JPH0389164A - Reagent for measurement of enzyme immunity and preparation thereof - Google Patents
Reagent for measurement of enzyme immunity and preparation thereofInfo
- Publication number
- JPH0389164A JPH0389164A JP22552389A JP22552389A JPH0389164A JP H0389164 A JPH0389164 A JP H0389164A JP 22552389 A JP22552389 A JP 22552389A JP 22552389 A JP22552389 A JP 22552389A JP H0389164 A JPH0389164 A JP H0389164A
- Authority
- JP
- Japan
- Prior art keywords
- enzyme
- antibody
- antibodies
- labeled
- alp
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 148
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 148
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 21
- 238000005259 measurement Methods 0.000 title claims abstract description 13
- 230000036039 immunity Effects 0.000 title abstract 3
- 238000002360 preparation method Methods 0.000 title description 9
- 238000000034 method Methods 0.000 claims abstract description 43
- 239000000427 antigen Substances 0.000 claims abstract description 32
- 102000036639 antigens Human genes 0.000 claims abstract description 32
- 108091007433 antigens Proteins 0.000 claims abstract description 32
- 125000005439 maleimidyl group Chemical group C1(C=CC(N1*)=O)=O 0.000 claims abstract description 7
- 238000003018 immunoassay Methods 0.000 claims description 22
- 238000002156 mixing Methods 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 4
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 4
- 238000000691 measurement method Methods 0.000 claims description 3
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 3
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 239000007853 buffer solution Substances 0.000 abstract description 3
- 241001465754 Metazoa Species 0.000 abstract description 2
- 239000003795 chemical substances by application Substances 0.000 abstract description 2
- 239000012634 fragment Substances 0.000 abstract description 2
- 230000003053 immunization Effects 0.000 abstract description 2
- 239000003550 marker Substances 0.000 abstract 6
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 129
- 238000001514 detection method Methods 0.000 description 20
- 230000000694 effects Effects 0.000 description 18
- 238000001179 sorption measurement Methods 0.000 description 16
- 238000000746 purification Methods 0.000 description 15
- 239000000203 mixture Substances 0.000 description 13
- 239000000872 buffer Substances 0.000 description 12
- 239000007790 solid phase Substances 0.000 description 12
- 239000012506 Sephacryl® Substances 0.000 description 11
- 239000003431 cross linking reagent Substances 0.000 description 11
- 238000012360 testing method Methods 0.000 description 11
- 241000283973 Oryctolagus cuniculus Species 0.000 description 10
- 238000002835 absorbance Methods 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 6
- 239000008363 phosphate buffer Substances 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 102100023635 Alpha-fetoprotein Human genes 0.000 description 5
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 5
- 238000010828 elution Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 241000283707 Capra Species 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 238000011088 calibration curve Methods 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 230000009870 specific binding Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- BCHIXGBGRHLSBE-UHFFFAOYSA-N (4-methyl-2-oxochromen-7-yl) dihydrogen phosphate Chemical compound C1=C(OP(O)(O)=O)C=CC2=C1OC(=O)C=C2C BCHIXGBGRHLSBE-UHFFFAOYSA-N 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 229920005654 Sephadex Polymers 0.000 description 3
- 239000012507 Sephadex™ Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 238000005227 gel permeation chromatography Methods 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical compound O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 3
- PVGATNRYUYNBHO-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-(2,5-dioxopyrrol-1-yl)butanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCN1C(=O)C=CC1=O PVGATNRYUYNBHO-UHFFFAOYSA-N 0.000 description 2
- VLARLSIGSPVYHX-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 6-(2,5-dioxopyrrol-1-yl)hexanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCCCN1C(=O)C=CC1=O VLARLSIGSPVYHX-UHFFFAOYSA-N 0.000 description 2
- 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
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- 239000008351 acetate buffer Substances 0.000 description 2
- 125000003172 aldehyde group Chemical group 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 238000004452 microanalysis Methods 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 2
- 229960002317 succinimide Drugs 0.000 description 2
- KFDNQUWMBLVQNB-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;sodium Chemical compound [Na].[Na].[Na].[Na].OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KFDNQUWMBLVQNB-UHFFFAOYSA-N 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102100035092 ER membrane protein complex subunit 5 Human genes 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 101000877400 Homo sapiens ER membrane protein complex subunit 5 Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 230000029918 bioluminescence Effects 0.000 description 1
- 238000005415 bioluminescence Methods 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- -1 carboxylic acid succinimide ester Chemical class 0.000 description 1
- 238000004976 chemiluminescence spectroscopy Methods 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- UFULAYFCSOUIOV-UHFFFAOYSA-N cysteamine Chemical compound NCCS UFULAYFCSOUIOV-UHFFFAOYSA-N 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 108010074605 gamma-Globulins Proteins 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 239000007986 glycine-NaOH buffer Substances 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 1
- 229960003151 mercaptamine Drugs 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- 238000013094 purity test Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- UEUXEKPTXMALOB-UHFFFAOYSA-J tetrasodium;2-[2-[bis(carboxylatomethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O UEUXEKPTXMALOB-UHFFFAOYSA-J 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
Ａ、産業上の利用分野
本発明は抗原抗体反応の定量的測定法に用いる酵素免疫
測定用試薬及びその調製方法に関する。DETAILED DESCRIPTION OF THE INVENTION A. Field of Industrial Application The present invention relates to an enzyme immunoassay reagent used for quantitative measurement of antigen-antibody reactions and a method for preparing the same.
