to 20 times, preferably 5 to 10 times. The concentration method is not particularly limited as long as it does not produce a precipitate, but an ultrafiltration method or a dialysis method is preferable. Next, the concentrated culture supernatant is salted out to collect a precipitate. The salting out can be performed by, for example, an ammonium sulfate fractionation method. That is, an equivalent amount of a saturated ammonium sulfate solution adjusted to pH 7 was added to the concentrated solution (50% saturation), and the mixture was allowed to stand overnight,
The resulting precipitate was dissolved in 0.5N carbonate buffer (pH 9.5),
A half amount of a saturated ammonium sulfate solution is added (33% saturation) and the resulting precipitate is collected.
次に得られた沈澱物を少量のpH6.0〜7.5の緩衝液、例
えば、0.05Mリン酸塩緩衝液（pH7.2）で洗浄した後、pH
8〜10の緩衝液、例えば0.5M炭酸塩緩衝液（pH9.5）に溶
解すれば、高純度の本モノクローナル抗体溶液が得られ
る。この段階で本モノクローナル抗体はかなり純度が高
いが、更に高純度にするためにはゲル濾過法によって精
製するのが好ましい。ここで他の精製方法、例えば弱イ
オン交換クロマトグラフィーなどの中性域、低塩濃度で
吸着させることが必要な精製法は適用が困難である。ま
た、吸着、溶出に酸性の状態が必要な精製法は安定性の
面から適用が困難である。ゲル濾過法の担体としてはIg
Gの分子量から適当な担体、例えばセファクリルS-300 H
Rを用い、0.5M炭酸塩緩衝液（pH9.5）にてゲル濾過を行
うのが好ましい。活性画分を集め、限外濾過法にて濃縮
し精製本モノクローナル抗体を得る。この精製抗体は電
気泳動で均一なバンドを示す。また、この精製抗体をpH
9より酸性側の緩衝液で透析すると、多量の沈澱物が生
じ、取扱いが困難になる。以上の操作は冷所で行うのが
好ましい。Next, the obtained precipitate is washed with a small amount of a buffer solution having a pH of 6.0 to 7.5, for example, a 0.05 M phosphate buffer solution (pH 7.2).
When dissolved in a buffer solution of 8 to 10, for example, a 0.5 M carbonate buffer solution (pH 9.5), a highly pure monoclonal antibody solution can be obtained. At this stage, the monoclonal antibody has considerably high purity, but it is preferable to purify the monoclonal antibody by a gel filtration method in order to further increase the purity. Here, it is difficult to apply other purification methods, for example, a purification method that requires adsorption in a neutral region and a low salt concentration such as weak ion exchange chromatography. Further, it is difficult to apply a purification method that requires an acidic state for adsorption and elution in terms of stability. Ig as a carrier for gel filtration
Suitable carriers based on the molecular weight of G, such as Sephacryl S-300H
It is preferable to perform gel filtration using 0.5M carbonate buffer (pH 9.5) using R. The active fraction is collected and concentrated by ultrafiltration to obtain a purified monoclonal antibody. This purified antibody shows a uniform band on electrophoresis. In addition, this purified antibody is
Dialysis with a buffer solution more acidic than 9 produces a large amount of precipitate, which makes handling difficult. The above operation is preferably performed in a cool place.
このようにして得られる本モノクローナル抗体は、M.
pに優れた特異性を持つことから、そのまま、あるいは
蛍光色素（例えばフルオレッセンス）、酵素（例えばペ
ルオキシダーゼ）、放射性物質（例えば125I）、金コロ
イド、ラテックスなどで標識して、蛍光抗体法、エンザ
イムイムノアッセイ、ラジオイムノアッセイ、金粒子免
疫測定法、凝集反応などの方法で、M.p感染症の診断薬
として適用可能であるが、本モノクローナル抗体は不安
定であり冷蔵保存が必要である。しかし、本モノクロー
ナル抗体又はその標識体を、当該抗体又は標識体に対し
て12.5〜250重量倍のアルブミンを含有するpH5.5〜8.
5、好ましくはpH6.0〜8.0の水性溶液に溶解するか、あ
るいは当該抗体又は標識体に対して62.5〜625重量倍の
アルブミンを含有するpH5.5〜8.5、好ましくはpH6.0〜
8.0の水性溶液に溶解後凍結乾燥すれば、本モノクロー
ナル抗体の安定性が高まり、室温保存も可能となる。The monoclonal antibody thus obtained is M.
Since p has excellent specificity, it can be used as is or labeled with a fluorescent dye (eg, fluorescens), enzyme (eg, peroxidase), radioactive substance (eg, 125 I), colloidal gold, latex, etc. Although it can be applied as a diagnostic agent for Mp infection by methods such as enzyme immunoassay, radioimmunoassay, gold particle immunoassay, and agglutination, the monoclonal antibody is unstable and requires refrigerated storage. However, the present monoclonal antibody or a labeled product thereof is pH 5.5 to 8 containing 12.5 to 250 times by weight of albumin with respect to the antibody or the labeled product.
5, preferably dissolved in an aqueous solution of pH 6.0 to 8.0, or containing 5.5 to 625 times by weight of albumin relative to the antibody or label, pH 5.5 to 8.5, preferably pH 6.0 to 8.5
If the monoclonal antibody is dissolved in an aqueous solution of 8.0 and freeze-dried, the stability of the present monoclonal antibody is enhanced, and storage at room temperature becomes possible.
本発明で使用されるアルブミンの種類は特に限定され
ないが、血清アルブミン、特に牛血清アルブミン（以
下、BSAと略す）が好ましい。The type of albumin used in the present invention is not particularly limited, but serum albumin, particularly bovine serum albumin (hereinafter abbreviated as BSA) is preferred.
ここで、アミブミンは本モノクローナル抗体の反応を
妨害しないので、前記安定化法により得られる組成物は
安定なM.p診断用試薬として用いることができる。Here, since amibmin does not interfere with the reaction of the present monoclonal antibody, the composition obtained by the stabilization method can be used as a stable reagent for diagnosing Mp.
