JPH0614052B2 - Antigen preparation for periodontal disease diagnosis - Google Patents
Antigen preparation for periodontal disease diagnosisInfo
- Publication number
- JPH0614052B2 JPH0614052B2 JP475885A JP475885A JPH0614052B2 JP H0614052 B2 JPH0614052 B2 JP H0614052B2 JP 475885 A JP475885 A JP 475885A JP 475885 A JP475885 A JP 475885A JP H0614052 B2 JPH0614052 B2 JP H0614052B2
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- antigen
- periodontal disease
- preparation
- antigen preparation
- pbs
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
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- Tropical Medicine & Parasitology (AREA)
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- Food Science & Technology (AREA)
- Virology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】 産業上の利用分野 本発明は歯周疾患診断用抗原調製品、さらに詳しくは、
抗原の保存安定性を向上させた、特異抗体検出による歯
肉疾患の診断に有用な抗原調製品に関する。TECHNICAL FIELD The present invention relates to an antigen preparation for periodontal disease diagnosis, more specifically,
The present invention relates to an antigen preparation having improved antigen storage stability and useful for diagnosing gingival diseases by detecting specific antibodies.
従来の技術 近年、歯周疾患の原因に歯肉溝内のグラム陰性嫌気性細
菌が関与していることが明らかにされ、その菌の存在を
判定する細菌学的方法や、その菌の感染有無を検定する
免疫学的方法を歯周疾患の診断に採用する試みがなされ
ている。このうち、免疫学的方法、とくに、患者の該細
菌に対する特異抗体を検出する方法は、比較的簡単な操
作で、比較的短時間に診断結果が得られるところから、
望ましい方法である。2. Description of the Related Art Recently, it has been clarified that gram-negative anaerobic bacteria in the gingival sulcus are involved in the cause of periodontal disease, and a bacteriological method for determining the presence of the bacterium and the presence or absence of infection of the bacterium Attempts have been made to employ immunoassays to assay for the diagnosis of periodontal disease. Among them, the immunological method, in particular, the method of detecting a specific antibody against the bacterium of a patient, is a relatively simple operation, and a diagnostic result can be obtained in a relatively short time.
This is the preferred method.
一方、特異抗体を検出する方法として、抗原を、被覆、
吸着、結合などにより固体担体に担持させた抗原調製品
を用いるラジオイムノアッセイ、酵素免疫測定法などが
知られている。しかしながら、このような抗原調製品は
非常に不安定であり、実験室レベルでの試験、研究に使
用するうえにおいてはあまり問題とされないものの、こ
れを病院等における日常の診断検査に応用できるように
するためには、安定化を図り、長期保存に耐えうる、試
薬として市販可能な調製品とする必要がある。On the other hand, as a method of detecting a specific antibody, an antigen is coated,
Radioimmunoassays, enzyme immunoassays, and the like that use an antigen preparation supported on a solid carrier by adsorption, binding, or the like are known. However, such an antigen preparation is extremely unstable, and although it is not a big problem when it is used for testing and research at the laboratory level, it can be applied to daily diagnostic tests in hospitals and the like. In order to do so, it is necessary to stabilize and prepare a commercially available preparation as a reagent that can withstand long-term storage.
従来、歯周疾患診断用にこのような安定化を図った抗原
調製品は見当らないが、固体担体に担持させた抗原をグ
リセリン、エチレングリコール、プロピレングリコー
ル、ヒドロキシプロピルメチルセルロース、メチルセル
ロースなどの安定化剤で処理後、凍結乾燥した安定化抗
原調製品が知られている（例えば、特開昭５８−１２３
４５９号等参照）。Conventionally, there is no antigen preparation that is designed for such stabilization for periodontal disease diagnosis, but the antigen supported on a solid carrier is a stabilizer such as glycerin, ethylene glycol, propylene glycol, hydroxypropyl methylcellulose, or methylcellulose. It is known that the stabilized antigen preparation is freeze-dried after being treated with (for example, JP-A-58-123).
459 etc.).
発明が解決しようとする問題点 本発明は、従来の安定化抗原調製品におけるような凍結
乾燥操作を必要としない、簡単な操作できわめて良好な
安定化が図れる、歯周疾患診断に適した抗原調製品を提
供することを目的とする。DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention The present invention is an antigen suitable for diagnosis of periodontal disease, which does not require freeze-drying operation as in conventional stabilized antigen preparations and can achieve extremely good stabilization by simple operation. The purpose is to provide prepared products.
