JPH0677018B2 - Complement measurement method - Google Patents
Complement measurement methodInfo
- Publication number
- JPH0677018B2 JPH0677018B2 JP15795186A JP15795186A JPH0677018B2 JP H0677018 B2 JPH0677018 B2 JP H0677018B2 JP 15795186 A JP15795186 A JP 15795186A JP 15795186 A JP15795186 A JP 15795186A JP H0677018 B2 JPH0677018 B2 JP H0677018B2
- Authority
- JP
- Japan
- Prior art keywords
- complement
- peroxidase
- immune complex
- measuring
- antibody
- 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
Links
- 230000000295 complement effect Effects 0.000 title claims description 39
- 238000000691 measurement method Methods 0.000 title 1
- 108040007629 peroxidase activity proteins Proteins 0.000 claims description 28
- 238000000034 method Methods 0.000 claims description 21
- 230000000694 effects Effects 0.000 claims description 17
- 150000002978 peroxides Chemical class 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 239000000427 antigen Substances 0.000 claims description 7
- 102000036639 antigens Human genes 0.000 claims description 7
- 108091007433 antigens Proteins 0.000 claims description 7
- 150000002989 phenols Chemical class 0.000 claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 230000007928 solubilization Effects 0.000 claims description 4
- 238000005063 solubilization Methods 0.000 claims description 4
- 230000001590 oxidative effect Effects 0.000 claims description 3
- 102000003992 Peroxidases Human genes 0.000 claims 6
- 102000013415 peroxidase activity proteins Human genes 0.000 description 22
- 210000002966 serum Anatomy 0.000 description 12
- 230000003381 solubilizing effect Effects 0.000 description 11
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 238000005119 centrifugation Methods 0.000 description 7
- 229940088598 enzyme Drugs 0.000 description 7
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 206010018910 Haemolysis Diseases 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 230000004154 complement system Effects 0.000 description 3
- 230000008588 hemolysis Effects 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- HFZWRUODUSTPEG-UHFFFAOYSA-N 2,4-dichlorophenol Chemical compound OC1=CC=C(Cl)C=C1Cl HFZWRUODUSTPEG-UHFFFAOYSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102000009109 Fc receptors Human genes 0.000 description 1
- 108010087819 Fc receptors Proteins 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 229920001202 Inulin Polymers 0.000 description 1
- 108010023244 Lactoperoxidase Proteins 0.000 description 1
- 102000045576 Lactoperoxidases Human genes 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229920000392 Zymosan Polymers 0.000 description 1
- 231100000354 acute hepatitis Toxicity 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 102000006834 complement receptors Human genes 0.000 description 1
- 108010047295 complement receptors Proteins 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 235000013681 dietary sucrose Nutrition 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000003617 erythrocyte membrane Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 239000000852 hydrogen donor Substances 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- JYJIGFIDKWBXDU-MNNPPOADSA-N