JPH0412274A - Indirect solid phase antibody method - Google Patents
Indirect solid phase antibody methodInfo
- Publication number
- JPH0412274A JPH0412274A JP11599190A JP11599190A JPH0412274A JP H0412274 A JPH0412274 A JP H0412274A JP 11599190 A JP11599190 A JP 11599190A JP 11599190 A JP11599190 A JP 11599190A JP H0412274 A JPH0412274 A JP H0412274A
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- JP
- Japan
- Prior art keywords
- antibody
- antigen
- reaction
- antibodies
- protein
- 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.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 46
- 239000007790 solid phase Substances 0.000 title claims abstract description 12
- 238000006243 chemical reaction Methods 0.000 claims abstract description 61
- 102000036639 antigens Human genes 0.000 claims abstract description 48
- 239000000427 antigen Substances 0.000 claims abstract description 47
- 108091007433 antigens Proteins 0.000 claims abstract description 47
- 238000007865 diluting Methods 0.000 claims abstract description 4
- 239000003085 diluting agent Substances 0.000 claims abstract description 4
- 101150079601 recA gene Proteins 0.000 claims abstract description 4
- 229920000136 polysorbate Polymers 0.000 claims description 10
- 102000008300 Mutant Proteins Human genes 0.000 claims description 2
- 108010021466 Mutant Proteins Proteins 0.000 claims description 2
- 238000004445 quantitative analysis Methods 0.000 claims description 2
- 229920000642 polymer Polymers 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 abstract description 15
- 108090000790 Enzymes Proteins 0.000 abstract description 15
- 230000007423 decrease Effects 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 abstract description 2
- 239000012295 chemical reaction liquid Substances 0.000 abstract 2
- 230000021615 conjugation Effects 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 32
- 102000001218 Rec A Recombinases Human genes 0.000 description 23
- 108010055016 Rec A Recombinases Proteins 0.000 description 23
- 238000010790 dilution Methods 0.000 description 15
- 239000012895 dilution Substances 0.000 description 15
- 102000004169 proteins and genes Human genes 0.000 description 15
- 108090000623 proteins and genes Proteins 0.000 description 15
- 238000011088 calibration curve Methods 0.000 description 8
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 230000005526 G1 to G0 transition Effects 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 102000007562 Serum Albumin Human genes 0.000 description 2
- 108010071390 Serum Albumin Proteins 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 238000007385 chemical modification Methods 0.000 description 2
- 238000001114 immunoprecipitation Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 102000013415 peroxidase activity proteins Human genes 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 101100355584 Mus musculus Rad51 gene Proteins 0.000 description 1
- 101100380699 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) atp-8 gene Proteins 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000002967 competitive immunoassay Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、間接固相抗体法に関する。さらに詳細には、
本発明は、溶液中の抗原・抗体反応の定量方法に関する
。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an indirect solid-phase antibody method. In more detail,
The present invention relates to a method for quantifying an antigen-antibody reaction in a solution.
従来、抗原・抗体反応の定量としては、固相酵素抗体法
、免疫沈降法、競合免疫法、及び阻害試験法等が知られ
ている。Conventionally, solid-phase enzyme antibody methods, immunoprecipitation methods, competitive immunoassays, inhibition test methods, and the like are known for quantifying antigen-antibody reactions.
しかるに固相抗体法では、抗原を反応容器の内側に固定
する際に抗原の抗原性が変化してしまうことが少なくな
く、更には抗原が全く変性してしまうこともあり、溶液
条件下における抗原抗体反応を正確に測定することは不
可能であった。However, in the solid-phase antibody method, the antigenicity of the antigen often changes when the antigen is immobilized inside the reaction container, and even the antigen may be completely denatured, so that It was not possible to accurately measure antibody responses.
また、免疫沈降法では、反応系に添加される二次抗体や
沈澱剤、それらに含まれる不純物が抗原の反応性や抗原
性に影響を与える可能性が大きく、溶液条件下での抗原
抗体反応を正確に測定することはできなかった。また、
高感度で沈澱中の抗原や抗体の量を測定することは容易
でないという間題もあった。In addition, in the immunoprecipitation method, there is a large possibility that the secondary antibodies and precipitants added to the reaction system, and the impurities contained in them, will affect the reactivity and antigenicity of the antigen, and the antigen-antibody reaction under solution conditions. could not be measured accurately. Also,
There was also the problem that it was not easy to measure the amount of antigen or antibody in the precipitate with high sensitivity.
競合免疫法においても同様に、反応液中の因子が測定系
の抗原抗体反応に影響を与えるので、溶液条件下での抗
原抗体反応の測定は不可能であり、さらに阻害試験法に
おいては、反応系、測定系の相互間の影響を除外できず
、正確な抗原抗体反応の定量ばできなかった。Similarly, in the competitive immunization method, factors in the reaction solution affect the antigen-antibody reaction in the measurement system, so it is impossible to measure the antigen-antibody reaction under solution conditions.Furthermore, in the inhibition test method, the reaction It was not possible to exclude the mutual influence of the system and measurement system, and it was not possible to accurately quantify the antigen-antibody reaction.