Ｂ８発明の概要
本発明は抗原抗体反応の定量的測定法に用いる酵素免疫
測定用試薬において、
前記酵素標識体がアルカリフォスファターゼ（以下、Ａ
ＬＰと略す）１分子に抗原又は抗体が４分子結合してい
る酵素免疫測定用試薬を用いることにより酵素免疫測定
法の感度及び再現性を向上させることを可能とする。B8 Summary of the Invention The present invention provides an enzyme immunoassay reagent used for quantitative measurement of antigen-antibody reactions, wherein the enzyme label is alkaline phosphatase (hereinafter referred to as A).
By using an enzyme immunoassay reagent in which four antigen or antibody molecules are bound to one molecule (abbreviated as LP), it is possible to improve the sensitivity and reproducibility of enzyme immunoassay.
また、本発明（よ酵素免疫測定用試薬の調製方法におい
て、
前記酵素かＡＬＰであり、該ＡＬＰ １モルに対して、
ｎ倍（但し、ｎは２以上の整数である）の割合で抗原又
は抗体を混合して、調製することにより前記酵素免疫測
定用試薬を高収率で得ることを可能とする。In addition, in the present invention (method for preparing a reagent for enzyme immunoassay), the enzyme is ALP, and for 1 mole of ALP,
By mixing and preparing antigens or antibodies at a ratio of n times (where n is an integer of 2 or more), the enzyme immunoassay reagent can be obtained in high yield.
Ｃ６従来の技術
臨床検査部門における高感度な微量分析法の開発は現在
盛んに行われており、酵素免疫測定法（以下ｒＥＩＡ法
」と略す）、化学発光分析法を組み合わせた測定方法も
多数報告されている。C6 Conventional technology The development of highly sensitive microanalysis methods in clinical laboratory departments is currently actively underway, and many measurement methods that combine enzyme immunoassay (rEIA) and chemiluminescence spectrometry have been reported. has been done.
この方法は抗原又は抗体に酵素を標識した酵素標識体を
被測定物質である抗原又は抗体と特異的に反応させた後
、標識酵素に基質を反応させ、このとき生成する物質を
化学発光法により検出し、間接的に被測定物質を測定す
る方法である。In this method, an enzyme-labeled antigen or antibody labeled with an enzyme is reacted specifically with the antigen or antibody, which is the substance to be measured, and then the labeled enzyme is reacted with a substrate. This is a method of detecting and indirectly measuring the substance to be measured.
ところで、ＥＩＡ法の代表例であるサンドイッチ広につ
いて述べると、まず抗原、例えばα−フェトプロティン
（以下、ｒＡＦＰＪという）をウサギに定期的に皮下注
射により投入し、このウサギの血清から前記抗原にもと
づいて生じた免疫グロブリンを得、これを例えばポリス
チレンボールよりなる固相に吸着させて固相抗体を調製
する。By the way, to describe the sandwich method that is a typical example of the EIA method, first, an antigen, such as α-fetoprotein (hereinafter referred to as rAFPJ), is injected periodically into a rabbit by subcutaneous injection, and then an antigen is extracted from the rabbit's serum based on the antigen. A solid-phase antibody is prepared by adsorbing the resulting immunoglobulin onto a solid phase made of, for example, a polystyrene ball.
