JPH0260982B2 - - Google Patents
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- Publication number
- JPH0260982B2 JPH0260982B2 JP54047490A JP4749079A JPH0260982B2 JP H0260982 B2 JPH0260982 B2 JP H0260982B2 JP 54047490 A JP54047490 A JP 54047490A JP 4749079 A JP4749079 A JP 4749079A JP H0260982 B2 JPH0260982 B2 JP H0260982B2
- Authority
- JP
- Japan
- Prior art keywords
- fluorescent antibody
- microorganisms
- antibody
- fluorescent
- specific
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
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- 244000005700 microbiome Species 0.000 claims description 72
- 239000000427 antigen Substances 0.000 claims description 30
- 102000036639 antigens Human genes 0.000 claims description 20
- 108091007433 antigens Proteins 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 18
- 238000006243 chemical reaction Methods 0.000 claims description 16
- 239000007850 fluorescent dye Substances 0.000 claims description 7
- 230000000813 microbial effect Effects 0.000 claims description 6
- 230000001678 irradiating effect Effects 0.000 claims description 2
- 239000000725 suspension Substances 0.000 description 28
- 230000005284 excitation Effects 0.000 description 14
- 241000894006 Bacteria Species 0.000 description 13
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 10
- 241000222122 Candida albicans Species 0.000 description 9
- 238000011002 quantification Methods 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- 239000002504 physiological saline solution Substances 0.000 description 7
- 239000008280 blood Substances 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 238000004020 luminiscence type Methods 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 5
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 5
- 241000283973 Oryctolagus cuniculus Species 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 241000589516 Pseudomonas Species 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 238000011481 absorbance measurement Methods 0.000 description 3
- 229940095731 candida albicans Drugs 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 108010074605 gamma-Globulins Proteins 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 101710135378 pH 6 antigen Proteins 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 1
- 241000183726 Echium albicans Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000064675 Nonnotus albicans Species 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000001032 anti-candidal effect Effects 0.000 description 1
- 108010090946 astrocytin Proteins 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 239000010802 sludge Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
本発明は螢光抗体抗原反応を利用した微生物の
定量法に関する。
より詳しくは、予め特定微生物の螢光抗体抗原
反応として螢光色素により標識された抗体と任意
の数からなる特定微生物を接触させ、得られた螢
光抗体抗原に例えば紫外線のような短い波長の光
線を照射し、螢光抗体抗原から発する励起光量を
電気的に計測し特定微生物の定量線を求めるこ
と、次いで前記方法により得られた特定微生物の
定量線と混合微生物系中より予め特定微生物の螢
光抗体抗原反応として調製された抗体と任意の数
からなる特定微生物を接触させ、得られた螢光抗
体抗原に例えば紫外線のような短い波長の光線を
照射し螢光抗体抗原から発する励起光量を電気的
に計測し得られた計測値を符合させ、混合微生物
系中より、特定微生物を定量する方法に関するも
のである。
本発明者らは、先にうさぎなどの脊椎動物の血
液中に特定の微生物を送り込むことにより送り込
まれた特定の微生物に対し、一定期間経過すると
その血液中に脊椎動物特有の抗体（γ―グロブリ
ン）が得られることを利用して、特定微生物を有
効に管理する方法を見出した。すなわち、うさぎ
等の脊椎動物の血液中に産成された抗体は特定微
生物特有の抗体となり、得られた抗体を螢光色素
により染色し標識後、一方の混合培養基より適量
の微生物をサンプリングし、前記の螢光色素によ
り染色し標識した抗体と接触させると螢光抗体反
応により混合培養基よりサンプリングした混合微
生物系中、特定微生物のみが螢光を発してその確
認及び定量ができることを見出し、活性汚泥中に
占める特定微生物の濃度を一定に維持する微生物