従って、上記方法を用いても溶液条件下での抗原抗体反
応を正確に定量することば不可能であった。特に反応溶
液中に種々の因子を含む反応溶液条件下では、これらの
因子の影響を考慮しなりればならず、抗体量の測定が一
層困難であった。例えば同相酵素抗体法（ＥＬＩＳＡ）
では、抗原抗体反応に影響を与える因子と酵素による発
色反応に影響を与える因子のいずれによってもシグナル
強度が変化するので、反応系の因子の影響を受は易く正
確な測定が不可能になる場合があった。Therefore, even using the above method, it has been impossible to accurately quantify antigen-antibody reactions under solution conditions. Particularly under reaction solution conditions in which the reaction solution contains various factors, the influence of these factors must be taken into account, making it even more difficult to measure the amount of antibody. For example, homologous enzyme immunoassay (ELISA)
In this case, signal intensity changes depending on factors that affect the antigen-antibody reaction and factors that affect the coloring reaction by the enzyme, so it is easily affected by factors in the reaction system, making accurate measurement impossible. was there.
従って本発明は、溶液状態における抗原抗体反応を正確
に定量する方法を提供することを目的とし、特には種々
の因子を含む溶液中でも正確に抗原抗体反応を定量する
ことを可能にする方法を提供することを目的とする。Therefore, an object of the present invention is to provide a method for accurately quantifying antigen-antibody reactions in a solution state, and in particular, to provide a method that allows accurately quantifying antigen-antibody reactions even in solutions containing various factors. The purpose is to
そこで、本発明者は鋭意研究した結果、抗原抗体反応の
反応溶液を希釈することによって抗原抗体反応を停止さ
せるとともに、反応系の因子が測定に与える影響を最小
限に抑えると抗原抗体反応を正確に定量できることを見
出し本発明を完成するに至った。本発明の方法によれば
、希釈の後、未反応の抗体量を測定することで、反応系
に含まれる因子が測定の際に与える影響を完全に除去す
ることができ、さらに、例えば同相酵素抗体法において
抗体量とシグナル強度が比例する条件を用いることによ
り、未反応の抗体の量を正確に定量することができる。Therefore, as a result of intensive research, the present inventor found that it is possible to stop the antigen-antibody reaction by diluting the reaction solution for the antigen-antibody reaction, and to minimize the influence of factors in the reaction system on the measurement to accurately measure the antigen-antibody reaction. The present invention was completed based on the discovery that it can be quantified. According to the method of the present invention, by measuring the amount of unreacted antibody after dilution, it is possible to completely eliminate the influence of factors contained in the reaction system on the measurement, and furthermore, for example, by measuring the amount of unreacted antibody. By using conditions in which the antibody amount and signal intensity are proportional to each other in the antibody method, the amount of unreacted antibody can be accurately quantified.
すなわち、本発明は、
抗原抗体反応を定量する方法において、■既知量の抗体
と抗原とを溶液中で反応させる工程、
■」−記溶液を希釈する工程、及び
■抗原と未反応の抗体を面相抗体法で定量する工程を含
む定量方法を提供するものである。That is, the present invention provides a method for quantifying an antigen-antibody reaction, which includes: (1) reacting a known amount of antibody with the antigen in a solution, (2) diluting the solution, and (2) reacting the unreacted antibody with the antigen. The present invention provides a quantitative method including a step of quantifying using a face-phase antibody method.
本発明の方法において使用される抗原と抗体の組合せの
例としては、ｒｅｃ Ａタンパク抗原と、ＡＲＭ３２１
抗体、ＡＲＭ４１４抗体、ＡＲＭ１９１抗体、Ａ１１Ｍ
１９３抗体からなる群より選ばれる抗体、ｒｅｃ Ａタ
ンパクの変異タンパクであるｒｅｃＡ４３０抗原と、Ａ
ＲＭ３２１抗体、ＡＲＭ４１４抗体、ＡＲＭ１９１抗体
、ＡＲＭ１９３抗体からなる群より選ばれる抗体等が挙
げられる。Examples of antigen and antibody combinations used in the methods of the invention include rec A protein antigen and ARM321
Antibody, ARM414 antibody, ARM191 antibody, A11M
An antibody selected from the group consisting of 193 antibodies, recA430 antigen, which is a mutant protein of rec A protein, and A
Examples include antibodies selected from the group consisting of RM321 antibody, ARM414 antibody, ARM191 antibody, and ARM193 antibody.
ｒｅｃ Ａタンパク、ＡＲＭ３２１抗体、ＡＲＭ４１４
抗体、ＡＲＭ１９１抗体、及びＡＲＭ １９３抗体は、
Ｔｈｅ Ｊｏｕｒｎａｌ ｏｆＢｉｏｌｏｇｉｃａｌ
Ｃｈｅｍｉｓｔｒｙ、 １９８５． ２６０． ｐｐ１
５４０２−１５４０５に、ｒｅｃ Ａ ４３０は、Ｔｈ
ｅ Ｊ。rec A protein, ARM321 antibody, ARM414
The antibody, ARM191 antibody, and ARM193 antibody are
The Journal of Biological
Chemistry, 1985. 260. pp1
5402-15405, rec A 430 Th
eJ.
ｕｒｎａｌ ｏｆ Ｂｉｏｌｏｇｉｃａｌ Ｃｈ
ｅｍｉｓｔｒｙ、 １ ９ ８ ９． ２ ６ ４
゜ｐｐ２１１６７−２１１７６に記載されている。Urnal of Biological Ch.
emistry, 1 9 8 9. 2 6 4
゜pp21167-21176.
このうち、ｒｅｃ Ａタンパク抗原と、ＡＲＭ３２１抗
体、ＡＲＭ４１４抗体、及びＡＲ，Ｍ１９１抗体からな
る群より選ばれる抗体を使用することが好ましい。Among these, it is preferable to use rec A protein antigen and an antibody selected from the group consisting of ARM321 antibody, ARM414 antibody, and AR, M191 antibody.