inulin Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@]1(OC[C@]2(OC[C@]3(OC[C@]4(OC[C@]5(OC[C@]6(OC[C@]7(OC[C@]8(OC[C@]9(OC[C@]%10(OC[C@]%11(OC[C@]%12(OC[C@]%13(OC[C@]%14(OC[C@]%15(OC[C@]%16(OC[C@]%17(OC[C@]%18(OC[C@]%19(OC[C@]%20(OC[C@]%21(OC[C@]%22(OC[C@]%23(OC[C@]%24(OC[C@]%25(OC[C@]%26(OC[C@]%27(OC[C@]%28(OC[C@]%29(OC[C@]%30(OC[C@]%31(OC[C@]%32(OC[C@]%33(OC[C@]%34(OC[C@]%35(OC[C@]%36(O[C@@H]%37[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O%37)O)[C@H]([C@H](O)[C@@H](CO)O%36)O)[C@H]([C@H](O)[C@@H](CO)O%35)O)[C@H]([C@H](O)[C@@H](CO)O%34)O)[C@H]([C@H](O)[C@@H](CO)O%33)O)[C@H]([C@H](O)[C@@H](CO)O%32)O)[C@H]([C@H](O)[C@@H](CO)O%31)O)[C@H]([C@H](O)[C@@H](CO)O%30)O)[C@H]([C@H](O)[C@@H](CO)O%29)O)[C@H]([C@H](O)[C@@H](CO)O%28)O)[C@H]([C@H](O)[C@@H](CO)O%27)O)[C@H]([C@H](O)[C@@H](CO)O%26)O)[C@H]([C@H](O)[C@@H](CO)O%25)O)[C@H]([C@H](O)[C@@H](CO)O%24)O)[C@H]([C@H](O)[C@@H](CO)O%23)O)[C@H]([C@H](O)[C@@H](CO)O%22)O)[C@H]([C@H](O)[C@@H](CO)O%21)O)[C@H]([C@H](O)[C@@H](CO)O%20)O)[C@H]([C@H](O)[C@@H](CO)O%19)O)[C@H]([C@H](O)[C@@H](CO)O%18)O)[C@H]([C@H](O)[C@@H](CO)O%17)O)[C@H]([C@H](O)[C@@H](CO)O%16)O)[C@H]([C@H](O)[C@@H](CO)O%15)O)[C@H]([C@H](O)[C@@H](CO)O%14)O)[C@H]([C@H](O)[C@@H](CO)O%13)O)[C@H]([C@H](O)[C@@H](CO)O%12)O)[C@H]([C@H](O)[C@@H](CO)O%11)O)[C@H]([C@H](O)[C@@H](CO)O%10)O)[C@H]([C@H](O)[C@@H](CO)O9)O)[C@H]([C@H](O)[C@@H](CO)O8)O)[C@H]([C@H](O)[C@@H](CO)O7)O)[C@H]([C@H](O)[C@@H](CO)O6)O)[C@H]([C@H](O)[C@@H](CO)O5)O)[C@H]([C@H](O)[C@@H](CO)O4)O)[C@H]([C@H](O)[C@@H](CO)O3)O)[C@H]([C@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@H](O)[C@@H](CO)O1 JYJIGFIDKWBXDU-MNNPPOADSA-N 0.000 description 1
- 229940029339 inulin Drugs 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 229940057428 lactoperoxidase Drugs 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- -1 parchlorophenol Chemical compound 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 201000000306 sarcoidosis Diseases 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 229960004793 sucrose Drugs 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical compound OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】 「産業上の利用分野」 本発明は血清等の試料中に存在する補体の免疫複合体可
溶化能を利用した補体の均一系分析方法に関する。TECHNICAL FIELD The present invention relates to a method for homogeneous analysis of complement utilizing the immune complex solubilizing ability of complement present in a sample such as serum.
「従来の技術」 補体系は、新鮮な血清中に含まれる20種類近いタンパク
質によって構成されている反応系で、抗原抗体反応物が
存在すると活性化される古典経路あるいは抗原抗体反応
物以外の物質、例えば酵母から分離した細胞壁であるザ
イモザン、植物性多糖類であるイヌリン、細菌から分離
した毒素などで活性化される二次経路によって、細胞溶
解をはじめ種々の生物活性を現わす。その中の一つに微
生物や感染細胞などの細胞表面を標的とする働きとは異
なる免疫複合体可溶化能がある。この作用で可溶化され
た免疫複合体は、分子量が小さくなるとともにそれ以上
補体系を活性化する能力がなくなり、FCレセプターや補
体レセプターとも反応しなくなることが知られている。
これは生体内では補体系由来の炎症を抑制したり局所に
沈着した不溶性免疫複合体を除去する働きにつながると
考えられる。よって免疫複合体可溶化能を利用して補体
を測定することは臨床検査分野において有用と考えられ
る。"Prior art" The complement system is a reaction system composed of nearly 20 kinds of proteins contained in fresh serum, and is a classical pathway activated by the presence of an antigen-antibody reaction product or a substance other than the antigen-antibody reaction product. For example, it exhibits various biological activities including cell lysis by a secondary pathway activated by zymosan, which is a cell wall isolated from yeast, inulin which is a plant polysaccharide, and toxin isolated from bacteria. One of these is the ability to solubilize immune complexes, which is different from the function of targeting the cell surface of microorganisms and infected cells. It is known that the immune complex solubilized by this action becomes smaller in molecular weight, loses the ability to activate the complement system further, and does not react with FC receptor or complement receptor.
