JPH01196299A - Bioassay technique - Google Patents
Bioassay techniqueInfo
- Publication number
- JPH01196299A JPH01196299A JP1850188A JP1850188A JPH01196299A JP H01196299 A JPH01196299 A JP H01196299A JP 1850188 A JP1850188 A JP 1850188A JP 1850188 A JP1850188 A JP 1850188A JP H01196299 A JPH01196299 A JP H01196299A
- Authority
- JP
- Japan
- Prior art keywords
- bacteria
- agar
- chemotaxis
- solution
- filter paper
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000012455 bioassay technique Methods 0.000 title 1
- 241000894006 Bacteria Species 0.000 claims abstract description 54
- 238000000034 method Methods 0.000 claims abstract description 46
- 229920001817 Agar Polymers 0.000 claims abstract description 29
- 239000000126 substance Substances 0.000 claims abstract description 25
- 238000003556 assay Methods 0.000 claims abstract description 17
- 230000035605 chemotaxis Effects 0.000 claims description 41
- 241000588724 Escherichia coli Species 0.000 claims description 26
- 239000008272 agar Substances 0.000 claims description 26
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 claims description 25
- 229960004373 acetylcholine Drugs 0.000 claims description 25
- 238000004166 bioassay Methods 0.000 claims description 8
- 239000000203 mixture Substances 0.000 abstract description 10
- 239000013543 active substance Substances 0.000 abstract description 8
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 241000206672 Gelidium Species 0.000 abstract 3
- 235000010419 agar Nutrition 0.000 abstract 3
- KWTSXDURSIMDCE-QMMMGPOBSA-N (S)-amphetamine Chemical compound C[C@H](N)CC1=CC=CC=C1 KWTSXDURSIMDCE-QMMMGPOBSA-N 0.000 abstract 2
- 241001646716 Escherichia coli K-12 Species 0.000 abstract 1
- 229940025084 amphetamine Drugs 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 35
- 230000001580 bacterial effect Effects 0.000 description 34
- 239000000243 solution Substances 0.000 description 28
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 18
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 16
- 235000003704 aspartic acid Nutrition 0.000 description 16
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 16
- 238000001514 detection method Methods 0.000 description 9
- 239000007788 liquid Substances 0.000 description 8
- 230000030786 positive chemotaxis Effects 0.000 description 7
- 230000005540 biological transmission Effects 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 239000005667 attractant Substances 0.000 description 5
- 230000031902 chemoattractant activity Effects 0.000 description 5
- 239000012530 fluid Substances 0.000 description 5
- 239000011521 glass Substances 0.000 description 5
- 230000033001 locomotion Effects 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 230000007246 mechanism Effects 0.000 description 5
- 239000002269 analeptic agent Substances 0.000 description 4
- 239000002858 neurotransmitter agent Substances 0.000 description 4
- 238000011002 quantification Methods 0.000 description 4
- 238000003127 radioimmunoassay Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- KWTSXDURSIMDCE-UHFFFAOYSA-N 1-phenylpropan-2-amine Chemical compound CC(N)CC1=CC=CC=C1 KWTSXDURSIMDCE-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 230000003698 anagen phase Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000012131 assay buffer Substances 0.000 description 3
- 210000001124 body fluid Anatomy 0.000 description 3
- 239000010839 body fluid Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000003399 chemotactic effect Effects 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 210000005036 nerve Anatomy 0.000 description 3
- 210000000653 nervous system Anatomy 0.000 description 3
- 239000012064 sodium phosphate buffer Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 244000063299 Bacillus subtilis Species 0.000 description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 150000002314 glycerols Chemical class 0.000 description 2
- 239000004519 grease Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 229960001252 methamphetamine Drugs 0.000 description 2
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 2
- 238000004452 microanalysis Methods 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 230000008035 nerve activity Effects 0.000 description 2
- 230000001537 neural effect Effects 0.000 description 2
- 229920001296 polysiloxane Polymers 0.000 description 2
- 239000008057 potassium phosphate buffer Substances 0.000 description 2
- 238000012207 quantitative assay Methods 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 239000005871 repellent Substances 0.000 description 2
- 230000002940 repellent Effects 0.000 description 2
- 239000000021 stimulant Substances 0.000 description 2
- 230000029305 taxis Effects 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241001138501 Salmonella enterica Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 210000004958 brain cell Anatomy 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000000739 chaotic effect Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 230000013439 flagellum movement Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- NJBKCLCEXIDHDR-OANDGCGGSA-N n-[(2z,3r,4r,5s,6r)-4,5-dihydroxy-2-hydroxyimino-6-(hydroxymethyl)oxan-3-yl]acetamide Chemical compound CC(=O)N[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O\C1=N/O NJBKCLCEXIDHDR-OANDGCGGSA-N 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 210000000608 photoreceptor cell Anatomy 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 230000000946 synaptic effect Effects 0.000 description 1
- 230000005062 synaptic transmission Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
［産業上の利用分野］
本発明は、化学的、物理的、あるいは免疫学的手法によ
らず、化学物質の受容、該化学物質の受容に関する情報
の細胞内伝達、該情報に対応した鞭毛運動からなる一連
の機構による生物現象である細菌が示す走化性を指標と
して利用する化学物質のバイオアッセイ法に関する。Detailed Description of the Invention [Industrial Field of Application] The present invention relates to the reception of chemical substances, the intracellular transmission of information related to the reception of the chemical substances, regardless of chemical, physical, or immunological methods. The present invention relates to a bioassay method for chemical substances that utilizes chemotaxis exhibited by bacteria, which is a biological phenomenon caused by a series of mechanisms consisting of flagellar movement corresponding to this information, as an indicator.
特に、本発明は、細菌等の走化性を示す生物の走化性を
、軟寒天プレートあるいは毛細管で検定することにより
、メタンフェタミン、アンフェタミンなどのアンフェタ
ミン類やアセチルコリン等の各種神経生理活性（神経作
用、神経伝達）物質の微量定量、微量検定に好適な方法
に関する。In particular, the present invention examines the chemotaxis of chemotactic organisms, such as bacteria, using soft agar plates or capillary tubes. The present invention relates to a method suitable for micro-quantification and micro-assay of (neurotransmitter) substances.
［従来の技術］
神経生理活性物質であるメタンフェタミン（Ｍｅｔｈａ
ｍｐｈｅｔａｍｉｎｅ、以下ＭＡと略記する）は日本に
おいて主に用いられている覚醒剤の一つである。なお、
ＭＡの類似化合物であるＤＬ−アンフェタミン（ＤＬ−
Ａｍｐｈｅｔａｍｉｎｅ）は、ヨーロッパやアメリカに
おいて覚醒剤として用いられている。[Prior art] Methamphetamine (Metha), a neurophysiologically active substance
Mphetamine (hereinafter abbreviated as MA) is one of the stimulants mainly used in Japan. In addition,
DL-amphetamine (DL-
Amphetamine) is used as a stimulant in Europe and America.
覚醒剤の事犯は戦後の混乱した社会情勢の中で急激に広
がったが、その後の厳しい取締や中毒者に対する医療処
置の徹底によって、−時鎮静化した。しかしながら、昭
和４５年頃から再び急激に増加し始め、昭和６０年には
若干減少したものの検挙者が２３．３４４名にのぼった
ことが厚生省の統計に示されている。Crimes related to stimulants rapidly spread in the chaotic post-war social situation, but were brought to a standstill by strict crackdowns and thorough medical treatment for addicts. However, from around 1970, the number of cases started to increase rapidly again, and in 1986, although there was a slight decrease, the number of people arrested reached 23,344, according to Ministry of Health and Welfare statistics.