実施例 以下実施例を挙げ、本発明を更に詳しく説明する。Examples Hereinafter, the present invention will be described in more detail with reference to Examples.
参考例 ハイブリドーマの調製法： （ｉ）抗体産生細胞の調製 マイコプラズマ・ニューモニエ（Mycoplasma pneumon
iae）Mac株をPPLO液体培地に接種して37℃で５日間前培
養を行った。この培養液を同培地1,000mlに接種し、各1
00mlを500ml容ルービンに移して37℃で５日間静置して
培養を行った。培養後、培養液を捨て、ガラス面に吸着
した菌体を、0.01Mリン酸緩衝食塩液（pH7.2）（以下、
PBSと略す）を加えてラバーポリスマンにてかき集め
た。この菌液について凍結融解を４回繰り返し、PBSで
３回洗浄しM.p膜抗原（１）とした。この抗原の蛋白濃
度をローリーらの方法に準じて測定した。（注） 同様に、Mac株を佐々木らが報告しているEY培地（卵
黄２％含有）に接種して37℃で５日間前培養を行った。
この培養液を、同培養液1,000mlに接種して、37℃で５
日間静置して培養を行った。培養後、培養液を捨てガラ
ス面に吸着した菌体を、PBSを加えてラバーポリスマン
にてかき集めた。この菌液について凍結融解を４回行
い、PBSで３回洗浄してM.p膜抗原（２）とした。この抗
原についてもローリーらの方法に準じて蛋白濃度を求め
た。Reference Example Hybridoma preparation method: (i) Preparation of antibody-producing cells Mycoplasma pneumon
iae) Mac strain was inoculated into a PPLO liquid medium and precultured at 37 ° C for 5 days. This culture is inoculated into 1,000 ml of the same medium,
00 ml was transferred to a 500-ml Rubin, and left at 37 ° C. for 5 days to culture. After the culture, the culture solution was discarded, and the cells adsorbed on the glass surface were removed using a 0.01 M phosphate buffered saline (pH 7.2)
PBS), and the mixture was scraped with a rubber policeman. This bacterial solution was repeatedly freeze-thawed four times and washed three times with PBS to obtain Mp membrane antigen (1). The protein concentration of this antigen was measured according to the method of Lowry et al. (Note) Similarly, the Mac strain was inoculated into an EY medium (containing 2% egg yolk) reported by Sasaki et al., And pre-cultured at 37 ° C for 5 days.
This culture is inoculated into 1,000 ml of the same culture,
The culture was performed by allowing to stand for a day. After the culture, the culture solution was discarded and the cells adsorbed on the glass surface were added to PBS and scraped with a rubber policeman. This bacterial solution was freeze-thawed four times and washed three times with PBS to obtain Mp membrane antigen (2). The protein concentration of this antigen was determined according to the method of Lowry et al.
M.p膜抗原（１）（蛋白として100μｇ相当）をそのま
ま、BALB/c系雌性マウス（６〜７週齢）の腹腔内に投与
した。１週後、同量を腹腔内に追加免疫した。更に６週
後、M.p膜抗原（２）を同量静脈内に追加免疫を施し
た。１〜２週後、血中抗体価が上昇していることを確認
した後、M.p膜抗原（２）を蛋白として10μｇ相当量を
静脈内に投与した。（注）Lowry.O.H,N.J.Rosenbrough,
A.L.Farr,and R.J.Randall,1951,J.Biol.Chem.193:265-
275 最終免疫３日後に脾臓を無菌的に採取した。採取した
脾臓はピンセットでほぐし、メッシュ（＃100）を通過
させて細胞浮遊液とした。トリス塩化アンモニウム緩衝
液を10倍量加えて氷冷中２〜３分放置して赤血球を除去
した。イーグルMEM培地（以下、MEMと略す）にて３回洗
浄後5.0×107個/mlに調製した。Mp membrane antigen (1) (corresponding to 100 μg as a protein) was directly administered intraperitoneally to BALB / c female mice (6 to 7 weeks old). One week later, the same amount was boosted intraperitoneally. After another 6 weeks, the same amount of Mp membrane antigen (2) was intravenously boosted. One to two weeks later, after confirming that the blood antibody titer was elevated, 10 μg equivalent of Mp membrane antigen (2) as a protein was intravenously administered. (Note) Lowry.OH, NJRosenbrough,
ALFarr, and RJRandall, 1951, J.Biol.Chem.193: 265-
275 days after the final immunization, the spleen was aseptically collected. The collected spleen was loosened with forceps and passed through a mesh (# 100) to obtain a cell suspension. Erythrocytes were removed by adding a 10-fold amount of a tris ammonium chloride buffer and allowing to stand for 2 to 3 minutes on ice. After washing three times with an Eagle MEM medium (hereinafter abbreviated as MEM), the mixture was adjusted to 5.0 × 10 7 cells / ml.
（ii）細胞融合及び抗体産生ハイブリドーマの調製 予め培養しておいたミエローマ細胞（X63-Ag 8.6.5.