問題点を解決するための手段 本発明者らは、意外にも、固体担体に担持させた歯周疾
患原因細菌またはそれ由来の抗原成分が凍結乾燥を施さ
なくても、特異的に、カルボキシメチルセルロースの塩
を含有する水溶液中で安定に保たれることを見出し、本
発明を完成するにいたった。Means for Solving the Problems The present inventors have, surprisingly, found that even when periodontal disease-causing bacteria or an antigen component derived therefrom carried on a solid carrier are not subjected to freeze-drying, specifically, carboxymethylcellulose. It has been found that it can be stably maintained in an aqueous solution containing the salt of 1), and has completed the present invention.
すなわち、本発明は、カルボキシメチルセルロースの塩
を含有する水溶液中に、固体担体に担持させた歯周疾患
原因細菌またはそれ由来の抗原成分を収容してなる歯周
疾患診断用抗原調製品を提供するものである。本発明の
抗原調製品においては、凍結乾燥のごとき面倒な操作を
施さなくても、単に、該細菌またはそれ由来の抗原成分
をカルボキシメチルセルロースの塩を含有する水溶液
（以下、保存液と称する）中で保存するだけで、これら
の抗原物質がきわめて安定に保たれ、特異抗体の検出も
簡単な操作で行なうことができる。That is, the present invention provides an antigen preparation for periodontal disease diagnosis, which comprises a periodontal disease-causing bacterium supported on a solid carrier or an antigen component derived therefrom, in an aqueous solution containing a salt of carboxymethyl cellulose. It is a thing. In the antigen preparation of the present invention, the bacterium or the antigen component derived therefrom is simply dissolved in an aqueous solution containing a salt of carboxymethyl cellulose (hereinafter, referred to as a preservation solution) without a troublesome operation such as freeze-drying. These antigenic substances can be kept extremely stable simply by storing them in, and the specific antibody can be detected by a simple operation.
保存液の必須成分として用いるカルボキシメチルセルロ
ースの塩としては、例えば、ナトリウム塩、カルシウム
塩などが挙げられ、保存液中の濃度は特に限定するもの
ではないが、その安定化効果および溶解性の観点から、
一般に、０．０１〜１０％（重量％、以下同じ）、好ま
しくは、０．１〜１％程度とする。また、保存液はカル
ボキシメチルセルロースの塩の単なる水溶液でもよい
が、緩衝液とすることが好ましく、特に、中性pH付近の
緩衝液、例えば、pH７のリン酸緩衝生理食塩水(PBS)と
することが好ましい。さらに、抗原物質の安定性をそこ
なわない成分、例えば、適当な界面活性剤、防腐剤等を
適宜保存液に添加してもよい。Examples of the salt of carboxymethyl cellulose used as an essential component of the storage solution include, for example, sodium salt, calcium salt, etc., and the concentration in the storage solution is not particularly limited, but from the viewpoint of its stabilizing effect and solubility. ,
Generally, it is 0.01 to 10% (wt%, the same applies hereinafter), preferably about 0.1 to 1%. The storage solution may be a simple aqueous solution of a salt of carboxymethyl cellulose, but it is preferable to use a buffer solution, particularly a buffer solution near neutral pH, for example, phosphate buffered saline (PBS) having a pH of 7. Is preferred. Further, a component which does not impair the stability of the antigenic substance, for example, a suitable surfactant or preservative may be appropriately added to the storage solution.
抗原物質を担持させる固体担体は通常用いられるものい
ずれでもよく、例えば、紙、ガラス、合成樹脂製のプレ
ート状、チューブ状、ビーズ状などの担体が用いられ、
ことに、ポリスチレン製のマイクロタイター・プレート
やチューブが好ましい。The solid carrier for supporting the antigenic substance may be any of those usually used, for example, paper, glass, synthetic resin plate-shaped, tube-shaped, beads-shaped carriers and the like,
In particular, polystyrene microtiter plates and tubes are preferred.