管理方法を確立した。（特願昭53―115005号）
しかしながら、この方法は、その都度、顕微鏡
により視認しなければならないという煩しさがあ
り且つ定量に至つては正確な定量は望めないもの
である。
他方、混合系の微生物から特定微生物のみを定
量しようとしても従来法の定量法では混合した総
懸濁度あるいは乾燥菌体総量しか測定できないた
め混合系の微生物については全く測定が不可能で
ある。
そこで、本発明者らはより簡便にし微生物を定
量する方法を検討した結果、螢光抗体抗原反応に
より抗体から発する螢光度を計測し結果、螢光度
に対し微生物の定量性があること及びその定量性
は任意の数からなる微生物に対しても常に一定で
あることを新たに見出し本発明を完成するに至つ
た。
以下、図によりその代表例を説明する。図に示
したのはアミンコ社製（米国）、J4型の螢光度計
の原理図であり、１は光源、２は増幅器、３は分
光器、４は螢光抗体抗原、５は分光器、６は増巾
器、７は測定器をそれぞれ示し、L1は分光器３
により分光された特定波長の光線、L2は螢光抗
体抗原４により発光する励起光線、L3は分光器
５により分光された特定波長励起光線を示してい
る。
光源１より発光された光線、（通常は紫外線を
利用）は増幅器２により所定の光量に増幅され分
光器３により特定波長L1として螢光抗体抗原４
に照射される。照射を受けた特定微生物の螢光抗
体抗原は励起光L2として発光し、分光器５に入
り特定波長の励起光L3として分光され、更に増
幅器６により所定の光量に増幅されて測定器７に
より電気的に測定することにより特定微生物の定
量線を求める。また、定量線を求めるにあたつて
は、求めようとする特定微生物を従来法に従い、
例えば乾燥菌体重量換算法等により定量してお
き、該定量結果と電気的による計測結果を符合さ
せ、計測値を定量値に換算する。特定微生物の定
量線は一般的には図２Ａ，Ｂのように縦軸に螢光
発光度（励起光）、横軸に微生物菌体の懸濁液容
量を示す図が用いられる。次いで、前記方法によ
り求めた特定微生物の定量線と予め特定微生物の
螢光抗体抗原反応として調製された抗体と任意の
数からなる特定微生物を接触させた螢光抗体抗原
４に光源１により発光された光線（通常は紫外線
を利用）を増巾器２により所定の光量に増巾し、
分光器３により所定波長L1とし照射される。照
射を受けた特定微生物の螢光抗体抗原は励起光
L2として発光し、分光器５に入り特定波長の励
起光L3として増巾器６により所定の光量に増巾
されて測定器７により電気的に計測し得られた計
測値を符合させることにより任意の数からなる特
定微生物を定量することができる。
なお、予め調製された螢光抗体抗原反応として
調製された抗体とは、特定の微生物を例えばうさ
ぎなどの脊椎動物の血液中に送り込み、送り込ま
れた微生物に対し一定期間経過すると血液中に形
成される特定の微生物特有の抗体を回収し、螢光
色素により標識したものを云う。
本発明方法により求められる微生物の定量線
は、微生物の懸濁濃度、混合微生物の有無には影
響されずに常に一定である。この理由は、微生物
の螢光抗体抗原から発生する光量を電気的に計測
するためである。従つて、一旦、特定微生物の定
量線を求めておけば実際の定量にあたり、この定
量線と螢光抗体抗原から発光する光量を電気的に
計測して得られた計測値を符合させれば懸濁濃
度、混合微生物の有無等に関係なく特定微生物の
定量ができる。また、抗体抗原反応は、予め反応
させようとする微生物に対応する同一微生物の螢
光抗体を予め用意し、螢光抗体抗原反応に備えな
ければならない。従つて、ここで特定微生物とは
脊椎動物の体内に築成する抗体の抗原となる微生
物全般を指し、同一の微生物の螢光抗体と同一の
微生物の定量に供される微生物を意味する。
また、本発明方法では、微生物の定量線は常に
一定であるので混合微生物中の特定微生物に対し
ても容易に定量が可能であるが係る場合は、螢光
抗体抗原反応により特定の微生物のみが励起光を
発することになる。故に、複数の微生物からなる
螢光抗体を予め調製混合しておき、混合微生物系
中の複数の特定微生物のみ反応させれば同時にし
て複数の特定微生物の定量が可能である。
特定微生物の定量線との計測値の符合は、その
都度、符合するより定量する方法が簡便である
が、微生物毎に定量線を求めておき、得られた定
量線を電機等に記憶させ、反応後直ちに定量する
方法も検討可能である。
次に使用される装置としては、前記のアミコ社
製（米国）の螢光度計を一般的例として示してあ
るが、その他FR―500／510型（島津製作所製）、
204―Ａ／Ｓ型、650―10M型、MPF―４型（い
ずれも日立製作所製）など、螢光抗体抗原反応を
定量可能な装置であれば、広範囲にわたる各種の
装置の利用が可能である。
以下、試験例および実施例により本発明を更に
具体的に説明する。
試験例
ノカルデア．エリスロボレスKR―Ｓ−１
（FERM―ＰNo.3530）をブイヨン液体培地に30℃
で５日間培養をおこなつた。培養後冷却遠心をお
こない前記菌体を得た。得られた菌体を生理食塩
水で洗滌後ホルマリンにて一昼夜菌をおこない再
び生理食塩水にて洗滌後、生理食塩水に懸濁し１
％懸濁液として抗原とした。
次に、前記の懸濁液を若成年の体重2.5Kgのウ
サギの耳静脈に第１日目１ml、第２日目２ml、第
３日目３mlを注射し、以後４日間休止し、このこ
の操作を３回繰り返し得られた血液から遠心法に
より抗血清を分離した。
次にこの抗血清を硫安分画法によりγ―グロブ
リン分画を調整し、更にこのγ―グロブリン画分
に螢光色素（フルオレツセインイソチオシアネー
ト英国BBL社製）を10℃で４時間反応させて標
識し、セフアデツクスＧ―50（生化学工業社製）
にてゲル過を行い、更にDEAE―セルロース
（生化学工学社製）カラムクロマトにて非特異性
因子を除去し螢光抗体液を作成した。
実施例 １
試験例により調整した螢光抗体を用いて目的と
する微生物を分別定量することを目的として次の
ような実験を行つた。
即ち、ブイヨン寒天培地にて30℃で２日間培養
した前記ノカルデア・エリスロボレスKR―Ｓ―
１株（FERM―Ｐ―No.3530）を白金耳にてかき
とり集菌し、生理食塩水にて懸濁を行つた。ここ
で分光光度計にて波長660nmにて菌の懸濁度を測
定した。このノカルデア．エリスロポレスKR―
Ｓ―１懸濁液0.5mlに対し、試験例により調整し
たノカルデア．エリスロポレスKR―Ｓ―１用の
螢光抗体液を0.5ml加え、30℃にて１時間反応さ
せる。
次に遠心を行つて反応物を集めた後、1.5mlの
生理食塩水に再懸濁をした後、螢光光度計（米国
アミコ社製）にて螢光発光度を測定した。
なお、螢光光度計の一次フイルターは紫外線透
過可視光吸収フイルターを、二次フイルター付シ
ヤープカツト黄フイルター（415nm以上の波長の
み光を透過させる）を用いた。
菌の懸濁度と螢光発光度との関係を表１に示
す。
表１ ノカルデア．エリスロボレスKR―Ｓ―１
株の懸濁度とＳ―１株用螢光抗体を反応させた後
の菌一螢光抗体物の螢光発光度
