JPH08178924A - Production of bacterium surface layer antigen - Google Patents

Abstract

PURPOSE: To simply produce a high purity antigen by using freeze-thawing for crushing bacteria in a bacteria suspension and using a surfactant in the solubilization of an insoluble component. CONSTITUTION: After bacteria have been crushed by freezing and thawing a bacteria suspension, an insoluble component is fractionated and, subsequently, the solubilization of the insoluble component to an aq. medium is performed by using a surfactant and a soluble component is fractionated as an antigen. The soluble component of impurities can be certainly removed by crushing and the mixing of impurities in the succeeding solubilization can be prevented and, therefore, the purity of the produced bacterium surface layer antigen can be markedly enhanced. Since the cell surface layers of bacteria are partially destructed by crushing operation, solubilization can be efficiently executed by the use of the surfactant of the insoluble component or the combination use of the surfactant and a modifier succeeding to fractionation.

Description

【発明の詳細な説明】Detailed Description of the Invention

【０００１】[0001]

【産業上の利用分野】本発明は感染症検査試薬に用いら
れる抗原の製造方法に関する。さらに詳しくは特異性が
高く、且つ高感度、特に初期感染を検出可能な感染症検
査試薬用として好適な菌表層抗原の製造方法に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing an antigen used as an infectious disease test reagent. More specifically, it relates to a method for producing a bacterial surface antigen, which has high specificity and high sensitivity, and is particularly suitable for an infectious disease test reagent capable of detecting an initial infection.

【０００２】[0002]

【従来の技術】感染症の臨床検査試薬としては、抗原抗
体反応を利用して患者血清、血漿中の抗体を検出する感
染症検査試薬が主流を占めている。この感染症検査試薬
にはマイクロタイター試薬、酵素免疫試薬、免疫比濁試
薬、ラテックス凝集試薬などがある。例えば、代表的な
感染症検査試薬である梅毒検査試薬には富沢らによって
開発されたＴＰＨＡテストがあり、鋭敏性、特異性、操
作性の利点があり、現在広く用いられている。ＴＰＨＡ
テストは梅毒トレポネーマ菌液を抗原として用い、患者
血清中の梅毒トレポネーマ抗体（以下、ＴＰ抗体と略記
する）を検出する検査試薬である。2. Description of the Related Art As a clinical test reagent for infectious diseases, an infectious disease test reagent which detects an antibody in patient serum or plasma by utilizing an antigen-antibody reaction is predominant. The infectious disease test reagents include microtiter reagents, enzyme immunoreagents, immunoturbidimetric reagents, latex agglutination reagents, and the like. For example, the syphilis test reagent that is a typical infectious disease test reagent includes the TPHA test developed by Tomizawa et al., Which has the advantages of sensitivity, specificity, and operability, and is currently widely used. TPHA
The test is a test reagent that uses Treponema pallidum bacterium solution as an antigen to detect Treponema pallidum antibody (hereinafter abbreviated as TP antibody) in patient serum.

【０００３】上記のような感染症検査試薬において、抗
原として超音波処理、ホモジナイザー処理などによって
感染症病原微生物（以下、菌とも略記する）、即ち、細
菌、ウイルス等を破砕し、直接あるいはこれをさらに可
溶化して抗原として用いている。感染症検査試薬に用い
る抗原の特性は菌の破砕方法及び可溶化方法により大き
く影響を受け、得られる抗原の特性は、その感染症検査
試薬の性能を左右する。例えば、破砕時に超音波破砕機
等で生じる熱で抗原性が著しく低下したり、抽出時に抗
原であるところのタンパク、脂質等の立体構造が変化
し、抗原特異性が変化することなどがある。従って、菌
の破砕方法及び可溶化方法の選択は抗原調製では重要な
要素である。In the infectious disease test reagent as described above, an infectious disease-causing microorganism (hereinafter abbreviated as a bacterium), that is, a bacterium, a virus, etc., is crushed by an ultrasonic treatment, a homogenizer treatment or the like as an antigen, and is directly or directly Further solubilized and used as an antigen. The characteristics of the antigen used for the infectious disease test reagent are greatly influenced by the crushing method and the solubilization method of the bacterium, and the property of the obtained antigen influences the performance of the infectious disease test reagent. For example, the antigenicity may be significantly reduced by heat generated by an ultrasonic crusher during crushing, or the three-dimensional structure of the protein, lipid or the like that is an antigen during extraction may change, resulting in a change in antigen specificity. Therefore, selection of the crushing method and the solubilizing method of the bacterium is an important factor in antigen preparation.

【０００４】一般に感染症検査試薬に用いる抗原は、菌
の表層のタンパク、糖類、脂質、糖タンパク、リポタン
パク等（以下、菌表層抗原とも略記する）に多く存在し
ている。さらに、感染症の治療には感染初期に発見する
ことが治療に有効であり、感染症検査試薬では初期抗体
（ＩｇＭ抗体）の検出が重要視されている。ＩｇＭ抗体
と反応性の高い抗原は特に菌の表層に局在化していると
考えられる。[0004] Generally, many antigens used for infectious disease test reagents are present in proteins, sugars, lipids, glycoproteins, lipoproteins, etc. on the surface layer of bacteria (hereinafter also abbreviated as bacterial surface antigens). Furthermore, for the treatment of infectious diseases, it is effective to detect them at the early stage of infection, and the detection of the initial antibody (IgM antibody) is regarded as important in the infectious disease test reagents. It is considered that the antigen highly reactive with the IgM antibody is particularly localized on the surface layer of the bacterium.

【０００５】従って、上記の菌表層抗原を効率よく回収
する破砕及び可溶化方法を確立し、特異性が高く、且つ
高感度、特に初期感染を効率よく検出可能な抗原を製造
する技術は、優れた感染症検査試薬を開発するための重
要である。[0005] Therefore, a technique for establishing a crushing and solubilizing method for efficiently recovering the above-mentioned bacterial surface antigen and for producing an antigen having high specificity and high sensitivity, particularly capable of efficiently detecting an initial infection is excellent. It is important to develop infectious disease test reagents.

【０００６】しかしながら、感染症検査試薬に用いる菌
表層抗原を簡便、高純度に得る製造方法は未だ確定して
ないのが現状である。[0006] However, the present situation is that the production method for obtaining the bacterial surface antigen used as a reagent for infectious disease test in a simple and highly purified manner has not yet been determined.

【０００７】これまでに本発明に類する技術として特開
昭５８−７１４５７公報に梅毒トレポネーマ菌培養物の
うち比重１．０１以下の画分を除去した抗原含有物を用
いる例が記載されている。しかし、上記の方法には菌の
破砕方法、可溶化方法の適切な記載がなく、適切な破砕
方法、可溶化方法を採用しなければ比重分画だけで純粋
な抗原を得ることは困難である。As a technique similar to the present invention, Japanese Patent Application Laid-Open No. 58-71457 discloses an example of using an antigen-containing substance obtained by removing a fraction having a specific gravity of 1.01 or less from a culture of Treponema pallidum bacterium. However, in the above method, there is no suitable description of the crushing method and the solubilizing method of the bacterium, and it is difficult to obtain a pure antigen only by the specific gravity fraction unless an appropriate crushing method and solubilizing method are adopted. .

【０００８】さらに別の類する技術として特開平２−２
３４０６３公報及び特開平３−２６７７６０公報に梅毒
トレポネーマ菌培養物由来タンパク抗原から陽イオン交
換体によって不純物を除去する方法、及び梅毒トレポネ
ーマ菌培養物由来タンパク抗原からハイドロキシアパタ
イトによって不純物を除去する方法がある。いずれの技
術にも十分な菌の破砕方法、可溶化方法の記載がなく、
さらに、陽イオン交換体、及びハイドロキシアパタイト
等で抗原を分画すると抗原性を有するタンパク成分を排
除してしまう可能性がある。As another similar technique, Japanese Patent Laid-Open No. 2-2
JP-A-34063 and JP-A-3-267760 have a method of removing impurities from a protein antigen derived from Treponema pallidum culture using a cation exchanger, and a method of removing impurities from a protein antigen derived from Treponema pallidum culture using hydroxyapatite. . There is no description of sufficient crushing method of bacteria or solubilization method in any technology,
Furthermore, when the antigen is fractionated with a cation exchanger, hydroxyapatite, etc., the protein component having antigenicity may be eliminated.

【０００９】[0009]

【発明が解決しようとする課題】本発明は、感染症検査
試薬に用いる菌表層抗原の製造方法において、簡便な高
純度抗原の製造方法を提供することを目的としている。DISCLOSURE OF THE INVENTION An object of the present invention is to provide a simple method for producing a high-purity antigen in a method for producing a surface antigen of a bacterium to be used as an infectious disease test reagent.

【００１０】[0010]

【課題を解決するための手段】本発明者らは上記技術課
題を解決すべく鋭意研究を行ってきた。その結果、菌懸
濁液中の菌の破砕方法として凍結融解を用い、且つ該不
溶性成分の可溶化方法として界面活性剤を用いることに
より、上記目的を達成されることを見い出した。さら
に、該不溶性成分の可溶化時に尿素等の変性剤を併用す
ることにより、抽出効率がより向上できる事実を見い出
し、本発明を完成するに至った。[Means for Solving the Problems] The present inventors have conducted extensive research to solve the above technical problems. As a result, they have found that the above-mentioned objects can be achieved by using freeze-thawing as a method for disrupting bacteria in a bacterial suspension and using a surfactant as a method for solubilizing the insoluble component. Furthermore, it was found that the extraction efficiency can be further improved by using a modifier such as urea together when the insoluble component is solubilized, and the present invention has been completed.

【００１１】即ち、本発明は、菌懸濁液を凍結融解して
菌を破砕したのち不溶性成分を分画し、次いで該不溶性
成分を界面活性剤を用いて水性溶媒への可溶化を行った
のち可溶性成分を抗原として分画することを特徴とする
菌表層抗原の製造方法である。That is, in the present invention, the insoluble component was fractionated after freeze-thawing the bacterial suspension to crush the bacteria, and then the insoluble component was solubilized in an aqueous solvent using a surfactant. After that, a soluble component is fractionated as an antigen, which is a method for producing a surface antigen of a bacterium.

【００１２】他の発明は、不溶性成分を変性剤の共存下
に界面活性剤を用いて可溶化することを特徴とする上記
菌表層抗原の製造方法である。以下、本発明を詳細に説
明する。[0012] Another invention is the above-mentioned method for producing a surface antigen of a bacterium, which comprises solubilizing an insoluble component with a surfactant in the presence of a denaturing agent. Hereinafter, the present invention will be described in detail.

【００１３】本発明に用いる菌としては感染症病原性を
持つ細菌、スピロヘータ、リケッチア、クラミジア、真
菌、ウイルス等の感染症病原微生物が挙げられる。細菌
にはグラム陽性菌としてブドウ球菌、クロストリジウム
属、マイコバクテリウム属等があり、グラム陰性菌とし
てリン菌、サルモネラ属、レジオネラ菌等がある。スピ
ロヘータには梅毒トレポネーマ、レプトスピラ、ボレリ
ア・ブルグドルフェリ等がある。さらに、リケッチアに
は発疹チフスリケッチア、ツツガムシ病リケッチア等が
あり、クラミジアにはオウム病クラミジア、トラコーマ
クラミジア等がある。真菌としてはカンジダ属、ヒスト
プラズマ属、クリプトコッカス属、ムコール属等であ
る。ウイルスには後天性免疫不全症ウイルス（ＨＩ
Ｖ）、成人Ｔ細胞白血病ウイルス（ＡＴＬＡ）、Ｂ型肝
炎ウイルス（ＨＢＶ）、Ｃ型肝炎ウイルス（ＨＣＶ）等
がある。Examples of the bacterium used in the present invention include infectious disease-causing microorganisms such as bacteria having infectious disease pathogenicity, spirochete, rickettsia, chlamydia, fungi and viruses. Bacteria include Staphylococcus, Clostridium, Mycobacterium, etc. as Gram-positive bacteria, and Phosphorus, Salmonella, Legionella, etc. as Gram-negative bacteria. Spirochetes include Treponema pallidum, Leptospira, Borrelia burgdorferi and others. Furthermore, rickettsia includes typhus rickettsia and tsutsugamushi disease rickettsia, and chlamydia includes parrot disease chlamydia and trachoma chlamydia. The fungi include Candida, Histoplasma, Cryptococcus, Mucor and the like. The viruses include acquired immunodeficiency virus (HI
V), adult T-cell leukemia virus (ATLA), hepatitis B virus (HBV), hepatitis C virus (HCV) and the like.