This is considered to lead to the function of suppressing inflammation derived from the complement system and removing locally deposited insoluble immune complexes in vivo. Therefore, it is considered useful to measure complement by utilizing the immune complex solubilizing ability in the clinical laboratory field.
血清中の補体について従来調べられているのは補体価で
あり、これは古典経路を介して赤血球膜を破壊する活性
から求められている。この補体価測定法は、メイヤーら
によって開発され感作ヒツジ赤血球と検体血清との反応
後に、溶血により遊離してきたヘモグロビン量を遠心上
清の波長541nmでの吸光度から求める方法であり、50％
溶血を起こした補体の量を一単位としている。高補体価
を示す疾患として悪性リンパ腫、糖尿病、急性肝炎、サ
ルコイドーシス等が知られ又低補体価を示す疾患として
SLE、DIC等が知られている。What has been previously investigated for complement in serum is complement titer, which is sought from the activity of destroying the erythrocyte membrane via the classical pathway. This method of measuring the complement value is a method developed by Meyer et al., Which is a method of obtaining the amount of hemoglobin released by hemolysis after the reaction between the sensitized sheep red blood cells and the sample serum from the absorbance of the centrifugal supernatant at a wavelength of 541 nm, and 50%.
One unit is the amount of complement that caused hemolysis. Malignant lymphoma, diabetes, acute hepatitis, sarcoidosis, etc. are known as diseases showing high complement value, and as diseases showing low complement value.
SLE, DIC, etc. are known.
一方、免疫複合体可溶化能を利用した補体の測定は一般
に不溶性免疫複合体を用い、検体血清との反応後、遠心
操作によって不溶性画分を除き上清に遊離してきた抗原
あるいは抗体の量を知ることから成り立っている。可溶
化した抗原あるいは抗体量を知るには以下に挙げる方法
が知られている。１つは、そのどちらか一方に放射性同
位元素を標識しておいて遠心後の上清をシンチレーショ
ンカウンターで測定する方法又は抗原あるいは抗体に酵
素を標識しておいて遠心上清に標識酵素の基質を加え、
酵素活性を測定する方法、あるいは酵素を抗原としてそ
の抗体との不溶性免疫複合体を用い遠心上清の酵素活性
を測定する方法である。On the other hand, the measurement of complement utilizing the solubilizing ability of immune complex generally uses an insoluble immune complex, and after the reaction with the sample serum, the amount of the antigen or antibody released in the supernatant after removing the insoluble fraction by centrifugation. It consists of knowing. The following methods are known to know the amount of solubilized antigen or antibody. One is a method in which either one of them is labeled with a radioisotope and the supernatant after centrifugation is measured by a scintillation counter, or an enzyme is labeled in the antigen or antibody and the centrifugation supernatant is labeled with a substrate for the labeled enzyme. And add
It is a method of measuring the enzyme activity, or a method of measuring the enzyme activity of the centrifugal supernatant using an insoluble immune complex with the antibody as an antigen.
免疫複合体可溶化能に基づく補体量はこの遠心上清中の
放射性同位元素量、あるいは酵素活性量と比例関係にあ
ることから求められている。The amount of complement based on the solubilizing ability of the immune complex is determined from the fact that it has a proportional relationship with the amount of radioisotope in this centrifugal supernatant or the amount of enzyme activity.
従来の技術では補体と免疫複合体との反応後、不溶性画
分を遠心で除くことによって初めて免疫複合体可溶化能
を利用した補体測定が可能である。この遠心の必要性が
測定操作を非常に煩雑にしていた。又同じ理由によって
自動分析機への適用も非常に困難である。In the conventional technique, the complement measurement using the immune complex solubilizing ability can be performed only by removing the insoluble fraction by centrifugation after the reaction between the complement and the immune complex. The necessity of this centrifugation made the measurement operation very complicated. Also, it is very difficult to apply to an automatic analyzer for the same reason.