The present invention relates to a method for quantifying microorganisms using a fluorescent antibody-antigen reaction. More specifically, as a fluorescent antibody-antigen reaction of a specific microorganism, an antibody labeled with a fluorescent dye and an arbitrary number of specific microorganisms are contacted in advance, and the obtained fluorescent antibody antigen is exposed to short wavelength light such as ultraviolet light. irradiate with a light beam and electrically measure the amount of excitation light emitted from the fluorescent antibody antigen to determine the quantitative line of the specific microorganism, and then compare the quantitative line of the specific microorganism obtained by the above method with the quantitative line of the specific microorganism in the mixed microbial system in advance. The amount of excitation light emitted from the fluorescent antibody antigen by bringing the antibody prepared as a fluorescent antibody antigen reaction into contact with an arbitrary number of specific microorganisms, and irradiating the obtained fluorescent antibody antigen with short wavelength light such as ultraviolet rays. The present invention relates to a method for quantifying specific microorganisms in a mixed microbial system by electrically measuring them and matching the measured values obtained. The present inventors first introduced specific microorganisms into the blood of vertebrates such as rabbits, and after a certain period of time, antibodies specific to vertebrates (γ-globulin) were detected in the blood of the animals. ), we have discovered a method to effectively manage specific microorganisms. In other words, antibodies produced in the blood of vertebrates such as rabbits become antibodies specific to specific microorganisms, and after staining and labeling the obtained antibodies with a fluorescent dye, an appropriate amount of microorganisms is sampled from one mixed culture medium. It was discovered that when brought into contact with an antibody dyed and labeled with the fluorescent dye described above, only specific microorganisms in a mixed microbial system sampled from a mixed culture medium emit fluorescence due to the fluorescent antibody reaction, making it possible to confirm and quantify activated sludge. We have established a microbial control method that maintains a constant concentration of specific microorganisms. (Japanese Patent Application No. 53-115005) However, this method is troublesome as it requires visual confirmation using a microscope each time, and accurate quantification cannot be expected. On the other hand, even if an attempt is made to quantify only specific microorganisms from a mixed system of microorganisms, conventional quantitative methods can only measure the total degree of suspension or the total amount of dry microbial cells in the mixture, making it impossible to measure the mixed system of microorganisms at all. Therefore, the present inventors investigated a simpler method for quantifying microorganisms, and as a result, they measured the fluorescence intensity emitted from antibodies through a fluorescent antibody-antigen reaction. The present inventors have newly discovered that the properties of microorganisms are always constant even for any number of microorganisms, leading to the completion of the present invention. Hereinafter, typical examples will be explained with reference to figures. The figure shows the principle of a J4 type fluorometer manufactured by Aminco (USA), where 1 is a light source, 2 is an amplifier, 3 is a spectrometer, 4 is a fluorescent antibody antigen, 5 is a spectrometer, 6 indicates the amplifier, 7 indicates the measuring device, and L 1 indicates the spectrometer 3.