3）をMEMにて３回洗浄した後、5.0×106個/mlに調製し
た。（ｉ）で調製した脾細胞5.0mlとミエローマ細胞
5.0mlを40mlの遠沈管にとり、遠心分離（1,100rpm、５
分間）して上清を除去して細胞を集めた。ペレットをよ
く解きほぐし、予め37℃に温めておいた50％ポリエチレ
ングリコール1000溶液0.5mlを約１〜２分間で徐々に加
えた。次に37℃に保温しておいたMEM10mlを約2ml/分の
速度でゆっくりと加えて反応を停止させた。遠心分離
（1,100rpm、５分間）して上清を除去した後、10％牛胎
児血清を含むRPMI-1640培地を加え、ミエローマ細胞が
5.0×105個/mlになるように調製し、96穴マイクロプレ
ートに200μl/well分注した。37℃で５％炭酸ガスを含
む炭酸ガス培養器中で１日培養した後、培地の半量を捨
てHAT培地（10％牛胎児血清を含むRPMI-1640培地にてヒ
ポキサンチン1.0×10-4Ｍ、アミノプテリン4.0×10
-7Ｍ、チミジン1.6×10-5Ｍを加えたもの）100μｌを添
加する。以後、２〜３日毎に半量をHAT培地で交換し
た。10〜14日後、細胞の増殖がみられたら、培地の半量
をHT培地（HAT培地からアミノプテリンを除いたもの）
で交換し、２日後HT培地で半量交換した後は、２〜３日
毎に10％牛胎児血清を含むRPMI-1640培地で交換した。
約２週間後、ELISA法及び間接赤血球凝集反応（セロデ
ィア−MYCO,富士レビオ（株）製を使用）により抗M.p抗
体産生ハイブリドーマをスクリーニングした。その結
果、融合細胞は100％のウエルに認められ、そのうち抗
体産生が認められたウエルは6.8％であったが、培養経
過後も再現性の認められたものは3.7％であった。(Ii) Cell fusion and preparation of antibody-producing hybridoma Myeloma cells (X63-Ag 8.6.5.
3) was washed three times with MEM, and then adjusted to 5.0 × 10 6 cells / ml. 5.0 ml of splenocytes prepared in (i) and myeloma cells
Transfer 5.0 ml to a 40 ml centrifuge tube, and centrifuge (1,100 rpm, 5
Min) and the supernatant was removed to collect the cells. The pellets were thoroughly disentangled, and 0.5 ml of a 50% polyethylene glycol 1000 solution pre-warmed to 37 ° C. was gradually added over about 1 to 2 minutes. Next, 10 ml of MEM kept at 37 ° C. was slowly added at a rate of about 2 ml / min to stop the reaction. After removing the supernatant by centrifugation (1,100 rpm, 5 minutes), RPMI-1640 medium containing 10% fetal calf serum was added, and the myeloma cells were removed.
It was adjusted to 5.0 × 10 5 cells / ml and dispensed into a 96-well microplate at 200 μl / well. After culturing at 37 ° C. for 1 day in a carbon dioxide incubator containing 5% carbon dioxide, half of the medium was discarded and HAT medium (hypoxanthine 1.0 × 10 −4 M in RPMI-1640 medium containing 10% fetal bovine serum) was added. , Aminopterin 4.0 × 10
-7 M and thymidine 1.6 × 10 -5 M) 100 μl. Thereafter, half of the HAT medium was replaced every two to three days. 10-14 days later, when cell growth is observed, half of the medium is replaced with HT medium (HAT medium minus aminopterin)
After two days, half of the medium was replaced with an HT medium, and then every two to three days, the medium was replaced with an RPMI-1640 medium containing 10% fetal bovine serum.
About two weeks later, anti-Mp antibody-producing hybridomas were screened by ELISA and indirect hemagglutination (using Cellodia-MYCO, manufactured by Fujirebio Inc.). As a result, fused cells were found in 100% of the wells, of which 6.8% showed antibody production, but 3.7% showed reproducibility even after the culturing.
抗体産生の認められたハイブリドーマは限界希釈法に
より、クローニングを行った。即ち、ハイブリドーマを
6.0個/mlに調製し、正常BALB/c系マウスの胸腺細胞を10
7個/mlに調製した液を1:1に混合して、96穴マイクロプ
レートに200μｌずつウエルに分注した。37℃で５％炭
酸ガスを含む炭酸ガス培養器中で培養し、約２〜３週後
に培養上清中の抗体産生を前記の方法にて検討した。そ
の結果抗体産生の良好なハイブリドーマについては再度
クローニングを繰り返した。２回クローニングをして得
られたハイブリドーマは、培養上清中の抗体を得ること
及び長期の培養でも安定に産生することを確認する目的
で、96穴マイクロプレートから24穴マルチプレートに移
しかえ、更に50ml組織培養フラスコに移しかえた。その
結果、抗体産生は長期の継代にても安定に産生された。Hybridomas in which antibody production was recognized were cloned by the limiting dilution method. That is, the hybridoma
Adjusted to 6.0 cells / ml, 10 thymocytes from normal BALB / c mice
The solutions prepared at 7 cells / ml were mixed at a ratio of 1: 1 and dispensed into a 96-well microplate at 200 μl / well. The cells were cultured at 37 ° C. in a carbon dioxide incubator containing 5% carbon dioxide, and about 2-3 weeks later, antibody production in the culture supernatant was examined by the above-mentioned method. As a result, the cloning was repeated again for the hybridomas with good antibody production. Hybridomas obtained by cloning twice were transferred from a 96-well microplate to a 24-well multiplate for the purpose of obtaining antibodies in the culture supernatant and confirming that they were produced stably even during long-term culture. Further transferred to a 50 ml tissue culture flask. As a result, antibody production was stable even after long-term passage.
以上の如くして、ハイブリドーマC2-G3、G1-B8及びG1
-E6を得た。As described above, hybridomas C2-G3, G1-B8 and G1
-I got E6.