「発明が解決しようとする問題点」 本発明者らは、上記問題点を解決すべく鋭意検討の結
果、ペルオキシダーゼの酵素活性が過剰の過酸化水素存
在下では抑制されるが、抗体との会合状態では抑制され
ないことに着目し、遠心操作の必要がない免疫複合体可
溶化能を利用した補体の均一系測定法を目的として本発
明に到達した。“Problems to be Solved by the Invention” As a result of intensive studies to solve the above problems, the present inventors have found that the enzymatic activity of peroxidase is suppressed in the presence of excess hydrogen peroxide, but the association with antibodies Focusing on the fact that it is not suppressed in the state, the present invention has been achieved for the purpose of a method for measuring a homogeneous system of complement utilizing the immune complex solubilizing ability that does not require centrifugation.
「問題点を解決するための手段」 すなわち、本発明はペルオキシダーゼとその抗体からな
る免疫複合体が補体により可溶化されることにより生じ
る、過剰過酸化物存在下のペルオキシダーゼ活性の変化
を検知することを特徴とする補体の測定法である。"Means for Solving Problems" That is, the present invention detects a change in peroxidase activity in the presence of excess peroxide, which is caused by solubilization of an immune complex consisting of peroxidase and its antibody by complement. The method for measuring complement is characterized by the following.
更に、本発明は対応する抗原と抗体の組合せのいずれか
一方にペルオキシダーゼを標識し、両者の反応により形
成される免疫複合体が補体により可溶化されることによ
り生じる過剰過酸化物存在下のペルオキシダーゼ活性の
変化を検知することを特徴とする補体の測定法にも関す
る。Furthermore, the present invention labels peroxidase on either one of the corresponding antigen and antibody combinations, and in the presence of excess peroxide generated by solubilization of the immune complex formed by the reaction of the two with complement. It also relates to a method for measuring complement, which is characterized by detecting a change in peroxidase activity.
以下、本発明を詳細に説明する。Hereinafter, the present invention will be described in detail.
本発明に用いられるペルオキシダーゼは、植物・細菌・
動物等に存在し、過酸化物による物質の酸化反応を触媒
する酵素であり、例えば西洋ワサビペルオキシダーゼ、
ラクトペルオキシダーゼ、細菌由来ペルオキシダーゼ等
である。ペルオキシダーゼ抗体とは、上記ペルオキシダ
ーゼを動物に免疫して得られる抗体であり、例えば抗ペ
ルオキシダーゼウサギ抗体、抗ペルオキシダーゼヤギ抗
体等である。The peroxidase used in the present invention includes plants, bacteria,
It is an enzyme that exists in animals and catalyzes the oxidation reaction of substances by peroxides, such as horseradish peroxidase,
Lactoperoxidase, bacterial peroxidase and the like. The peroxidase antibody is an antibody obtained by immunizing an animal with the above-mentioned peroxidase, and examples thereof include anti-peroxidase rabbit antibody and anti-peroxidase goat antibody.
本発明に用いられる免疫複合体は上記のようなペルオキ
シダーゼと抗ペルオキシダーゼ抗体を反応させて調整す
る。この免疫複合体に含まれるペルオキシダーゼは会合
状態にあるため、過酸化物過剰下においても酵素活性を
発現する。この免疫複合体は、補体を含む試料との反応
によって定量的に可溶化される。ところが、この可溶化
によって遊離してきたペルオキシダーゼは、過酸化物過
剰下で活性が抑制されるのでペルオキシダーゼの活性を
測定することにより補体を定量することができる。The immune complex used in the present invention is prepared by reacting the above-mentioned peroxidase with an anti-peroxidase antibody. Since the peroxidase contained in this immune complex is in an associated state, it exhibits enzyme activity even in the presence of excess peroxide. This immune complex is quantitatively solubilized by reaction with a sample containing complement. However, since the activity of peroxidase released by this solubilization is suppressed in the presence of excess peroxide, complement can be quantified by measuring the activity of peroxidase.