抗原物質として用いる歯周疾患原因細菌としては、歯肉
溝内に見られるグラム陰性嫌気性細菌、とりわけ、バク
テロイデス、ジンジバリス(Bacteroides gingivalis)、
アクチノバチルス・アクチノマイセテムコミタンス(Act
inobacillus actin-omycetemcomitanc)が挙げられ、こ
れらは、バクテロイデス・ジシジバリス ＡＴＣＣ３３
３２７株、アクチノバチルス・アクチノマイセテムコミ
タンス ＡＴＣＣ２９５２２株〜ＡＴＣＣ２９５２４株
として、アメリカン・タイプ・カルチャー・コレクショ
ン(ATCC)より入手可能であり、公知の嫌気的培養法によ
り増殖させることができる。本発明においては、これら
の細菌の培養菌体自体を抗原物質として用いてもよく、
また、常法に従って調整した菌体破壊物、その遠心上清
をゲル過等で精製したものなどのこれらの細菌由来の
抗原成分を用いてもよい。As periodontal disease-causing bacteria used as an antigenic substance, gram-negative anaerobic bacteria found in the gingival sulcus, especially Bacteroides, gingivalis (Bacteroides gingivalis),
Actinobacillus actinomycetem comitance (Act
inobacillus actin-omycetemcomitanc), and these are Bacteroides dysigevarius ATCC33.
327 strains, Actinobacillus actinomycetemcomitans ATCC 29522 strain to ATCC 29524 strain are available from American Type Culture Collection (ATCC) and can be grown by a known anaerobic culture method. In the present invention, the cultured bacterial cells of these bacteria may be used as the antigenic substance,
In addition, an antigen component derived from these bacteria, such as a disrupted bacterial cell product prepared by a conventional method or a product obtained by purifying the centrifugal supernatant by gel filtration or the like, may be used.
かくして、本発明の抗原調製品は、常法に従って、固体
担体に抗原を担持させた後、これに保存液を添加し、抗
原を保存液で被覆または浸漬した状態に保存することに
より製造することができ、作用機序は不明であるが、保
存液の作用により、そのまま、歯周疾患診断時まで、抗
原を安定に保存することができる。Thus, the antigen preparation of the present invention can be produced by loading an antigen on a solid carrier according to a conventional method, adding a preservative solution to the solid carrier, and storing the antigen in a state of being coated with or immersed in the preservative solution. Although the mechanism of action is unknown, the action of the preservation solution allows the antigen to be stably preserved as it is until the periodontal disease diagnosis.
本発明の抗原調製品を用いて歯周疾患の診断を行なうに
は、患者から採取した唾液、血液、歯肉溝滲出液などの
検体と、例えば、ラジオイムノアッセイ、酵素免疫測定
法などの公知の方法で抗原抗体反応を行なわせ、検体中
の特異抗体の有無を判定する。この際、検体は、保存液
を含有しているままの抗原調製品に直接添加しても、ま
た、保存液を除去し、他の適当な溶媒と置換した後に抗
原調製品に添加してもよい。検体は遠心分離、抽出等の
処理を施しても、適宜に稀釈してもよい。例えば、酵素
免疫測定法を用いる場合、所定量の検体またはその希釈
物を本発明の抗原調製品に直接または保存液を除去し、
他の溶媒と置換した後に添加し、例えば、２０〜４０℃
で１〜１８時間反応させる。反応終了後、洗浄し、酵素
標識抗抗体（例えば、アルカリホスファターゼ、パーオ
キシダーゼ等で標識された抗ヒト抗体）を添加し、２０
〜４０℃で１〜１８時間反応させる。ついで、再度洗浄
し、酵素に対する基質（例えば、ｐ−ニトロフェニルリ
ン酸、ｏ−フェニレンジアミンなど）を添加し、同温度
で３０〜９０分間呈色反応させ、吸光度を測定し、力価
既知の標準抗体について同様に操作して得られた標準曲
線から、検体中の特異抗体量を算出することができ、こ
れにより、歯周疾患の診断を行なうことができる。For diagnosing periodontal disease using the antigen preparation of the present invention, samples such as saliva, blood, and gingival crevicular fluid collected from a patient and known methods such as radioimmunoassay and enzyme immunoassay are used. The antigen-antibody reaction is carried out with and the presence or absence of specific antibody in the sample is determined. At this time, the sample may be added directly to the antigen preparation as it is containing the preservation solution, or may be added to the antigen preparation after removing the preservation solution and replacing it with another suitable solvent. Good. The sample may be subjected to processing such as centrifugation and extraction, or may be diluted appropriately. For example, when using the enzyme immunoassay, a predetermined amount of the sample or a dilution thereof is directly added to the antigen preparation of the present invention or the preservation solution is removed,
Add after replacing with another solvent, for example, 20 to 40 ° C.