既知量の抗体量と抗原量は、抗原と抗体の組合せにより
異なるか、抗体量に対する抗原量の比率は抗体結合価と
等しいかそれより大きいことか望ましい。ここで抗体結
合価とは、１分子の抗体と結合しうる抗原の最大分子数
をいい、抗体に対して抗原が大過剰に存在する条件下で
の抗原・抗体複合体中の抗原モル数を抗体モル数で割っ
た値で示される。例えば、ＡＲＭ３２１抗体、ＡＲＭ４
１４抗体、及びＡＲＭ１９１抗体は、ＩｇＧに属し抗体
結合価は２である。そこでこれらの抗体に対するｒａｃ
Ａタンパクの量は、２．５〜５が好ましい。It is desirable that the known amounts of antibody and antigen differ depending on the combination of antigen and antibody, or that the ratio of the amount of antigen to the amount of antibody is equal to or greater than the antibody binding value. Here, antibody binding value refers to the maximum number of molecules of antigen that can bind to one molecule of antibody, and refers to the number of moles of antigen in the antigen-antibody complex under conditions where the antigen is present in large excess relative to the antibody. It is expressed as the value divided by the number of moles of antibody. For example, ARM321 antibody, ARM4
14 antibody and ARM191 antibody belong to IgG and have an antibody binding value of 2. Therefore, rac against these antibodies
The amount of A protein is preferably 2.5 to 5.
抗原と抗体を反応させる溶液は、いかなるものも可能で
あり、抗原性を変える因子の存在する任意の溶液を選ぶ
ことができる。例えば、ｒｅｃ Ａタンパクを抗原とし
て用いる場合は、ｒｅｃ Ａタンパクの抗原性に影響を
与える可能性のあるＡＴＰやＤＮＡを含む溶液中で抗原
抗体反応を定量することができる。その他、Ｉｌｌ、イ
オン強度などを変化させる因子を含有する任意の溶液を
選ぶことができる。Any solution can be used to react the antigen and antibody, and any solution containing a factor that changes antigenicity can be selected. For example, when rec A protein is used as an antigen, the antigen-antibody reaction can be quantified in a solution containing ATP and DNA that may affect the antigenicity of rec A protein. In addition, any solution containing factors that change Ill, ionic strength, etc. can be selected.
反応の条件としては、反応温度は任意に選ぶことかでき
、反応時間は抗原と抗体の反応が飽和に達するまでの任
意の時間を選ぶことができる。Regarding the reaction conditions, the reaction temperature can be arbitrarily selected, and the reaction time can be arbitrarily selected until the reaction between the antigen and the antibody reaches saturation.
抗原・抗体反応後、抗原・抗体の結合を解離させない希
釈剤を用いて反応溶液の希釈を行うことか好ましい。こ
の様な希釈剤としてはＰＢＳを挙げることかできる。Ｐ
ＢＳとは、リン酸緩衝液を含有する生理食塩水である。After the antigen-antibody reaction, it is preferable to dilute the reaction solution using a diluent that does not dissociate the antigen-antibody bond. PBS may be mentioned as such a diluent. P
BS is physiological saline containing phosphate buffer.
該希釈工程は抗原・抗体反応を効果的に停止させ、かつ
抗体以外の物質が後の固相抗体法の検出限界以下になる
様に行う。例えば、ｒｅｃ ＡタンパクとＡＲＭ３２１
抗体、ＡＲＭ４１４抗体、及びＡＲＭ１９Ｌ抗体からな
る群より選ばれる抗体を用いる場合には、ＰＢＳ−ツイ
ーンで約１、５００倍に希釈することが好ましい。ここ
でＰＢＳツイーンとは、０．０５％ツイーンをＰＢＳに
溶かした溶液をいう。さらに、該希釈工程は室温（約２
５℃）で、５分以内に行うことが好ましい。The dilution step is carried out so that the antigen-antibody reaction is effectively stopped and substances other than the antibody are below the detection limit of the subsequent solid-phase antibody method. For example, rec A protein and ARM321
When using an antibody selected from the group consisting of antibodies, ARM414 antibodies, and ARM19L antibodies, it is preferably diluted approximately 1,500 times with PBS-Tween. Here, PBS Tween refers to a solution in which 0.05% Tween is dissolved in PBS. Furthermore, the dilution step is carried out at room temperature (approximately 2
5°C) within 5 minutes.
上記の様に希釈した反応液中の抗体量を測定するための
固相抗体法としては、酵素抗体法、蛍光抗体法、ラジオ
イムノアッセイ等が挙げられる。Solid-phase antibody methods for measuring the amount of antibodies in the reaction solution diluted as described above include enzyme antibody methods, fluorescent antibody methods, radioimmunoassays, and the like.
このうち、酵素抗体法か好ましい。具体的には、マイク
ロタイタープレートのウェルに抗原を固定し、このウェ
ルに抗原抗体反応を行った反応液を希釈した液を加え未
反応の抗体とウェルに固定した抗原を反応させ、その後
上記希釈した反応液を洗い流し、さらにこのウェルに酵
素で標識した二次抗体を加えウェルに固定した抗原と反
応した抗体と反応させ、次いてこのウェルに上記酵素の
基質となる物質を加え、酵素反応によって生成した物質
の比色により抗原抗体複合体を定量することができる。Among these, enzyme antibody method is preferred. Specifically, an antigen is immobilized in a well of a microtiter plate, a diluted solution of the reaction solution used for antigen-antibody reaction is added to the well, and the unreacted antibody is allowed to react with the antigen immobilized in the well. The reaction solution was washed away, and a secondary antibody labeled with an enzyme was added to this well to react with the antibody that had reacted with the antigen immobilized on the well. Next, a substance that would serve as a substrate for the enzyme was added to this well, and the enzyme reaction was performed. The antigen-antibody complex can be quantified by colorimetry of the produced substance.