本発明に用いられる過酸化物としては、例えば過酸化水
素、過酸化尿素等である。Examples of the peroxide used in the present invention include hydrogen peroxide and urea peroxide.
又、ペルオキシダーゼと抗ペルオキシダーゼ抗体を用い
て免疫複合体を形成させる代わりに免疫複合体として、
例えばウシ血清アルブミン（BSA）と抗BSAウサギ抗体の
ような、抗原と抗体の組合せで形成させ、そのどちらか
一方にペルオキシダーゼを標識しておいてもよい。例え
ば、BSAにペルオキシダーゼを標識しておくことによっ
て免疫複合体中にはペルオキシダーゼが会合状態で含ま
れることになる。この免疫複合体と補体を含む試料を反
応させてから、ペルオキシダーゼの活性を測定すること
により、ペルオキシダーゼとその抗体からなる免疫複合
体を用いた場合と同様の原理で補体を定量することがで
きる。Also, instead of forming an immune complex using peroxidase and anti-peroxidase antibody, as an immune complex,
For example, a combination of an antigen and an antibody such as bovine serum albumin (BSA) and anti-BSA rabbit antibody may be formed, and either one of them may be labeled with peroxidase. For example, by labeling BSA with peroxidase, peroxidase is contained in the immune complex in an associated state. By reacting a sample containing this immune complex with complement and then measuring the activity of peroxidase, complement can be quantified by the same principle as in the case of using the immune complex consisting of peroxidase and its antibody. it can.
ペルオキシダーゼの活性測定用試薬中には、発色剤とし
てフェノール類を含むがこの発色剤は過剰の過酸化物存
在下での会合状態のペルオキシダーゼを遊離状態でのペ
ルオキシダーゼの活性の違いを、検知するのに好ましい
水素供与体であり、フェノール、パルクロロフェノー
ル、2,4−ジクロロフェノール等がある。The reagent for measuring the activity of peroxidase contains phenols as a color-forming agent, and this color-forming agent detects peroxidase in the associated state in the presence of excess peroxide and detects the difference in the activity of peroxidase in the free state. Preferred hydrogen donors are phenol, parchlorophenol, and 2,4-dichlorophenol.
フェノール類による発色に際しては、通常用いられるフ
ェノール類の酸化縮合剤が用いられ、例えば４−アミノ
アンチピリン等が好適に用いられる。In the case of color development with phenols, a commonly used oxidative condensing agent for phenols is used, and for example, 4-aminoantipyrine and the like are preferably used.
「実施例」 以下、本発明を実施例により更に具体的に説明する。"Examples" Hereinafter, the present invention will be described in more detail with reference to Examples.
１）ａ免疫複合体の調整法 西洋ワサビペルオキシダーゼ（東洋紡社製、以下HRPと
記す。）を緩衝液Ａ（0.15mM CaCl2,0.5mM MgCl2,0.15M
NaClを含む20mMトリス塩酸緩衝液pH7.5）で６μg/mlに
調整した。又緩衝液Ａで抗HRPウサギ血清を20倍に希釈
し上記のHRP液と等量混合した。４℃において２日間反
応して免疫複合体を形成させた後、緩衝液Ｂ（10％サッ
カロースと４％フィコール400を含む20mMトリス塩酸緩
衝液pH7.5）と等量混合し、2mlずつ分注して凍結乾燥し
た。この凍結乾燥品を緩衝液A6mlで溶解し、免疫複合体
試薬として用いた。1) Method for preparing a immune complex Horseradish peroxidase (manufactured by Toyobo Co., Ltd., hereinafter referred to as HRP) was added to buffer A (0.15 mM CaCl 2 , 0.5 mM MgCl 2 , 0.15 M).