【００１４】本発明の菌懸濁液は上記菌と生理食塩水、
ＰＢＳ等の水性溶媒との混合物をいう。すなわち、一般
に菌を増殖させるためには炭素源、窒素源等生育に必要
な栄養素が必要である。菌を増殖させるための培養法に
は大きく分類して２つがある。第１は栄養素を人為的に
添加した液体であるところの培地を培養用フラスコ、試
験管等に入れて菌を接種、増殖させる生体外培養法であ
り、第２は生体外培養法での増殖されることが困難な梅
毒トレポネーマのような菌を家兎等の実験動物生体内に
注入して体液成分中で増殖させる生体内菌培養法であ
る。上記のようにして増殖させた菌と培地あるいは体液
成分の懸濁物を培養液と呼ぶ。それぞれの菌に適した培
養法を選択する必要がある。どちらの培養法を用いても
菌を増殖させた培地及び体液には抗原性を持たない多く
の不純物が含まれているが、特に生体外培養法に比較し
て生体内菌培養法では不純物を多く含有する。培養液中
の菌を遠心分離、遠心洗浄等を施して可溶性不純物を簡
易的に除去したのち緩衝液等の生理食塩水、ＰＢＳ等の
水性溶媒に懸濁させて菌懸濁液として用いる。The bacterial suspension of the present invention comprises the above bacteria and physiological saline,
It refers to a mixture with an aqueous solvent such as PBS. That is, generally, nutrients necessary for growth such as a carbon source and a nitrogen source are required to grow bacteria. There are roughly two types of culture methods for growing bacteria. The first is an in vitro culture method in which a medium, which is a liquid to which nutrients are artificially added, is placed in a culture flask, a test tube, etc. to inoculate and grow the bacteria, and the second is an in vitro culture method. It is an in-vivo bacterial culture method in which a bacterium such as Treponema pallidum, which is difficult to be treated, is injected into a living body of an experimental animal such as a rabbit and proliferated in a body fluid component. A suspension of the bacteria grown as described above and a medium or body fluid component is called a culture medium. It is necessary to select a culture method suitable for each bacterium. Both culture methods use a large amount of impurities that do not have antigenicity in the medium and body fluid in which the bacteria are grown. Contains a lot. The bacteria in the culture solution are subjected to centrifugation, centrifugal washing, etc. to easily remove soluble impurities, and then suspended in physiological saline such as a buffer solution or an aqueous solvent such as PBS to be used as a bacterial suspension.

【００１５】例えば、梅毒トレポネーマについては以下
のような方法で菌懸濁液を調製する。ミラーらの方法
（Ｍｉｌｌｅｒ ｅｔ ａｌ．，Ｖｏｌ．９６，ｐ４５
０（１９６６））に従って培養集菌すればよい。すなわ
ち、家兎睾丸に梅毒トレポネーマ菌（以下、ＴＰ菌と略
す）を１１〜１４日間、接種し増殖させ、家兎睾丸をハ
サミで切断して０．１５Ｍ塩化ナトリウム水溶液（以
下、生理食塩水と記す）で懸濁してＴＰ菌を抽出する。
抽出液を２００×ｇで１５分間遠心して睾丸組織の大き
な破片を沈澱除去する。続いて、１９，０００×ｇで９
０分間遠心してＴＰ菌を沈澱させる。このＴＰ菌液を生
理食塩水に懸濁したものをＴＰ菌の菌懸濁液と称する。For example, for Treponema pallidum, a bacterial suspension is prepared by the following method. Miller et al. (Miller et al., Vol. 96, p45)
0 (1966)). That is, a rabbit testis was inoculated with Treponema pallidum (hereinafter abbreviated as TP) for 11 to 14 days to grow, and the rabbit testis was cut with scissors to prepare a 0.15 M sodium chloride aqueous solution (hereinafter, referred to as saline solution). (Note) to suspend TP bacteria.
The extract is centrifuged at 200 xg for 15 minutes to precipitate out large debris from the testis tissue. Then 9 at 19,000 × g
Centrifuge for 0 minutes to precipitate TP bacteria. A suspension of this TP bacterium solution in physiological saline is referred to as a TP bacterium suspension.

【００１６】本発明においては先ず菌懸濁液を凍結融解
して破砕を行う。該破砕はこれに続く不溶性成分の分
画、及び該不溶性成分の界面活性剤又は界面活性剤と変
性剤の併用により行う可溶化を効率的に実施するために
必須の操作である。In the present invention, first, the bacterial suspension is freeze-thawed and crushed. The crushing is an essential operation in order to efficiently carry out the subsequent fractionation of the insoluble component and the solubilization of the insoluble component by using a surfactant or a combination of a surfactant and a denaturing agent.

【００１７】破砕を十分且つ均一に行えば、これに続く
抗原性を有するタンパク、糖質、脂質、糖タンパク、リ
ポタンパク等を含む不溶性成分と、夾雑物であるところ
の可溶性成分を確実に分画することができる。すなわ
ち、破砕を行うことにより夾雑物を確実に除去でき、続
く可溶化での夾雑物の混入を防ぐことができるため、製
造される菌表層抗原の純度を著しく向上させることが可
能である。もし破砕が十分に行わなければ、細胞内に存
在する可溶性成分が続く可溶化で混入して最終的に製造
される菌表層抗原の純度が低下する。また、該破砕操作
は菌の細胞表層を部分的に破壊するので分画に続く不溶
性成分の界面活性剤又は界面活性剤と変性剤の併用によ
り行う可溶化を効率的に実施することができる。If the crushing is carried out sufficiently and uniformly, it is possible to reliably separate the insoluble components such as proteins, carbohydrates, lipids, glycoproteins, lipoproteins, etc. having antigenicity and the soluble components which are contaminants. Can be drawn. That is, since the contaminants can be reliably removed by crushing and the contaminants can be prevented from being mixed in during the subsequent solubilization, it is possible to remarkably improve the purity of the bacterial surface antigen produced. If the crushing is not performed sufficiently, the soluble components existing in the cells are contaminated by the subsequent solubilization, and the purity of the bacterial surface antigen finally produced is lowered. Further, since the crushing operation partially destroys the cell surface layer of the bacterium, the solubilization of the insoluble component subsequent to the fractionation can be efficiently carried out by using the surfactant or the combined use of the surfactant and the denaturing agent.

【００１８】上記凍結融解による破砕は細菌、真菌（酵
母、カビ等）、ウイルス等の細胞の物理的な破砕方法の
一つで、菌懸濁物を凍結し、細胞内に氷の結晶を形成さ
せた後、融解する操作を数回繰り返すことで破砕する方
法である。The crushing by freeze-thawing is one of the physical crushing methods for cells such as bacteria, fungi (yeast, mold, etc.), viruses, etc. Freezing a suspension of bacteria to form ice crystals in the cells. This is a method of crushing by repeating the melting operation several times after the reaction.

【００１９】凍結はドライアイス−アセトン、液体窒
素、液体ヘリウム等冷却剤で行えば良く、できるだけ急
速に冷却できる方が好ましい。凍結に要する時間は１分
以内が好ましく、３０秒以内がさらに好適である。温度
は−２０℃以下が好ましく、−４０℃以下が特に好適に
用いられる。例えば、液体窒素を用いた凍結が挙げられ
る。融解はできるだけ急速に行うことが望ましく、要す
る時間は５分以内が好ましく、３分以内がさらに好適で
ある。融解は室温下、水槽内等で行う。融解に用いる温
度は２０℃以上６０℃以下が好ましく、３０℃以上５０
℃が特に好ましい。凍結融解に供する菌数濃度は任意で
よい。処理に要する容量が少ないほど簡便に行えるの
で、菌数は１０⁹細胞／ｍｌオーダー以上が好ましい。The freezing may be carried out with a cooling agent such as dry ice-acetone, liquid nitrogen, liquid helium, etc., and it is preferable that it can be cooled as rapidly as possible. The time required for freezing is preferably within 1 minute, more preferably within 30 seconds. The temperature is preferably −20 ° C. or lower, and −40 ° C. or lower is particularly preferably used. For example, freezing with liquid nitrogen can be mentioned. Melting is preferably performed as quickly as possible, and the time required is preferably 5 minutes or less, more preferably 3 minutes or less. The melting is performed at room temperature in a water tank or the like. The temperature used for melting is preferably 20 ° C or higher and 60 ° C or lower, and 30 ° C or higher and 50 ° C or higher.
C is especially preferred. The concentration of the number of bacteria used for freeze-thawing may be arbitrary. Since the smaller the volume required for the treatment, the easier the treatment, the number of bacteria is preferably 10 ⁹ cells / ml or more.

【００２０】凍結融解は菌懸濁液に施すが、凍結融解を
２回以上行えば菌の細胞を有効に破砕することができ
る。凍結融解による破砕の対象は前記の菌であれば、特
に限定なく、例えば、対象が細菌である場合、比較的細
胞壁の厚いグラム陽性菌と、細胞壁が比較的薄いグラム
陰性菌に対しても凍結融解により有効に破砕できる。さ
らに破砕対象がスピロヘータ、リケッチア、クラミジア
等の原核生物である場合でも同様に有効に作用する。真
菌である酵母等、あるいはウイルス等にも有効に作用す
る。特に梅毒トレポネーマにおいて好適に採用できる。Freezing and thawing is applied to the bacterial suspension, but if the freezing and thawing is performed twice or more, the bacterial cells can be effectively disrupted. The target of disruption by freezing and thawing is not particularly limited as long as it is the above-mentioned bacteria.For example, when the target is bacteria, it is also frozen against Gram-positive bacteria with relatively thick cell walls and Gram-negative bacteria with relatively thin cell walls. Can be effectively crushed by melting. Furthermore, even if the crushing target is a procaryote such as spirochete, rickettsia, chlamydia, etc., the same effect is exerted. It also effectively acts on fungi such as yeast and viruses. Especially, it can be preferably used in Treponema pallidum.

【００２１】細胞の破砕方法には他に超音波処理、ホモ
ジナイザー処理、ガラスビーズ破砕等があるが、凍結融
解は他の方法に比べて良好な破砕方法である。他の方法
である超音波処理では破砕時に生じる温度上昇により、
抗原性を有するタンパク、糖質、脂質、糖タンパク、リ
ポタンパク等の立体構造に障害を受け、得られる菌表層
抗原の抗原性が低下する可能性がある。さらに超音波破
砕では破砕対象であるところの菌の形状により破砕の程
度が異なり、桿菌の細胞に比べ、球菌の細胞では破砕さ
れにくい。さらに他の破砕方法であるところのホモジナ
イザー処理はホモジナイザー内壁面と菌の摩擦による物
理的破砕であり、菌を均一に破砕することが困難であ
る。破砕されなかった菌が存在することにより、可溶化
時に菌内に存在する抗原性の低い可溶性成分の混入によ
る純度の低下、及び可溶化効率の低下で抗原成分の回収
率が下がる欠点がある。さらに他の破砕方法であるとこ
ろのガラスビーズ破砕はガラスビーズと菌との衝突頻度
に起因するところが大きくホモジナイザー破砕同様に破
砕程度を均一にすることは困難である。Other methods of cell disruption include ultrasonic treatment, homogenizer treatment, glass bead disruption, and freeze-thawing is a better disruption method than other methods. In ultrasonic treatment, which is another method, due to the temperature rise that occurs during crushing,
There is a possibility that the three-dimensional structure of proteins, carbohydrates, lipids, glycoproteins, lipoproteins, etc. having antigenicity will be damaged, and the antigenicity of the resulting bacterial surface antigen will be reduced. Furthermore, in ultrasonic crushing, the degree of crushing varies depending on the shape of the bacterium to be crushed, and it is less crushed by coccus cells than by bacillus cells. The homogenizer treatment, which is another crushing method, is physical crushing by friction between the inner wall surface of the homogenizer and the bacterium, and it is difficult to uniformly crush the bacterium. The presence of non-crushed bacteria has a drawback that the purity is lowered due to the inclusion of soluble components having low antigenicity present in the bacteria at the time of solubilization, and the solubilization efficiency is lowered, so that the recovery rate of the antigen components is lowered. Further, as another crushing method, glass bead crushing is largely due to the frequency of collision between glass beads and bacteria, and it is difficult to make the crushing degree uniform like homogenizer crushing.