上記固相抗体法では、抗原と未反応の抗体か定量される
ので、この値を抗原抗体反応前の抗体量から差し引くこ
とにより、抗原抗体反応を定量することができる。In the solid-phase antibody method described above, the amount of antibody that has not reacted with the antigen is quantified, so the antigen-antibody reaction can be quantified by subtracting this value from the amount of antibody before the antigen-antibody reaction.
本発明の方法により、溶液状態下での抗原抗体反応を、
極めて容易にかつ正確に定量することが可能になった。By the method of the present invention, antigen-antibody reactions in a solution state can be
It has become possible to quantify extremely easily and accurately.
さらに、抗原を含むタンパク質内のアミノ酸変異や化学
修飾による抗原抗体反応の変化を知ることが可能になっ
た。尚、このアミノ酸変異や化学修飾により抗原性が変
化しても、本発明の定量は可能である。Furthermore, it has become possible to learn about changes in antigen-antibody reactions due to amino acid mutations and chemical modifications in proteins containing antigens. Note that even if antigenicity changes due to amino acid mutations or chemical modifications, quantification according to the present invention is possible.
以下に本発明の実施態様として実施例をあげるか、これ
は本発明の説明のためのものであって、本発明の範囲に
制限するものではない。Examples will be given below as embodiments of the present invention, but these are for illustrating the present invention and are not intended to limit the scope of the present invention.
以下の実施例は、特に断らない限り室温（２５°Ｃ）下
で行った。The following examples were conducted at room temperature (25°C) unless otherwise specified.
また、本実施例で用いたｒｅｃ Ａタンパク及びＤＮＡ
φ×１７４は、Ｊ、Ｂｉｏｌ、Ｃｈｅｍ、 １９８１
、 ２５６ｐｐ７５５７−７５６４に、ＡＲＭ３２１抗
体、ＡＲＭ ４１４抗体、ＡＲＭ１９１抗体、及びＡＲ
Ｍ１９３抗体は、Ｊ、 Ｂｉｏｌ、 Ｃｈａｍ１９８５
，２６０、ｐｐ１５４０２−１５４０４に、ｒｅｃＡ４
３０は、Ｊ、Ｂｉｏｌ、Ｃｈｅｍ、 １９８９２６４、
ｐｐ２１１６７−２１１７６に記載の方法によって得た
。In addition, the rec A protein and DNA used in this example
φ×174 is J. Biol. Chem. 1981
, 256pp7557-7564, ARM321 antibody, ARM414 antibody, ARM191 antibody, and AR
M193 antibody was prepared by J. Biol, Cham1985.
, 260, pp 15402-15404, recA4
30 J, Biol, Chem, 1989264,
Obtained by the method described in pp. 21167-21176.
〔参考例 １〕
固定相の作製
マイクロタイタープレート（９６穴）のウェルに５０μ
ｌのｒｅｃ Ａタンパク（２μｇ／−）溶液を加えて４
°Ｃで１５時間放置して、更に２００μｌのＰＢＳ（５
０ｍＭリン酸緩衝液、ｐＨ７，２，１５０ｍＭ ＮａＣ
１）で３回洗い、ウェルの壁をｒｅｃ Ａタンパクで覆
い固定した。更にブロッキングの為に、１００μｌの小
牛血清アルブミン溶液（１％）をウェルに入れ、６時間
はと放置してから１３０μｍのＰＢＳでウェルを洗った
。[Reference Example 1] Preparation of stationary phase 50μ in the wells of a microtiter plate (96 wells)
Add 1 of rec A protein (2 μg/-) solution and
Leave at °C for 15 hours and add 200 μl of PBS (5
0mM phosphate buffer, pH 7,2, 150mM NaC
After washing three times with 1), the walls of the wells were covered with rec A protein and fixed. Furthermore, for blocking, 100 μl of calf serum albumin solution (1%) was added to the wells, left for 6 hours, and then the wells were washed with 130 μm PBS.