実施例１ 本モノクローナル抗体の精製 参考例で得られたハイブリドーマ、G1-E6をGIT培地
（日水製薬（株）製）を用いて、37℃で５％炭酸ガスを
含む炭酸ガス培養器中で培養を行い、４〜５日目に培養
上清を採取した。得られた培養上清（1,500ml）をpH8に
調整後、遠心分離（10,000rpm、20分間）しメンブラン
フィルターで濾過した。濾液を限外濾過により５倍に濃
縮した。この濃縮液に等量のpH7に調整した飽和硫安溶
液（SAS）を加え（50％飽和）、冷所にて一夜放置後、
遠心分離（10,000rpm、20分間）により沈澱を集めた。
この沈澱物を0.5M炭酸塩緩衝液（pH9.5）200mlに溶解
し、これに100mlのSASを加え（33％飽和）、冷所に静置
後、生じた沈澱を遠心分離（10,000rpm、20分間）によ
り集めた。この沈澱物を10mlの0.05Mリン酸塩緩衝液（p
H7.2）にて洗浄し、遠心分離（10,000rpm、20分間）に
より沈澱を集めた。この沈澱物を0.5M炭酸塩緩衝液（pH
9.5）8mlに懸濁し、0.5M炭酸塩緩衝液（pH9.5）で一夜
透析した。ごく僅かに残った不溶物を遠心分離（10,000
rpm、20分間）で除いた後、0.5M炭酸塩緩衝液（pH9.5）
で充填したセファクリルS-300 HR（ファルマシア社製）
のカラム（直径26mm、長さ1,000mm）を用いてゲル濾過
法によって分画した。活性画分を集め、限外濾過により
濃縮し精製抗体を得た（収量35.6mg）。この精製抗体は
電気泳動的に均一であった。Example 1 Purification of the present monoclonal antibody The hybridoma, G1-E6, obtained in Reference Example was used in a GIT medium (manufactured by Nissui Pharmaceutical Co., Ltd.) at 37 ° C. in a carbon dioxide incubator containing 5% carbon dioxide. The culture was performed, and the culture supernatant was collected on the 4th to 5th days. The resulting culture supernatant (1,500 ml) was adjusted to pH 8, centrifuged (10,000 rpm, 20 minutes), and filtered through a membrane filter. The filtrate was concentrated 5-fold by ultrafiltration. To this concentrated solution, an equal amount of a saturated ammonium sulfate solution (SAS) adjusted to pH 7 was added (50% saturation), and allowed to stand overnight in a cool place.
The precipitate was collected by centrifugation (10,000 rpm, 20 minutes).
This precipitate was dissolved in 200 ml of 0.5 M carbonate buffer (pH 9.5), 100 ml of SAS was added thereto (33% saturation), the mixture was allowed to stand in a cool place, and the resulting precipitate was centrifuged (10,000 rpm, 20 minutes). The precipitate is added to 10 ml of 0.05M phosphate buffer (p
H7.2), and the precipitate was collected by centrifugation (10,000 rpm, 20 minutes). The precipitate is washed with 0.5M carbonate buffer (pH
9.5) Suspended in 8 ml and dialyzed overnight against 0.5 M carbonate buffer (pH 9.5). Centrifuge (10,000)
rpm, 20 minutes), then 0.5M carbonate buffer (pH9.5)
Sephacryl S-300 HR (Pharmacia) filled with
(26 mm in diameter, 1,000 mm in length) by a gel filtration method. The active fraction was collected and concentrated by ultrafiltration to obtain a purified antibody (yield 35.6 mg). This purified antibody was electrophoretically uniform.
比較例１ 本モノクローナル抗体の精製（特開昭63-184
064号の方法） 実施例１と同様のハイブリドーマ培養上清（1,500m
l）に等量のpH7に調整した飽和硫安溶液（SAS）を加
え、冷所にて一夜静置後、遠心分離（10,000rpm、20分
間）により沈澱を集めた。この沈澱物を0.05Mトリス緩
衝食塩液（pH8.6）250mlに溶解し、一夜透析した。不溶
物を遠心分離（10,000rpm、20分間）で除いた後、0.05M
トリス緩衝食塩液（pH8.6）で充填したプロテインＡ−
セファロース4B（ファルマシア社製）のカラム（直径26
mm、長さ400mm）を用いたアフィニティークロマトグラ
フィーによって分画し、活性画分を集め、限外濾過によ
り濃縮し精製抗体を得た（収量1.3mg）。Comparative Example 1 Purification of the present monoclonal antibody (JP-A-63-184)
No. 064) Hybridoma culture supernatant (1,500 m
To l), an equal volume of a saturated ammonium sulfate solution (SAS) adjusted to pH 7 was added, and the mixture was allowed to stand overnight in a cool place, and then the precipitate was collected by centrifugation (10,000 rpm, 20 minutes). The precipitate was dissolved in 250 ml of 0.05 M Tris buffered saline (pH 8.6) and dialyzed overnight. After removing insoluble matter by centrifugation (10,000 rpm, 20 minutes), 0.05M
Protein A- filled with Tris buffered saline (pH 8.6)
Sepharose 4B (Pharmacia) column (diameter 26
The active fractions were collected and concentrated by ultrafiltration to obtain a purified antibody (yield 1.3 mg).
実施例２ 実施例１に示した培養上清を限外濾過法により種々の
濃度に濃縮し、これに２分の１量または等量のSASを加
え（33％飽和または55％飽和）、生じた沈澱物中の抗体
回収率を比較した。結果を表−１に示した。Example 2 The culture supernatant shown in Example 1 was concentrated to various concentrations by an ultrafiltration method, and a half or equal amount of SAS was added thereto (33% saturation or 55% saturation) to produce The antibody recoveries in the precipitated precipitates were compared. The results are shown in Table 1.
実施例３ 実施例１に示した本モノクローナル抗体の精製過程
で、培養上清から得られた33％飽和硫安沈澱物を中性緩
衝液で洗浄することによる精製効果を検討した。 Example 3 In the purification process of the present monoclonal antibody shown in Example 1, the purification effect of washing a 33% saturated ammonium sulfate precipitate obtained from the culture supernatant with a neutral buffer was examined.
50％飽和硫安沈澱物をpH9.5炭酸塩緩衝液に溶解した
液を２等分し、それぞれにSAS 2分の１量を加え、33％
飽和硫安沈澱物を得た。一方はこれをそのままpH9.5炭
酸塩緩衝液に溶解し（１）、もう一方はpH7.2リン酸塩
緩衝液で洗浄し（２）、その後pH9.5炭酸塩緩衝液に溶
解した（３）。それぞれの抗体比活性と回収率を比較し
た。結果を表−２に示した。A solution prepared by dissolving a 50% saturated ammonium sulfate precipitate in a pH 9.5 carbonate buffer solution was divided into two equal parts, and one half of SAS was added to each of them to obtain 33%
A saturated ammonium sulfate precipitate was obtained. One was directly dissolved in a pH 9.5 carbonate buffer (1), the other was washed with a pH 7.2 phosphate buffer (2), and then dissolved in a pH 9.5 carbonate buffer (3). ). The specific activity of each antibody and the recovery rate were compared. The results are shown in Table-2.