【００２２】上記凍結融解で破砕操作を終了した時点
で、菌懸濁液は、抗原性を有する不溶性成分と夾雑物で
あるところの可溶性成分との混合物となる。通常該混合
物をそのまま次の抗原性を有する不溶性成分のみを回収
する分画操作に使用する。At the time when the disruption operation is completed by freeze-thawing, the bacterial suspension becomes a mixture of an insoluble component having antigenicity and a soluble component which is a contaminant. Usually, the mixture is directly used for the fractionation operation for collecting only the insoluble component having the following antigenicity.

【００２３】抗原性を有する不溶性成分の該分画操作は
代表的には遠心分離によって行う。該遠心分離によっ
て、可溶性成分と不溶性成分の懸濁液を入れた遠心用ロ
ーターを回転することにより遠心力場をつくり、これに
より該懸濁液中に含まれる不溶性成分のみを回収する。
遠心分離に採用する遠心力は不溶性成分を沈殿できる遠
心力であれば良い。例えば、梅毒トレポネーマ菌の凍結
融解後の不溶性成分と可溶性成分の懸濁液より細胞壁、
細胞膜等の抗原性を有する不溶性成分のみを分離するに
は１０，０００×ｇ、３０分間で十分である。The operation of fractionating the insoluble component having antigenicity is typically performed by centrifugation. By the centrifugal separation, a centrifugal force field is created by rotating a centrifugal rotor containing a suspension of a soluble component and an insoluble component, whereby only the insoluble component contained in the suspension is recovered.
The centrifugal force used for centrifugation may be any centrifugal force capable of precipitating insoluble components. For example, the cell wall from a suspension of insoluble and soluble components after freeze-thawing of Treponema pallidum,
For separating only insoluble components having antigenicity such as cell membranes, 10,000 × g for 30 minutes is sufficient.

【００２４】得られる不溶性成分の主な成分は菌の細胞
壁、細胞膜、真菌の核、ミトコンドリア、リボソーム等
であるが、大部分は細胞膜と細胞壁である。菌の抗原性
を有する成分は細胞表層に局在しているので、不溶性成
分には抗原性を有するタンパク、脂質、糖質、糖タンパ
ク、リポタンパク等が多く含まれていると考えられる。The main components of the obtained insoluble components are the cell wall of fungus, cell membrane, nucleus of fungus, mitochondria, ribosome, etc., but most of them are cell membrane and cell wall. Since the antigenic component of the bacterium is localized on the cell surface, it is considered that the insoluble component contains a large amount of antigenic protein, lipid, carbohydrate, glycoprotein, lipoprotein and the like.

【００２５】上記分画操作では、凍結融解による破砕後
の抗原性を有する不溶性成分を回収し、抗原性の低い可
溶性成分を除去することを目的とする。該分画により簡
単な１ステップの操作で最終的に得られる抗原の純度を
向上されることができる。The above-mentioned fractionation operation aims at recovering insoluble components having antigenicity after crushing by freeze-thawing and removing soluble components having low antigenicity. By the fractionation, the purity of the finally obtained antigen can be improved by a simple one-step operation.

【００２６】次いで、得られた不溶性成分は界面活性
剤、又は界面活性剤と変性剤を用いて水性溶媒に可溶化
される。該水性溶媒としては生理食塩水、リン酸緩衝液
（ｐＨ７．２）含有生理食塩水〔以下ＰＢＳという〕等
が用いられる。Then, the obtained insoluble component is solubilized in an aqueous solvent by using a surfactant or a surfactant and a modifier. As the aqueous solvent, physiological saline, physiological saline containing a phosphate buffer (pH 7.2) [hereinafter referred to as PBS] and the like are used.

【００２７】不溶性成分は界面活性剤、又は界面活性剤
と変性剤を添加することにより、水性溶媒に溶解が可能
となる。不溶性成分は通常、水性溶媒中では凝集してい
るか又は脂質２重層などに埋め込まれている場合が多い
が、該可溶化により水性溶媒中に移行する。すなわち、
可溶化により抗原性を有するタンパク、脂質、糖質、糖
タンパク、リポタンパク等が水性溶媒へ溶出される。可
溶化を終了した段階では、抗原性を有する可溶性成分
と、抗原性を有さない不要な不溶性成分の懸濁液として
存在する。続く遠心分離に代表される分画操作により抗
原性を有するを可溶性成分を抗原として回収することが
できる。The insoluble component can be dissolved in an aqueous solvent by adding a surfactant or a surfactant and a modifier. The insoluble component is usually agglomerated in an aqueous solvent or embedded in a lipid bilayer or the like in many cases, but is transferred to the aqueous solvent by the solubilization. That is,
Upon solubilization, proteins, lipids, carbohydrates, glycoproteins, lipoproteins, etc. having antigenicity are eluted in an aqueous solvent. When the solubilization is completed, it exists as a suspension of a soluble component having antigenicity and an unnecessary insoluble component having no antigenicity. A soluble component having an antigenicity can be recovered as an antigen by a fractionation operation typified by subsequent centrifugation.

【００２８】該可溶化は本製造方法で必須の操作であ
る。本可溶化操作を実施しなければ、抗原性を有さない
不溶性成分が混在し、その結果、得られる抗原の純度、
特異性が低下する。The solubilization is an essential operation in the present production method. If this solubilization operation is not carried out, insoluble components having no antigenicity are mixed, and as a result, the purity of the obtained antigen,
Specificity decreases.

【００２９】不溶性成分を可溶化する際、変性剤の共存
下に界面活性剤を用いて可溶化することが好ましい。変
性剤を共存させることで界面活性剤単独の可溶化に比べ
てタンパク、脂質、糖質、糖タンパク、リポタンパク等
の抗原の可溶化効率が著しく上昇する。When solubilizing the insoluble component, it is preferable to solubilize the insoluble component with a surfactant in the presence of a modifier. The coexistence of the denaturant significantly increases the solubilization efficiency of antigens such as proteins, lipids, carbohydrates, glycoproteins, lipoproteins, etc., as compared with the solubilization of the surfactant alone.

【００３０】本発明の可溶化で用いる界面活性剤の種類
としては、非イオン性界面活性剤又はイオン性界面活性
剤を用いることができる。As the kind of the surfactant used for the solubilization of the present invention, a nonionic surfactant or an ionic surfactant can be used.

【００３１】ほとんどすべての非イオン性界面活性剤は
親水基部分に糖またはポリオキシエチレンをもつ。非イ
オン性界面活性剤はタンパク変性作用が弱く膜タンパク
等を変性させにくく可溶化に適している。非イオン界面
活性剤としてはオクチルグルコシド、デシルグルコシ
ド、ヘプチルチオグルコシド、オクチルチオグルコシ
ド、ノニルチオグルコシド、オクチルマルトシド、ドデ
シルマルトシド、デカノイル−Ｎ−メチルグルカミド、
トリトンＸ４５、トリトンＸ１１４、ノニデットＰ４
０、トリトンＸ１００、トリトンＸ１０２、トリトンＸ
１６５、トリトンＸ３０５、ブリッジ７６、ブリッジ９
６、ブリッジ５６、ブリッジ５８、トリトンＮ１０１、
スクロースモノヘキサン酸エステル、スクロースモノラ
ウリン酸エステル、モノステアリン酸ソルビトール、モ
ノラウリン酸ソルビトール等を用いることができる。Almost all nonionic surfactants have a sugar or polyoxyethylene in the hydrophilic moiety. The nonionic surfactant has a weak protein denaturing effect and is not suitable for denaturing membrane proteins and is suitable for solubilization. Nonionic surfactants include octyl glucoside, decyl glucoside, heptyl thioglucoside, octyl thioglucoside, nonyl thioglucoside, octyl maltoside, dodecyl maltoside, decanoyl-N-methylglucamide,
Triton X45, Triton X114, Nonidet P4
0, Triton X100, Triton X102, Triton X
165, Triton X305, bridge 76, bridge 9
6, bridge 56, bridge 58, Triton N101,
Sucrose monohexanoate, sucrose monolaurate, sorbitol monostearate, sorbitol monolaurate and the like can be used.

【００３２】イオン性界面活性剤には陰イオン性界面活
性剤、陽イオン性界面活性剤、両性界面活性剤がある。
陰イオン性界面活性剤のうち長鎖アルキル基を持つもの
は一般的に界面活性作用が強く抗原調製に適する。The ionic surfactants include anionic surfactants, cationic surfactants and amphoteric surfactants.
Among the anionic surfactants, those having a long-chain alkyl group generally have a strong surfactant action and are suitable for antigen preparation.

【００３３】陰イオン性界面活性剤としてはドデシル硫
酸ナトリウム（ＳＤＳ）、テトラデシル硫酸ナトリウ
ム、ドデシルスルホン酸ナトリウム、テトラデシルスル
ホン酸ナトリウム、コール酸ナトリウム、デオキシコー
ル酸ナトリウム、タウロコール酸ナトリウム、タウロデ
オキシコール酸ナトリウム等を用いることができる。As the anionic surfactant, sodium dodecyl sulfate (SDS), sodium tetradecyl sulfate, sodium dodecyl sulfonate, sodium tetradecyl sulfonate, sodium cholate, sodium deoxycholate, sodium taurocholate, taurodeoxycholic acid. Sodium or the like can be used.

【００３４】陽イオン性界面活性剤としてはセチルトリ
メチルアンモニウムブロミド、テトラジシルトリメチル
アンモニウムクロリド、ドデシルピリジニウムブロミ
ド、セチルピリジニウムクロリド等を用いることができ
る。As the cationic surfactant, cetyltrimethylammonium bromide, tetradicyltrimethylammonium chloride, dodecylpyridinium bromide, cetylpyridinium chloride and the like can be used.

【００３５】両性界面活性剤としては３−［（３−コラ
ミドプロピル）ジメチルアンモニオ］−１−プロパンス
ルホン酸、３−［（コラミドプロピル）ジメチルアンモ
ニオ］−２−ヒドロキシ−１−プロパンスルホン酸等を
用いることができる。Examples of the amphoteric surfactant include 3-[(3-colamidopropyl) dimethylammonio] -1-propanesulfonic acid and 3-[(colamidopropyl) dimethylammonio] -2-hydroxy-1-propane. Sulfonic acid or the like can be used.

【００３６】これら界面活性剤のうち、特に非イオン性
界面活性剤の親水基部分に糖をもつオクチルグルコシ
ド、デシルグルコシド、ヘプチルチオグルコシド、オク
チルチオグルコシド、ノニルチオグルコシド、オクチル
マルトシド、ドデシルマルトシド、デカノイル−Ｎ−メ
チルグルカミドを好適に使用できる。Of these surfactants, octyl glucoside, decyl glucoside, heptyl thioglucoside, octyl thioglucoside, nonyl thioglucoside, octyl maltoside, dodecyl maltoside, among others, which have a sugar in the hydrophilic group of the nonionic surfactant. , Decanoyl-N-methylglucamide can be preferably used.