ＭＡの一般社会への浸透にともなう学術及び医療以外で
の使用やＭＡ中毒者の増加による社会的影響は深刻であ
り、それに対するより効果的な予防対策の確立が急務と
なっている。The social impact of MA's use in non-academic and non-medical fields and the increase in the number of MA addicts as MA has spread into general society is serious, and there is an urgent need to establish more effective preventive measures against it.
ＭＡによる社会汚染に対する対策の一環として、ＭＡ使
用者やＭＡ中毒患者の鑑定と、これらへの適切な医療処
置がある。As part of the measures against social contamination caused by MA, there is a need to identify MA users and MA addicts and provide appropriate medical treatment for them.
ＭＡ使用者やＭＡ中毒患者の鑑定には、これらの尿など
からＭＡを検出する方法がもっばら利用されてきている
。These methods of detecting MA from urine have been widely used to identify MA users and MA addicts.
従来のＭＡの検出法としては、マスフラグメントグラフ
ィー法（Ｎｉｗａｇｕｃｈｉ、Ｔ、 ｅｔ ａｌ、 Ａ
ｒｃｈ。Conventional MA detection methods include mass fragmentography (Niwaguchi, T. et al., A.
rch.
Ｔｏｘｉｃｏｌ、、５２．１５７−１６４（１９８３）
；及び鈴木真−ら、衛生化学、廷（１） 、２３−２
６（１９８４） ］ 、ラジオイムノアッセイ法［神田
征夫ら、科学警察研究所報告、旦（３） 、１５８−１
６１（１９７８） ；及び三井利幸、分析、４ 、１０
３−１０５（１９８５）］及び発色法［庄山正敏ら、衛
生化学、３１ （６）、４１０−４１３（１９８５）］
等が利用されてきた。Toxicol, 52.157-164 (1983)
; and Makoto Suzuki et al., Sanitary Chemistry, Tei (1), 23-2.
6 (1984) ], Radioimmunoassay method [Yukio Kanda et al., Scientific Police Research Institute Report, Dan (3), 158-1
61 (1978); and Toshiyuki Mitsui, Analysis, 4, 10.
3-105 (1985)] and color method [Masatoshi Shoyama et al., Sanitary Chemistry, 31 (6), 410-413 (1985)]
etc. have been used.
［発明が解決しようとする課題］
しかしながら、ＭＡ使用者やＭＡ中毒患者の鑑定件数は
年間約４万件（昭和６０年）にも達しており、多大な時
間と労力が必要とされてきた。[Problems to be Solved by the Invention] However, the number of appraisals of MA users and MA addicts has reached approximately 40,000 cases per year (1985), requiring a great deal of time and effort.
そこで、より迅速かつ簡便であり、しかも実用的なＭＡ
検出方法の開発が要望されている。Therefore, MA is faster, simpler, and more practical.
There is a need to develop a detection method.
ところが、上述したような従来のＭＡ検出方法］よその
実用性において必ずしも十分なものとは言えなかった。However, the conventional MA detection method as described above was not necessarily sufficient in terms of practicality.
例えば、マスフラグメントグラフィー法は、検出感度が
Ｏ，ｌｌｐｍｏｌと極めて高いという利点を有している
が、用いる装置が非常に高価であり、広く一般に利用で
きない。For example, mass fragmentography has the advantage of extremely high detection sensitivity of 0,11 pmol, but the equipment used is very expensive and cannot be widely used.
また、ラジオイムノアッセイ法の検出感度は１０ｐｍｏ
ｌと高いが、ＩｔｓＩＭＡや３Ｈ−Ｍ Ａなどのラジ
オアイソトープを利用するので、特別な施設と検出器が
必要とされる。In addition, the detection sensitivity of radioimmunoassay method is 10 pmo.
However, since it uses radioisotopes such as ItsIMA and 3H-MA, special facilities and detectors are required.
更に、イオン会合性試薬を用いる発色法は簡便であると
いう利点を有するが、試薬のＭＡに対する特異性に欠け
る場合が多くその信頼性が低く、また検出感度が５．４
ｎｍｏ１程度と低く、低濃度のＭＡの測定ができないな
どの欠点を有する。Furthermore, the color method using an ion-associative reagent has the advantage of being simple, but the reagent often lacks specificity for MA, and its reliability is low, and the detection sensitivity is 5.4.
It has drawbacks such as being as low as about nmol, making it impossible to measure low concentrations of MA.
本発明者らは、上記問題点に鑑み、より実用的なＭＡ検
出法を確立するために種々の検討を行なう過程で、魚類
等のを推動物から細菌に至る運動性を有する生物が、環
境の変化（外部からの光、重力、温度または化学物質な
どの刺激）を感知することにより起す一定の方向性を持
つ移動運動［低次の生得的行動（本能）］として定暑さ
れる走性（ｔａｘｉｓ）のなかで、化学物質が刺激とな
る走化性に注目し、なかでも細菌が示す走化性を利用し
てＭＡのみならずアセチルコリン等の各種神経生理活性
（神経作用、神経伝達）物質の微量定量、微量検定が、
簡便かつ迅速に実施できるとの新たな知見を得るに至っ
た。In view of the above-mentioned problems, the present inventors conducted various studies to establish a more practical MA detection method. Taxis is defined as locomotion with a certain direction [lower innate behavior (instinct)] caused by sensing changes in the environment (external stimuli such as light, gravity, temperature, or chemical substances). (taxis), we focus on chemotaxis stimulated by chemical substances, and in particular, utilize chemotaxis exhibited by bacteria to not only MA but also various neurophysiological activities (nerve action, nerve transmission) such as acetylcholine. Micro-quantification and micro-analysis of substances,
We have obtained new knowledge that it can be carried out easily and quickly.
すなわち、まず、本発明者らは、高等生物の神経系にお
けるシナプスの情報伝達機構と細菌の走化性における鞭
毛駆動機構の比較検討により、高等生物の神経系におけ
るシナプスの情報伝達機構におけるレセプターでの神経
伝達物質の受容が情報伝達の引金となっている事実と、
細菌類の走化性における鞭毛駆動機構において、走化性
レセプターが化学物質を受容することが情報伝達の引金
である事実とが類似・対応するとの認識を得た。That is, first, the present inventors conducted a comparative study of the synaptic information transmission mechanism in the nervous system of higher organisms and the flagellar drive mechanism in bacterial chemotaxis. The fact that the reception of neurotransmitters is the trigger for information transmission,
It was recognized that the flagellar drive mechanism of chemotaxis in bacteria is similar to, and corresponds to, the fact that the reception of chemical substances by chemotactic receptors is the trigger for information transmission.
更に、大腸菌が走化性を示す各種アミノ酸、例えばグリ
シンは神経末梢で産生された中枢神経系における抑制性
の伝達物質として、またアスパラギン酸やグルタミン酸
などはを推動物の脳、視細胞などで同定され、神経伝達
物質として働くだけでなく、神経末端から遊離して拡散
し、局所的に多くの細胞に働く局所性化学仲介物質（神
経調節物質）として知られている点に注目し、細菌の走
化性が高次な情報伝達機構である神経系の起源である可
能性があるとの考えを持つに至った。Furthermore, various amino acids that E. coli exhibits chemotaxis, such as glycine, which is produced in the nerve periphery and acts as an inhibitory transmitter in the central nervous system, and aspartic acid and glutamic acid, have been identified in the brain and photoreceptor cells of mammals. We focused on the fact that it not only acts as a neurotransmitter, but is also known as a local chemical mediator (neuromodulator) that diffuses from nerve terminals and acts locally on many cells. This led to the idea that chemotaxis may be the origin of the nervous system, which is a higher-order information transmission mechanism.