また、中性緩衝液で洗浄した硫安分画（３）と、洗浄
しない硫安分画（１）をそれぞれゲル濾過法で精製した
ときの分離パターンをそれぞれ図−１及び図−２に示し
た。図−１及び図−２中、PHA価はセロディア−MYCO-II
（富士レビオ（株）製）を用いた凝集活性を示す。中性
緩衝液で洗浄した硫安分画を用いた方が抗体がきれいに
分離していた。 In addition, FIGS. 1 and 2 show separation patterns when the ammonium sulfate fraction (3) washed with a neutral buffer and the unwashed ammonium sulfate fraction (1) were respectively purified by gel filtration. In FIG. 1 and FIG. 2, the PHA value is cellodia-MYCO-II.
(Aggregation activity using Fujirebio Inc.) is shown. Antibodies were more clearly separated when the ammonium sulfate fraction washed with a neutral buffer was used.
実施例４ 蛍光標識本モノクローナル抗体の作製 実施例１で得た精製抗体（5mg）にモル比で４倍量のF
ITC（Fluorescein isothiocyanate）を加え、0.5M炭酸
塩緩衝液（pH9.5）中で４℃、４時間反応した。反応終
了後、遠心分離（10,000rpm、10分間）により不溶物を
除き、0.01M炭酸塩緩衝液（pH9.5）、150mM塩化ナトリ
ウムで充填したセファデックスG-25（ファルマシア社
製）のカラム（直径10mm、長さ400mm）を用いてゲル濾
過法により未反応のFITCを除き、精製蛍光標識本モノク
ローナル抗体を得た（収量4.5mg）。抗体１分子当りのF
ITC結合比（F/P比）は1.1であった。またFITCはそのほ
とんどが抗体分子中のＨ鎖に結合していることがSDS−
電気泳動によって確認された。Example 4 Preparation of Fluorescently Labeled Monoclonal Antibody Fourfold molar amount of F was added to the purified antibody (5 mg) obtained in Example 1.
ITC (Fluorescein isothiocyanate) was added and reacted in a 0.5 M carbonate buffer (pH 9.5) at 4 ° C. for 4 hours. After completion of the reaction, insoluble substances were removed by centrifugation (10,000 rpm, 10 minutes), and a column of Sephadex G-25 (manufactured by Pharmacia) packed with 0.01 M carbonate buffer (pH 9.5) and 150 mM sodium chloride was used. Unreacted FITC was removed by gel filtration using a gel of 10 mm in diameter and 400 mm in length) to obtain a purified fluorescent-labeled monoclonal antibody (yield 4.5 mg). F per antibody molecule
The ITC binding ratio (F / P ratio) was 1.1. Also, most of FITC is bound to the H chain in the antibody molecule by SDS-
Confirmed by electrophoresis.
実施例５ ペルオキシダーゼ標識本モノクローナル抗体
の作製 西洋ワサビ・ペルオキシダーゼ水溶液（4mg/ml）に、
4mgの過ヨウ素酸ナトリウムを加え室温で10分間反応し
た。反応終了後、1mM酢酸塩緩衝液（pH4.0）に対し、４
℃で一晩透析した。得られたアルデヒド・ペルオキシダ
ーゼ溶液を、0.2M炭酸塩緩衝液（pH9.5）によりpH9.3に
修正後、直ちに、実施例１で得た精製抗体5mg（0.01M炭
酸塩緩衝液pH9.5に対し、４℃で一晩透析したもの）を
加えた。室温で２時間反応した後、水素化ホウ素ナトリ
ウム0.4mgを加え、４℃で２時間反応後、遠心分離（10,
000rpm、10分間）により不溶物を除いた。0.01M炭酸塩
緩衝液（pH9.5）・150mM塩化ナトリウムで充填したウル
トロゲルACA-44（LKB社製）のカラム（直径16mm、長さ7
00mm）を用いて、ゲル濾過法により、ペルオキシダーゼ
と抗体の高分子重合体、未反応のペルオキシダーゼ及び
抗体を除き、精製ペルオキシダーゼ標識本モノクローナ
ル抗体を得た（収量4.5mg）。Example 5 Preparation of Peroxidase-Labeled Monoclonal Antibody An aqueous solution of horseradish peroxidase (4 mg / ml)
4 mg of sodium periodate was added and reacted at room temperature for 10 minutes. After completion of the reaction, 4 mM against 1 mM acetate buffer (pH 4.0)
Dialysis at 0 ° C. overnight. Immediately after correcting the obtained aldehyde / peroxidase solution to pH 9.3 with 0.2 M carbonate buffer (pH 9.5), 5 mg of the purified antibody obtained in Example 1 (0.01 M carbonate buffer pH 9.5) Was dialyzed overnight at 4 ° C.). After reacting at room temperature for 2 hours, add 0.4 mg of sodium borohydride, react at 4 ° C for 2 hours, and centrifuge (10,
000 rpm for 10 minutes) to remove insolubles. Ultrogel ACA-44 (manufactured by LKB) column packed with 0.01 M carbonate buffer (pH 9.5) and 150 mM sodium chloride (diameter 16 mm, length 7)
The purified peroxidase-labeled monoclonal antibody was obtained by removing the high molecular weight polymer of peroxidase and antibody, the unreacted peroxidase and the antibody by gel filtration method (yield 4.5 mg).