【００３７】可溶化時の界面活性剤の濃度は該界面活性
剤を水性溶媒に溶解したときにミセルを形成する濃度、
すなわち、臨界ミセル濃度（以下、ＣＭＣとも略記す
る）以上であれば有効である。さらにＣＭＣ以上または
ＣＭＣの１０倍までの濃度で好適に用いられ、特にＣＭ
ＣからＣＭＣの５倍までの濃度で可溶化を実施すると好
適に作用する。界面活性剤には親水基と疎水基の大小と
強弱のバランスを示す値としてＨＬＢ（ｈｙｄｒｏｐｈ
ｉｌｅ−ｌｉｐｏｐｈｉｌｅ ｂａｒａｎｃｅ）があ
る。本発明で使用する界面活性剤はＨＬＢ１０〜１７の
ものを好適に用いることができる。The concentration of the surfactant during solubilization is the concentration at which micelles are formed when the surfactant is dissolved in an aqueous solvent,
That is, it is effective if it is at or above the critical micelle concentration (hereinafter also abbreviated as CMC). Further, it is preferably used at a concentration of CMC or more or up to 10 times the concentration of CMC, especially CM
Solubilization at a concentration of up to 5 times C to CMC works well. For the surfactant, HLB (hydroph) is used as a value indicating the balance between the strength and strength of hydrophilic and hydrophobic groups.
There is an ile-lipophile balance). As the surfactant used in the present invention, those having HLB 10 to 17 can be preferably used.

【００３８】本発明で界面活性剤と共に用いる変性剤と
はタンパク変性を起こす物質であり、タンパク変性と
は、生理条件でとっている固有の１次構造は変化せずに
高次構造のみが破壊されることをいう。従って、変性剤
は水性溶媒に不溶性成分を可溶化する作用がある。変性
剤の具体的な例示をすれば、４Ｍ以上の尿素、２Ｍ以上
の塩酸グアニジンが用いられる。好適にはタンパクが完
全変性する８Ｍ以上の尿素、６Ｍ以上の塩酸グアニジン
が用いられる。The denaturant used in the present invention together with the surfactant is a substance that causes protein denaturation. Protein denaturation means that only the higher-order structure is destroyed without changing the inherent primary structure under physiological conditions. Is said to be done. Therefore, the modifier has a function of solubilizing the insoluble component in the aqueous solvent. As a specific example of the denaturant, 4M or more urea and 2M or more guanidine hydrochloride are used. Preferably, 8 M or more urea and 6 M or more guanidine hydrochloride that completely denature the protein are used.

【００３９】好ましい界面活性剤と変性剤の組合せは、
界面活性剤として非イオン性界面活性剤の親水基部分に
糖をもつものと、変性剤として尿素が特に好適に用いら
れる。例えば、８Ｍ尿素と２％オクチルグルコシド、８
Ｍ尿素と２％オクチルチオグルコシド、８Ｍ尿素と２％
デシルグルコシド、８Ｍ尿素と２％ヘプチルチオグルコ
シドなどが採用できる。A preferred combination of surfactant and modifier is
As the surfactant, those having a sugar in the hydrophilic group portion of the nonionic surfactant and urea as the modifier are particularly preferably used. For example, 8M urea and 2% octyl glucoside, 8
M urea and 2% octyl thioglucoside, 8M urea and 2%
Decyl glucoside, 8M urea and 2% heptyl thioglucoside can be used.

【００４０】界面活性剤又は界面活性剤と変性剤を併用
しての可溶化の条件としては、可溶化温度は１℃から１
００℃、さらに好適には２０℃から６０℃が有効であ
る。可溶化の時間は３０分以上で効果を発揮し、好まし
くは３０分以上２４時間以下で作用させれば効果的であ
る。The solubilization temperature is 1 ° C. to 1 ° C. under the conditions of solubilization using a surfactant or a surfactant and a modifier in combination.
00 ° C., more preferably 20 ° C. to 60 ° C. is effective. The solubilization time is effective for 30 minutes or more, preferably 30 minutes or more and 24 hours or less.

【００４１】界面活性剤又は界面活性剤と変性剤を併用
して可溶化する具体的な方法としては、凍結融解による
破砕、次いで分画操作により得られた不溶性成分に直
接、界面活性剤又は界面活性剤と変性剤を水性溶媒に溶
解した溶液を添加するか、或は、得られた不溶性成分を
水性溶媒に予め懸濁した後に、界面活性剤又は界面活性
剤と変性剤を水性溶媒に溶解した溶液を添加してもよ
い。また添加後、懸濁液を十分に攪拌することで得られ
る可溶性成分の抽出効率は向上する。As a specific method of solubilizing a surfactant or a surfactant and a denaturant in combination, the insoluble component obtained by crushing by freeze-thawing and then fractionation operation is directly added to the surfactant or the interface. Add a solution of the active agent and the modifier in an aqueous solvent, or suspend the obtained insoluble component in the aqueous solvent in advance, and then dissolve the surfactant or the surfactant and the modifier in the aqueous solvent. The above solution may be added. In addition, the extraction efficiency of the soluble component obtained by sufficiently stirring the suspension after the addition is improved.

【００４２】上記の可溶化操作で不溶性成分から水性溶
媒中に溶出された可溶性成分は、残渣であるところの可
溶化後の不溶性成分を除去して分画される。この分画さ
れた可溶性成分には抗原性を有するタンパク、脂質、糖
質、糖タンパク、リポタンパク等の成分が含まれてお
り、純度が高く、特異性が高い、特に初期感染との反応
性が高い抗原として利用することができる。The soluble component eluted from the insoluble component into the aqueous solvent by the above solubilization operation is fractionated by removing the insoluble component after solubilization, which is the residue. The fractionated soluble component contains components such as proteins, lipids, carbohydrates, glycoproteins and lipoproteins having antigenicity, and has high purity and high specificity, especially reactivity with initial infection. Can be used as a high antigen.

【００４３】可溶性成分を分画するための方法として
は、通常、遠心分離を用いる。遠心分離に採用する遠心
力は不溶性成分を沈殿除去できる遠心力であれば良い。
例えば、梅毒トレポネーマ菌の可溶化後の不溶性成分と
可溶性成分の懸濁液より抗原性を有する可溶性成分のみ
を分離するには１０，０００×ｇ、３０分間で十分であ
る。 不溶性成分は抗原性を有するタンパク、脂質、糖
質、糖タンパク、リポタンパク等を溶出させた残渣で、
抗原性はほとんど存在しない。この分画により、最終的
に得られる菌表層抗原の純度、特異性を向上させること
ができる。もし、該分画を行わなければ、得られる菌表
層抗原の純度は著しく低下する。上記のように分画して
得られた可溶性成分の溶液は通常この溶液状態のまま抗
原溶液として用い、必要に応じて生理食塩水、ＰＢＳ等
で希釈して後述の不溶性担体の感作操作に供する。又該
抗原溶液には可溶化に用いた界面活性剤や変性剤を含ん
でいてもよいが、必要ならば透析等で除去すればよい。Centrifugation is usually used as a method for fractionating soluble components. The centrifugal force used for centrifugation may be any centrifugal force capable of removing insoluble components by precipitation.
For example, 10,000 × g, 30 minutes is sufficient to separate only the soluble component having antigenicity from the suspension of the insoluble component and the soluble component after solubilization of Treponema pallidum. The insoluble component is a residue obtained by eluting proteins, lipids, carbohydrates, glycoproteins, lipoproteins, etc. having antigenicity,
There is little antigenicity. By this fractionation, the purity and specificity of the bacterial surface antigen finally obtained can be improved. If the fractionation is not carried out, the purity of the obtained bacterial surface antigen is remarkably reduced. The solution of the soluble component obtained by fractionation as described above is usually used as an antigen solution in this solution state, and may be diluted with physiological saline, PBS or the like, if necessary, for the sensitization operation of the insoluble carrier described below. To serve. The antigen solution may contain the surfactant or denaturant used for solubilization, but if necessary, it may be removed by dialysis or the like.

【００４４】本発明でいう菌表層抗原とは病原性微生物
由来の抗原で、感染症患者の体液中に存在する特異抗体
と抗原抗体反応で反応する抗原を指す。菌表層抗原は上
記菌の感染に起因する疾病の診断用検査試薬の抗原とし
て用いることができる。The bacterial surface antigen referred to in the present invention is an antigen derived from a pathogenic microorganism and refers to an antigen that reacts with a specific antibody present in the body fluid of an infectious disease patient in an antigen-antibody reaction. The bacterium surface antigen can be used as an antigen of a test reagent for diagnosing a disease caused by infection with the bacterium.

【００４５】診断用検査試薬とは抗原抗体反応を応用し
た免疫学的診断薬であり、例えば、マイクロタイター試
薬、ラテックス凝集試薬、免疫比濁試薬、酵素免疫試薬
等が含まれる。本発明によって得られる菌表層抗原は、
特にマイクロタイター試薬、ラテックス凝集試薬などの
抗原抗体反応を凝集反応で捉える試薬の抗原として好適
である。The diagnostic test reagent is an immunological diagnostic reagent to which an antigen-antibody reaction is applied, and includes, for example, a microtiter reagent, a latex agglutination reagent, an immunoturbidimetric reagent, an enzyme immunoreagent and the like. Bacterial surface antigen obtained by the present invention,
In particular, it is suitable as an antigen of a reagent that captures an antigen-antibody reaction by an agglutination reaction such as a microtiter reagent and a latex agglutination reagent.

【００４６】本発明の製造方法によって得られる菌表層
抗原は不溶性担体に担持して検査用試薬を成す。該不溶
性担体粒子としては公知の免疫学的凝集法の診断試薬に
用いることができる担体が制限なく使用できる。例え
ば、核部となる無機質化合物に染料を被覆させた高比重
複合粒子（特開昭６２ー１１５３６６参照、以下、ＨＤ
Ｐとも略記する）、羊赤血球、ポリスチレン粒子、ゼラ
チン粒子等が挙げられる。中でもＨＤＰ及び赤血球は抗
原あるいは抗体の吸着量が多く好適に用いられる。さら
に人工担体であるところのＨＤＰは表面官能基を人工的
に付加することができ、抗原あるいは抗体の種類に応じ
て表面状態を変化させることができるので免疫学的凝集
法の感染症検査試薬の担体粒子としてはさらに好適に用
いることができる。The bacterial surface antigen obtained by the production method of the present invention is carried on an insoluble carrier to form a test reagent. As the insoluble carrier particles, carriers that can be used for a diagnostic reagent of a known immunological agglutination method can be used without limitation. For example, high specific gravity composite particles obtained by coating a dye on a core inorganic compound (see JP-A-62-115366, hereinafter HD
Abbreviated as P), sheep red blood cells, polystyrene particles, gelatin particles and the like. Among them, HDP and erythrocytes are suitable because they have a large amount of antigen or antibody adsorption. Furthermore, HDP, which is an artificial carrier, can be artificially added with a surface functional group, and its surface state can be changed depending on the type of antigen or antibody. The carrier particles can be more preferably used.

【００４７】また、不溶性担体の粒子径も免疫学的凝集
法診断試薬として用いうる公知の範囲のものならば特に
制限されないが、通常、粒子径０．０１μｍから３μｍ
までのものが好適に用いられる。The particle size of the insoluble carrier is not particularly limited as long as it is within a known range that can be used as a diagnostic reagent for immunological agglutination method, but usually the particle size is 0.01 μm to 3 μm.
Those up to are preferably used.

【００４８】不溶性担体の比重は１．０以上であればよ
いが、特に沈降反応を利用したマイクロタイター試薬用
担体粒子は比重が大きい程、沈降速度が速く判定時間が
短縮化できるので１．５以上のものが好適に用いられ
る。The specific gravity of the insoluble carrier may be 1.0 or more. Especially, the larger the specific gravity of the carrier particles for microtiter reagent utilizing the precipitation reaction, the faster the sedimentation speed and the shorter the determination time. The above is preferably used.

【００４９】本発明でいう担持とは、不溶性担体に感作
物質としての抗原剤等を吸着させることである。具体的
な担持方法としては、感作物質を溶解した緩衝溶液中に
不溶性担体を分散、混合して感作物質を不溶性担体上に
担持したのち余剰の感作物質を遠心洗浄で除去し次いで
ブロッキング剤を同様にして担持する方法が用いられ
る。The carrying in the present invention means that an antigen agent or the like as a sensitizing substance is adsorbed on an insoluble carrier. As a specific supporting method, an insoluble carrier is dispersed and mixed in a buffer solution in which the sensitizing substance is dissolved, and the sensitizing substance is supported on the insoluble carrier, and then excess sensitizing substance is removed by centrifugal washing and then blocked. A method of supporting the agent in the same manner is used.