〔参考例 ２〕
検量線の作製
参考例１で作製した固定相のウェル中に、ＰＢＳ−ツイ
ーン２０ （０，０５％ツイーン２０（バイオラッド社
製）をＰＢＳに溶かした溶液）に溶かした濃度０．０２
５ｎＭの抗ｒｅｃ Ａタンパク抗体であるＡＲＭ３２１
抗体を５０μｌ加え１時間放置した。そのウェルを１３
０μｌのＰＢＳ−ツイーンで３回洗った。次に、そのウ
ェルに５０μｌのペルオキシダーゼで標識した５００希
釈ヤギに由来する抗マウス［ｇＧ抗体（ＫＰＬ社製）を
加え１時間放置した後、１３０μｌのＰＢＳ−ツイーン
で３回、次いで２００μｍのＰＢＳで洗った。そこへ、
１００μｌの０−フェニレンジアミン（１ｍｇ／ｍｌ）
、０．０５Ｍクエン酸、０．０５Ｍクエン酸ナトリウ
ム、０．０１５％過酸化水素溶液を加え３０分放置し、
呈色反応を行った。３２μｍの４．５Ｍ硫酸を加えその
反応を停めた。呈色反応は、４９０ｎｍの吸光度を測る
ことによって測定した。以上の操作を、ＡＲＭ３２１抗
体の濃度を０．０３８ｎＭ 、 ０．０５ｎＭ、 ０．
０６３ｎＭ、 ０．０９４ｎＭ 。[Reference Example 2] Preparation of a calibration curve In the stationary phase well prepared in Reference Example 1, the concentration of PBS-Tween 20 (a solution of 0.05% Tween 20 (manufactured by Bio-Rad) dissolved in PBS) was determined. 0.02
ARM321, an anti-rec A protein antibody at 5 nM
50 μl of antibody was added and left for 1 hour. 13 that well
Washed 3 times with 0 μl of PBS-Tween. Next, 50 μl of peroxidase-labeled 500-diluted goat-derived anti-mouse [gG antibody (manufactured by KPL) was added to the well and left for 1 hour. washed. There,
100μl 0-phenylenediamine (1mg/ml)
, 0.05M citric acid, 0.05M sodium citrate, and 0.015% hydrogen peroxide solution were added and left for 30 minutes.
A color reaction was performed. The reaction was stopped by adding 32 μm of 4.5M sulfuric acid. The color reaction was measured by measuring absorbance at 490 nm. The above operation was performed with the concentration of ARM321 antibody being 0.038 nM, 0.05 nM, and 0.05 nM.
063 nM, 0.094 nM.
０．１２５ｎＭ 、 ０．１５６ｎＭ 、 ０．１８８
ｎＭＳ０．２１９ｎＭ 、 ０．２５０ｎＭ、 ０．２
８１ｎＭ 、 ０．３１３ｎＭに変えて繰り返すことに
より、検量線を作製した。0.125nM, 0.156nM, 0.188
nMS 0.219nM, 0.250nM, 0.2
A calibration curve was prepared by changing the concentration to 81 nM and 0.313 nM and repeating the experiment.
以上の操作を、ＡＲＭ４１４抗体、ＡＲＭ１９１抗体、
ＡＲＭＩ９３抗体を用いて繰り返した。The above operations were performed for ARM414 antibody, ARM191 antibody,
Repeated with ARMI93 antibody.
結果を第１図に示した。この結果より、酵素抗体法のシ
グナル（４９０ｎｍでの吸光度）の強さが抗ｒｅｃ Ａ
タンパク抗体の量に比例しており、未反応の抗ｒｅｃ
Ａタンパク抗体を０．２μＭまでなら酵素抗体法によっ
て正確に定量できることがわかる。そこで、本実施例で
は、希釈操作の後、未反応の抗体濃度が０．１３ｎＭに
なるようにした。The results are shown in Figure 1. From this result, the strength of the signal (absorbance at 490 nm) of the enzyme antibody method was determined by the anti-rec A
It is proportional to the amount of protein antibody, and unreacted anti-rec
It can be seen that A protein antibodies up to 0.2 μM can be accurately quantified by the enzyme antibody method. Therefore, in this example, after the dilution operation, the unreacted antibody concentration was set to 0.13 nM.
〔参考例 ３〕
抗ｒｅｃタンパク抗体（ＩｇＧ）の濃度と酵素抗体２１
μ＋の反応液Ａ （３１ｍＭ）リス緩衝液、ｐＨ７５，
１３ｍＭ塩化マグネシウム、２．８ ｍＭジチオスレイ
トール、８８μｇ／ｍｎ小牛血清アルブミン、２％グリ
セリン）の中で、０．２μＭの抗ｒｅｃ Ａタンパク単
りローンＩｇＧのひとつであるＡＲＭ３２１抗体と濃度
０．２μＭ Ｓｏ、 ５μＭ、ｌμＭ、２μＭの抗原で
あるｒｅｃ Ａタンパクとを３７°Ｃで１０分反応させ
た。該反応液から４μＩを取り出し１．５００倍容Ｃ６
−）のＰＢＳ−ツイーンを加えて抗原抗体反応を停止し
てから、室温中で希釈後５分以内に５０μｌの希釈溶液
をすてにｒｅｃタンパクをウェルの壁に固定しである参
考例１のマイクロタイタープレートのウェルに入れ、１
時間放置した。そのウェルを１３０μｌのＰＢＳ−ツイ
ーンで３回洗った。次に、５０μｍのペルオキシダーゼ
で標識した１１５００希釈ヤギに由来する抗マウスＩｇ
Ｇ抗体（ＫＰＬ社製）を加え１時間放置した後、１３０
μｍのＰＢＳ−ツイーンで３回、次いで２００μｍのＰ
ＢＳで洗った。そこへ、１００μｍの０フエニレンジア
ミン（１ｍｇ／ ｍｌｌ ） 、０．０５ Ｍクエン酸
、０．０５Ｍクエン酸ナトリウム、０．０１５％過酸化
水素溶液を加え３０分放置して発色させた後、３２μｍ
の４．５Ｍ硫酸を加えその反応を停めた。[Reference Example 3] Concentration of anti-rec protein antibody (IgG) and enzyme antibody 21
μ+ reaction solution A (31mM) Liss buffer, pH 75,
13 mM magnesium chloride, 2.8 mM dithiothreitol, 88 μg/mn calf serum albumin, 2% glycerin) with 0.2 μM of anti-rec A protein ARM321 antibody, which is one of the clone IgGs, and a concentration of 0. 2 μM So, 5 μM, 1 μM, and 2 μM of the antigen rec A protein were reacted at 37° C. for 10 minutes. Take out 4μI from the reaction solution and 1.500 times volume of C6
-) PBS-Tween was added to stop the antigen-antibody reaction, and within 5 minutes after dilution at room temperature, the rec protein was immobilized on the wall of the well by discarding 50 μl of the diluted solution. Place into the wells of a microtiter plate and add 1
I left it for a while. The wells were washed three times with 130 μl of PBS-Tween. Next, 11,500 diluted goat-derived anti-mouse Ig labeled with peroxidase of 50 μM
After adding G antibody (manufactured by KPL) and leaving it for 1 hour,
3 times with µm PBS-Tween, then 200 µm PBS-Tween
I washed it with BS. To this, 100 μm 0-phenylenediamine (1 mg/ml), 0.05 M citric acid, 0.05 M sodium citrate, and 0.015% hydrogen peroxide solution were added and left to stand for 30 minutes to develop color.