なお、細菌が走化性を示す化学物質については、Ａｄｌ
ｅｒらにより数多く調べられ、大腸菌、サルモネラ菌、
枯草菌などの有鞭毛細菌類は、糖、アミノ酸などの栄養
物質（誘因物質）には引きつけられ、各種有害物質など
の忌避物質から遠ざかることが明らかにされている（
Ａｄｌｅｒ、 Ｊ。Regarding chemical substances that cause chemotaxis to bacteria, Adl
Many studies have been conducted by E. coli, Salmonella,
It has been shown that flagellated bacteria such as Bacillus subtilis are attracted to nutritional substances (attracting substances) such as sugars and amino acids, and are kept away from repellent substances such as various harmful substances (
Adler, J.
：５ｃｉｅｎｃｅ、 １５３ 、７０８−７１６（１９
６６）； Ｔｓｏ、 Ｗ、 Ｗ、＆Ａｄｌｅｒ、 Ｊ、
： Ｊ、 Ｂａｃｔｅｒｉｏｌｏｇｙ、旦８．５６０−
５７６（１９７４） 、及びＡｄｌｅｒ、 Ｊ、： Ａ
ｎｎ、 Ｒｅｖ、 Ｂｉｏｃｈｅｍ、。:5science, 153, 708-716 (19
66); Tso, W., & Adler, J.
: J, Bacteriology, Dan 8.560-
576 (1974), and Adler, J.: A.
nn, Rev. Biochem.
月、 ３４１−３５６ （１９７５）等参照］。See Moon, 341-356 (1975), etc.].
また、Ａｄｌｅｒらの用いた毛細管法によれば、大腸菌
は誘因物質であるアスパラギン酸に対して、１μβの容
量において１０−’Ｍ Ｎ１０−８Ｍの濃度から走化性
を示すことが報告されている［Ａｄｌｅｒ、 Ｊ、：Ａ
ｎｎ、 Ｒｅｖ、 Ｂｉｏｃｈｅｍ、、４４．３４１−
３５６ （１９７５） ］ 。Furthermore, according to the capillary method used by Adler et al., it has been reported that E. coli exhibits chemotaxis toward aspartic acid, an attractant, from a concentration of 10-'M N10-8M in a volume of 1μβ. [Adler, J.:A
nn, Rev. Biochem, 44.341-
356 (1975)].
しかしながらこれらの報告においては、本発明者らが想
定した上述の走化性と神経系との関連については全く示
唆されてｄ）ない。However, in these reports, there is no suggestion at all about the relationship between the above-mentioned chemotaxis and the nervous system, which was assumed by the present inventors d).
このような背景から、本発明者らは、神経生理活性物質
であるＭＡやアセチルコリンなども高い感度で、細菌の
走化性を惹起させたり、あるいは細菌の走化性に影響を
与える可能性を想到した。Against this background, the present inventors investigated with high sensitivity the possibility that neurophysiologically active substances such as MA and acetylcholine may induce or affect bacterial chemotaxis. I came up with the idea.
本発明者らは、この推論に基づき、種々検討を重ねた結
果、大腸菌等がＭＡに対して正の走化性（刺激源に向っ
て進む）を示すことを見い出した。Based on this inference, the present inventors conducted various studies and found that Escherichia coli and the like exhibit positive chemotaxis (progress toward a stimulus source) toward MA.
更に、Ａｄｌｅｒらの細菌の走化性のキャピラリー測定
系の改良により、より簡便なスクリーニング、検定方法
を確立し［岩本昌之、篠沢隆雄ら：生物教材としての細
菌工、大腸菌の簡便な取り扱いと走化性の観察、科学教
育研究、１１． Ｎｏ、３．１０８−１１３（１９８７
）および福永晋哉、篠沢隆雄ら：生物教材としての細菌
■、大腸菌の走化性の定量的検定法、科学教育研究、１
１． Ｎｏ、４（１９８７）］ 、並びに、ろ紙法も採
用してＭＡやアセチルコリンを定性的、定量的にバイオ
アッセイすることに成功し、１０−’Ｍ以上のＭＡ濃度
でのＭＡの検出が可能であり、しかも試料の量が１μβ
で十分であることからラジオアイソトープを用いること
なく、ラジオイムノアッセイ法なみの１０ｐｍｏ ｌと
いう微量のＭＡを検出できることを見い出し、本発明を
完成した。Furthermore, by improving the capillary measurement system for bacterial chemotaxis by Adler et al., a simpler screening and assay method was established [Masayuki Iwamoto, Takao Shinozawa et al.: Bacteriology as a biological teaching material, simple handling and chemotaxis of E. coli]. Observation of chemical nature, science education research, 11. No. 3.108-113 (1987
) and Shinya Fukunaga, Takao Shinozawa et al.: Bacteria as biological teaching materials■, Quantitative assay method for chemotaxis of Escherichia coli, Science Education Research, 1
1. No. 4 (1987)] and also adopted the filter paper method to qualitatively and quantitatively bioassay MA and acetylcholine, making it possible to detect MA at MA concentrations of 10-'M or higher. Yes, and the amount of sample is 1μβ
The present invention was completed based on the discovery that it is possible to detect a trace amount of MA as small as 10 pmol, which is equivalent to radioimmunoassay, without using a radioisotope.
なお、ＤＬ−アンフェタミンについてはＣｈｅｔらがシ
ュードモナス フルオレセンス（Ｐｓｅｕｄｏｍｏｎａ
ｓｆｌｕｏｒｅｓｅｎｓ ）の走化性を利用して検定し
ている（Ｃｈｅｔ、 １． ｅｔ ａｌ ： Ｊ、 Ｂ
ａｃｔｅｒｉｏｌｏｇｙ、 １１５（３）。Regarding DL-amphetamine, Chet et al.
sfluoresens) chemotaxis (Chet, 1. et al: J, B
acteriology, 115(3).
１２１５−１２１８ （１９７３）。しかしながら、こ
の検定は簡便なアッセイ法である軟寒天プレート法で行
なわれたものでない。1215-1218 (1973). However, this assay was not conducted using the soft agar plate method, which is a simple assay method.
本発明の目的は、ＭＡ等のアンフェタミン類やアセチル
コリンなどの各種神経生理活性（神経作用、神経伝達）
物質の微量定量、微量検定に好適であり、簡便かつ迅速
に実施できる極めて実用的な方法を提供することにある
。The purpose of the present invention is to promote various neurophysiological activities (nerve action, neurotransmission) of amphetamines such as MA and acetylcholine.
The object of the present invention is to provide an extremely practical method that is suitable for microquantity determination and microanalysis of substances, and can be carried out easily and quickly.
［課題を解決するための手段］
本発明のバイオアッセイ法は、細菌の走化性を利用する
ことを特徴とする。[Means for Solving the Problems] The bioassay method of the present invention is characterized by utilizing chemotaxis of bacteria.