L 2 is an excitation light beam emitted by the fluorescent antibody antigen 4 , and L 3 is an excitation light beam of a specific wavelength that is separated by the spectroscope 5 . The light beam (usually using ultraviolet rays) emitted from the light source 1 is amplified to a predetermined light intensity by the amplifier 2, and then converted to a specific wavelength L1 by the spectroscope 3, which is then detected by the fluorescent antibody antigen 4.
is irradiated. The fluorescent antibody antigen of the irradiated specific microorganism emits light as excitation light L2 , which enters the spectrometer 5 and is spectrally dispersed as excitation light L3 with a specific wavelength, which is further amplified to a predetermined light intensity by the amplifier 6 and sent to the measuring device 7. Quantitative curve of specific microorganisms is determined by electrical measurement. In addition, when determining the quantitative curve, the specific microorganism to be determined is determined according to the conventional method.
For example, it is quantified using a dry bacterial weight conversion method or the like, and the quantification result and electrical measurement result are matched to convert the measured value into a quantified value. For the quantitative determination of specific microorganisms, diagrams such as those shown in FIGS. 2A and 2B are generally used, in which the vertical axis shows the fluorescence luminescence intensity (excitation light) and the horizontal axis shows the suspension volume of the microorganism cells. Next, the light source 1 emits light from the fluorescent antibody antigen 4 which has been brought into contact with the quantitative line of the specific microorganism obtained by the above method, an antibody prepared in advance as a fluorescent antibody antigen reaction of the specific microorganism, and an arbitrary number of specific microorganisms. The beam of light (usually using ultraviolet rays) is amplified to a predetermined amount of light by amplifier 2,
It is irradiated with a predetermined wavelength L1 by the spectroscope 3. Fluorescent antibody antigens of specific microorganisms that have been irradiated are exposed to excitation light.
It emits light as L2 , enters the spectroscope 5, becomes excitation light L3 of a specific wavelength, is amplified to a predetermined light intensity by an amplifier 6, and is electrically measured by a measuring device 7, and the obtained measurement value is matched. It is possible to quantify specific microorganisms consisting of an arbitrary number. In addition, antibodies prepared as pre-prepared fluorescent antibody-antigen reactions are produced by introducing specific microorganisms into the blood of a vertebrate animal such as a rabbit, and forming them in the blood against the introduced microorganisms after a certain period of time. Antibodies unique to specific microorganisms are collected and labeled with fluorescent dyes. The quantitative line for microorganisms determined by the method of the present invention is always constant, regardless of the suspended concentration of microorganisms or the presence or absence of mixed microorganisms. The reason for this is to electrically measure the amount of light generated from the fluorescent antibody antigen of the microorganism. Therefore, once the quantification curve for a specific microorganism has been determined, for actual quantification, it is necessary to match this quantification curve with the measurement value obtained by electrically measuring the amount of light emitted from the fluorescent antibody antigen. Specific microorganisms can be quantified regardless of turbidity concentration, presence or absence of mixed microorganisms, etc. In addition, for the antibody-antigen reaction, it is necessary to prepare in advance a fluorescent antibody of the same microorganism that corresponds to the microorganism to be reacted, and prepare for the fluorescent antibody-antigen reaction. Therefore, the term "specific microorganisms" as used herein refers to all microorganisms that serve as antigens for antibodies built in the bodies of vertebrates, and refers to microorganisms that are used for quantitative determination of fluorescent antibodies of the same microorganisms and the same microorganisms. In addition, in the method of the present invention, since the quantification line for microorganisms is always constant, it is possible to easily quantify specific microorganisms in a mixed microorganism. This will emit excitation light. Therefore, it is possible to simultaneously quantify a plurality of specific microorganisms by preparing and mixing fluorescent antibodies composed of a plurality of microorganisms in advance and allowing only the plurality of specific microorganisms in the mixed microorganism system to react. It is easier to determine the agreement of the measurement value with the quantification line of a specific microorganism, rather than matching it each time. It is also possible to consider a method of quantifying the amount immediately after the reaction. As for the equipment to be used next, the above-mentioned fluorophotometer made by Amico (USA) is shown as a general example, but other devices include FR-500/510 type (made by Shimadzu Corporation),
A wide variety of devices can be used as long as they are capable of quantifying fluorescent antibody-antigen reactions, such as the 204-A/S type, 650-10M type, and MPF-4 type (all manufactured by Hitachi). . The present invention will be explained in more detail below using test examples and examples. Test example Nocaldea. Erythroboles KR-S-1
(FERM-PNo.3530) in broth liquid medium at 30℃
Culture was carried out for 5 days. After culturing, cooling centrifugation was performed to obtain the bacterial cells. The obtained bacterial cells were washed with physiological saline, cultured overnight in formalin, washed again with physiological saline, and suspended in physiological saline.