一方、前記免疫グロブリンに標識体、例えば酸化酵素で
あるグルコースオキシダーゼ（以下「Ｇ。On the other hand, the immunoglobulin is labeled with a label, such as glucose oxidase (hereinafter referred to as "G"), which is an oxidizing enzyme.
Ｄ」という。）を結合させて酵素標識抗体を調製する。D”. ) to prepare an enzyme-labeled antibody.
ここで、被測定物質である人体内の前記抗原となるＡＦ
Ｐの量を調べるためには、先ず測定対象となる抗原を固
相抗体に接触させると、抗原は固相抗体に特異的に吸着
される。次いで、これに酵素標識抗体を作用させろと、
酵素標識抗体は抗原に特異的に吸着される。即ち抗原は
固相抗体と酵素標識抗体に挟まれた状態になる。次いで
、酵素標識抗体のＣＯＤの量を測定する。この測定は、
ＣＯＤにグルコースを作用させて過酸化水素を発生させ
、この過酸化水素にルミノールを作用させて発光量を調
べるいわゆる化学発光法により行われ、被測定物質であ
る抗原即ち、ＡＰ’Ｐの固相抗体への吸着量はＣＯＤの
測定量から間接的に求まる。そのためＥＩＡ法の感度上
昇を望むには、優れた酵素標識抗体の調製が必要とされ
る。Here, AF, which is the antigen in the human body that is the substance to be measured,
To examine the amount of P, first, the antigen to be measured is brought into contact with a solid-phase antibody, and the antigen is specifically adsorbed to the solid-phase antibody. Next, they decided to apply an enzyme-labeled antibody to this.
Enzyme-labeled antibodies are specifically adsorbed to antigens. That is, the antigen is sandwiched between the solid-phase antibody and the enzyme-labeled antibody. Next, the amount of COD of the enzyme-labeled antibody is measured. This measurement is
The so-called chemiluminescence method is used to react COD with glucose to generate hydrogen peroxide, and then react with luminol to measure the amount of light emitted. The amount of adsorption to the antibody can be determined indirectly from the measured amount of COD. Therefore, in order to increase the sensitivity of the EIA method, it is necessary to prepare excellent enzyme-labeled antibodies.
一方、ＥＩＡ法に用いる酵素標識抗体を得る方法として
次のものかある。On the other hand, the following methods are available for obtaining enzyme-labeled antibodies used in the EIA method.
（りまず、第一に、酵素を架橋剤として反応させろこと
によりアルデヒド基を導入し、このアルデヒド基と抗体
のもつアミノ基とを反応させて酵素を抗体に標識させる
二段階グルタルアルデヒド法及び過ヨーソ酸法である。(First, the two-step glutaraldehyde method involves introducing an aldehyde group by reacting with an enzyme as a crosslinking agent, and then reacting this aldehyde group with the amino group of the antibody to label the enzyme with the antibody. This is the iosoacid method.
（２）第二に、酵素とマレイミド基を有する架橋剤とを
反応させることにより酵素にマレイミド基を導入し、こ
のマレイミド基と抗体のもつチオール基とを反応させて
、酵素を抗体に標識させるマレイミド法である。(2) Second, a maleimide group is introduced into the enzyme by reacting the enzyme with a crosslinking agent having a maleimide group, and this maleimide group is reacted with a thiol group of the antibody to label the enzyme with the antibody. This is the maleimide method.
（３）第三に、酵素とピリジルチオール基とを反応させ
ることにより酵素にピリジルチオール基を導入し、この
ピリジルチオール基と抗体のもつチオール基とを反応さ
せて、酵素を抗体に標識させるビリジルフスルフィド法
である。(3) Third, a pyridylthiol group is introduced into the enzyme by reacting the enzyme with a pyridylthiol group, and this pyridylthiol group is reacted with a thiol group of an antibody to label the enzyme with an antibody. This is the dilfsulfide method.
現在、上記の方法の中でマレイミド法が最も広く使用さ
れている。これはマレイミド法が他の方法に比し高収率
で酵素標識抗体を得ることができ、しかも自己重合など
により高重合体が形成されないことから抗体活性の低下
を防止でき、さらに抗原抗体反応における非特異的吸着
も少ないためである。Currently, the maleimide method is the most widely used among the above methods. This is because the maleimide method can obtain enzyme-labeled antibodies in a higher yield than other methods, and since high polymers are not formed due to self-polymerization, it can prevent a decrease in antibody activity, and furthermore, it can prevent a decrease in antibody activity. This is because there is less non-specific adsorption.