【００５０】不溶性担体に感作物質を担持する方法とし
ては疎水吸着法等の物理的吸着法、塩化クロム法等の化
学的吸着法等の公知の方法が採用されが、感作物質は不
溶性担体上に担持されたのち被検体中の測定目的物質と
抗原抗体反応により結合をするため感作物質はできるだ
け穏やかな条件で担持することが必要であり、そのため
疎水的吸着法が特に好適に用いられる。As a method for supporting the sensitizing substance on the insoluble carrier, a known method such as a physical adsorption method such as a hydrophobic adsorption method or a chemical adsorption method such as a chromium chloride method is adopted. The sensitizer must be supported under mild conditions as much as possible because it binds to the measurement target substance in the analyte by the antigen-antibody reaction after being supported on it, and therefore the hydrophobic adsorption method is particularly preferably used. .

【００５１】上記疎水的吸着法による担持は、緩衝作用
のある緩衝液中に不溶性担体と吸着物質を分散させた状
態で行う。該緩衝液は燐酸緩衝液、グリシン緩衝液、ト
リス緩衝液、酢酸緩衝液等など緩衝作用のあるものなら
ば制限なく用いることができる。緩衝液のｐＨについて
は担持させる吸着物質によりそれぞれ最適な範囲を選択
して採用するが、一般に中性領域ｐＨ６．０〜ｐＨ８．
０が望ましい。不溶性担体に吸着物質を担持させる時間
は吸着物質が均一的に不溶性担体表面に担持されるのに
十分な時間であればよいが、例えば梅毒抗原をＨＤＰの
疎水吸着法で行う場合は３０分間以上で十分である。さ
らに温度に関しても担持する吸着物質が熱変性を受けな
い範囲即ち１℃以上８０℃以下が採用される。The loading by the above-mentioned hydrophobic adsorption method is carried out in a state where the insoluble carrier and the adsorbent are dispersed in a buffer solution having a buffering action. The buffer solution can be used without limitation as long as it has a buffering action such as a phosphate buffer solution, a glycine buffer solution, a Tris buffer solution, an acetate buffer solution and the like. Regarding the pH of the buffer solution, an optimum range is selected and adopted depending on the adsorbing substance to be supported, but generally, the neutral range pH 6.0 to pH 8.
0 is desirable. The time for supporting the adsorbent on the insoluble carrier may be a time sufficient to allow the adsorbent to be uniformly supported on the surface of the insoluble carrier. For example, when the syphilis antigen is subjected to the HDP hydrophobic adsorption method, it is 30 minutes or more. Is enough. Further, regarding the temperature, a range in which the adsorbed material to be supported is not subjected to thermal denaturation, that is, 1 ° C. or more and 80 ° C. or less is adopted.

【００５２】さらに不溶性担体へのブロッキング剤の担
持も上述の感作物質の担持と同様の方法を採用すること
ができる。The blocking agent may be carried on the insoluble carrier by the same method as that for carrying the sensitizing substance.

【００５３】[0053]

【実施例】以下、実施例により本発明を具体的に説明す
る。ただし、これらの実施例により本発明の技術的範囲
が限定されるものではない。The present invention will be described below in detail with reference to examples. However, the technical scope of the present invention is not limited by these examples.

【００５４】参考例１ タンパク濃度の定量方法 タンパク濃度の定量方法はローリー法（Ｌｏｗｒｙ ｅ
ｔ ａｌ，Ｊ．Ｂｉｏ．Ｃｈｅｍ．，Ｖｏｌ．１９３，
ｐｐ２６５−２７５（１９５１））で行った。すなわ
ち、タンパク量５〜１００μｇを含む溶液０．２ｍｌを
試験管にとり、炭酸ナトリウム−アルカリ性銅試薬１ｍ
ｌを加えて混合した。室温で１０分間放置後、水で２倍
に希釈したフェノール試薬を０．１ｍｌを加えて直ちに
混和し、３０分間放置後、７５０ｎｍでの吸光度を測定
した。標準曲線は牛血清アルブミンを用いて作成した。Reference Example 1 Method for Quantifying Protein Concentration The method for quantifying protein concentration was the Lowry method.
t al, J .; Bio. Chem. , Vol. 193
pp265-275 (1951)). That is, 0.2 ml of a solution containing 5 to 100 μg of protein was placed in a test tube, and 1 m of sodium carbonate-alkaline copper reagent
1 was added and mixed. After standing at room temperature for 10 minutes, 0.1 ml of a phenol reagent diluted 2-fold with water was added and immediately mixed, and after standing for 30 minutes, the absorbance at 750 nm was measured. The standard curve was prepared using bovine serum albumin.

【００５５】参考例２ 抗原価の測定方法 抗原性の評価はドットブロット法で行った。測定手順は
以下の通り。Reference Example 2 Method for Measuring Antigen Titer The antigenicity was evaluated by the dot blot method. The measurement procedure is as follows.

【００５６】（１） 被検体である抗原液をＰＢＳで
１，２，５，１０，２０，５０，１００，２００，５０
０倍に希釈してニトロセルロースフィルター（ミリポア
社製、HAHY00010）上に２μｌずつスポットした（２枚
用意する）。(1) The antigen solution which is the subject is 1, 2, 5, 10, 20, 50, 100, 200, 50 in PBS
It was diluted 0 times and spotted in 2 μl portions on a nitrocellulose filter (HAHY00010, manufactured by Millipore) (two sheets are prepared).

【００５７】（２） フィルターを風乾したのち、１０
％スキムミルクを０．１５Ｍ塩化ナトリウム含有０．１
Ｍトリス緩衝液，ｐＨ７．５（以下、ＴＢＳと略記す
る）に溶解したブロッキング溶液に浸し、３７℃、１時
間ブロッキングを行った。(2) After air-drying the filter, 10
% Skim milk containing 0.15 M sodium chloride 0.1
It was immersed in a blocking solution dissolved in M Tris buffer, pH 7.5 (hereinafter abbreviated as TBS), and blocked at 37 ° C. for 1 hour.

【００５８】（３） １次抗体として健常者血清、及び
患者血清をＴＢＳでそれぞれ２５倍希釈して血清希釈液
を作製し、これにブロッキングをしたニトロセルロース
を１枚ずつ浸し、３７℃、１時間反応させた。(3) Serum of healthy subjects and serum of patients were respectively diluted 25 times with TBS as primary antibodies to prepare serum diluted solutions, and one block of blocked nitrocellulose was soaked in each of the diluted solutions at 37 ° C and 1 ° C. Reacted for hours.

【００５９】（４） （３）のニトロセルロースフィル
ターを０．１％トゥイーン８０（ｔｗｅｅｎ８０）含有
ＴＢＳで３回洗浄した。(4) The nitrocellulose filter of (3) was washed 3 times with TBS containing 0.1% tween 80.

【００６０】（５） ２次抗体としてアビジン標識抗ヒ
トＩｇＧ（フナコシ社製）とアビジン標識抗ヒトＩｇＭ
（フナコシ社製）をそれぞれＴＢＳで４００倍希釈して
２次抗体液を調製し（４）の洗浄済みニトロセルロース
フィルターを浸して３７℃、１時間反応させた。(5) Avidin-labeled anti-human IgG (Funakoshi) and avidin-labeled anti-human IgM as secondary antibodies
(Manufactured by Funakoshi Co., Ltd.) was diluted 400 times with TBS to prepare a secondary antibody solution, and the washed nitrocellulose filter of (4) was dipped and reacted at 37 ° C. for 1 hour.

【００６１】（６） （４）と同様に洗浄操作を行っ
た。(6) A washing operation was performed in the same manner as (4).

【００６２】（７） 酵素標識としてビオチン化パーオ
キシダーゼをＴＢＳで２００倍希釈して（６）の洗浄済
みニトロセルロースフィルターを浸して３７℃、３０分
間反応させた。(7) Biotinylated peroxidase as an enzyme label was diluted 200-fold with TBS, and the washed nitrocellulose filter of (6) was dipped and reacted at 37 ° C. for 30 minutes.

【００６３】（８） （４）と同様に洗浄を行った。(8) Washing was performed in the same manner as (4).

【００６４】（９） ０．１％ジアミノベンチジン水溶
液に３０％過酸化水素液を１／２０添加しニトロセルロ
ースフィルターに作用させ発色させた。１次抗体に患者
血清を用いたニトロセルロースフィルターにおいて発色
の確認できた抗原液の最高希釈倍率を抗原価とし、抗原
性の強さの指標とした。なお、１次抗体で健常者血清を
用いたニトロセルロースフィルターにおいて発色した最
高希釈倍率を対照値とし、夾雑物による非特異反応の程
度を表す指標とした。(9) 1/20 of a 30% hydrogen peroxide solution was added to a 0.1% diaminobenzidine aqueous solution to act on a nitrocellulose filter for color development. The highest dilution ratio of the antigen solution whose color was confirmed in the nitrocellulose filter using patient serum as the primary antibody was defined as the antigen titer, which was used as an index of the antigenicity. In addition, the highest dilution ratio that developed the color in a nitrocellulose filter using the serum of a healthy person with the primary antibody was used as a control value, and was used as an index showing the degree of nonspecific reaction due to contaminants.

【００６５】実施例１ 凍結融解法により菌を破砕した
ときの梅毒検査試薬用抗原の製造、及び梅毒検査試薬の
製造、評価（１） （１）ＴＰ菌の培養 ＴＰ菌の培養は以下のようなミラーらの方法（Ｍｉｌｌ
ｅｒ ｅｔ ａｌ．，Ｖｏｌ．９６，ｐ４５０（１９６
６））に従って、培養集菌を行った。すなわち、家兎睾
丸にＴＰ菌を１１〜１４日間、接種し増殖させ、家兎睾
丸をハサミで切断して生理食塩水で懸濁してＴＰ菌を抽
出した。抽出液を２００×ｇで１５分間遠心して睾丸組
織の大きな破片を沈澱除去した。続いて、１９，０００
×ｇで９０分間遠心してＴＰ菌を沈澱させ、このＴＰ菌
を生理食塩水の懸濁したものをＴＰ菌の菌懸濁液とし
た。Example 1 Production of antigen for syphilis test reagent when strains were crushed by freeze-thaw method, and production and evaluation of syphilis test reagent (1) (1) Cultivation of TP bacterium Cultivation of TP bacterium was as follows. Method of Miller et al. (Mill
er et al. , Vol. 96, p450 (196
According to 6)), the culture was collected. That is, the rabbit testis was inoculated with the TP bacterium for 11 to 14 days to grow, and the rabbit testis was cut with scissors and suspended in physiological saline to extract the TP bacterium. The extract was centrifuged at 200 xg for 15 minutes to precipitate out large debris of testicular tissue. Then, 19,000
The TP bacterium was precipitated by centrifugation at × g for 90 minutes, and the TP bacterium was suspended in physiological saline to obtain a TP bacterium suspension.

【００６６】（２）凍結融解によるＴＰ菌の破砕 上記ＴＰ菌の菌数を細菌計数盤（Ｃ．Ａ．Ｈａｕｓｓｅ
ｒ＆Ｓｏｎ社製細菌計数器）、暗視野顕微鏡にて測定し
たところ、３×１０⁹細胞／ｍｌであった。ＴＰ菌液１
ｍｌをスピッツ管（栄研機材）に入れた。続いて、上記
スピッツ管を液体窒素に３０秒間接触させて凍結したの
ち、３７℃恒温水槽に３分間入れて融解した。この操作
を３回繰り返して凍結融解によりＴＰ菌を破砕し、破砕
物懸濁液を得た。(2) Crushing of TP bacterium by freeze-thawing The number of the above-mentioned TP bacterium was determined by a bacterial counter (CA Hausse).
It was 3 × 10 ⁹ cells / ml when measured with a dark field microscope (a bacterial counter manufactured by r & Son). TP bacterium liquid 1
ml was put in a Spitz tube (Eiken Equipment). Subsequently, the Spitz tube was brought into contact with liquid nitrogen for 30 seconds to freeze it, and then put in a 37 ° C. constant temperature water bath for 3 minutes to melt. This operation was repeated 3 times to crush the TP bacterium by freeze-thawing to obtain a crushed product suspension.