実施例６ ビオチン標識本モノクローナル抗体の作製 Ｎ−ハイドロキシ・スクシンイミドビオチン（フナコ
シ薬品社製）1mgを50〜100μｌのジメチルホルムアミド
に溶解し、実施例１で得た精製抗体5mg（1mg/mlに調整
後、0.1M炭酸水素ナトリウムに対し、４℃で一晩透析し
たもの）を加え、室温で４時間反応した。反応終了後、
遠心分離（10,000rpm、10分間）により不溶物を除去
後、0.01M炭酸塩緩衝液（pH9.5）・0.15M塩化ナトリウ
ムで充填したウルトロゲルACA-44（LKB社製）のカラム
（直径16mm、長さ400mm）を用いて、ゲル濾過法によ
り、未反応のＮ−ハイドロキシ・スクシンイミドビオチ
ンを除き、精製ビオチン標識本モノクローナル抗体を得
た（収量4.1mg）。Example 6 Preparation of Biotin-Labeled Monoclonal Antibody 1 mg of N-hydroxysuccinimidobiotin (manufactured by Funakoshi Pharmaceutical Co., Ltd.) was dissolved in 50 to 100 μl of dimethylformamide, and the purified antibody 5 mg obtained in Example 1 (adjusted to 1 mg / ml) , Dialyzed against 0.1 M sodium bicarbonate at 4 ° C overnight) and reacted at room temperature for 4 hours. After the reaction,
After removing insolubles by centrifugation (10,000 rpm, 10 minutes), a column (16 mm in diameter, Ultrogel ACA-44 (manufactured by LKB)) packed with 0.01 M carbonate buffer (pH 9.5) and 0.15 M sodium chloride was used. The unreacted N-hydroxysuccinimide biotin was removed by gel filtration using a gel of 400 mm in length to obtain a purified biotin-labeled monoclonal antibody (yield 4.1 mg).
実施例７ 蛍光標識抗体の熱安定性に対するpHの影響 1ml当り、80μｇの実施例４で得られたFITC標識本モ
ノクローナル抗体と、20mgのBSA、及び8.5mgの塩化ナト
リウムを含む溶液を種々のpHに調整した。これらの溶液
を50℃で12時間又は40℃で１週間加熱し、安定性を比較
した。結果を表−３に示した。Example 7 Effect of pH on Thermal Stability of Fluorescently Labeled Antibody A solution containing 80 μg of the FITC-labeled monoclonal antibody obtained in Example 4, 20 mg of BSA, and 8.5 mg of sodium chloride per ml was prepared at various pH values. Was adjusted. These solutions were heated at 50 ° C. for 12 hours or at 40 ° C. for one week, and the stability was compared. The results are shown in Table-3.
実施例８ 蛍光標識抗体溶液の熱安定性に対するBSAの
影響 pH6.5の溶液中、1ml当り、80μｇの実施例４で得たFI
TC標識本モノクローナル抗体と、1.0mg〜50mgのBSA、及
び8.5mgの塩化ナトリウムを含む溶液を各々調製した。
これらの溶液を50℃で12時間又は40℃で１週間加熱し、
安定性をBSAを含まない溶液と比較した。結果を表−４
に示した。その結果本モノクローナル抗体は1.0mg〜20m
g/mlのBSAを含む溶液中で安定であり、2.0mg〜5.0mg/ml
のBSAを含む溶液中で特に安定であった（すなわち、本
モノクローナル抗体に対して12.5〜250重量倍のBSAで安
定、25〜62.5重量倍のBSAで特に安定）。 Example 8 Effect of BSA on Thermal Stability of Fluorescently Labeled Antibody Solution 80 μg of FI obtained in Example 4 per ml in a solution of pH 6.5
Solutions containing the TC-labeled monoclonal antibody, 1.0 mg to 50 mg of BSA, and 8.5 mg of sodium chloride were each prepared.
Heat these solutions at 50 ° C for 12 hours or at 40 ° C for 1 week,
Stability was compared to solutions without BSA. Table 4 shows the results.
It was shown to. As a result, this monoclonal antibody
stable in solutions containing g / ml BSA, 2.0 mg-5.0 mg / ml
(I.e., stable at 12.5 to 250 times by weight BSA and particularly stable at 25 to 62.5 times by weight BSA with respect to the present monoclonal antibody).
実施例９ 蛍光標識抗体凍結乾燥品の熱安定性に対する
BSAの影響 pH6.5の溶液中、1ml当り、80μｇの実施例４で得たFI
TC標識本モノクローナル抗体と、5.0mg〜50mgのBSA、及
び8.5mgの塩化ナトリウムを含む溶液を各々調製し、こ
れらの溶液を0.5mlずつ褐色のバイアル瓶に分注し、凍
結乾燥後、窒素ガス置換を行った（１バイアル当り、40
μｇのFITC標識本モノクローナル抗体と、2.5mg〜25mg
のBSA、及び4.25mgの塩化ナトリウムを含む）。これら
のバイアル瓶を、50℃で１週間加熱し、安定性をBSAを
含まないバイアル瓶と比較した。結果を表−５に示し
た。 Example 9 On the thermal stability of a freeze-dried product of a fluorescently labeled antibody
Effect of BSA 80 ml of FI obtained in Example 4 per ml in a solution of pH 6.5
A solution containing the TC-labeled monoclonal antibody, 5.0 mg to 50 mg of BSA, and 8.5 mg of sodium chloride was prepared.These solutions were dispensed in 0.5 ml aliquots into brown vials, freeze-dried, and nitrogen gas was added. Substitution was performed (40 per vial
μg of FITC-labeled monoclonal antibody and 2.5 mg to 25 mg
BSA, and 4.25 mg of sodium chloride). The vials were heated at 50 ° C. for one week and the stability was compared to vials without BSA. The results are shown in Table-5.
その結果、本モノクローナル抗体は2.5mg〜25mg/バイ
アルのBSAを含むバイアル中で安定であり、5.0mg〜25mg
/バイアルのBSAを含む溶液中で特に安定であった（すな
わち、本モノクローナル抗体に対して62.5〜625重量倍
のBSAで安定、125〜625重量倍のBSAで特に安定）。As a result, the monoclonal antibody is stable in vials containing 2.5 mg to 25 mg / vial BSA, and 5.0 mg to 25 mg
/ Vial was particularly stable in the solution containing BSA (ie, stable at 62.5-625 weight times BSA, particularly stable at 125-625 weight times BSA relative to the monoclonal antibody).