しかしながら、上記マレイミド法により調製され、精製
して得られた酵素標識抗体は各ロット毎に性能に差が見
られ、そのためこの酵素標識抗体をＥＩＡ法に用いた場
合、その結果に再現性を得ることが困難であった。However, the performance of the enzyme-labeled antibodies prepared and purified by the above-mentioned maleimide method differs from lot to lot, and therefore, when this enzyme-labeled antibody is used in the EIA method, it is difficult to obtain reproducible results. It was difficult.
従って、このことからＥＩＡ法による被測定物質の検出
限界も比較的高い濃度にとどまらざるを得ないという問
題があった。Therefore, there is a problem in that the detection limit of the substance to be measured by the EIA method has to remain at a relatively high concentration.
Ｄ０発明が解決しようとする課題
本発明は酵素標識抗体の性能が酵素標識抗体中の抗体数
のバラツキから生ずることに着目して創案されたもので
あって、酵素標識抗体がＡ Ｌ Ｐ １分子に抗原又は
抗体が４分子結合していることを特徴とする酵素免疫測
定用試薬、及び前記酵素がＡＬＰであり、該ＡＬＰ１モ
ルに対して、ｎ（身（但し、ｎは２以上の整数である）
の割合で、抗原又は抗体と混合することを特徴とする前
記酵素免疫測定用試薬の調製方法を提供するものである
。D0 Problems to be Solved by the Invention The present invention was created focusing on the fact that the performance of enzyme-labeled antibodies arises from the variation in the number of antibodies in the enzyme-labeled antibodies. An enzyme immunoassay reagent characterized in that 4 molecules of antigen or antibody are bound to be)
The present invention provides a method for preparing the reagent for enzyme immunoassay, characterized in that the reagent is mixed with an antigen or an antibody at a ratio of .
８０課題を解決するための手段及び作用即ち、本発明は
抗原抗体反応の定量的測定法に用いる酵素免疫測定用試
薬において、前記酵素標識体がＡＩ、ｐｔ分子に抗原又
は抗体が４分子結合していること及び酵素免疫測定用試
薬の調製方法において、前記酵素がＡＬＰであり、該Ａ
ＬＰ１モルに対してｎ倍（但し、ｎは２以上の整数であ
る）の割合で抗原又は抗体を混合することを、その解決
手段としている。80 Means and Effects for Solving the Problems Namely, the present invention provides an enzyme immunoassay reagent used for a quantitative measurement method of an antigen-antibody reaction, in which the enzyme label is AI, and 4 antigen or antibody molecules are bound to a pt molecule. and the method for preparing a reagent for enzyme immunoassay, wherein the enzyme is ALP, and the ALP
The solution is to mix antigens or antibodies at a ratio of n times (where n is an integer of 2 or more) to 1 mole of LP.
以下、本発明をさらに詳細に説明する。The present invention will be explained in more detail below.
まず、本発明では標識抗体としてＡＬＰを用いる。この
酵素は分子量ｌＯ万であり、抗体標識として用いた場合
に安定性にすぐれるため、従来から酵素抗体法に用いら
れている。また、この酵素を標識する肢体は免疫反応の
特質から抗原、抗体のいずれでも使用することができる
が通常抗体を用いる。従って、本発明の酵素免疫測定用
試薬においてら酵素標識の肢体として抗体を用いる場合
のみ説明する。First, in the present invention, ALP is used as a labeled antibody. This enzyme has a molecular weight of 10,000,000 and has excellent stability when used as an antibody label, so it has been conventionally used in enzyme antibody methods. Further, as the limb for labeling this enzyme, either an antigen or an antibody can be used due to the characteristics of the immune reaction, but antibodies are usually used. Therefore, only the case where an antibody is used as an enzyme label in the enzyme immunoassay reagent of the present invention will be explained.
そして、この抗体としては通常使用されるもの、例えば
、被測定物質である抗原を動物１例えばウサギ、ヤギ、
モルモットなどに免疫して得られる抗体、さらにこれら
の抗体にタンパク質分解酵素を作用させて得られる抗原
結合活性のあるフラグメント（Ｆａｂ）などを使用する
ことができる。The antibody is one that is commonly used, for example, the antigen that is the substance to be measured is used in an animal such as a rabbit, a goat, or
Antibodies obtained by immunizing guinea pigs, etc., and fragments (Fab) with antigen-binding activity obtained by treating these antibodies with proteolytic enzymes can be used.