本発明の方法に用いることができる細菌としは、Ｅｓｃ
ｈｅｒｉｃｈｉａ ｃｏｌｉ ｋ１２ Ｗ３１１０ （
野生株、東京大学応用微生物研究所あるいは発酵研究所
等から一般に容易に入手できる）などの大腸菌、サルモ
ネラ菌、枯草菌などを挙げることができるが、なかでも
大腸菌はその遺伝学的および生化学的性質が最も良く解
明されているので、取り扱い易い。Bacteria that can be used in the method of the present invention include Esc.
herichia coli k12 W3110 (
E. coli, Salmonella enterica, and Bacillus subtilis (wild strains, generally easily available from the Institute of Applied Microbiology or Fermentation Research Institute, University of Tokyo, etc.) can be mentioned. is the best understood and therefore easy to handle.
また、これらの耐熱性菌も有用である。These heat-resistant bacteria are also useful.
本発明の方法は、細菌を含む系に試料を添加した際の細
菌の挙動（走化性）を検定することにより行なうことが
できる。The method of the present invention can be carried out by assaying the behavior (chemotaxis) of bacteria when a sample is added to a system containing bacteria.
本発明で用いる細菌を含む系は、例えば以下に示すよう
な液体あるいは軟寒天プレート等として必要に応じて種
々の形態とすることができる。The system containing bacteria used in the present invention can be in various forms as required, for example as a liquid or a soft agar plate as shown below.
以下本発明の方法に適用し得る軟寒天プレート法及び毛
細管法について説明する。The soft agar plate method and capillary tube method applicable to the method of the present invention will be explained below.
本発明に用い得る軟寒天プレート法としては、ａ）細菌
を含有させた軟寒天プレート上の所定位置に試料を容易
に拡散しない状態で添加し、所定時間放置後、該プレー
ト中での菌体の移動状態を観察して試料中に細菌が走化
性を示すＭＡやアセチルコリン等の各種神経生理活性物
質が存在するかどうか検定する方法、
ｂ）試料を含有する軟寒天プレート上の所定位置に細菌
を含有させた溶液を滴下し、所定時間放置後、該プレー
ト中での菌体の移動状態を観察して試料中に細菌が走化
性を示すＭＡやアセチルコリン等の各種神経生理活性物
質が存在するかどうか検定する方法、
などが利用できる。The soft agar plate method that can be used in the present invention is as follows: a) A sample is added to a predetermined position on a soft agar plate containing bacteria in a state that does not easily diffuse, and after being left for a predetermined time, bacterial cells in the plate are added. b) A method of observing the state of movement of bacteria to determine whether there are various neurophysiologically active substances such as MA and acetylcholine that cause chemotaxis in the sample; After dropping a solution containing bacteria and leaving it for a predetermined period of time, the state of movement of bacterial cells in the plate was observed, and various neurophysiologically active substances such as MA and acetylcholine, which show bacteria chemotaxis, were detected in the sample. You can use methods to test whether it exists.
例えば、試料中に細菌を誘因する物質、すなわち細菌が
刺激源に向って進む正の走化性を示す物質が存在する場
合には、上記ａ）の方法においては、試料添加位置周辺
に菌体が集合し、菌体密度の高い部分が形成され、それ
が白いリングとして目視できるようになる。For example, if there is a substance in the sample that attracts bacteria, that is, a substance that exhibits positive chemotaxis that causes bacteria to move toward the stimulus source, in method a) above, bacteria will be present around the sample addition position. The cells aggregate to form areas with high bacterial cell density, which become visible as white rings.
軟寒天プレート法に用いるプレートは、操作上の取り扱
いが容易であり、かつプレート内の細菌を良好に維持で
き、更に該細菌の走化性が効率良く現われるようにその
組成を適宜選択して調整することができる。The plate used for the soft agar plate method is easy to handle, can maintain the bacteria within the plate well, and has a composition that is appropriately selected and adjusted so that the chemotaxis of the bacteria is efficiently exhibited. can do.
例えば、通常用いられている各種細菌用培地や細菌を取
り扱う際に用いる各種溶液から、用いる細菌に応じて選
択したものに、寒天を０．２８重量％程度加えて調整す
ることができる。For example, it can be prepared by adding about 0.28% by weight of agar to a solution selected from various commonly used bacterial culture media and various solutions used when handling bacteria, depending on the bacteria used.
なお、該プレートは菌の増殖を目的としたものでないの
で、培地の組成を菌体の良好な維持が可能なものに適宜
変更すると良い。Note that, since the plate is not intended for the growth of bacteria, the composition of the medium may be appropriately changed to one that allows good maintenance of the bacteria.
具体的には、後述の実施例で用いているし一グロスやポ
リペプトンブロスを用いた軟寒天プレートが好適に利用
できる。Specifically, a soft agar plate using Shiichi gloss or polypeptone broth used in the Examples described later can be suitably used.
軟寒天プレート上への試料の添加は、試料を含ませた適
当な大きさのろ紙をプレート上に載置して行ない、プレ
ートは３７℃で保温する。A sample is added onto a soft agar plate by placing a filter paper of an appropriate size containing the sample on the plate, and the plate is kept warm at 37°C.
一方、毛細管法は、その一端を閉管して試料を含む溶液
を封入し毛細管の他方の開口端を、スライドガラス等の
適当な基体上に用意した細菌含有液に接触させ、所定時
間静置後、菌体の移動状態を観察して試料中に細菌が走
化性を示すＭＡやアセチルコリン等の各種神経生理活性
物質が存在するかどうか検定するものである。On the other hand, in the capillary tube method, one end of the tube is closed and a solution containing the sample is sealed, and the other open end of the capillary tube is brought into contact with a bacteria-containing solution prepared on a suitable substrate such as a slide glass, and after being allowed to stand still for a predetermined period of time. This test examines the state of movement of bacterial cells to determine whether there are various neurophysiologically active substances such as MA and acetylcholine, which cause chemotaxis in bacteria, in the sample.
なお、第１図に毛細管内に誘因物質、すなわち大腸菌が
正の走化性を示す物質を含有させた場合における菌体の
動きを示した
該毛細管法に用いる菌体な含む溶液は、細菌を良好に維
持でき、かつ該細菌の走化性が効率良く現われるように
その組成を適宜選択して調製することができ、例えば、
通常用いられている各種細菌用培地や細菌を取り扱う際
に用いる各種溶液から、用いる細菌に応じて選択したも
のを使用できる。Figure 1 shows the movement of bacterial cells when the capillary tube contains an attractant, that is, a substance that shows positive chemotaxis for E. coli. The composition can be appropriately selected and prepared so that it can be maintained well and the chemotaxis of the bacterium is efficiently exhibited. For example,
It is possible to use one selected from among various commonly used bacterial culture media and various solutions used when handling bacteria, depending on the bacteria to be used.
な都、該溶液もまた菌の増殖を目的としたものでないの
で、培地の組成を菌体の良好な維持が可能なものに適宜
変更すると良い。However, since this solution is not intended for the growth of bacteria, it is preferable to change the composition of the medium as appropriate to one that allows good maintenance of the bacteria.
具体的には、後述の実施例で用いている溶液が好適に利
用できる。Specifically, the solutions used in the Examples described later can be suitably used.
細菌含有液をスライドガラス等の上に用意するには、例
えば、第２図および第３図に示すようにスライドガラス
上に直管やＵ字管を置きその中に細菌含有液を入れ、更
に、直管またはＵ字管上にカバーガラスを載せる方法、
第４図に示すようにホールスライドガラスの凹部表面に
シリコングリス等の疎水性を示す物質を塗布し、そこに
細菌含有液を滴下する方法などを用いることができる。To prepare a bacteria-containing solution on a slide glass, for example, place a straight tube or U-shaped tube on the slide glass, pour the bacteria-containing solution into it, and then , a method of placing a cover glass on a straight tube or a U-shaped tube,
As shown in FIG. 4, a method can be used in which a hydrophobic substance such as silicone grease is applied to the surface of the recessed portion of the hole slide glass, and a bacteria-containing liquid is dropped thereon.