And react for 1 to 18 hours. After completion of the reaction, the plate is washed, and an enzyme-labeled anti-antibody (for example, an anti-human antibody labeled with alkaline phosphatase, peroxidase, etc.) is added,
React at -40 ° C for 1-18 hours. Then, it is washed again, a substrate for the enzyme (for example, p-nitrophenyl phosphate, o-phenylenediamine, etc.) is added, color reaction is carried out at the same temperature for 30 to 90 minutes, and the absorbance is measured. The amount of specific antibody in the sample can be calculated from the standard curve obtained by carrying out the same operation for the standard antibody, and thus the periodontal disease can be diagnosed.
実施例 つぎに実施例を挙げて本発明をさらに詳しく説明する。EXAMPLES Next, the present invention will be described in more detail with reference to Examples.
実施例１ バクテロイデス・ジンジバリス ＡＴＣＣ３３３２７株
をトリプチケース・ソイ・ブロス培地（トリプチケース
・ソイ・ブロス１５．０ｇ、酵母エキス０．５ｇ、ヘミ
ン２．５mg、０．２５％ビタミンＫ水溶液０．１mlを水
で５００mlとする。pH７．２）中、ＣＯ2５％、Ｎ2８５
％、Ｈ2１０％の嫌気性条件下、３７℃で２日時間培養
する。培養した菌体を集め、ＰＢＳ（pH７．０）で洗浄
後、１×１０個／mの濃度でＰＢＳ（pH７．０）に懸
濁する。この懸濁液１mを０．１Ｍ炭酸緩衝液（pH
９．６）９mlと混合し、ポリスチレン製マイクロタイタ
ー・プレートの各孔に１００μづつ分注し、３７℃で
１時間放置する。放置後、１％ウシ血清アルブミンを含
有する０．１Ｍ炭酸緩衝液１００μずつを各孔に分注
し、３７℃で１時間放置する。ついで、各孔の液体を除
去し、０．０５％ツイーン(Tween)２０を含有するＰＢ
Ｓ（pH７．０）で３回洗浄する。洗浄後、マイクロタイ
ター・プレートの各孔を、０．５％カルボキシメチルセ
ルロース・ナトリウム、０．０５％ツイーン２０および
０．０２％三窒化ナトリウムを含有するＰＢＳ（pH７．
０）からなる保存液で満たし、密封して、所望の抗原調
製品を得る。Example 1 Bacteroides gingivalis ATCC 33327 strain was treated with Trypticase soy broth medium (10.5 g of trypticase soy broth, 0.5 g of yeast extract, 2.5 mg of hemin, 0.1 ml of 0.25% vitamin K aqueous solution). To 500 ml with water, pH 7.2), CO 2 5%, N 2 85
%, H 2 10% under anaerobic conditions, the cells are cultured at 37 ° C. for 2 days. The cultured cells are collected, washed with PBS (pH 7.0), and then suspended in PBS (pH 7.0) at a concentration of 1 × 10 5 cells / m 2. 1m of this suspension was added to 0.1M carbonate buffer (pH
9.6) Mix with 9 ml, dispense 100 μ each into each hole of a polystyrene microtiter plate, and leave at 37 ° C. for 1 hour. After standing, 100 μ each of 0.1 M carbonate buffer containing 1% bovine serum albumin is dispensed into each hole and left at 37 ° C. for 1 hour. The liquid in each hole was then removed and PB containing 0.05% Tween 20 was added.
Wash 3 times with S (pH 7.0). After washing, each well of the microtiter plate was washed with PBS containing 0.5% sodium carboxymethylcellulose, 0.05% Tween 20 and 0.02% sodium trinitride (pH 7.
Fill with a preservative solution consisting of 0) and seal to obtain the desired antigen preparation.