The antigen was prepared as a % suspension. Next, 1 ml of the suspension was injected into the ear vein of a young adult rabbit weighing 2.5 kg on the first day, 2 ml on the second day, and 3 ml on the third day. The antiserum was separated from the blood obtained by repeating the procedure three times by centrifugation. Next, a γ-globulin fraction was prepared from this antiserum by ammonium sulfate fractionation, and this γ-globulin fraction was further reacted with a fluorescent dye (fluorescein isothiocyanate manufactured by BBL, UK) at 10°C for 4 hours. Labeled with Sephadex G-50 (manufactured by Seikagaku Corporation)
A fluorescent antibody solution was prepared by gel filtration using DEAE-Cellulose (Seikagaku Kogyo Co., Ltd.) column chromatography to remove non-specific factors. Example 1 The following experiment was conducted for the purpose of differentially quantifying a target microorganism using a fluorescent antibody prepared according to the test example. That is, the Nocaldea erythroboles KR-S- was cultured on a bouillon agar medium at 30°C for 2 days.
One strain (FERM-P-No. 3530) was collected by scraping with a platinum loop, and suspended in physiological saline. Here, the degree of bacterial suspension was measured using a spectrophotometer at a wavelength of 660 nm. This Nocaldea. Erythropores KR―
Nocaldea prepared according to the test example was added to 0.5 ml of S-1 suspension. Add 0.5 ml of fluorescent antibody solution for Erythropores KR-S-1 and react at 30°C for 1 hour. Next, the reaction product was collected by centrifugation, and then resuspended in 1.5 ml of physiological saline, and the fluorescence intensity was measured using a fluorophotometer (manufactured by Amico, USA). The primary filter of the fluorophotometer was an ultraviolet transmitting and visible light absorbing filter, and a sharp cut yellow filter with a secondary filter (transmits only light with wavelengths of 415 nm or more). Table 1 shows the relationship between the degree of suspension of bacteria and the degree of fluorescence. Table 1 Nocaldea. Erythroboles KR-S-1
Suspension of strain and fluorescence luminescence of bacteria and fluorescent antibody after reacting with fluorescent antibody for strain S-1
【表】
表１に示す如く、菌の懸濁度と菌一螢光抗体物
の螢光発光度は同じように直線関係を示し、定量
性のあることが認められた。
実施例 ２
実施例１のノカルデア．エリスロポレスKR―
Ｓ―１株の無希釈の懸濁液にブイヨン寒天培地か
らかきとつた。シユウドモナスKR―256―１株
（FERM Ｐ―No.3529）を追加懸濁したところ、
混合懸濁液の懸濁度は3900になつた。この混合懸
濁度を表１における希釈割合と同じように２倍、
５倍、10倍希釈した後、表１にて行つたのと同じ
方法により、Ｓ―１株用の螢光抗体と反応させた
後、同じ方法を用い菌一螢光抗体の螢光発光度を
調べた。もし、シユウドモナス．KR―256―１
株に妨げられずノカルデア．エリスロボレスＳ―
１株のみを確認し、分別定量できるならば螢光抗
体と反応せた後の螢光発光度の値は表１と同じ値
をしめすはずである。
表２ ノカルデア．エリスロポレスKR―Ｓ―１
株とシユウドモナス．アシドボランスKR―256
―１株の混合系における混合懸濁度とＳ―１株用
の螢光抗体を用いた菌―螢光抗体の螢光発光度の
関係を示す。[Table] As shown in Table 1, the degree of suspension of the bacteria and the fluorescence luminescence intensity of the bacteria-fluorescent antibody similarly showed a linear relationship, and it was confirmed that the results were quantitative. Example 2 Nocaldea of Example 1. Erythropores KR―
An undiluted suspension of strain S-1 was scraped from the broth agar medium. When Pseudomonas KR-256-1 strain (FERM P-No. 3529) was additionally suspended,
The suspension degree of the mixed suspension was 3900. This mixed suspension degree was doubled in the same manner as the dilution ratio in Table 1.