The reaction was stopped by adding 4.5M sulfuric acid.
４９０ｎｍの吸光度を測定し、吸光度から参考例２の検
量線を用いて未反応の抗体量を求めた。The absorbance at 490 nm was measured, and the amount of unreacted antibody was determined from the absorbance using the calibration curve of Reference Example 2.
以上の操作をＡＲＭ４１４抗体、ＡＲＭ１９１抗体につ
いて繰り返した。The above operations were repeated for the ARM414 antibody and the ARM191 antibody.
結果を第２図に示した。上記抗体については、ｒｅｃ
Ａタンパクの濃度が高くなるにつれて未反応の抗体の量
が少なくなることを示している。大体０．５μＭ前後の
ｒｅｃ Ａタンパクで未反応の抗体がなくなった。The results are shown in Figure 2. For the above antibodies, rec
It is shown that as the concentration of A protein increases, the amount of unreacted antibody decreases. Unreacted antibodies disappeared with approximately 0.5 μM of rec A protein.
〔参考例 ４〕
抗原抗体複合体の１．５００倍希釈に対する安定性０．
２μＭの抗体（ＡＲＭ４１４抗体、ＡＲＭ３２１抗体、
ＡＲＭ１９１抗体、ＡＲＭ１９３抗体）と１μＭのｒｅ
ｃ Ａタンパクとを参考例３と同じ条件で反応させてか
ら、１５００倍容の倍容Ｓ−ツイーンで希釈し、室温で
放置する時間を変えて、酵素抗体法で抗原と結合してい
ない抗体の量を定量した。[Reference Example 4] Stability of antigen-antibody complex against 1.500-fold dilution: 0.
2 μM of antibodies (ARM414 antibody, ARM321 antibody,
ARM191 antibody, ARM193 antibody) and 1 μM re
c After reacting with A protein under the same conditions as in Reference Example 3, diluted with 1500 times the volume of S-Tween and left at room temperature for different times, the enzyme antibody method was used to determine the antibody that did not bind to the antigen. The amount of was quantified.
結果を第３図に示す。第３図中、・は抗原、抗体の入っ
た完全系を示し：△は抗体を除いたコントロールを示し
；○は抗原（ｒｅｃＡ“タンパク）を除いたコントロー
ルを示す。この結果から、ＡＲＭ４１４抗体、ＡＲＭ３
２１抗体、ＡＲＭ１９１抗体については、希釈後６０分
にわたって複合体は安定に存在していたが、ＡＲＭＩ９
３抗体は希釈直後でも全てシグナルを与えることがわか
る。この事は、ＡＲＭ１９３抗体とｒｅｃＡタンパクと
の結合はこの希釈に耐えず、この抗体については本発明
の方法は使用できないことを示している。The results are shown in Figure 3. In Figure 3, . indicates a complete system containing antigen and antibody; △ indicates a control without the antibody; ○ indicates a control without the antigen (recA "protein). From this result, ARM414 antibody, ARM3
For ARM191 antibody and ARM191 antibody, the complex existed stably for 60 minutes after dilution, but for ARMI9 antibody, the complex existed stably for 60 minutes after dilution.
It can be seen that all three antibodies give signals even immediately after dilution. This indicates that the binding between the ARM193 antibody and the recA protein does not survive this dilution, and the method of the present invention cannot be used with this antibody.
〔参考例 ５〕
抗原抗体反応の１．５００倍希釈による停止処置の効果
６−のＰＢＳ−ツイーンに１μＭのｒｅｃ Ａタンパク
を４μｍ加え、直後に０．２μＭの抗体（ＡＲＭ４１４
抗体、ＡＲＭ３２１抗体、ＡＲＭ１９１抗体）溶液を４
μｍ加え室温で放置する時間を変えて、酵素抗体法によ
り抗原と結合してしない抗体の量を定量した。[Reference Example 5] Effect of stopping treatment by 1.500-fold dilution of antigen-antibody reaction
antibody, ARM321 antibody, ARM191 antibody) solution
The amount of antibody that did not bind to the antigen was quantified by enzyme-linked antibody method by varying the time of adding μm and leaving it at room temperature.