実施例２ 実施例１と同様に、嫌気性条件下で培養したバクテロイ
デス・ジンジバリス ＡＴＣＣ３３３２７株を集菌し、
洗浄後、０．２ｇ（湿重量）／mの濃度でＰＢＳ（pH
７．０）に懸濁する。この懸濁液をガラス・ビーズと共
に振とうして菌体を破砕し、ついで１２０００×ｇで２
０分間遠心分離する。得られた上清を０．１Ｍ炭酸緩衝
液（pH９．６）で５０μｇ蛋白／mとなるように稀釈
し、ポリスチレン製マイクロタイター・プレートの各孔
に１００μづつ分注し、３７℃で１時間放置する。放
置後、各孔の液体を除去し、０．０５％ツイーン２０を
含有するＰＢＳ（pH７．０）で３回洗浄する。ついで、
０．５％カルボキシメチルセルロース・ナトリウム、
０．０５％ツイーン２０および０．０２％三窒化ナトリ
ウムを含有するＰＢＳ（pH７．０）からなる保存液で各
孔を満たし、密封して、所望の抗原調製品を得る。Example 2 As in Example 1, Bacteroides gingivalis ATCC 33327 strain cultured under anaerobic conditions was collected,
After washing, PBS (pH) at a concentration of 0.2 g (wet weight) / m
7.0). The suspension was shaken with glass beads to crush the cells, and then the cells were mixed with 12000 × g for 2
Centrifuge for 0 minutes. The resulting supernatant was diluted with 0.1 M carbonate buffer (pH 9.6) to 50 μg protein / m, 100 μl was dispensed into each hole of a polystyrene microtiter plate, and the mixture was incubated at 37 ° C. for 1 hour. put. After standing, the liquid in each hole is removed, and the wells are washed three times with PBS (pH 7.0) containing 0.05% Tween 20. Then,
0.5% sodium carboxymethyl cellulose,
Fill each hole with a stock solution consisting of PBS (pH 7.0) containing 0.05% Tween 20 and 0.02% sodium trinitride and seal to obtain the desired antigen preparation.
実施例３ 実施例１と同様にして得られたバクテロイデス・ジンジ
バリス ＡＴＣＣ３３３２７株のＰＢＳ（pH７．０）中
懸濁液を超音波処理して菌体を破砕し、１２０００×ｇ
で２０分間遠心分離する。得られた上清を０．１Ｍ炭酸
緩衝液（pH９．６）で５０μｇ蛋白／mとなるように
稀釈し、ポリスチレン製チューブに１mづつ分注し、
３７℃で１時間放置する。放置後、チューブ中の液体を
除去し、０．０５％ツイーン２０を含有するＰＢＳ（pH
７．０）で３回洗浄する。ついで、０．５％カルボキシ
メチルセルロース・ナトリウム、０．０５％ツイーン２
０および０．０２％三窒化ナトリウムを含有するＰＢＳ
（pH７．０）からなる保存液をチューブに入れ、密封し
て、所望の抗原調製品を得る。Example 3 A suspension of Bacteroides gingivalis strain ATCC 33327 strain obtained in the same manner as in Example 1 in PBS (pH 7.0) was sonicated to crush the cells, and 12000 × g.
Centrifuge for 20 minutes at. The obtained supernatant was diluted with 0.1 M carbonate buffer (pH 9.6) to 50 μg protein / m and dispensed into polystyrene tubes in 1 m increments.
Leave at 37 ° C for 1 hour. After standing, the liquid in the tube was removed and PBS containing 0.05% Tween 20 (pH
Wash 3 times with 7.0). Next, 0.5% sodium carboxymethylcellulose, 0.05% Tween 2
PBS containing 0 and 0.02% sodium trinitride
A preservation solution consisting of (pH 7.0) is put in a tube and sealed to obtain a desired antigen preparation.
実施例４ 実施例１と同様に、嫌気性条件下で培養したバクテロイ
デス・ジシジバリス ＡＴＣＣ３３３５７株を集菌し、
菌体１ｇ（湿重量）当り５mMのＥＤＴＡを含有するＰＢ
Ｓ（pH７．０）１０mを用いて菌体分散液を調製す
る。これに直径０．１３mmのグラスビーズ２mを加
え、４℃で２日間振とうして菌体を破砕する。ついで、
１２０００×ｇで２０分間遠心分離し、上清を得る。こ
れを０．１Ｍ炭酸緩衝液（pH９．６）で５０μｇ蛋白／
mとなるように稀釈し、ついで、実施例２と同様にし
て、ポリスチレン製マイクロタイター・プレートの孔に
担持させ、０．５％カルボキシメチルセルロース・ナト
リウムおよび０．０５％ツイーン２０を含有するＰＢＳ
（pH７．０）からなる保存液を用いて所望の抗原調製品
を得る。Example 4 In the same manner as in Example 1, Bacteroides desigibalis ATCC33357 strain cultured under anaerobic conditions was collected,
PB containing 5 mM EDTA per 1 g (wet weight) of cells
A cell dispersion liquid is prepared using 10 m of S (pH 7.0). To this, 2 m of glass beads having a diameter of 0.13 mm is added and shaken at 4 ° C. for 2 days to crush the cells. Then,
Centrifuge at 12000 xg for 20 minutes to obtain the supernatant. 50 μg protein / 0.1M carbonate buffer (pH 9.6)
PBS containing 0.5% sodium carboxymethylcellulose and 0.05% Tween 20 was added to the pores of a polystyrene microtiter plate in the same manner as in Example 2, and then diluted to give m.