本発明に用いる細菌は、必要に応じて前培養する等の方
法によりその菌体活性な走化性の検出に適した状態にし
ておくと良い。The bacteria used in the present invention may be brought into a state suitable for detection of cell activity and chemotaxis by pre-culturing or the like, if necessary.
なお、本発明の方法は、例えば岩本昌之、篠沢隆雄ら：
生物教材としての細菌工、大腸菌の簡便な取り扱いと走
化性の観察、科学教育研究、Ｕ、Ｎｏ、 ３．１０Ｂ−
１１３＜１９８７）および福永晋哉、篠沢隆雄ら：生物
教材としての細菌■、大腸菌の走化性の定量的検定法、
科学教育研究、貝、Ｎｏ、 ４ （１９８７）等を参考
として実施することができる。The method of the present invention is described, for example, by Masayuki Iwamoto, Takao Shinozawa, et al.
Bacteriology as a biological teaching material, simple handling of Escherichia coli and observation of chemotaxis, science education research, U, No. 3.10B-
113<1987) and Shinya Fukunaga, Takao Shinozawa et al.: Bacteria as biological teaching materials ■, Quantitative assay method for chemotaxis of Escherichia coli,
This can be carried out by referring to Science Education Research, Kai, No. 4 (1987), etc.
［実施例］ 以下、実施例により本発明を更に詳細に説明する。[Example] Hereinafter, the present invention will be explained in more detail with reference to Examples.
実施例１
（軟寒天プレートの調製）
乾熱殺菌したアルミキャップ付きの３０ｍｆ２試験管（
１８ｍｍＸ １８ｍｍ）にオートクレーブ処理しである
下記組成のグリセリン塩類培地５ｍβを入れ、Ｅｓｃｈ
ｅｒｉｃｈｉａ ｃｏｌｉ ｋ１２ Ｗ３１１０ （野
生株、東京大学応用微生物研究所より入手）接種し、３
７℃、７０〜８０回振盪／分の条件で一晩振盪培養し、
培養の定常期状態を得た。なお、培養終了時の菌体濃度
は、２〜４Ｘ１０”個／ｍｌであった。Example 1 (Preparation of soft agar plate) A 30mf2 test tube with a dry heat sterilized aluminum cap (
18 mm
Erichia coli k12 W3110 (wild strain, obtained from the Institute of Applied Microbiology, University of Tokyo) was inoculated, and
Shaking culture was carried out overnight at 7°C and 70 to 80 shakes/min.
A stationary phase state of the culture was obtained. The bacterial cell concentration at the end of the culture was 2 to 4 x 10'' cells/ml.
グリセリン塩類培地組成：
グリセリン ５．０ｇ（他の組成分
とは別に煮
沸して使用直前に混合）
ＮａＣＩ ５． ＯｇＫ
、ＨＰＯ，１１，２ｇ
Ｋ）１２ＰＯ４４，８ｇ
（Ｎ）１４） ２Ｓ０４ ２．
０ｇＭｇ５０４・ＨＪ ０．
２５ｇＦｅｗ （ＳＯ４） ｓ・ｘＨ２Ｏ０，５ｍｇ全
容量１０００ ＋ｎＪ２ （ｐＨ７，０）なお、この培
養菌体液は４℃で２〜３週間保存可能であった。Glycerin salt medium composition: Glycerin 5.0g (boiled separately from other ingredients and mixed immediately before use) NaCI 5. OgK
, HPO, 11,2g K)12PO44,8g (N)14) 2S04 2.
0gMg504・HJ 0.
25gFew (SO4) s.xH2O0.5mg Total volume 1000 +nJ2 (pH 7.0) This cultured cell fluid could be stored at 4°C for 2 to 3 weeks.
次に、該培養菌体液の０．２５ｍＩ２を４ｍＩ２のグリ
セリン塩類培地に接種し、３７℃、７０〜８０回振盪／
分で２時間培養し、最も運動性が高い対数増殖期の菌を
得た。なお、得られた培養菌体液の５９０ｎｍでの濁度
（光路１ ｃｍ）は０．２〜０．４であり、菌体数は３
〜４Ｘ１０”個／ｍＩ２であった。Next, 0.25 mI2 of the cultured bacterial body fluid was inoculated into 4 mI2 glycerin salt medium, and shaken 70 to 80 times at 37°C.
After culturing for 2 hours, bacteria in the logarithmic growth phase, which has the highest motility, was obtained. The turbidity at 590 nm (light path 1 cm) of the obtained cultured bacterial cell fluid was 0.2 to 0.4, and the number of bacterial cells was 3.
˜4×10”/mI2.
更に、この対数増殖期の菌を含む培養菌体液１．０ｍｌ
を３０００ｒｐｍ 、１５分の遠心分離で集菌し、得ら
れた菌体に１．０ｍｇの洗浄液［１０−’Ｍのエチレン
ジアミン四酢酸カリウム塩（Ｋ−ＥＤＴＡ）を含む１０
−”Ｍのリン酸カリウム緩衝液（ｐＨ７，０） ］を加
えてよく攪拌して菌体を洗浄し、更に上記と同様の条件
の遠心分離により集菌し、再度同様の操作を繰り返した
。Furthermore, 1.0 ml of cultured bacterial body fluid containing this logarithmic growth phase bacteria
The cells were collected by centrifugation at 3,000 rpm for 15 minutes, and the resulting cells were mixed with 1.0 mg of a washing solution [10-'M potassium ethylenediaminetetraacetic acid (K-EDTA)].
-''M potassium phosphate buffer (pH 7.0)] was added and stirred well to wash the bacterial cells, and the bacteria were collected by centrifugation under the same conditions as above, and the same operation was repeated again.
次に、洗浄菌体に１．０ｍｇの検定用緩衝液［１０１Ｍ
のリン酸カリウム緩衝液（ｐＨ７，０） ］を加え検定
用として用いた。なお、この菌体液は氷冷しておくこと
によって１日は十分に使用できた。Next, 1.0 mg of assay buffer [101M
Potassium phosphate buffer (pH 7.0)] was added and used for assay. Note that this bacterial body fluid could be used for one day by keeping it ice-cooled.
これとは別に、ろ紙を利用した集菌、洗浄を行なって検
定用菌体の調製を行なった。Separately, bacteria were collected using filter paper and washed to prepare bacterial cells for assay.
すなわち、上記対数増殖期の菌を含む培養菌体液（２０
ｍｇ）を、予め乾熱殺菌（１６０℃、３時間）したろ紙
（Ｎｏ、２、東洋ろ紙株式会社）でろ過し、ろ紙上に残
った菌体を洗浄液１０ ｍＩ２で２度洗浄し、ろ紙の中
心部を半径的２ｃｍに切り取り検定用緩衝液と接触させ
て、ろ紙上の菌体を検定用緩衝液に懸濁し、その菌体濃
度を６．８Ｘ１０’個７ｍ１２に調製し検定用として用
いた。That is, the cultured bacterial fluid containing the bacteria in the logarithmic growth phase (20
mg) was filtered through a filter paper (No. 2, Toyo Roshi Co., Ltd.) that had been dry heat sterilized (160°C, 3 hours) in advance, and the bacterial cells remaining on the filter paper were washed twice with 10 mI2 of washing solution. The center was cut to a radius of 2 cm and brought into contact with an assay buffer, and the bacterial cells on the filter paper were suspended in the assay buffer, and the bacterial cell concentration was adjusted to 6.8 x 10' cells (7 m12) and used for assay. .