次に、ＡＬＰを抗体に標識する際の架橋剤としては、従
来のマレイミド法で使用されているマレイミド基をもつ
もの、例えばＮ−（ｒ−マレイミドブチルオキシ）サク
シンイミド（以下、Ｇ Ｍ ＢＳと略す。）、
Ｎ−（ε−マレイミドカプロルオキシ）ザクンンイミド
（以下、ＥＭＣ５と略す。）、４−（マレイミドメヂル
）シクロヘキサン−Ｉカルボン酸すクシンイミドエステ
ル（以下、ＣＨＭと略す。）などが挙げられる。Next, as a crosslinking agent for labeling an antibody with ALP, one having a maleimide group used in the conventional maleimide method, such as N-(r-maleimidobutyloxy)succinimide (hereinafter abbreviated as GM BS), is used. ), N-(ε-maleimidocaproloxy)zacunnimide (hereinafter abbreviated as EMC5), 4-(maleimidomedyl)cyclohexane-I carboxylic acid succinimide ester (hereinafter abbreviated as CHM), and the like.
更に、酵素標識抗体は次のように調製、精製により得る
ことができる。Furthermore, enzyme-labeled antibodies can be prepared and purified as follows.
即ち、ＡＬＰ １分子に導入される抗体数と同じ割合で
酵素と抗体とを混合して調製する。例えば、抗体数２．
３．４の酵素標識抗体を得るには酵素と抗体とをそれぞ
れｌ：２，１＋３，１：４の割合（モル比）で混合調製
する。そして、この割合で調製することにより、主とし
て抗体数２．３゜４の酵素標識抗体が高い収率で、しか
も再現性よく得られる。That is, it is prepared by mixing enzymes and antibodies in the same proportion as the number of antibodies introduced into one ALP molecule. For example, the number of antibodies is 2.
In order to obtain the enzyme-labeled antibody of 3.4, the enzyme and antibody are mixed and prepared at a ratio (molar ratio) of 1:2, 1+3, and 1:4, respectively. By preparing at this ratio, enzyme-labeled antibodies mainly containing 2.3.4 antibodies can be obtained in high yield and with good reproducibility.
なお、標識となる酵素に結合する抗体数が多いほど酵素
免疫測定法における検出感度及び再現性の向上を図るこ
とができることから、本発明の酵素免疫測定用試薬はＡ
ＬＰ１モルに対してｎモルの抗体を混合して調製し、Ａ
ＬＰ１分子に対し、抗体が４分子結合した酵素標識抗体
を用いるのが望ましい。Note that the detection sensitivity and reproducibility in enzyme immunoassay can be improved as the number of antibodies that bind to the labeled enzyme increases.
Prepared by mixing n moles of antibody to 1 mole of LP, A
It is desirable to use an enzyme-labeled antibody in which four molecules of the antibody are bound to the LP1 molecule.
このようにして抗体数４を有する調製された酵素標識抗
体は通常用いられている精製性、例えば高速ゲルクロマ
トグラフィー用カラム、好ましくは排除限界６０万以上
、理論段数１５，０００ 以上の高速ゲルクロマトグラ
フィー用カラムを使用することにより未反応の抗体（分
子Ｍ４．５万）、さらに未反応の酵素や副反応として生
じる抗体数の異なる酵素標識抗体から分離される。なお
、高速ゲルクロマトグラフィー用カラムとしてＴＳＫＧ
−３０００ｓｗ（東ソー製）、スペロース（Ｓｕｐｅｒ
ｏｓｅ）６ＨＲ１０／３０ （ファルマシア社製）、Ｇ
５−６２０ （旭化成社製）、プロティン（ＰＲＯＴＥ
ＩＮ）ｗｓ−８０３（昭和電工社製）を挙げることかで
きる。The enzyme-labeled antibody thus prepared having 4 antibodies can be purified using a commonly used column for purification, such as a high-speed gel chromatography column, preferably a high-speed gel chromatography column with an exclusion limit of 600,000 or more and a theoretical plate number of 15,000 or more. By using a column for graphics, it is separated from unreacted antibodies (molecules M: 45,000), unreacted enzymes, and enzyme-labeled antibodies with different numbers of antibodies generated as side reactions. In addition, TSKG is a column for high-speed gel chromatography.