A desired antigen preparation is obtained using a preservation solution consisting of (pH 7.0).
実施例５ アクチノバチルス・アクチノマイセテムコミタンス Ａ
ＴＣＣ２９５２２株をチオグリコレート培地（酵母エキ
ス５．０ｇ、トリプチケース・ペプトン１５．０ｇ、デ
キストロース５．０ｇ、塩化ナトリウム２．５ｇ、Ｌ−
システイン塩酸塩０．７５ｇ、チオグリコール酸ナトリ
ウム０．５ｇを水で１０００mlにする。pH７．０）中、
ＣＯ2２０％、空気８０％の嫌気性条件下、３７℃で２
日間培養する。培養した菌体を集め、実施例２と同様に
処理して、アクチノバチルス・アクチノマイセテムコミ
タンスの菌体破砕物を抗原とし、０．５％カルボキシメ
チルセルロース・ナトリウム、０．０５％ツイーン２０
および０．０２％三窒化ナトリウムを含有するＰＢＳ
（pH７．０）からなる保存液を用いたポリスチレン製マ
イクロタイター・プレートに担持された所望の抗原調製
品を得る。Example 5 Actinobacillus actinomycetemcomitance A
TCC29522 strain was added to thioglycollate medium (yeast extract 5.0 g, trypticase peptone 15.0 g, dextrose 5.0 g, sodium chloride 2.5 g, L-
0.75 g of cysteine hydrochloride and 0.5 g of sodium thioglycolate are brought to 1000 ml with water. pH 7.0),
2 at 37 ° C under anaerobic conditions of 20% CO 2 and 80% air
Incubate for a day. The cultured cells were collected and treated in the same manner as in Example 2, and the cell disrupted product of Actinobacillus actinomycetemcomitans was used as an antigen, and 0.5% carboxymethylcellulose sodium and 0.05% Tween 20 were used.
And PBS containing 0.02% sodium trinitride
A desired antigen preparation supported on a polystyrene microtiter plate using a preservation solution of (pH 7.0) is obtained.
実施例６ 実施例５と同様に、嫌気性条件下で培養したアクチノバ
チルス・アクチノマイセテムコミタンス ＡＴＣＣ２９
５２２株を集菌し、実施例４と同様に処理して、ポリス
チレン製マイクロタイター・プレートの孔に菌体抽出成
分を担持させ、０．５％カルボキシメチルセルロース・
ナトリウムおよび０．０５％ツイーン２０を含有するＰ
ＢＳ（pH７．０）からなる保存液を用いて所望の調製品
を得る。Example 6 Actinobacillus actinomycetemcomitans ATCC29 cultured under anaerobic conditions in the same manner as in Example 5.
The 522 strain was collected and treated in the same manner as in Example 4 so that the pores of the polystyrene microtiter plate were loaded with the bacterial cell extraction component, and 0.5% carboxymethyl cellulose.
P containing sodium and 0.05% Tween 20
The desired preparation is obtained using a stock solution consisting of BS (pH 7.0).