The concentration was adjusted to 6 μg / ml with 20 mM Tris-HCl buffer (pH 7.5) containing NaCl. Also, anti-HRP rabbit serum was diluted 20 times with buffer solution A and mixed with the above HRP solution in the same amount. After reacting at 4 ° C for 2 days to form an immune complex, an equal volume of buffer B (20 mM Tris-HCl buffer pH7.5 containing 10% saccharose and 4% Ficoll 400) was mixed and dispensed in 2 ml aliquots. And lyophilized. This lyophilized product was dissolved in 6 ml of buffer A and used as an immune complex reagent.
ｂ免疫複合体可溶化能に基づく補体量の標準曲線作成法 プール新鮮血清をセントリコン−10（アミコン社製）で
２倍に濃縮し標準補体用血清とした。これを非働化血清
（56℃、30分間熱処理）で希釈し標準物を調整した。こ
の標準物50μｌにａで調整した免疫複合体試薬200μｌ
を加え37℃において20分間反応した。次に基質溶液（0.
75mM4−アミノアンチピリン、25mMフェノール、40mM過
酸化水素を含む緩衝液Ａ）1mlを加え、37℃において10
分間反応した。更に反応停止液（1.8％ホルムアルデヒ
ドを含む20mMリン酸緩衝液pH7.0）2mlを加えた後、波長
500nmにおける吸光度を測定した。横軸に標準補体用血
清濃度をとり、縦軸に波長500nmにおける吸光度をとっ
て標準曲線を作製した。この結果を第１図に示す。b Standard curve preparation method of complement amount based on solubilizing ability of immune complex Pool fresh serum was concentrated 2-fold with Centricon-10 (manufactured by Amicon) to obtain standard complement serum. This was diluted with inactivated serum (heat treated at 56 ° C for 30 minutes) to prepare a standard. 200 μl of immune complex reagent prepared in a to 50 μl of this standard
Was added and reacted at 37 ° C. for 20 minutes. Then the substrate solution (0.
Add 1 ml of buffer A) containing 75 mM 4-aminoantipyrine, 25 mM phenol, 40 mM hydrogen peroxide, and add 10 ml at 37 ° C.
Reacted for minutes. After adding 2 ml of reaction stop solution (20 mM phosphate buffer pH 7.0 containing 1.8% formaldehyde),
The absorbance at 500 nm was measured. The standard complement serum concentration was plotted on the horizontal axis and the absorbance at a wavelength of 500 nm was plotted on the vertical axis to prepare a standard curve. The results are shown in FIG.
２）調整検体の本法と50％溶血法との相関 種々の正常ヒト血清をセントリコン−10で濃縮あるいは
非働化血清で希釈して補体価の異なる検体を調整した。
次にこの検体の免疫複合体可溶化能に基づく補体量を実
施例１）ｂと同様の実験操作で測定した。更に各検体の
得られた吸光度に対する補体量標準曲線から読みとり％
表示した。2) Correlation between this method and 50% hemolysis method of prepared samples Various normal human sera were concentrated with Centricon-10 or diluted with inactivated serum to prepare samples with different complement values.
Next, the amount of complement based on the immune complex solubilizing ability of this sample was measured by the same experimental procedure as in Example 1) b. Furthermore, read from the standard curve of the amount of complement to the obtained absorbance of each sample,
displayed.
一方、調製した検体の補体価をメイヤーらの方法に従っ
て測定した。得られたCH50と可溶化能に基づく補体量の
関係は、第２図に示すごとく相関係数γ＝0.94とよく相
関することがわかった。On the other hand, the complement value of the prepared sample was measured according to the method of Meyer et al. The relationship between the obtained CH 50 and the amount of complement based on the solubilizing ability was found to correlate well with the correlation coefficient γ = 0.94, as shown in FIG.