【００６７】（３）遠心分離による不溶性成分の分画 上記破砕物懸濁液に１０，０００×ｇ、３０分間の遠心
分離を施し、夾雑物質を含む可溶性成分と分画してＴＰ
菌を含む不溶性成分を得た。(3) Fractionation of Insoluble Components by Centrifugation The suspension of the crushed material was centrifuged at 10,000 × g for 30 minutes to fractionate soluble components containing contaminants to TP.
An insoluble component containing bacteria was obtained.

【００６８】（４）遠心洗浄による不溶性成分の洗浄 この不溶性成分にＰＢＳ、２ｍｌを添加して１０，００
０×ｇ、３０分間遠心分離を行い、ＴＰ菌含有可溶性成
分を遠心洗浄した。この遠心洗浄操作を３回繰り返しの
ち、洗浄済み不溶性成分とした。 （５）界面活性剤のよる不溶性成分の可溶化 上記洗浄済み不溶性成分にＰＢＳに溶解した１％オクチ
ルグルコシドを２ｍｌ添加して３７℃、１時間可溶化を
行った。可溶化後、１０，０００×ｇ、３０分間遠心分
離を行い、遠心上清の可溶性成分２ｍｌを梅毒検査試薬
用抗原として得た。(4) Washing of insoluble component by centrifugal washing PBS (2 ml) was added to this insoluble component to obtain 10,000
Centrifugation was performed at 0 × g for 30 minutes, and the soluble components containing TP bacteria were washed by centrifugation. This centrifugal washing operation was repeated 3 times to obtain a washed insoluble component. (5) Solubilization of insoluble components with a surfactant 2 ml of 1% octyl glucoside dissolved in PBS was added to the washed insoluble components to solubilize them for 1 hour at 37 ° C. After solubilization, centrifugation was performed at 10,000 × g for 30 minutes, and 2 ml of the soluble component of the centrifugation supernatant was obtained as an antigen for a syphilis test reagent.

【００６９】（６）抗原の評価結果 参考例１に従いタンパク定量を行ったところ、０．７ｍ
ｇ／ｍｌであった。さらに、参考例２に従って抗原価を
測定したところ、抗原価は１００倍、対照値は１倍であ
った。抗原性の指標である抗原価は１００倍と高く、一
方、非特異凝集の指標となる対照値は１倍と低いことよ
り、特異性が高く、不純物を含まない抗原が得られたと
判断できる。(6) Evaluation result of antigen When the protein was quantified according to Reference Example 1, it was 0.7 m.
It was g / ml. Furthermore, when the antigen titer was measured according to Reference Example 2, the antigen titer was 100 times and the control value was 1 time. Since the antigen titer, which is an index of antigenicity, is as high as 100 times, and the control value, which is an index of non-specific aggregation, is as low as 1 time, it can be judged that an antigen having high specificity and containing no impurities was obtained.

【００７０】（７）感作 直径１．８μｍのＨＤＰ（(株)トクヤマ製品）をＰＢＳ
で２．５（ｗ／ｗ）％になるように懸濁し、ＨＤＰ懸濁
液とした。上記実施例３、４、及び比較例１で調製した
３種類の梅毒検査試薬用抗原を各１０倍希釈して抗原溶
液とした。上記ＨＤＰ懸濁液１ｍｌと抗原溶液１ｍｌを
試験管内で混合して室温で１時間放置してＨＤＰ表面に
疎水的に担持させた（以下、この吸着操作を感作とも略
記する）。(7) Sensitization HDP having a diameter of 1.8 μm (manufactured by Tokuyama Corporation) was used in PBS.
Was suspended at 2.5 (w / w)% to obtain an HDP suspension. Each of the three types of syphilis test reagent antigens prepared in Examples 3 and 4 and Comparative Example 1 was diluted 10 times to prepare an antigen solution. The HDP suspension (1 ml) and the antigen solution (1 ml) were mixed in a test tube and left at room temperature for 1 hour to be hydrophobically supported on the HDP surface (hereinafter, this adsorption operation is also referred to as sensitization).

【００７１】（８）洗浄操作 その後、余剰の梅毒検査試薬用抗原を除去するために、
上記混合液に２，５００ｒｐｍ、５分間遠心分離を施
し、遠心上清を除去した。その遠心沈澱物に洗浄のた
め、ＰＢＳ２ｍｌを添加、懸濁後２，５００ｒｐｍ、５
分間遠心後上清を除去した。上記梅毒検査試薬用抗原を
吸着させたＨＤＰをＴＰ抗原感作粒子とした。(8) Washing operation After that, in order to remove the excess antigen for syphilis test reagent,
The above mixed solution was centrifuged at 2,500 rpm for 5 minutes to remove the centrifugal supernatant. To the centrifugal precipitate, 2 ml of PBS was added for washing, and after suspension, 2,500 rpm, 5
After centrifugation for a minute, the supernatant was removed. HDP having the antigen for a syphilis test reagent adsorbed thereon was used as a TP antigen-sensitized particle.

【００７２】（９）ブロッキング操作 ＴＰ抗原感作粒子（５０ｍｇ）に１％牛血清アルブミン
（以下、ＢＳＡとも略記する）、２ｍｌを添加して３７
℃で３時間ブロッキングを行った。ブロッキング後、上
記懸濁液に２，５００ｒｐｍ、５分間遠心分離を施し、
遠心上清を除去した。上記操作でブロッキング操作を完
了し、梅毒検査試薬を調製した。この梅毒検査試薬を３
（ｖｏｌ／ｖｏｌ）％正常ウサギ血清含有ＰＢＳ（以
下、Ａ液とも略記する）に０．５（ｗ／ｖｏｌ）％にな
るように懸濁して梅毒検査試薬懸濁液として以下の測定
操作に供した。(9) Blocking Operation To TP antigen-sensitized particles (50 mg), 2 ml of 1% bovine serum albumin (hereinafter also abbreviated as BSA) was added and 37
Blocking was performed at 3 ° C for 3 hours. After blocking, the suspension is centrifuged at 2,500 rpm for 5 minutes,
The centrifugation supernatant was removed. The blocking operation was completed by the above operation, and a syphilis test reagent was prepared. This syphilis test reagent 3
Suspended in (vol / vol)% normal rabbit serum-containing PBS (hereinafter also abbreviated as A solution) to 0.5 (w / vol)%, and used as the syphilis test reagent suspension in the following measurement operation. did.

【００７３】（１０）測定操作 一方、検査に用いる検体をＡ液で５倍より順次倍数希釈
した。次に、検体の希釈液を９６穴マイクロタイタープ
レートに各々２５μｌずつ１穴から１２穴まで滴下し
た。ついで、上記で梅毒検査試薬懸濁液を各穴２５μｌ
を滴下した。滴下後、プレートミキサーで振とうして３
０分間静置したのち、管底凝集像を観察した。(10) Measurement Operation On the other hand, the specimen used for the test was serially diluted with the solution A from 5 times. Next, 25 μl each of the diluted solution of the sample was dropped from 1 to 12 holes on a 96-well microtiter plate. Then, add 25 μl of the syphilis test reagent suspension to each hole.
Was dripped. After dripping, shake with plate mixer 3
After standing still for 0 minutes, a tube bottom aggregation image was observed.

【００７４】（１１）梅毒検査試薬の評価結果 上記のように調製した梅毒検査試薬の性能評価を健常者
検体１検体、梅毒患者検体３検体、Ａ、Ｂ、Ｃを用いて
行った。結果を表２に示した。健常者検体は２０倍希釈
でも陽性像が検出されなかったのに対して梅毒患者検体
では陽性検体Ａで１６０倍希釈、陽性検体Ｂで３２０倍
希釈、陽性検体Ｃで１２８０倍希釈まで陽性像が検出で
きた。ドットブロット法による抗原の評価同様、試薬化
の検討でも健常者検体で凝集反応を示さず、梅毒患者検
体で高希釈倍率でも凝集反応を示すことより、特異性が
高い抗原が調製できたことが証明できた。(11) Evaluation results of syphilis test reagent The performance of the syphilis test reagent prepared as described above was evaluated using 1 healthy subject sample, 3 syphilis patient samples, A, B and C. The results are shown in Table 2. No positive image was detected in the 20-fold diluted sample of healthy subjects, whereas 160-fold diluted sample of positive sample A, 320-fold diluted sample of positive specimen B, and 1280-fold diluted sample of positive sample C in syphilis patients. I was able to detect it. Similar to the evaluation of the antigen by the dot blot method, it was possible to prepare an antigen with high specificity by showing no agglutination reaction in healthy subject samples in the examination of reagentization and agglutination reaction in syphilis patient samples even at a high dilution ratio. I was able to prove it.

【００７５】実施例２ 凍結融解法により菌破砕したと
きの梅毒検査試薬用抗原の製造、及び梅毒検査試薬の製
造、評価（２） （１）ＴＰ菌の培養、（２）凍結融解によるＴＰ菌の破
砕、（３）遠心分離による不溶性成分の分画、（４）遠
心洗浄による不溶性成分の洗浄は実施例１と同様。Example 2 Production of antigen for syphilis test reagent when crushed by freeze-thaw method, and production and evaluation of syphilis test reagent (2) (1) Culture of TP bacterium, (2) TP bacterium by freeze-thawing Crushing, (3) fractionation of insoluble components by centrifugation, and (4) washing of insoluble components by centrifugal washing are the same as in Example 1.

【００７６】（５）界面活性剤のよる不溶性成分の可溶
化 上記洗浄済み不溶性成分にＰＢＳに溶解した１％ヘプチ
ルチオグルコシドを２ｍｌ添加して３７℃、１時間可溶
化を行った。可溶化後、１０，０００×ｇ、３０分間遠
心分離を行い、遠心上清の可溶性成分２ｍｌを梅毒検査
試薬用抗原を得た。(5) Solubilization of insoluble components with a surfactant 2 ml of 1% heptylthioglucoside dissolved in PBS was added to the washed insoluble components to solubilize them at 37 ° C for 1 hour. After solubilization, centrifugation was performed at 10,000 × g for 30 minutes, and 2 ml of the soluble component of the centrifugation supernatant was obtained as an antigen for a syphilis test reagent.

【００７７】（６）抗原の評価結果 参考例１に従いタンパク定量を行ったところ、０．８ｍ
ｇ／ｍｌであった。さらに、参考例２に従って抗原価を
測定したところ、抗原価は１００倍、対照値は１倍であ
った。実施例１と同様に抗原性の指標である抗原価は１
００倍と高く、一方、非特異反応の指標となる対照値は
１倍と低いことより、特異性が高く、不純物を含まない
抗原が得られたと判断できる。(6) Evaluation result of antigen When the protein was quantified according to Reference Example 1, it was 0.8 m.
It was g / ml. Furthermore, when the antigen titer was measured according to Reference Example 2, the antigen titer was 100 times and the control value was 1 time. The antigen titer, which is an index of antigenicity, is 1 as in Example 1.
Since it is as high as 00 times and the control value as an index of non-specific reaction is as low as 1 time, it can be judged that an antigen having high specificity and containing no impurities was obtained.

【００７８】（７）感作、（８）洗浄操作、（９）ブロ
ッキング操作、（１０）測定操作は実施例１と同様。(7) Sensitization, (8) Washing operation, (9) Blocking operation, (10) Measuring operation are the same as in Example 1.

【００７９】（１１）梅毒検査試薬の評価結果 上記のように調製した梅毒検査試薬の性能評価を健常者
検体１検体、梅毒患者検体３検体、Ａ、Ｂ、Ｃを用いて
行った。結果を表２に示した。健常者検体は２０倍希釈
でも陽性像が検出されなかったのに対して梅毒患者検体
では陽性検体Ａで１６０倍希釈、陽性検体Ｂで３２０倍
希釈、陽性検体Ｃで１２８０倍希釈まで陽性像が検出で
きた。実施例１と同様、試薬化の検討でも健常者検体で
凝集反応を示さず、梅毒患者検体で高希釈倍率でも凝集
反応を示すことより、特異性が高い抗原が調製できたこ
とが証明できた。(11) Evaluation result of syphilis test reagent The performance of the syphilis test reagent prepared as described above was evaluated using 1 healthy subject sample, 3 syphilis patient samples, A, B and C. The results are shown in Table 2. No positive image was detected in the 20-fold diluted sample of healthy subjects, whereas 160-fold diluted sample of positive sample A, 320-fold diluted sample of positive specimen B, and 1280-fold diluted sample of positive sample C in syphilis patients. I was able to detect it. As in Example 1, it was proved that an antigen with high specificity could be prepared by the fact that no agglutination reaction was observed in the healthy subject sample in the examination of reagentization and the agglutination reaction was exhibited in the syphilis patient sample even at a high dilution ratio. .