発明の効果 (1)保存試験 実施例４および６で得られた抗原調製品およびその保存
液中のカルボキシメチルセルロース・ナトリウムの量を
変化させ、あるいはカルボキシメチルセルロース・ナト
リウムの代りに抗原を安定化させうると考えられる物質
を用いて得られた抗原調製品を、各々、４０℃で１カ月
および３カ月保存した。実施例４または６で調製した抗
原に充分な力価を示す血清を標準抗体として用い、アル
カリホスファターゼ標識抗ヒト抗体およびｐ−ニトロフ
ェニルリン酸を用いる酵素免疫測定法により、４０５nm
の吸光度を測定して抗体価を算出し、保存前の抗体価か
ら、次式により、抗原活性の残存率（％）を算出した。EFFECTS OF THE INVENTION (1) Storage test It is possible to change the amount of sodium carboxymethylcellulose / sodium in the antigen preparations and the storage solutions thereof obtained in Examples 4 and 6, or to stabilize the antigen instead of carboxymethylcellulose / sodium. The antigen preparations obtained with the substances considered to have been stored at 40 ° C. for 1 month and 3 months, respectively. 405 nm was obtained by an enzyme immunoassay using an alkaline phosphatase-labeled anti-human antibody and p-nitrophenyl phosphate, using a serum showing a sufficient titer for the antigen prepared in Example 4 or 6 as a standard antibody.
The antibody titer was calculated by measuring the absorbance of the, and the residual rate (%) of the antigen activity was calculated from the antibody titer before storage by the following formula.
対照として、安定化剤を含有しない保存液(0.０５％ツ
イーン２０含有ＰＢＳ（pH７．０）を用いて得られた抗
原調製品についても、同様に試験した。結果を第１表に
示す。 As a control, an antigen preparation obtained by using a stock solution containing no stabilizer (PBS containing 0.05% Tween 20 (pH 7.0) was also tested in the same manner. The results are shown in Table 1.
表１に示すとおり、カルボキシメチルセルロースの塩を
用いると、特異的に抗原活性の安定化が図れる。 As shown in Table 1, when a salt of carboxymethyl cellulose is used, the antigen activity can be specifically stabilized.
(2)歯周疾患の診断 実施例２および実施例５で得られた抗原調製品を、各
々、４０℃で３カ月保存後、この抗原調製品で、被検者
２０名の血清検体(0.０５％ツイーン２０および0.5％ウ
シ血清アルブミン含有ＰＢＳ（pH７．０）にて１００倍
に稀釈）および歯肉溝滲出液検体（ペーパーストリップ
により採取、血清検体と同じ希釈液にて１００倍に稀
釈）の抗体価を酵素免疫測定法により算出した。(2) Diagnosis of periodontal disease Each of the antigen preparations obtained in Example 2 and Example 5 was stored at 40 ° C. for 3 months, and then the antigen preparations were used to obtain serum samples (0 Diluted 100 times with PBS (pH 7.0) containing 0.05% Tween 20 and 0.5% bovine serum albumin) and gingival crevicular fluid sample (collected with a paper strip, diluted 100 times with the same diluent as the serum sample) The antibody titer was calculated by enzyme immunoassay.
すなわち、マイクロタイター・プレートの孔内の保存液
を排出し、検体１００μを加え、３７℃で１時間放置
した。ついで、０．０５％ツイーン２０を含有するＰＢ
Ｓ（pH７．０）で３回洗浄し、アルカリホスファターゼ
標識抗ヒト抗体および０．０５％ツイーン２０を含有す
るＰＢＳ（pH７．０）１００μを各孔に添加し、３７
℃で１時間放置し、０．０５％ツイーン２０を含有する
ＰＢＳ（pH７．０）で３回洗浄した。１mMの塩化マグネ
シウムを含む５０mM炭酸緩衝液（pH９．８）で１mg/ml
の濃度となるように溶解したｐ−ニトロフェニルリン酸
１００μを各孔に加え、３７℃で３０分間呈色反応さ
せ、４０５nmにて吸光度を測定した。検体の代りに標準
抗体（酵素免疫測定法により、予め、該抗原に対して充
分な抗体価を示すことが判明している血清）を用いて同
様に操作して作成した検量線より抗体価を算出した。な
お、検体採取時に各被検者の歯肉炎症指数(GＩ、Ｈ．Ｌ
ｅおよびＪ．Silness,Acta.Odont.Scand.,２１ ５３
３−５５１（１９６３）参照）および歯肉溝のポケット
の深さ（ＰＤ）を測定した。結果を第２表に示す。That is, the stock solution in the holes of the microtiter plate was discharged, 100 μm of the sample was added, and the mixture was left at 37 ° C. for 1 hour. Then PB containing 0.05% Tween 20
After washing 3 times with S (pH 7.0), 100 μl of PBS (pH 7.0) containing alkaline phosphatase-labeled anti-human antibody and 0.05% Tween 20 was added to each well, and 37
The plate was left at 1 ° C. for 1 hour and washed 3 times with PBS (pH 7.0) containing 0.05% Tween 20. 1 mg / ml with 50 mM carbonate buffer (pH 9.8) containing 1 mM magnesium chloride
100 μ of p-nitrophenylphosphoric acid dissolved so as to have a concentration of was added to each hole, a color reaction was performed at 37 ° C. for 30 minutes, and the absorbance was measured at 405 nm. The antibody titer was determined from the standard curve prepared in the same manner using a standard antibody (serum that was previously shown to have a sufficient antibody titer against the antigen by enzyme immunoassay) instead of the sample. It was calculated. Note that the gingival inflammation index (GI, HL
e and J. Silness, Acta.Odont.Scand., 21 53
3-551 (1963)) and gingival sulcus pocket depth (PD) was measured. The results are shown in Table 2.