なお、上記の遠心分離法による方法で調製した検定用菌
体液、およびろ紙性により検定用菌体液はともに後述の
検定に良好に用いることができた。In addition, both the bacterial cell liquid for assay prepared by the above-mentioned centrifugation method and the bacterial cell liquid for assay due to the filter paper properties could be used satisfactorily in the assay described below.
次に、シャーレ中へ０．２８％の寒天を溶解させた下記
組成のポリペプトンブロス９ｍβと上記のようにして検
定用に調製した菌体液（菌体濃度を２〜４Ｘ１０８個／
ｍＩ２に調製）の１．０＋＋＋Ｊ２とを加え、十分に混
合した後静置し、寒天を固化し軟寒天プレートを得た。Next, polypeptone broth 9mβ with the following composition in which 0.28% agar was dissolved was added to a petri dish, and the bacterial cell liquid prepared for the assay as described above (the bacterial cell concentration was adjusted to 2 to 4 x 108 cells/
1.0+++J2 (prepared to mI2) was added, thoroughly mixed, and then allowed to stand to solidify the agar to obtain a soft agar plate.
ポリペプトンブロス組成：
ポリペプトン ｌｏｇ
ＮａＣ１５ｇ
全量１０００ ｍＩ２（ｐＨ７，０）
実施例２
（軟寒天プレート法によるＭＡに対する大腸菌の走化性
の検定）
実施例１で得た軟寒天プレート上の中央に１０ｍＭＭＡ
（大日本製薬社製）を含む１０ｍＭリン酸ナトリウム緩
衝液（ｐＨ７，５） ２０μβをしみ込ませた円形ろ紙
（ペーパーディスク、厚手、東洋ろ紙株式会社）を置き
、３７℃、１２時間静置した。Polypeptone broth composition: Polypeptone log NaC 15g Total amount 1000 mI2 (pH 7,0) Example 2 (Assessment of E. coli chemotaxis against MA by soft agar plate method) 10mMMA was placed in the center on the soft agar plate obtained in Example 1.
A round filter paper (paper disk, thick, Toyo Roshi Co., Ltd.) impregnated with 20 μβ of 10 mM sodium phosphate buffer (pH 7.5) containing (manufactured by Dainippon Pharmaceutical Co., Ltd.) was placed and allowed to stand at 37° C. for 12 hours.
その結果、ろ紙の周辺に白いリングが形成された。この
白いリングは、大腸菌が集合し、菌の濃度があがったた
めにできたものであった。As a result, a white ring was formed around the filter paper. This white ring was caused by aggregation of E. coli and an increased concentration of bacteria.
これはろ紙からしみ出したＭＡに対して大腸菌が正の走
化性を示したためである。すなわち、ＭＡが大腸菌が正
の走化性を示す誘因物質であることが明らかとされた。This is because E. coli showed positive chemotaxis toward MA seeped out from the filter paper. In other words, it was revealed that MA is an attractant that causes E. coli to exhibit positive chemotaxis.
実施例３
（毛細管法によるＭＡの定量）
まず、各種濃度のＭＡを１０ｍＭリン酸ナトリウム緩衝
液（ｐＨ７，５）に溶解して、濃度の異なるＭＡ温溶液
調製し、以下の操作に用いた。Example 3 (Quantification of MA by capillary tube method) First, various concentrations of MA were dissolved in 10 mM sodium phosphate buffer (pH 7,5) to prepare warm MA solutions with different concentrations, which were used in the following operations.
１μ℃の毛細管［両端開口、内径０．２ｍｍ、長さ３．
０ＣＩ１１％商品名Ｍｉｃｒｏｃａｐｓ、叶ｕｍｎｏｄ
５ｃｉｅｎｔｉｆｉｃ社製コの一方の先端にＭＡ温溶
液つけ、管内へ毛細管現象を利用して該溶液を吸い上げ
た後、その−方の開口を溶融により封じた。1μ℃ capillary tube [both ends open, inner diameter 0.2mm, length 3.
0CI11%Product name Microcaps, umnod
A hot MA solution was applied to one end of a tube manufactured by 5Ccientific, and the solution was sucked up into the tube using capillary action, and then the opening on the other end was sealed by melting.
一方、ホールスライドガラスの凹部にシリコングリスを
塗布し、その上に実施例１で検定用に調製した菌体液（
菌体濃度２〜４Ｘ１０”個／ｍβ）の０．８ｍβを滴下
し、第５図に示すように先に調整した毛細管を、その開
口が菌体液の液滴と接触するようにセットした。On the other hand, apply silicone grease to the concave part of the hole slide glass, and apply the bacterial cell liquid prepared for assay in Example 1 (
0.8 mβ with a bacterial cell concentration of 2 to 4×10” cells/mβ) was dropped, and as shown in FIG. 5, the previously prepared capillary tube was set so that its opening was in contact with the droplet of bacterial fluid.
その状態で、これらを３０℃で５０分間静置した後、毛
細管を取り出し、菌体液と接触していた開口部を洗浄液
で洗浄し毛細管外壁に付着した菌を除去した。In this state, these were allowed to stand at 30° C. for 50 minutes, and then the capillary tube was taken out, and the opening that had been in contact with the bacterial cell fluid was washed with a cleaning solution to remove bacteria adhering to the outer wall of the capillary tube.
次に、閉じている端を破壊し、マイクロとペラターで毛
細管中の液を洗浄液内に押し出し、１０’倍に希釈した
。Next, the closed end was broken and the liquid in the capillary was forced out into the washing liquid using a micro-pelleter and diluted 10' times.
得られた希釈液中の菌体数を重層寒天法により測定した
。The number of bacterial cells in the obtained diluted solution was measured by the layered agar method.
すなわち、まず第５図に示すようにシャーレ中に以下に
示す組成のし一ブロスに１．７％の寒天を含有させたプ
レートを調製し、その上に希釈液０、１＋ｎＩ２をのせ
、更に０．３％寒天水溶液をのせた後、加えた寒天を固
化させ、上下を逆にして３７℃で一晩放置し、プレート
に現われるコロニー数を計測した。That is, first, as shown in Figure 5, a plate containing 1.7% agar in Shiichi broth with the composition shown below is prepared in a petri dish, diluted solutions 0 and 1 + nI2 are placed on it, and then 0 and 1 + nI2 are placed on top of the plate. After placing a 3% agar solution on the plate, the added agar was solidified, left upside down at 37°C overnight, and the number of colonies appearing on the plate was counted.
Ｌ−ブロス組成：
ポリペプトン ｌＯ，０ｇ酵母エキス
トラクト ５．０ｇブドウ糖
１．０ｇＮａＯＨ２Ｎ
３．Ｏｎ＋ｊ２全量１０００ ｍ１２ （
ｐＨ７，２）ＭＡ濃度と毛細管へ移動した菌体数との関
係を第６図に示す。L-broth composition: Polypeptone 1O, 0g yeast extract 5.0g glucose
1.0gNaOH2N
3. On+j2 total volume 1000 m12 (
Figure 6 shows the relationship between the pH 7, 2) MA concentration and the number of bacterial cells that migrated to the capillary tube.