【００８０】比較例１ 超音波処理により菌破砕した時
の梅毒検査試薬用抗原の製造、及び梅毒検査試薬の製
造、評価 （１）ＴＰ菌の培養 実施例１と同様である。Comparative Example 1 Production of Antigen for Syphilis Test Reagent When Microbial Crushed by Ultrasonication, and Production and Evaluation of Syphilis Test Reagent (1) Cultivation of TP Bacteria The same as in Example 1.

【００８１】（２）超音波処理によるＴＰ菌の破砕 ＴＰ菌の菌数を細菌計数盤（Ｃ．Ａ．Ｈａｕｓｓｅｒ＆
Ｓｏｎ社製細菌計数器）、暗視野顕微鏡にて測定したと
ころ、３×１０⁹細胞／ｍｌであった。ＴＰ菌液１ｍｌ
を循環冷却しながら超音波処理（クボタ社製、インソナ
ーＭ２０１、２００Ｗ、１０分間）により破砕し、破砕
物懸濁液を得た。(2) Crushing of TP bacterium by ultrasonic treatment The number of TP bacterium was determined by a bacterial counter (CA Hausser &
It was 3 × 10 ⁹ cells / ml when measured with a dark field microscope (Son Bacteria Counter). TP bacterial solution 1 ml
Was crushed by ultrasonic treatment (Insonar M201, 200W, manufactured by Kubota Co., Ltd., 10 minutes) while being circulated and cooled to obtain a crushed product suspension.

【００８２】（３）遠心分離による不溶性成分の分画、
（４）遠心洗浄による不溶性成分の洗浄、（５）界面活
性剤のよる不溶性成分の可溶化は実施例１と同様であ
る。(3) Fractionation of insoluble components by centrifugation
(4) The washing of the insoluble component by centrifugal washing and (5) the solubilization of the insoluble component by the surfactant are the same as in Example 1.

【００８３】（６）抗原の評価結果 参考例１に従いタンパク定量を行ったところ、０．５ｍ
ｇ／ｍｌであった。さらに、参考例２に従って抗原価を
測定したところ、抗原価は５０倍、対照値は１倍であっ
た。実施例１と比べ、抗原性の指標である抗原価が５０
倍と低く超音波処理により菌の破砕を行うと得られる抗
原の抗原性が低下する。(6) Evaluation result of antigen When the protein was quantified according to Reference Example 1, it was 0.5 m.
It was g / ml. Furthermore, when the antigen titer was measured according to Reference Example 2, the antigen titer was 50 times and the control value was 1 time. Compared to Example 1, the antigenicity, which is an index of antigenicity, was 50.
When the bacteria are disrupted by sonication, the antigenicity of the obtained antigen decreases.

【００８４】（７）感作、（８）洗浄操作、（９）ブロ
ッキング操作、（１０）測定操作は実施例１と同様。(7) Sensitization, (8) Washing operation, (9) Blocking operation, (10) Measuring operation were the same as in Example 1.

【００８５】（１１）梅毒検査試薬の評価結果 上記のように調製した梅毒検査試薬の性能評価を健常者
検体１検体、梅毒患者検体３検体、Ａ、Ｂ、Ｃを用いて
行った。結果を表２に示した。健常者検体は２０倍希釈
でも陽性像が検出されなかったのに対して梅毒患者検体
では陽性検体Ａで４０倍希釈、陽性検体Ｂで１６０倍希
釈、陽性検体Ｃで３２０倍希釈までしか陽性像が検出で
きなかった。ドットブロット法の評価と同様、実施例１
よりも調製した試薬の感度が低いことが判る。(11) Evaluation results of syphilis test reagent The performance of the syphilis test reagent prepared as described above was evaluated using 1 healthy subject sample, 3 syphilis patient samples, A, B and C. The results are shown in Table 2. No positive image was detected in the 20-fold diluted sample of healthy subjects, whereas in the syphilis patient sample, the 40-fold diluted positive sample A, 160-fold diluted positive sample B, and 320-fold diluted positive sample C Could not be detected. Similar to the dot blot method evaluation, Example 1
It can be seen that the sensitivity of the prepared reagent is lower than that of the above.

【００８６】比較例２ 菌破砕を実施しないときの梅毒
検査試薬用抗原の製造、及び梅毒検査試薬の製造、評価 （１）ＴＰ菌の培養は実施例１と同様である。Comparative Example 2 Production of antigen for syphilis test reagent without crushing of bacterium, and production and evaluation of syphilis test reagent (1) Culture of TP bacterium is the same as in Example 1.

【００８７】（２）ＴＰ菌計数 上記ＴＰ菌の菌数を細菌計数盤（Ｃ．Ａ．Ｈａｕｓｓｅ
ｒ＆Ｓｏｎ社製細菌計数器）、暗視野顕微鏡にて測定し
たところ、３×１０⁹細胞／ｍｌであった。ＴＰ菌液１
ｍｌをスピッツ管（栄研機材）に入れた。(2) Counting of TP bacteria The number of the above-mentioned TP bacteria was calculated by using a bacteria counting board (CA Hausse).
It was 3 × 10 ⁹ cells / ml when measured with a dark field microscope (a bacterial counter manufactured by r & Son). TP bacterium liquid 1
ml was put in a Spitz tube (Eiken Equipment).

【００８８】（３）遠心分離による不溶性成分の分画 上記ＴＰ菌液に１０，０００×ｇ、３０分間の遠心分離
を施し、夾雑物質を含む可溶性成分と分画してＴＰ菌を
含む不溶性成分を得た。(3) Fractionation of Insoluble Components by Centrifugation The above TP bacterium solution was subjected to centrifugation at 10,000 × g for 30 minutes to fractionate soluble components containing contaminants and insoluble components containing TP bacteria. Got

【００８９】（４）遠心洗浄による不溶性成分の洗浄、
（５）界面活性剤のよる不溶性成分の可溶化は実施例１
と同様。(4) Washing of insoluble components by centrifugal washing,
(5) The solubilization of the insoluble component by the surfactant was carried out in Example 1.
same as.

【００９０】（６）抗原の評価結果 参考例１に従いタンパク定量を行ったところ、０．９ｍ
ｇ／ｍｌであった。さらに、参考例２に従って抗原価を
測定したところ、抗原価は２０倍、対照値は５倍であっ
た。ＴＰ菌の破砕方法として実施例１の凍結融解を用い
て調製した梅毒検査試薬用抗原より破砕方法として超音
波処理を行った比較例１の梅毒検査試薬用抗原の方が抗
原性が低かった。(6) Antigen evaluation results When the protein was quantified according to Reference Example 1, it was 0.9 m.
It was g / ml. Furthermore, when the antigen titer was measured according to Reference Example 2, the antigen titer was 20 times and the control value was 5 times. The antigen for syphilis test reagent of Comparative Example 1, which was subjected to ultrasonic treatment as a disruption method, was lower in antigenicity than the antigen for syphilis test reagent prepared by freeze-thawing of Example 1 as a method for crushing TP bacteria.

【００９１】（７）感作、（８）洗浄操作、（９）ブロ
ッキング操作、（１０）測定操作は実施例１と同様。(7) Sensitization, (8) Washing operation, (9) Blocking operation, (10) Measuring operation were the same as in Example 1.

【００９２】（１１）梅毒検査試薬の評価結果 上記のように調製した梅毒検査試薬の性能評価を健常者
検体１検体、梅毒患者検体３検体、Ａ、Ｂ、Ｃを用いて
行った。結果を表２に示した。健常者検体では４０倍希
釈、梅毒患者検体では陽性検体Ａで８０倍希釈、陽性検
体Ｂで１６０倍希釈、陽性検体Ｃで３２０倍希釈までし
か陽性像が検出できなかった。実施例１、２より試薬感
度が低いことが判る。さらに夾雑物の混入で非特異凝集
反応も強くなっていることが判る。(11) Evaluation results of syphilis test reagent The performance of the syphilis test reagent prepared as described above was evaluated using 1 healthy subject sample, 3 syphilis patient samples, A, B and C. The results are shown in Table 2. The positive image could be detected only up to 40 times in the healthy sample, 80 times in the positive sample A in the syphilis patient sample, 160 times in the positive sample B, and 320 times in the positive sample C. It can be seen that the reagent sensitivity is lower than in Examples 1 and 2. Furthermore, it can be seen that the nonspecific agglutination reaction is strengthened due to the inclusion of contaminants.

【００９３】比較例３ 凍結融解法により菌破砕したの
ち遠心分離による分画操作を実施しなかった場合の梅毒
検査試薬用抗原の製造、及び梅毒検査試薬の製造、評価 （１）ＴＰ菌の培養、（２）凍結融解によるＴＰ菌の破
砕は実施例１と同様。Comparative Example 3 Production of antigen for syphilis test reagent when crushing the bacterium by freeze-thaw method and then performing no fractionation operation by centrifugation, and production and evaluation of syphilis test reagent (1) Culture of TP bacterium (2) The disruption of TP bacteria by freeze-thawing is the same as in Example 1.

【００９４】（３）界面活性剤のよる不溶性成分の可溶
化も実施例１と同様。(3) The solubilization of the insoluble component by the surfactant is the same as in Example 1.

【００９５】（４）抗原の評価結果 参考例１に従いタンパク定量を行ったところ、２．０ｍ
ｇ／ｍｌであった。さらに、参考例２に従って抗原価を
測定したところ、抗原価は１００倍、対照値は２０倍で
あった。対象値が高いことより非特異的反応が実施例１
と比べ高い。(4) Results of evaluation of antigen When the protein was quantified according to Reference Example 1, it was 2.0 m.
It was g / ml. Furthermore, when the antigen titer was measured according to Reference Example 2, the antigen titer was 100 times and the control value was 20 times. Since the target value is high, the non-specific reaction is Example 1
Higher than

【００９６】（５）感作、（６）洗浄操作、（７）ブロ
ッキング操作、（８）測定操作は実施例１と同様。(5) Sensitization, (6) Washing operation, (7) Blocking operation, (8) Measuring operation were the same as in Example 1.

【００９７】（９）梅毒検査試薬の評価結果 上記のように調製した梅毒検査試薬の性能評価を健常者
検体１検体、梅毒患者検体３検体、Ａ、Ｂ、Ｃを用いて
行った。結果を表２に示した。健常者検体は８０倍希釈
まで陽性像が検出された。梅毒患者検体では陽性検体Ａ
で１６０倍希釈、陽性検体Ｂで６４０倍希釈、陽性検体
Ｃで２５６０倍希釈まで陽性像が検出できた。健常者検
体で陽性像が検出されたことより、非特異凝集反応が高
いといえる。(9) Evaluation Results of Syphilis Test Reagent The performance of the syphilis test reagent prepared as described above was evaluated using 1 healthy subject sample, 3 syphilis patient samples, A, B and C. The results are shown in Table 2. A positive image was detected up to 80-fold dilution in the healthy subject sample. Positive sample A in syphilis patient samples
The positive image could be detected up to 160-fold dilution with, positive sample B up to 640-fold dilution, and positive sample C up to 2560-fold dilution. It can be said that the non-specific agglutination reaction is high because the positive image was detected in the healthy subject sample.