結果を第４図に示す。第４図中、・は抗原、抗体の入っ
た完全系を示し；△は抗体を除いたコントロールを示し
；Ｏは抗原（ｒｅｃ Ａタンパク）を除いたコントロー
ルを示す。この結果は、適度の希釈の後では、抗原と抗
体はたとえ共存してもその抗原抗体反応はおこらないか
または極めてゆっくりとしか起こらないことを示してい
る。よって、この希釈によって抗原抗体反応が効果的に
停止できることがわかる。The results are shown in Figure 4. In FIG. 4, . indicates a complete system containing antigen and antibody; Δ indicates a control without antibody; O indicates a control without antigen (rec A protein). This result shows that after moderate dilution, even if antigen and antibody coexist, the antigen-antibody reaction does not occur or occurs only very slowly. Therefore, it can be seen that the antigen-antibody reaction can be effectively stopped by this dilution.
〔実施例 １〕
参考例３の反応液Ａ中で１μＭのｒｅｃ へタンパクと
０．２μＭのＡＲＭ３２１抗体とを、ＡＴＰ （ヤマザ
醤油（株）製）ａ度０．０．１．０．３．１，３．２．
６ｍＭの存在下で３７℃で反応時間を変えて反応させた
後、酵素抗体法で未反応の抗体量を測定した。[Example 1] In reaction solution A of Reference Example 3, 1 μM rec protein and 0.2 μM ARM321 antibody were mixed with ATP (manufactured by Yamaza Soy Sauce Co., Ltd.) at 0.0.1.0.3. 1,3.2.
After reacting in the presence of 6mM at 37°C for different reaction times, the amount of unreacted antibody was measured by enzyme antibody method.
結果を第５図に示す。ＡＴＰ８度は、・ＯｍＭ。The results are shown in Figure 5. ATP 8 degrees is ・OmM.
１０．１ｍＭ、◆０．３ｍＭ、ムＬ３ｍＭ、マ２．６ｍ
Ｍである。10.1mM, ◆0.3mM, M L3mM, M2.6m
It is M.
○はｒｅｃ Ａタンパクを除いたコントロール（ＡＴＰ
非存在下）を示し、はｒｅｃ へタンパクを除いたコン
トロール（ＡＴＰ２．６ｍＭ存在）を示す。この結果か
ら、ＡＴＰの存在によってｒｅｃ へタンパクがΔＲＭ
３２１抗体に対する抗原性を失うことがわかる。○ indicates control excluding rec A protein (ATP
(in the absence of ATP), and indicates a control in which the protein was removed from rec (in the presence of 2.6 mM ATP). From this result, the presence of ATP causes the protein to rec to ΔRM.
It can be seen that the antigenicity against the 321 antibody is lost.
〔実施例２〕
参考例３の反応液Ａ中で１μＭのｒｅｃΔタンパクと０
．２μＭのＡＲＭ４１４抗体を、八ＴＰｒＳ （ベー
リンガーマンハイム社製） （１，，３ｍＭ）及び単
鎖ＤＮＡφｘ １．７４（５０μＭ）の存在下、八ＴＰ
ｒＳ （１，３ｍＭ）の存在下、単鎖ＤＮΔφＸ１７
４ （１５０μＭ）の存在下、３７℃で反応時間を変え
て反応゛させた後、酵素抗体法で未反応の抗体量を測定
した。[Example 2] In reaction solution A of Reference Example 3, 1 μM recΔ protein and 0
．． 2 μM of ARM414 antibody was injected into 8TP in the presence of 8TPrS (Boehringer Mannheim) (1,3mM) and single-stranded DNAφx 1.74 (50μM).
Single-stranded DNAΔφX17 in the presence of rS (1,3mM)
4 (150 μM) at 37° C. for different reaction times, and then the amount of unreacted antibody was measured by enzyme antibody method.
結果を第６図に示す。ムは八ＴＰｒＳ及び単鎖ＤＮＡφ
×１７４の存在下、は＾ＴＰｒＳの存在下、・は単鎖Ｄ
ＮＡφＸ１７４の存在下を示す。○はｒｅＣＡタンパク
のみのコントロール（ＡＴＰ 及ヒＤＮＡ 非存在下）
、☆はｒｅｃ Ａタンパクを除いた抗体のみのコントロ
ール（ＡＴＰ及び（ｌＮＡ非存在下）を示す。The results are shown in Figure 6. The system consists of eight TPrS and single-stranded DNAφ
In the presence of ×174, is in the presence of ^TPrS, ・ is single-chain D
The presence of NAφX174 is shown. ○ is a control with only reCA protein (in the absence of ATP and human DNA)
, ☆ indicates an antibody-only control (in the absence of ATP and (lNA)) excluding the rec A protein.
この結果から、八ＴＰｒＳ及び単鎖ＤＮ八φ×１７４の
存在によってｒｅｃ ＡタンパクがＡＲＭ４１４抗体に
対する抗原性を失うことがわかる。This result shows that the rec A protein loses its antigenicity to the ARM414 antibody due to the presence of 8TPrS and single-chain DN 8φ×174.
〔実施例３〕
抗原にｒｅｃ Ａ ４３０を、抗体にＡＲＭ４１４を用
いて実施例２の方法を繰り返した。[Example 3] The method of Example 2 was repeated using rec A 430 as the antigen and ARM414 as the antibody.