After diluting 5-fold and 10-fold, react with the fluorescent antibody for strain S-1 using the same method as shown in Table 1. Examined. If it is Pseudomonas. KR-256-1
Nocaldea unhindered by stocks. Erythroboles S-
If only one strain can be identified and separately quantified, the value of fluorescence luminescence after reacting with a fluorescent antibody should show the same value as in Table 1. Table 2 Nocaldea. Erythropores KR-S-1
Strains and pseudomonas. Acidoborance KR-256
This figure shows the relationship between the degree of mixing suspension in a mixed system of the S-1 strain and the fluorescence luminescence intensity of the bacteria-fluorescent antibody using the fluorescent antibody for the S-1 strain.
【表】
螢光抗体を反応させた後の螢光発光度をみると
表１と表２の値は完全と一致していることより、
本発明により目的とする微生物のみを混合系にお
いても確認し分別定量できたことがわかつた。
なお、従来の定量法において乾燥菌体量を測定
しても、混合した総菌体量のみを測定できるにす
ぎず、分別定量は不可能であつた。
実施例 ３
実施例１と同じ方法を用いて、人間に対してカ
ンジダ病としての病原性を示す。カンジダ．アル
ビカンスＮ―500（IAM4080）株を用いて、Ｎ―
500株用の螢光体を調整した。このカンジダ、ア
ルビカンスＮ―500株の生理食塩水の懸濁液の懸
濁度を分光光度計で660nmにおいて測定したとこ
ろ0.35を示した。この懸濁液１ml、0.5ml、0.1ml
に対して前記Ｎ―500株用の螢光抗体液0.5を加え
37℃にて１時間反応させた後実施例１と同じよう
に生理食塩水3.5mlに再懸濁を行い菌一螢光抗体
の螢光発光度を測定した。
更に、このカンジタ．アルビカンスＮ―500の
懸濁液0.1ml、0.5ml、１mlに大腸菌、IFM3281株
の懸濁液（660nmで測定した懸濁度0.48）0.5mlを
各々に加えた後、前記Ｎ―500株用の螢光抗体液
0.5mlを反応させ、反応後の菌―螢光抗体の螢光
発光度を測定した。
これらの結果をまとめて図２に示す。
なお、図２において、グラフＡは、加えたカン
ジダ．アルビカンス菌体の懸濁度と抗カンジダ．
アルビカンスアストロシチン抗体とを反応させた
後の螢光発光量の関係を示しており、カンジダ．
アルビカンスのための定量線が求められた。又、
グラフＢは従来、菌の定量するために用いられて
いた吸光度測定と加えたカンジダ．アルビカンス
の懸濁度との関係を示している。グラフＡ，Ｂよ
り螢光発光量を測定することにより定量線を示
す。
次に、カンジダ．アルビカンスと大腸菌の混合
系に本法を適用したところ、従来の吸光度を測定
する方法ではグラフＤの如く、全体の懸濁度が測
定されるのみでカンジダ．アルビカンスがその混
合系においてどの位存在するか定量できないこと
は明らかである。これに対し、グラフＣに示す如
く螢光発光量を測定するとカンジダ．アルビカン
スのみを混合系において分別確認し、あらかじめ
求めておいた定量線（グラフＡ）と対比させるこ
とによつて容易に分別定量できた。
この結果、実施例１と同じようにカンジダ病と
して名高い病原微生物であるカンジダ．アルビカ
ンスＮ―500を本発明によれば分別確認し、分別
定量できることが認められた。[Table] Looking at the fluorescence intensity after reacting with the fluorescent antibody, the values in Tables 1 and 2 are in perfect agreement.