【００９８】比較例４ 凍結融解法により菌破砕し、遠
心分離により得た不溶性成分を可溶化せずに、そのまま
梅毒検査試薬用抗原とした時の抗原製造、及び梅毒検査
試薬の製造、評価 （１）ＴＰ菌の培養、（２）凍結融解によるＴＰ菌の破
砕、（３）遠心分離による不溶性成分の分画は実施例１
と同様である。Comparative Example 4 Antigen production when the bacteria were disrupted by the freeze-thaw method and the insoluble component obtained by centrifugation was directly used as an antigen for a syphilis test reagent without solubilization, and production and evaluation of a syphilis test reagent ( Example 1 was carried out by culturing TP bacteria, (2) crushing TP bacteria by freeze-thawing, and (3) fractionating insoluble components by centrifugation.
Is the same as

【００９９】（４）遠心洗浄による不溶性成分の洗浄 この不溶性成分にＰＢＳ、２ｍｌを添加して１０，００
０×ｇ、３０分間遠心分離を行い、ＴＰ菌含有可溶性成
分を遠心洗浄した。この遠心洗浄操作を３回繰り返しの
ち、洗浄済み不溶性成分を梅毒検査試薬用抗原とした。(4) Washing of insoluble component by centrifugation washing 2 ml of PBS was added to this insoluble component to obtain 10,000
Centrifugation was performed at 0 × g for 30 minutes, and the soluble components containing TP bacteria were washed by centrifugation. After repeating this centrifugal washing operation three times, the washed insoluble component was used as an antigen for a syphilis test reagent.

【０１００】（５）抗原の評価結果 参考例１に従いタンパク定量を行ったところ、０．７ｍ
ｇ／ｍｌであった。参考例１のタンパク定量法では不溶
性タンパクも定量可能である。さらに、参考例２に従っ
て抗原価を測定したところ、抗原価は１００倍、対照値
は１倍であった。(5) Antigen evaluation results When the protein was quantified according to Reference Example 1, it was 0.7 m.
It was g / ml. Insoluble proteins can also be quantified by the protein quantification method of Reference Example 1. Furthermore, when the antigen titer was measured according to Reference Example 2, the antigen titer was 100 times and the control value was 1 time.

【０１０１】（６）感作、（７）洗浄操作、（８）ブロ
ッキング操作、（９）測定操作は実施例１と同様であ
る。(6) Sensitization, (7) Washing operation, (8) Blocking operation, (9) Measuring operation are the same as in Example 1.

【０１０２】（１０）梅毒検査試薬の評価結果 上記のように調製した梅毒検査試薬の性能評価を健常者
検体１検体、梅毒患者検体３検体、Ａ、Ｂ、Ｃを用いて
行った。結果を表２に示した。健常者検体、梅毒患者陽
性検体Ａ、Ｂ、Ｃは２０倍希釈でも陽性像が検出されな
かった。ドットブロットによる抗原評価では良好な結果
を得たが、試薬化では良好な結果が得られなかった。抗
原成分が可溶化されてないことが原因と考えられる。(10) Evaluation results of syphilis test reagent The performance of the syphilis test reagent prepared as described above was evaluated using 1 healthy subject sample, 3 syphilis patient samples, A, B and C. The results are shown in Table 2. No positive image was detected in the healthy subject samples and syphilis patient positive samples A, B, and C even when diluted 20-fold. Good results were obtained in the antigen evaluation by dot blot, but not good results in reagent preparation. It is considered that the cause is that the antigen component is not solubilized.

【０１０３】比較例５ 凍結融解法により菌破砕し、遠
心分離により得た不溶性成分を可溶化後、遠心分画せず
に、そのまま梅毒検査試薬用抗原としたときの抗原製
造、及び梅毒検査試薬の製造、評価 （１）ＴＰ菌の培養、（２）凍結融解によるＴＰ菌の破
砕 （３）遠心分離による不溶性成分の分画、（４）遠心洗
浄による不溶性成分の洗浄は実施例１と同様である。Comparative Example 5 Antigen production and syphilis test reagent when the bacteria were crushed by the freeze-thaw method and the insoluble component obtained by centrifugation was solubilized and then directly used as the antigen for syphilis test reagent without centrifugation fractionation. (1) Culturing of TP bacterium, (2) Crushing of TP bacterium by freeze-thawing (3) Fractionation of insoluble component by centrifugation, (4) Washing of insoluble component by centrifugal washing is the same as in Example 1. Is.

【０１０４】（５）界面活性剤のよる不溶性成分の可溶
化 上記洗浄済み不溶性成分にＰＢＳに溶解した１％オクチ
ルグルコシドを２ｍｌ添加して３７℃、１時間可溶化を
行った。可溶化後の懸濁液２ｍｌをそのまま梅毒検査試
薬用抗原を得た。(5) Solubilization of insoluble component with surfactant A 2% portion of 1% octyl glucoside dissolved in PBS was added to the washed insoluble component and solubilized at 37 ° C. for 1 hour. 2 ml of the solubilized suspension was directly used as an antigen for a syphilis test reagent.

【０１０５】（６）抗原の評価結果 参考例１に従いタンパク定量を行ったところ、１．２ｍ
ｇ／ｍｌであった。さらに、参考例２に従って抗原価を
測定したところ、抗原価は１００倍、対照値は１倍であ
った。抗原性の指標である抗原価は１００倍と高く、一
方、非特異凝集の指標となる対照値は１倍と低いことよ
り、特異性が高く、不純物を含まない抗原が得られたと
判断できる。(6) Antigen evaluation results When the protein was quantified according to Reference Example 1, it was 1.2 m.
It was g / ml. Furthermore, when the antigen titer was measured according to Reference Example 2, the antigen titer was 100 times and the control value was 1 time. Since the antigen titer, which is an index of antigenicity, is as high as 100 times, and the control value, which is an index of non-specific aggregation, is as low as 1 time, it can be judged that an antigen having high specificity and containing no impurities was obtained.

【０１０６】（７）感作、（８）洗浄操作、（９）ブロ
ッキング操作、（１０）測定操作は実施例１と同様であ
る。(7) Sensitization, (8) Washing operation, (9) Blocking operation, (10) Measuring operation are the same as in Example 1.

【０１０７】（１１）梅毒検査試薬の評価結果 上記のように調製した梅毒検査試薬の性能評価を健常者
検体１検体、梅毒患者検体３検体、Ａ、Ｂ、Ｃを用いて
行った。結果を表２に示した。健常者検体、梅毒患者検
体Ａ、Ｂ、Ｃのいずれも２０倍希釈でも陽性像が検出さ
れなかった。ドットブロット法による抗原の評価では良
好な結果を得たが、試薬化の検討では健常者検体、梅毒
患者検体いずれでも凝集反応を示さず、試薬化に適した
抗原が調製できなかったといえる。抗原中に可溶化され
てない夾雑物が存在していることが原因と考えられる。(11) Evaluation results of syphilis test reagent The performance of the syphilis test reagent prepared as described above was evaluated using 1 healthy subject sample, 3 syphilis patient samples, A, B and C. The results are shown in Table 2. No positive image was detected in any of the healthy subject samples and the syphilis patient samples A, B, and C even when diluted 20-fold. Although good results were obtained in the evaluation of the antigen by the dot blot method, in the examination of the reagent, neither the healthy subject sample nor the syphilis patient sample showed the agglutination reaction, and it can be said that the antigen suitable for the reagent could not be prepared. It is considered that the presence of unsolubilized impurities in the antigen is the cause.

【０１０８】実施例３ 凍結融解法により菌破砕したの
ち、界面活性剤と変性剤の共存下で可溶化を行い製造し
た梅毒検査試薬用抗原の製造 （１）ＴＰ菌の培養、（２）凍結融解によるＴＰ菌の破
砕、（３）遠心分離による不溶性成分の分画、（４）遠
心洗浄による不溶性成分の洗浄は実施例１と同様。Example 3 Production of antigen for syphilis test reagent produced by lysing the cells by freeze-thaw method and solubilizing in the presence of a surfactant and a denaturant (1) culturing TP bacterium, (2) freezing The disruption of TP bacteria by thawing, (3) fractionation of insoluble components by centrifugation, and (4) washing of insoluble components by centrifugal washing are the same as in Example 1.

【０１０９】（５）界面活性剤、変性剤のよる不溶性成
分の可溶化） 洗浄済み不溶性成分に２％オクチルグルコシド、８Ｍ尿
素を含むＰＢＳを添加して３７℃、１時間可溶化を行っ
た。可溶化後、１０，０００×ｇ、３０分間遠心分離を
行い、遠心上清の可溶性成分２ｍｌを梅毒検査試薬用抗
原として得た。(5) Solubilization of Insoluble Component by Surfactant and Denaturant) PBS containing 2% octyl glucoside and 8M urea was added to the washed insoluble component and solubilized at 37 ° C. for 1 hour. After solubilization, centrifugation was performed at 10,000 × g for 30 minutes, and 2 ml of the soluble component of the centrifugation supernatant was obtained as an antigen for a syphilis test reagent.

【０１１０】（６）抗原の評価結果 参考例１に従いタンパク定量を行ったところ、１．２ｍ
ｇ／ｍｌであった。さらに、参考例２に従って抗原価を
測定したところ、抗原価は２００倍、対照値は２倍であ
った。(6) Antigen evaluation results When the protein was quantified according to Reference Example 1, it was 1.2 m.
It was g / ml. Furthermore, when the antigen titer was measured according to Reference Example 2, the antigen titer was 200 times and the control value was 2 times.

【０１１１】ＴＰ菌の破砕物懸濁液の可溶化方法として
実施例１の界面活性剤だけを用いる可溶化より実施例３
の変性剤の共存下で界面活性剤を用いる方がさらに抗原
性が高く、タンパク回収率も良好であった。As a method of solubilizing the suspension of crushed TP bacteria, solubilization using only the surfactant of Example 1
The use of a surfactant in the coexistence of the denaturant was more antigenic and the protein recovery rate was better.

【０１１２】（７）感作、（８）洗浄操作、（９）ブロ
ッキング操作、（１０）測定操作は実施例１と同様。(7) Sensitization, (8) Washing operation, (9) Blocking operation, (10) Measuring operation were the same as in Example 1.

【０１１３】（１１）梅毒検査試薬の評価結果 上記のように調製した梅毒検査試薬の性能評価を健常者
検体１検体、梅毒患者検体３検体、Ａ、Ｂ、Ｃを用いて
行った。結果を表２に示した。健常者検体は２０倍希釈
でも陽性像が検出されなかったのに対して梅毒患者検体
では陽性検体Ａで３２０倍希釈、陽性検体Ｂで６４０倍
希釈、陽性検体Ｃで２５６０倍希釈まで陽性像が検出で
きた。ドットブロット法による抗原性評価と同様、試薬
化での評価でも感度が高いことが判る。(11) Evaluation Results of Syphilis Test Reagent The performance of the syphilis test reagent prepared as described above was evaluated using 1 healthy subject sample, 3 syphilis patient samples, A, B and C. The results are shown in Table 2. No positive image was detected in the 20-fold diluted sample of healthy subjects, whereas in the syphilis patient sample, a positive image was obtained by 320-fold dilution with positive sample A, 640-fold diluted with positive sample B, and 2560-fold diluted with positive sample C. I was able to detect it. Similar to the antigenicity evaluation by the dot blot method, it can be seen that the sensitivity in the reagent evaluation is high.

【０１１４】[0114]

【表１】 [Table 1]

【０１１５】[0115]

【表２】 [Table 2]

Claims (2)

【特許請求の範囲】[Claims] 【請求項１】 菌懸濁液を凍結融解して菌を破砕したの
ち不溶性成分を分画し、次いで該不溶性成分を界面活性
剤を用いて水性溶媒への可溶化を行ったのち可溶性成分
を抗原として分画することを特徴とする菌表層抗原の製
造方法。1. An insoluble component is fractionated after freeze-thawing a microbial suspension to disrupt the bacterium, and the insoluble component is solubilized in an aqueous solvent using a surfactant, and then the soluble component is removed. A method for producing a bacterial surface antigen, which comprises fractionating as an antigen. 【請求項２】 不溶性成分を変性剤の共存下に界面活性
剤で可溶化することを特徴とする請求項１記載の菌表層
抗原の製造方法。2. The method for producing a bacterial surface antigen according to claim 1, wherein the insoluble component is solubilized with a surfactant in the presence of a denaturing agent.