実施例10 蛍光標識本モノクローナル抗体によるマイコ
プラズマ・ニューモニエ診断薬 実施例４で得たFITC標識本モノクローナル抗体8mg、B
SA 500mg、塩化ナトリウム850mg、及びアジ化ナトリウ
ム50mgを50mMリン酸塩緩衝液（pH7.0）100mlに溶解して
褐色ガラス瓶に1mlずつ分注した。 Example 10 Mycoplasma pneumoniae diagnostic agent using fluorescently labeled present monoclonal antibody FITC-labeled present monoclonal antibody obtained in Example 4 8 mg, B
500 mg of SA, 850 mg of sodium chloride, and 50 mg of sodium azide were dissolved in 100 ml of 50 mM phosphate buffer (pH 7.0), and dispensed into brown glass bottles in a volume of 1 ml.
実施例４で得たFITC標識本モノクローナル抗体2mg、B
SA 500mg、塩化ナトリウム850mg、及びアジ化ナトリウ
ム50mgを50mMリン酸塩緩衝液（pH8.0）100mlに溶解して
褐色ガラス瓶に1mlずつ分注した。FITC-labeled monoclonal antibody 2 mg obtained in Example 4, B
500 mg of SA, 850 mg of sodium chloride, and 50 mg of sodium azide were dissolved in 100 ml of a 50 mM phosphate buffer (pH 8.0) and dispensed into brown glass bottles in an amount of 1 ml.
実施例４で得たFITC標識本モノクローナル抗体8mg、B
SA2,000mg、塩化ナトリウム850mg、及びアジ化ナトリウ
ム50mgを50mMリン酸塩緩衝液（pH8.0）100mlに溶解して
褐色ガラス瓶に0.5mlずつ分注し、凍結乾燥後、窒素ガ
ス置換を行った。8 mg of FITC-labeled monoclonal antibody obtained in Example 4, B
2,000 mg of SA, 850 mg of sodium chloride, and 50 mg of sodium azide were dissolved in 100 ml of 50 mM phosphate buffer (pH 8.0), dispensed into brown glass bottles in an amount of 0.5 ml each, lyophilized, and replaced with nitrogen gas. .
、、の方法で製造した蛍光標識本モノクローナ
ル抗体によるマイコプラズマ・ニューモニエ診断薬の安
定性を表−６に示す。Table 6 shows the stability of the Mycoplasma pneumoniae diagnostic agent using the fluorescent-labeled monoclonal antibody produced by the methods described in the above items.
比較例BSA無添加pH9.5の緩衝液に抗体を80μg/ml溶解
させた液。Comparative Example BSA was prepared by dissolving 80 μg / ml of an antibody in a buffer solution without pH at 9.5.
比較例pH9.5の緩衝液にBSA 5mg/ml、抗体を80μg/ml
溶解させた液を褐色ガラス瓶に0.5mlずつ分注し、凍結
乾燥後、窒素ガス置換を行った。Comparative Example BSA 5 mg / ml in buffer of pH 9.5, antibody 80 μg / ml
The dissolved liquid was dispensed into brown glass bottles in an amount of 0.5 ml each, freeze-dried, and then replaced with nitrogen gas.
産業上の利用可能性 本発明によりマイコプラズマ・ニューモニエに優れた
特異性を持つ抗マイコプラズマ・ニューモニエモノクロ
ーナル抗体を、安価、簡便、高純度、高収率で大量に得
られるようになった。またこの抗マイコプラズマ・ニュ
ーモニエモノクローナル抗体又はその標識体の安定性を
著しく高めることができ、診断薬として極めて有用とな
った。 INDUSTRIAL APPLICABILITY According to the present invention, an anti-Mycoplasma pneumoniae monoclonal antibody having excellent specificity for Mycoplasma pneumoniae can be obtained in a large amount at low cost, simply, with high purity and high yield. In addition, the stability of the anti-Mycoplasma pneumoniae monoclonal antibody or its labeled product can be remarkably enhanced, and it has become extremely useful as a diagnostic agent.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 (Ｃ１２Ｐ 21/08 Ｃ１２Ｒ 1:91） ──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. 6 Identification number Agency reference number FI Technical indication (C12P 21/08 C12R 1:91)
Claims (4)
ーナル抗体又はその標識体を、当該抗体又は標識体に対
して12.5〜250重量倍のアルブミンを含有し糖類を含有
しないpH5.5〜8.5の水性溶液に溶解することにより得ら
れるマイコプラズマ・ニューモニエ診断用試薬。1. An anti-Mycoplasma pneumoniae monoclonal antibody or a labeled product thereof is dissolved in an aqueous solution containing albumin 12.5 to 250 times by weight of the antibody or the labeled product and containing no saccharide and having a pH of 5.5 to 8.5. A diagnostic reagent for Mycoplasma pneumoniae obtained by the above method.
ーナル抗体又はその標識体を、当該抗体又は標識体に対
して62.5〜625重量倍のアルブミンを含有し糖類を含有
しないpH5.5〜8.5の水性溶液に溶解した後凍結乾燥する
ことより得られるマイコプラズマ・ニューモニエ診断用
試薬。2. An anti-Mycoplasma pneumoniae monoclonal antibody or a labeled product thereof is dissolved in an aqueous solution containing albumin in an amount of 62.5 to 625 times by weight of the antibody or the labeled product and containing no saccharide and having a pH of 5.5 to 8.5. Mycoplasma pneumoniae diagnostic reagent obtained by freeze-drying afterwards.