It was found that, according to the present invention, only the target microorganisms could be identified and separately quantified even in a mixed system. Note that even if the amount of dry bacterial cells is measured using the conventional quantitative method, only the total amount of mixed bacterial cells can be measured, and fractional quantification is not possible. Example 3 Using the same method as in Example 1, pathogenicity as a Candida disease was demonstrated to humans. Candida. Using N. albicans N-500 (IAM4080) strain, N-
I prepared a phosphor for 500 plants. The suspension degree of this suspension of Candida albicans N-500 strain in physiological saline was measured with a spectrophotometer at 660 nm and found to be 0.35. This suspension 1ml, 0.5ml, 0.1ml
Add 0.5 of the fluorescent antibody solution for the N-500 strain to the
After reacting at 37°C for 1 hour, the suspension was resuspended in 3.5 ml of physiological saline in the same manner as in Example 1, and the fluorescence intensity of the bacteria-fluorescent antibody was measured. Furthermore, this candida. After adding 0.5 ml of a suspension of Escherichia coli and IFM3281 strain (suspension degree 0.48 measured at 660 nm) to 0.1 ml, 0.5 ml, and 1 ml of the suspension of E. albicans N-500, Fluorescent antibody solution
0.5 ml was reacted, and the fluorescence intensity of the bacteria-fluorescent antibody after the reaction was measured. These results are summarized in FIG. 2. In addition, in FIG. 2, graph A shows the added Candida. Suspension degree of C. albicans cells and anti-Candida.
It shows the relationship between the amount of fluorescent light emitted after reacting with Candida albicans astrocytin antibody.
A quantitative line for C. albicans was determined. or,
Graph B shows Candida. It shows the relationship with the degree of suspension of C. albicans. A quantitative line is shown by measuring the amount of fluorescence from graphs A and B. Next, candida. When this method was applied to a mixed system of Candida albicans and E. coli, the conventional method of measuring absorbance only measured the overall degree of suspension, as shown in graph D. It is clear that it is not possible to quantify how much C. albicans is present in the mixed system. On the other hand, when the amount of fluorescent light emission was measured as shown in graph C, Candida. Only C. albicans was separated and confirmed in the mixed system, and by comparison with a predetermined quantitative curve (graph A), it was possible to easily separate and quantify. As a result, as in Example 1, Candida, which is a pathogenic microorganism famous for Candida disease. It has been confirmed that C. albicans N-500 can be separately confirmed and quantified according to the present invention.
図１は螢光度計の計測機構図を示し、１は光
源、２は増幅器、３は分光器、４は微生物の螢光
抗体抗原、５は分光器、６は増幅器、L1は光源
１より発光された特定波長、L2は励起光、L3は
特定波長の励起光を示している。図２は菌体を螢
光抗体抗原としたときの螢光発光度による微生物
の定量線及び吸光度測定による微生物の定量線を
それぞれ示している。図２中のＡはカンジダ菌を
螢光抗体抗原としたときの螢光発光度による同菌
の定量線を示し縦軸に螢光発光度（励起光）、横
軸に同菌の懸濁度を示している。Ｂは同菌の従
来、菌の定量用として用いられている吸光度測定
の結果を示し、縦軸に吸光度、横軸に同菌の懸濁
度を示している。Ｃは同菌と大腸菌との混合系に
おける同菌の螢光抗体抗原としたときの螢光発光
度による計測結果を示し、縦軸に螢光発光度（励
起光）、横軸に同菌の懸濁度を示している。Ｄは
同菌と大腸菌との混合系において従来より使用さ
れている吸光度測定の結果を示し、縦軸に吸光
度、横軸に同菌と大腸菌との懸濁度を示してい
る。
Figure 1 shows the measurement mechanism of a fluorometer, where 1 is a light source, 2 is an amplifier, 3 is a spectrometer, 4 is a fluorescent antibody antigen of microorganisms, 5 is a spectrometer, 6 is an amplifier, and L 1 is from light source 1. The specific wavelength of light emitted, L 2 indicates excitation light, and L 3 indicates excitation light of a specific wavelength. FIG. 2 shows a quantitative line of microorganisms based on fluorescence luminescence and a quantitative line of microorganisms based on absorbance measurement when bacterial cells are used as a fluorescent antibody antigen. A in Figure 2 shows the quantitative line of Candida bacteria based on the fluorescence intensity when the fluorescent antibody antigen is used. The vertical axis is the fluorescence intensity (excitation light), and the horizontal axis is the suspension degree of the same bacteria. It shows. B shows the result of absorbance measurement of the same bacterium, which is conventionally used for quantifying the bacterium, with the vertical axis showing the absorbance and the horizontal axis showing the degree of suspension of the same bacterium. C shows the measurement results of the fluorescence intensity when using the same bacteria as a fluorescent antibody antigen in a mixed system of the same bacteria and E. coli, where the vertical axis is the fluorescence intensity (excitation light) and the horizontal axis is the fluorescence of the same bacteria. It shows the degree of suspension. D shows the results of absorbance measurement conventionally used in a mixed system of the same bacteria and E. coli, with the vertical axis showing the absorbance and the horizontal axis showing the degree of suspension between the same bacteria and E. coli.