第１図は、ｒｅｃ Ａタンパク抗原に対する抗体の検量
線である。第１図Ａは、ＡＲＭ３２１の検量線であり、
第１図Ｂは、ＡＲＭ４１４の検量線であり、第１図Ｃは
、ＡＲＭ１９１の検量線であり、第１図りは、ＡＲＭＩ
９３の検量線である。
第２図は、一定量の抗体に対しｒｅｃ Ａタンパク抗原
の量を変化させたときの４９０ｎｍの吸光度である。第
２図Ａは、抗体にＡＲＭ３２１を、第２図Ｂは、抗体に
ＡＲＭ４１４を、第２図Ｃは、抗体にＡＲＭ１９１を用
いたものである。
第３図は、ｒｅｃ Ａタンパク抗原・抗体複合体の１、
５００倍希釈に対する安定性を示すものである。
第３図Ａは、抗体にＡＲＭ３２１を、第３図Ｂは、抗体
にＡＲＭ４１４を、第３図Ｃは、抗体にＡＲＭ１９１を
、第３図りは、抗体にＡＲＭ１９３を用いたものである
。
第４図は、ｒｅｃ Ａタンパク抗原・抗体反応の１５０
０倍希釈による停止処置の効果を示すものである。第４
図Ａは、抗体にＡＲＭ３２１を、第４図Ｂは、抗体にＡ
ＲＭ４１４を、第４図Ｃは、抗体にＡＲＭ１９１を用い
たものである。
第５図は、ＡＴＰによるｒｅｃ Ａタンパクの抗原性の
変化を定量したものである。
第６図はＡＴＰ７Ｓ、単鎖ＤＮＡφ×１７４によるｒｅ
ｃＡタンパクの抗原性の変化を定量したものである。
図面の浄書（内容に変更なし）
第１図
１２２７４、（６）
第３図
第４図
手
続
補
正
書
（方式）
１、事件の表示
平成２年特許願第１
１５９９１号
２、発明の名称
間接固相抗体法
３、補正をする者
事件との関係
出
願人
名
称
理
化
学
研
究
所
４、代
理
人
５、補正命令の日付
平成２年７月３１日FIG. 1 is a standard curve of antibodies against the rec A protein antigen. FIG. 1A is the calibration curve of ARM321,
Figure 1B is the calibration curve of ARM414, Figure 1C is the calibration curve of ARM191, and the first diagram is the calibration curve of ARM414.
93 calibration curve. FIG. 2 shows the absorbance at 490 nm when the amount of rec A protein antigen was varied with respect to a fixed amount of antibody. FIG. 2A uses ARM321 as the antibody, FIG. 2B uses ARM414 as the antibody, and FIG. 2C uses ARM191 as the antibody. Figure 3 shows rec A protein antigen/antibody complex 1,
This shows stability against 500-fold dilution. Figure 3A uses ARM321 as the antibody, Figure 3B uses ARM414 as the antibody, Figure 3C uses ARM191 as the antibody, and Figure 3 uses ARM193 as the antibody. Figure 4 shows the 150 rec A protein antigen/antibody reaction.
This shows the effect of stopping treatment by 0-fold dilution. Fourth
Figure A shows ARM321 as an antibody, and Figure 4B shows A as an antibody.
RM414 was used as the antibody, and ARM191 was used as the antibody in FIG. 4C. FIG. 5 shows quantification of changes in antigenicity of rec A protein due to ATP. Figure 6 shows re using ATP7S and single-stranded DNAφ×174.
This is a quantification of changes in antigenicity of cA protein. Engraving of the drawings (no change in content) Figure 1 12274, (6) Figure 3 Figure 4 Procedural amendment (formality) 1. Indication of the case 1990 Patent Application No. 1 15991 2. Indirect confirmation of the title of the invention Composite antibody method 3, Person making the amendment Name of applicant related to the case: RIKEN 4, Agent 5, Date of amendment order: July 31, 1990
Claims (6)
既知量の抗体と抗原とを溶液中で反応させる工程、 ［２］上記溶液を希釈する工程、及び ［３］抗原と未反応の抗体を固相抗体法で定量する工程
を含む定量方法。(1) In a method for quantifying an antigen-antibody reaction, [1]
A quantitative method comprising: reacting a known amount of antibody and antigen in a solution; [2] diluting the solution; and [3] quantifying unreacted antibody with the antigen using a solid-phase antibody method.
体反応を定量する請求項［１］記載の方法。(2) The method according to claim [1], wherein the antigen-antibody reaction is quantified in a solution containing a factor that changes antigenicity.
方法。(3) The method according to claim [1], wherein the antibody is a monoclonal antibody.
、抗体がＡＲＭ３２１抗体、ＡＲＭ４１４抗体、及びＡ
ＲＭ１９１抗体からなる群より選ばれ、希釈剤がＰＢＳ
−ツイーンである請求項（１）記載の方法。(6) The antigen is recA protein or mutant protein, and the antibodies are ARM321 antibody, ARM414 antibody, and A
selected from the group consisting of RM191 antibodies, and the diluent was PBS.
- The method according to claim 1, wherein the method is a tween.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11599190A JP2915486B2 (en) | 1990-05-02 | 1990-05-02 | Indirect solid-phase antibody method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11599190A JP2915486B2 (en) | 1990-05-02 | 1990-05-02 | Indirect solid-phase antibody method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0412274A true JPH0412274A (en) | 1992-01-16 |
| JP2915486B2 JP2915486B2 (en) | 1999-07-05 |
Family
ID=14676157
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP11599190A Expired - Fee Related JP2915486B2 (en) | 1990-05-02 | 1990-05-02 | Indirect solid-phase antibody method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2915486B2 (en) |
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1990
- 1990-05-02 JP JP11599190A patent/JP2915486B2/en not_active Expired - Fee Related
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| Publication number | Publication date |
|---|---|
| JP2915486B2 (en) | 1999-07-05 |
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