「発明の効果」 実施例に示したごとく、本発明によって免疫複合体可溶
化能に基づく補体量が、短時間に遠心操作の必要なく定
量することが可能となった。又、分離操作の不必要な点
から自動分析機への応用も考えられ、多検体測定も困難
なく行なうことができる。更にCH50との相関も良いの
で、煩雑な検体希釈を必要とし、感作ヒツジ赤血球を使
用していることから試薬としての安定性が悪い従来の補
体価測定に代わり、臨床データの蓄積に伴いその測定の
意義が増すことが期待できる。"Effects of the Invention" As shown in the examples, the present invention makes it possible to quantify the amount of complement based on the immune complex solubilizing ability in a short time without the need for centrifugation. In addition, since the separation operation is unnecessary, it can be considered to be applied to an automatic analyzer, and multi-sample measurement can be performed without difficulty. Furthermore, since it has a good correlation with CH 50, it requires complicated sample dilution, and because it uses sensitized sheep erythrocytes, it has poor stability as a reagent. Therefore, the significance of the measurement can be expected to increase.
第１図は、本発明の方法により標準補体用血清の希釈列
を測定した場合に得られる結果で、横軸に標準補体濃度
を％表示し縦軸に波長500nmにおける吸光度を示すグラ
フ図であり、第２図は、本発明の測定法で得られた免疫
複合体可溶化能に基づく補体量と、メイヤー等の方法で
得られたCH50との相関を示すグラフ図である。FIG. 1 is a graph showing the results obtained when measuring the dilution series of serum for standard complement by the method of the present invention, showing the standard complement concentration in% on the horizontal axis and the absorbance at a wavelength of 500 nm on the vertical axis. FIG. 2 is a graph showing the correlation between the amount of complement based on the immune complex solubilizing ability obtained by the assay method of the present invention and the CH 50 obtained by the method of Mayer et al.
Claims (4)
複合体が補体により可溶化されることにより生じる、過
剰過酸化物存在下のペルオキシダーゼ活性の変化を検知
することを特徴とする補体の測定法。1. A method for measuring complement, which comprises detecting a change in peroxidase activity in the presence of excess peroxide, which is caused by solubilization of an immune complex consisting of peroxidase and its antibody by complement. .
過剰の過酸化物、フェノール類及びフェノール類に対す
る酸化縮合剤を用いる特許請求の範囲第１項記載の測定
法。2. The measuring method according to claim 1, wherein excess peroxide, phenols and an oxidative condensing agent for phenols are used as a reagent for measuring the activity of peroxidase.
方にペルオキシダーゼを標識し、両者の反応により形成
される免疫複合体が補体により可溶化されることにより
生じる過剰過酸化物存在下のペルオキシダーゼ活性の変
化を検知することを特徴とする補体の測定法。3. A peroxidase is labeled on either one of a combination of a corresponding antigen and an antibody, and an immune complex formed by the reaction of the both is solubilized by complement. A method for measuring complement, which comprises detecting a change in peroxidase activity.
過剰の過酸化物、フェノール類及びフェノール類に対す
る酸化縮合剤を用いる特許請求の範囲第３項記載の測定
法。4. The measuring method according to claim 3, wherein excess peroxide, phenols and an oxidative condensing agent for phenols are used as a reagent for measuring the activity of peroxidase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15795186A JPH0677018B2 (en) | 1986-07-07 | 1986-07-07 | Complement measurement method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15795186A JPH0677018B2 (en) | 1986-07-07 | 1986-07-07 | Complement measurement method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6315162A JPS6315162A (en) | 1988-01-22 |
| JPH0677018B2 true JPH0677018B2 (en) | 1994-09-28 |
Family
ID=15661026
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP15795186A Expired - Lifetime JPH0677018B2 (en) | 1986-07-07 | 1986-07-07 | Complement measurement method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0677018B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3811426B2 (en) | 2002-05-15 | 2006-08-23 | 株式会社日立製作所 | Permanent magnet rotating electric machine |
| CN110780082B (en) * | 2019-11-04 | 2023-05-12 | 上海昆涞实业发展有限公司 | Total complement activity quality control product, reagent, preparation method and application |
-
1986
- 1986-07-07 JP JP15795186A patent/JPH0677018B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6315162A (en) | 1988-01-22 |
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