Claims (1)
て、 １） 予め特定微生物の螢光抗体抗原反応とし
て、螢光色素により標識された抗体と特定微生
物を接触させ、得られた前記抗体と特定微生物
の螢光抗体抗原に光線を照射し、螢光抗体抗原
から発生する光量を電気的に計測して特定微生
物の定量線を求めること、 ２） 前記方法により得られた特定微生物の定量
線と、混合微生物系中予め特定微生物の螢光抗
体抗原反応として、螢光色素により標識された
抗体と特定微生物を接触させ、得られた前記抗
体と特定微生物の螢光抗体原に光線を照射し螢
光抗体抗から発光する光量を電気的に計測し、
得られた計測値とを符号させること、 を特徴とする螢光抗体抗原反応を利用した微生物
の定量法。[Scope of Claims] 1. In the quantitative determination of microorganisms in a mixed microorganism system, 1) the above-mentioned microorganism obtained by contacting an antibody labeled with a fluorescent dye with a specific microorganism in advance as a fluorescent antibody-antigen reaction of the specific microorganism; irradiating the antibody and the fluorescent antibody antigen of the specific microorganism with light, and electrically measuring the amount of light generated from the fluorescent antibody antigen to determine the quantitative line of the specific microorganism; 2) determining the quantitative line of the specific microorganism obtained by the above method; As a fluorescent antibody-antigen reaction of a specific microorganism in a mixed microbial system, an antibody labeled with a fluorescent dye and a specific microorganism are brought into contact with the quantitative line, and a light beam is applied to the obtained antibody and the fluorescent antibody antigen of the specific microorganism. The amount of light emitted from the fluorescent antibody is measured electrically.
A method for quantifying microorganisms using a fluorescent antibody-antigen reaction, characterized in that the obtained measured value is coded.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4749079A JPS55140151A (en) | 1979-04-18 | 1979-04-18 | Quantitative measurement of microorganism using fluorescent anti-body-antigen reaction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4749079A JPS55140151A (en) | 1979-04-18 | 1979-04-18 | Quantitative measurement of microorganism using fluorescent anti-body-antigen reaction |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS55140151A JPS55140151A (en) | 1980-11-01 |
| JPH0260982B2 true JPH0260982B2 (en) | 1990-12-18 |
Family
ID=12776551
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP4749079A Granted JPS55140151A (en) | 1979-04-18 | 1979-04-18 | Quantitative measurement of microorganism using fluorescent anti-body-antigen reaction |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS55140151A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0439802U (en) * | 1990-07-24 | 1992-04-03 |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02170053A (en) * | 1988-12-23 | 1990-06-29 | Meiji Seika Kaisha Ltd | Method and device for detecting microorganism |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5016597A (en) * | 1973-05-07 | 1975-02-21 | ||
| JPS52149195A (en) * | 1976-06-02 | 1977-12-12 | Beckman Instruments Inc | Method of and apparatus for generating signal for introduction of chemical reactive component into chemical analysis apparatus |
| US4100416A (en) * | 1977-03-02 | 1978-07-11 | Block Engineering, Inc. | Serum fluorescence suppression |
| US4133873A (en) * | 1975-05-26 | 1979-01-09 | Noller Hans G | Method of determining extracellular antigens and antibodies |
-
1979
- 1979-04-18 JP JP4749079A patent/JPS55140151A/en active Granted
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5016597A (en) * | 1973-05-07 | 1975-02-21 | ||
| US4133873A (en) * | 1975-05-26 | 1979-01-09 | Noller Hans G | Method of determining extracellular antigens and antibodies |
| JPS52149195A (en) * | 1976-06-02 | 1977-12-12 | Beckman Instruments Inc | Method of and apparatus for generating signal for introduction of chemical reactive component into chemical analysis apparatus |
| US4100416A (en) * | 1977-03-02 | 1978-07-11 | Block Engineering, Inc. | Serum fluorescence suppression |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0439802U (en) * | 1990-07-24 | 1992-04-03 |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS55140151A (en) | 1980-11-01 |
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