ーナル抗体又はその標識体を、当該抗体又は標識体に対
して12.5〜250重量倍のアルブミンを含有し糖類を含有
しないpH5.5〜8.5の水性溶液に溶解することを特徴とす
る抗マイコプラズマ・ニューモニエモノクローナル抗体
の安定化法。3. An anti-Mycoplasma neumonier monoclonal antibody or a labeled product thereof is dissolved in an aqueous solution containing albumin 12.5 to 250 times the weight of the antibody or the labeled product and containing no saccharide and having a pH of 5.5 to 8.5. A method for stabilizing an anti-Mycoplasma pneumoniae monoclonal antibody, comprising:
ーナル抗体又はその標識体を、当該抗体又は標識体に対
して62.5〜625重量倍のアルブミンを含有し糖類を含有
しないpH5.5〜8.5の水性溶液に溶解した後凍結乾燥する
ことを特徴とする抗マイコプラズマ・ニューモニエモノ
クローナル抗体の安定化法。4. An anti-Mycoplasma pneumoniae monoclonal antibody or a labeled product thereof is dissolved in an aqueous solution containing albumin in an amount of 62.5 to 625 times by weight of the antibody or the labeled product and containing no saccharide and having a pH of 5.5 to 8.5. A method for stabilizing an anti-Mycoplasma pneumoniae monoclonal antibody, which is followed by freeze-drying.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2510212A JP2627681B2 (en) | 1990-07-18 | 1990-07-18 | Mycoplasma pneumoniae diagnostic reagent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2510212A JP2627681B2 (en) | 1990-07-18 | 1990-07-18 | Mycoplasma pneumoniae diagnostic reagent |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2627681B2 true JP2627681B2 (en) | 1997-07-09 |
Family
ID=18527426
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2510212A Expired - Lifetime JP2627681B2 (en) | 1990-07-18 | 1990-07-18 | Mycoplasma pneumoniae diagnostic reagent |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2627681B2 (en) |
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|---|---|---|---|---|
| JPS57131232A (en) * | 1981-02-06 | 1982-08-14 | Toray Ind Inc | Sheet for stamping |
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| JPS5922722A (en) * | 1982-07-24 | 1984-02-06 | ロ−ルス・ロイス・ピ−エルシ− | Manufacture of article by composite material |
| JPS59215813A (en) * | 1983-05-24 | 1984-12-05 | Unitika Ltd | Electric conductive sheet and its formed product |
| JPS60112420A (en) * | 1983-11-07 | 1985-06-18 | インペリアル ケミカル インダストリーズ パブリツク リミテイド カンパニー | Method of molding reinforced thermoplastic composite material |
| JPS60146833A (en) * | 1984-01-10 | 1985-08-02 | Green Cross Corp:The | Monoclonal antibody pharmaceutical |
| JPS60146883A (en) * | 1983-12-17 | 1985-08-02 | ドクトル．カール トーメー ゲゼルシャフト ミット ベシュレンクテル ハフツンク | Novel benzothiazole compound |
| JPS6176423A (en) * | 1984-08-09 | 1986-04-18 | アボツト ラボラトリ−ズ | Stabilization of monoclonal antibody |
| JPS61252130A (en) * | 1985-03-21 | 1986-11-10 | インペリアル ケミカル インダストリ−ズ パブリツク リミテイド カンパニ− | Manufacture of molded shape from reinforced composite material |
| JPS61252131A (en) * | 1985-03-21 | 1986-11-10 | インペリアル ケミカル インダストリ−ズ パブリツク リミテイド カンパニ− | Manufacture of molded shape from reinforced composite material |
| JPS62289523A (en) * | 1986-06-09 | 1987-12-16 | Green Cross Corp:The | Heat treatment of immunoglobulin for intravenous administration |
| JPS63184064A (en) * | 1986-07-29 | 1988-07-29 | Ss Pharmaceut Co Ltd | Anti-mycoplasma-pneumonere monoclonal antibody, its preparation and reagent for identification and diagnosis of mycoplasma utilizing the same |
| JPS643439A (en) * | 1987-06-25 | 1989-01-09 | Matsushita Electric Works Ltd | Space cooling and heating floor panel |
-
1990
- 1990-07-18 JP JP2510212A patent/JP2627681B2/en not_active Expired - Lifetime
Patent Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57131232A (en) * | 1981-02-06 | 1982-08-14 | Toray Ind Inc | Sheet for stamping |
| JPS58162312A (en) * | 1982-03-08 | 1983-09-27 | イ−・アイ・デユポン・デ・ニモアス・アンド・カンパニ− | Method of thermally working reinforced polymer sheet |
| JPS5922722A (en) * | 1982-07-24 | 1984-02-06 | ロ−ルス・ロイス・ピ−エルシ− | Manufacture of article by composite material |
| JPS59215813A (en) * | 1983-05-24 | 1984-12-05 | Unitika Ltd | Electric conductive sheet and its formed product |
| JPS60112420A (en) * | 1983-11-07 | 1985-06-18 | インペリアル ケミカル インダストリーズ パブリツク リミテイド カンパニー | Method of molding reinforced thermoplastic composite material |
| JPS60146883A (en) * | 1983-12-17 | 1985-08-02 | ドクトル．カール トーメー ゲゼルシャフト ミット ベシュレンクテル ハフツンク | Novel benzothiazole compound |
| JPS60146833A (en) * | 1984-01-10 | 1985-08-02 | Green Cross Corp:The | Monoclonal antibody pharmaceutical |
| JPS6176423A (en) * | 1984-08-09 | 1986-04-18 | アボツト ラボラトリ−ズ | Stabilization of monoclonal antibody |
| JPS61252130A (en) * | 1985-03-21 | 1986-11-10 | インペリアル ケミカル インダストリ−ズ パブリツク リミテイド カンパニ− | Manufacture of molded shape from reinforced composite material |
| JPS61252131A (en) * | 1985-03-21 | 1986-11-10 | インペリアル ケミカル インダストリ−ズ パブリツク リミテイド カンパニ− | Manufacture of molded shape from reinforced composite material |
| JPS62289523A (en) * | 1986-06-09 | 1987-12-16 | Green Cross Corp:The | Heat treatment of immunoglobulin for intravenous administration |
| JPS63184064A (en) * | 1986-07-29 | 1988-07-29 | Ss Pharmaceut Co Ltd | Anti-mycoplasma-pneumonere monoclonal antibody, its preparation and reagent for identification and diagnosis of mycoplasma utilizing the same |
| JPS643439A (en) * | 1987-06-25 | 1989-01-09 | Matsushita Electric Works Ltd | Space cooling and heating floor panel |
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