第６図に示した結果から明らかなように、ＭＡ濃度１０
−’Ｍから毛細管中への大腸菌の移動個数が増加し始め
、ＭＡ濃度ｌ０−３〜１０−”Ｍで誘因された大腸菌数
が最大となり、ＭＡ濃度１０−’Ｍから大腸菌がＭＡに
対して正の走化性を示すことが明らかとされた。As is clear from the results shown in Figure 6, MA concentration 10
The number of E. coli transferred into the capillary from -'M begins to increase, and the number of E. coli induced at MA concentrations of 10-3 to 10-'M reaches its maximum, and from MA concentration of 10-'M, E. coli moves to MA. It was revealed that the larvae exhibited positive chemotaxis.
なお、この毛細管法で用いた毛細管の容量は１μｎであ
るので、ＭＡ濃度１０−’Ｍは１０ｐｍｏｌに相当する
。Note that since the capillary tube used in this capillary tube method has a capacity of 1 .mu.n, the MA concentration of 10-'M corresponds to 10 pmol.
実施例４
（軟寒天プレート法によるアセチルコリンに対する大腸
菌の走化性の検定）
実施例１で調製した軟寒天プレート上に、１０ｍＭアセ
チルコリン（和光純薬社製）を含む１０ｍＭリン酸ナト
リウム緩衝液（ｐＨ７，５） ２０μβをしみ込ま。Example 4 (Assessment of E. coli chemotaxis toward acetylcholine by soft agar plate method) On the soft agar plate prepared in Example 1, 10mM sodium phosphate buffer (pH 7) containing 10mM acetylcholine (manufactured by Wako Pure Chemical Industries, Ltd.) was placed. , 5) Soak 20μβ.
せたろ紙（実施例２で用いたのと同様）及び１０ｍＭリ
ン酸緩衝液（ｐｉ（７，５）のみ（２０μβ）をしみ込
ませたろ紙（実施例２で用いたのと同様）を置き、３７
℃、１２時間静置した。Place filter paper (same as used in Example 2) and filter paper impregnated with 10 mM phosphate buffer (pi(7,5) only (20 μβ) (same as used in Example 2). 37
The mixture was allowed to stand at ℃ for 12 hours.
その結果、アセチルコリンに対して大腸菌は走化性を示
さなかった。As a result, E. coli did not show chemotaxis toward acetylcholine.
更に、アスパラギン酸１０ｍＭとアセチルコリン１０ｍ
Ｍの混合液をろ紙にしみ込ませた場合と、アスパラギン
酸１０ｍＭのみをしみ込ませた場合とで、軟寒天プレー
ト法を実施例２と同様にして実施した。Furthermore, aspartic acid 10mM and acetylcholine 10mM
The soft agar plate method was carried out in the same manner as in Example 2, in which the filter paper was impregnated with a mixed solution of M and in the case where only 10 mM of aspartic acid was impregnated.
その結果、アスパラギン酸のみでは、ろ紙上の周辺に走
化した大腸菌が明確に観察できたが、アセチルコリンと
の混合液では、大腸菌の走化性が抑えられることがわか
った。The results showed that when using aspartic acid alone, E. coli chemotaxis could be clearly observed around the filter paper, but when mixed with acetylcholine, the chemotaxis of E. coli was suppressed.
実施例５
（毛細管法によるアスパラギン酸とアセチルコリンの定
量）
ｌＯ−６〜１０−”Ｍ濃度のアスパラギン酸溶液（０，
１ｍｌ２）と、１０−’ 〜１０−”Ｍ濃度のアスパラ
ギン酸溶液のそれぞれに１０ｍＭのアセチルコリンを混
合した溶液（０，１ｍβ）を各々調製し、それらを個々
に用いて、実施例３と同様にして、毛細管に移動する菌
体数を求めた。Example 5 (Quantification of aspartic acid and acetylcholine by capillary method) Aspartic acid solution (0,
1ml2) and a solution (0.1mβ) in which 10mM acetylcholine was mixed with an aspartic acid solution with a concentration of 10-' to 10-''M were prepared, and these were used individually in the same manner as in Example 3. The number of bacterial cells that migrated to the capillary tube was determined.
得られた結果を第７図に示す。なお、・はアスパラギン
酸溶液での結果を、Ｏはアスパラギン酸とアセチルコリ
ンの混合液の結果を示す。The results obtained are shown in FIG. Note that * indicates the results for an aspartic acid solution, and O indicates the results for a mixed solution of aspartic acid and acetylcholine.
第７図に示されているように、１０”’Ｍ付近からアス
パラギン酸への大腸菌の走化性が抑えられた。As shown in FIG. 7, chemotaxis of E. coli toward aspartic acid from around 10''M was suppressed.
実施例６
実施例５においてアスパラギン酸溶液濃度を１０ｍＭに
一定し、アセチルコリン溶液濃度を１０−８〜１０−”
Ｍ濃度に変えて、同様に毛細管法により検定した。Example 6 In Example 5, the aspartic acid solution concentration was kept constant at 10 mM, and the acetylcholine solution concentration was varied from 10-8 to 10-''
The assay was performed in the same manner using the capillary method, except for the concentration of M.
その結果、アセチルコリンは１０−’Ｍ付近からアスパ
ラギン酸への大腸菌の走化性を制御した。As a result, acetylcholine regulated the chemotaxis of E. coli toward aspartic acid from around 10-'M.
実施例７
覚醒剤患者（１０人）及び健康人（１０人）の尿（１（
ｌｒ＋＋Ｊ２）をそれぞれベンゼン１ ＋＋ｌに加え
抽出処理し、ベンゼン画分を分離後、ベンゼンを蒸発さ
せて、ベンゼン抽出物を得た。Example 7 Urine (1) of stimulant drug patients (10 people) and healthy people (10 people)
lr++J2) was added to 1 ++l of benzene for extraction treatment, the benzene fraction was separated, and the benzene was evaporated to obtain a benzene extract.
得られた各ベンゼン抽出物に水０．０５ｍβを加えて溶
解させた溶液を実施例２で用いたろ紙にしみ込ませた。The filter paper used in Example 2 was impregnated with a solution prepared by adding 0.05 mβ of water to each of the obtained benzene extracts.
こうして得られたろ紙のそれぞれを実施例１で調製した
軟寒天プレート上にのせ、実施例２と同様にして走化性
を検定した。Each of the filter papers thus obtained was placed on the soft agar plate prepared in Example 1, and chemotaxis was assayed in the same manner as in Example 2.
その結果、覚醒剤患者１０人中７人が陽性と判定され、
また健康人１０人中２人が陽性と判定された。As a result, 7 out of 10 stimulant drug patients tested positive.
Two out of 10 healthy people tested positive.
この覚醒剤患者と健康人とでの結果の差は、有為な差で
あり、本発明の方法が有効であることが確認された。This difference in results between stimulant drug patients and healthy people was significant, and it was confirmed that the method of the present invention is effective.
更に、上記ベンゼン抽出物溶液を用いて実施例３の毛細
管法を行なったところ、覚醒剤患者１０人からの試料で
走化性を示した菌体数の平均が、健康人１０人からの試
料で走化性を示した菌体数の平均の２倍の値を示した。Furthermore, when the capillary tube method of Example 3 was carried out using the above benzene extract solution, the average number of bacteria that showed chemotaxis in samples from 10 stimulant drug patients was lower than that in samples from 10 healthy subjects. The number of cells showing chemotaxis was twice the average number.