第２表に示すごとく、本発明の抗原調製品は40℃、３カ
月保存後も、臨床所見に相関した抗原抗体反応を生じ、
抗原活性の安定性が充分に維持されている。なお、保存
液として、０．０５％ツイーン２０および０．０２％三
窒化ナトリウムのみを含有するＰＢＳ（pH７．０）や、
これに０．５％ウシ血清アルブミンを添加した液を用い
た抗原調製品について同様な試験を行なったが、臨床所
見と相関した結果が得られなかった。 As shown in Table 2, the antigen preparation of the present invention produces an antigen-antibody reaction correlated with clinical findings even after storage at 40 ° C for 3 months,
The stability of antigen activity is sufficiently maintained. As a storage solution, PBS (pH 7.0) containing only 0.05% Tween 20 and 0.02% sodium trinitride,
A similar test was conducted on an antigen preparation using a solution to which 0.5% bovine serum albumin was added, but no results correlating with clinical findings were obtained.
Claims (5)
る水溶液中に、固体担体に担持させた歯周疾患原因細菌
またはそれ由来の抗原成分を収容してなることを特徴と
する歯周疾患診断用抗原調製品。1. An antigen preparation for diagnosing periodontal disease, characterized in that a periodontal disease-causing bacterium carried on a solid carrier or an antigen component derived therefrom is contained in an aqueous solution containing a salt of carboxymethylcellulose. Product.
acteroides gingivalis)である前記第(1)項の調製品。2. The bacterium is Bacteroides gingivalis (B
The preparation of paragraph (1) above, which is acteroides gingivalis).
イセテムコミタンス(Actinobacillus actinomycetemcom
itans)である前記第(1)項の調製品。3. The bacterium is Actinobacillus actinomycetemcomit
The preparation of paragraph (1) above, which is an itans).
ウム塩である前記第(1)項〜第(3)項いずれか１つの調製
品。4. The preparation according to any one of items (1) to (3), wherein the carboxymethyl cellulose salt is a sodium salt.
る前記第(1)項〜第(4)項いずれか１つの調製品。5. The preparation according to any one of items (1) to (4), wherein the aqueous solution is a phosphate buffered saline solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP475885A JPH0614052B2 (en) | 1985-01-14 | 1985-01-14 | Antigen preparation for periodontal disease diagnosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP475885A JPH0614052B2 (en) | 1985-01-14 | 1985-01-14 | Antigen preparation for periodontal disease diagnosis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS61162753A JPS61162753A (en) | 1986-07-23 |
| JPH0614052B2 true JPH0614052B2 (en) | 1994-02-23 |
Family
ID=11592790
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP475885A Expired - Lifetime JPH0614052B2 (en) | 1985-01-14 | 1985-01-14 | Antigen preparation for periodontal disease diagnosis |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0614052B2 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4741999A (en) * | 1986-02-26 | 1988-05-03 | The Research Foundation Of State University Of New York | Monoclonal antibodies useful in the identification of microorganisms causing periodontal disease |
| WO1990004789A1 (en) * | 1988-10-17 | 1990-05-03 | Meito Sangyo Kabushiki Kaisha | Reagent and method for immunological diagnosis of periodontal disease |
| JPH0797395A (en) * | 1993-09-28 | 1995-04-11 | Kyowa Medex Co Ltd | Peptides containing sequence of porphyromonas gingivalis cilium protein and its use |
-
1985
- 1985-01-14 JP JP475885A patent/JPH0614052B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPS61162753A (en) | 1986-07-23 |
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