［発明の効果］
本発明によれば、簡便かつ迅速な操作で、ラジオイムノ
アッセイなみの１０ｐｍｏｌという感度でのＭＡの検定
が可能となった。[Effects of the Invention] According to the present invention, MA can be assayed with a sensitivity of 10 pmol, which is comparable to radioimmunoassay, with simple and quick operations.
すなわち、本発明は細菌の極めて高度な走化性を利用し
たので検出感度が高く、また特殊な分析機器、分析用施
設を用いる必要がなく、かつ用いる材料も安価であり、
低コストでの検定をどこでも容易に実施できる。更に、
操作が簡単であり、多数の試料を効率良く短時間で処理
可能である。That is, since the present invention utilizes the extremely high chemotaxis of bacteria, the detection sensitivity is high, there is no need to use special analysis equipment or analysis facilities, and the materials used are inexpensive.
Tests can be easily carried out at low cost anywhere. Furthermore,
It is easy to operate and can efficiently process a large number of samples in a short time.
また、本発明によれば、アセチルコリンは大腸菌が走化
性を示す誘因物質や忌避物質ではないが、アセチルコリ
ンは誘引物質であるアスパラギン酸に対する大腸菌の正
の走化性を制御することが明らかとされた。このことよ
り、神経生理活性物質間の相互作用におけるアセチルコ
リンの役割、位置付けが明確にされるきっかけが与えら
れた。Furthermore, according to the present invention, although acetylcholine is neither an attractant nor a repellent for E. coli to exhibit chemotaxis, it has been shown that acetylcholine controls the positive chemotaxis of E. coli toward aspartic acid, which is an attractant. Ta. This provided an opportunity to clarify the role and position of acetylcholine in the interaction between neurophysiologically active substances.
また、本発明者らの新たな知見からアセチルコリンの定
量に困難さがある場合に、本発明の方法のように走化性
受容体（レセプター）を利用する方法がアセチルコリン
の検定に有用性を発揮する可能性が出てきた。In addition, the new findings of the present inventors indicate that a method using chemotactic receptors, such as the method of the present invention, is useful for assaying acetylcholine when it is difficult to quantify acetylcholine. There is a possibility to do so.
更に、種々の神経生理活性物質に特異性を有する菌株を
開発することにより、簡便で確実な検定が可能となる。Furthermore, by developing bacterial strains that have specificity for various neurophysiologically active substances, simple and reliable assays become possible.
第１図〜第４図は毛細管法の操作を説明するための模式
図、第５図は重層寒天法の操作を示した模式図、第６図
は実施例３で得られたＭＡ（メタンフェタミン）濃度と
毛細管内への移動菌体数との関係を示すグラフ、第７図
は、アセチルコリンと毛細管内への移動菌体数との関係
を示すグラフである。なお、第７図において、・はアス
パラギン酸溶液での結果を、Ｏはアスパラギン酸とアセ
チルコリンの混合液の結果を示す。
特許出願人 三井東圧化学株式会社
三井製薬工業株式会社
篠沢隆雄Figures 1 to 4 are schematic diagrams for explaining the operation of the capillary tube method, Figure 5 is a schematic diagram showing the operation of the layered agar method, and Figure 6 is the MA (methamphetamine) obtained in Example 3. FIG. 7 is a graph showing the relationship between the concentration and the number of microbial cells transferred into the capillary tube. FIG. 7 is a graph showing the relationship between acetylcholine and the number of microbial cells transferred into the capillary tube. In FIG. 7, * indicates the results for an aspartic acid solution, and O indicates the results for a mixed solution of aspartic acid and acetylcholine. Patent applicant Mitsui Toatsu Chemical Co., Ltd. Mitsui Pharmaceutical Industries, Ltd. Takao Shinozawa
Claims (1)
該試料中の化学物質を検出することを特徴とするバイオ
アッセイ法。 ２）前記細菌が大腸菌である請求項１のバイオアッセイ
法。 ３）前記走化性の検定法を、毛細管法により行なう請求
項１または２のバイオアッセイ法。 ４）前記走化性の検定法を、軟寒天プレート法により行
なう請求項１または２のバイオアッセイ法。 ５）前記化学物質がアンフェタミン類またはアセチルコ
リンである請求項１〜４のいずれかに記載のバイオアッ
セイ法。[Claims] 1) By assaying the chemotaxis of bacteria toward a sample,
A bioassay method characterized by detecting a chemical substance in the sample. 2) The bioassay method according to claim 1, wherein the bacterium is Escherichia coli. 3) The bioassay method according to claim 1 or 2, wherein the chemotaxis assay method is performed by a capillary tube method. 4) The bioassay method according to claim 1 or 2, wherein the chemotaxis assay method is performed by a soft agar plate method. 5) The bioassay method according to any one of claims 1 to 4, wherein the chemical substance is amphetamines or acetylcholine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1850188A JPH01196299A (en) | 1988-01-30 | 1988-01-30 | Bioassay technique |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1850188A JPH01196299A (en) | 1988-01-30 | 1988-01-30 | Bioassay technique |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH01196299A true JPH01196299A (en) | 1989-08-08 |
Family
ID=11973374
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1850188A Pending JPH01196299A (en) | 1988-01-30 | 1988-01-30 | Bioassay technique |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01196299A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5853638A (en) * | 1997-06-27 | 1998-12-29 | Samsung General Chemicals Co., Ltd. | Process for producing stretched porous film |
| USRE38863E1 (en) | 1995-02-03 | 2005-11-01 | Ruy Tchao | Chemotaxis assay procedure |
| JP2010252745A (en) * | 2009-04-28 | 2010-11-11 | Nippon Telegr & Teleph Corp <Ntt> | Bacterium analyzer |
| JP2010252746A (en) * | 2009-04-28 | 2010-11-11 | Nippon Telegr & Teleph Corp <Ntt> | Bacterium analyzer |
| JP2010252744A (en) * | 2009-04-28 | 2010-11-11 | Nippon Telegr & Teleph Corp <Ntt> | Bacterium analyzer |
| JP2012503975A (en) * | 2009-04-01 | 2012-02-16 | 湖南省天騎医学新技▲術▼有限公司 | Drug sensitivity test method and drug sensitivity test apparatus |
-
1988
- 1988-01-30 JP JP1850188A patent/JPH01196299A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| USRE38863E1 (en) | 1995-02-03 | 2005-11-01 | Ruy Tchao | Chemotaxis assay procedure |
| USRE40747E1 (en) | 1995-02-03 | 2009-06-16 | Ruy Tchao | Chemotaxis assay procedure |
| US5853638A (en) * | 1997-06-27 | 1998-12-29 | Samsung General Chemicals Co., Ltd. | Process for producing stretched porous film |
| JP2012503975A (en) * | 2009-04-01 | 2012-02-16 | 湖南省天騎医学新技▲術▼有限公司 | Drug sensitivity test method and drug sensitivity test apparatus |
| JP2010252745A (en) * | 2009-04-28 | 2010-11-11 | Nippon Telegr & Teleph Corp <Ntt> | Bacterium analyzer |
| JP2010252746A (en) * | 2009-04-28 | 2010-11-11 | Nippon Telegr & Teleph Corp <Ntt> | Bacterium analyzer |
| JP2010252744A (en) * | 2009-04-28 | 2010-11-11 | Nippon Telegr & Teleph Corp <Ntt> | Bacterium analyzer |
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