-3000sw (manufactured by Tosoh), Superose (Super
ose) 6HR10/30 (manufactured by Pharmacia), G
5-620 (manufactured by Asahi Kasei), protein (PROTE)
IN)ws-803 (manufactured by Showa Denko).
上記した調製法により、ＡＬＰを標識酵素としこのＡＬ
Ｐ １分子に抗体が４分子結合した酵素標識抗体を得る
ことができ、得られる酵素標識抗体は溶液又は粉末状い
ずれでも使用しうるが、ｐ Ｈ２〜８の緩衝液例えば、
リン酸緩衝液、クエン酸緩衝液、酢酸緩衝液、コハク緩
衝液などに適当な濃度に調製して使用するのが好ましい
。Using the above-mentioned preparation method, ALP is used as a labeling enzyme and this AL
An enzyme-labeled antibody in which four molecules of antibody are bound to one P molecule can be obtained, and the obtained enzyme-labeled antibody can be used in either a solution or powder form, but it can be used in a buffer solution with a pH of 2 to 8, for example.
It is preferable to use a phosphate buffer, a citrate buffer, an acetate buffer, an acetate buffer, a succinic buffer, etc. to prepare an appropriate concentration.
このようにして得られる本発明の酵素免疫測定用試薬は
、ユツト法などの均−法又はサンドイツチ法、二抗体法
などの不均一法のいずれの酵素免疫測定法にも使用する
ことができ、また酵素反応により生じた被検出物は化学
発光法、生物発光法、比色法、蛍光法など通常の方法に
より容易に検出することができる。The reagent for enzyme immunoassay of the present invention thus obtained can be used in any enzyme immunoassay method, such as a homogeneous method such as the Utt method, or a heterogeneous method such as the Sand-Deutsch method or the double-antibody method. Further, the analyte produced by the enzymatic reaction can be easily detected by conventional methods such as chemiluminescence, bioluminescence, colorimetry, and fluorescence.
なお、本発明の酵素免疫測定用試薬により検出できろ被
測定物質である抗原としてはＡＦＰ、カルチツエンプリ
オニツク抗原などのいわゆるガンマ−カーが挙げられる
。Incidentally, examples of the antigen which is the substance to be measured which can be detected by the reagent for enzyme immunoassay of the present invention include so-called gamma markers such as AFP and Kaltizen Prionik antigen.
Ｆ、実施例
以下、本発明に係る酵素免疫測定用試薬及びその調製方
法の詳細な説明を実施例に基づいて説明する。F. Examples Hereinafter, a detailed explanation of the enzyme immunoassay reagent and its preparation method according to the present invention will be explained based on Examples.
実施例１
（１）マレイミド化ＡＬＰの調製及びその精製１５ｍｇ
のＡＬＰを含む０．１Ｍ）リス緩衝液（ｐＨ７，６）ｌ
皮Ｑに５．７５朽のＣＭＢＳを含むジメチルホルムアミ
ド溶液６０μｇを加え、３０°Ｃで１時間撹拌した。次
にその混合物をセファデツクスＧ−２５（Ｓｅｐｈａｒ
３ｅｘＧ−２５）を充てんしたＰｏ−１０カラム（ファ
ルマシア社製）で脱塩、濃縮した。Example 1 (1) Preparation of maleimidized ALP and its purification 15 mg
0.1M) Lys buffer (pH 7,6) containing ALP
60 μg of a dimethylformamide solution containing 5.75% CMBS was added to Peel Q, and the mixture was stirred at 30° C. for 1 hour. The mixture was then mixed with Sephadex G-25 (Sephadex G-25).
The mixture was desalted and concentrated using a Po-10 column (manufactured by Pharmacia) packed with 3exG-25).
（２）マレイミド化ＡＬＰの調製及びその精製ＧＭＢＳ
に代えてＥＭＣＳを用いる以外は実施例１ （１）と同
様の方法でマレイミド化ＡＬＰの調製及びその精製を行
った。(2) Preparation of maleimidized ALP and its purification GMBS
Maleimidated ALP was prepared and purified in the same manner as in Example 1 (1) except that EMCS was used instead of EMCS.
（３）マレイミド化ＡＬＰの調製及びその精製Ｇ Ｍ
Ｂ Ｓに代えてＣＨＭを用いる以外は実施例１（１）と
同様の方法でマレイミド化ＡＬＰの調製及びその精製を
行った。(3) Preparation of maleimidized ALP and its purification GM
Maleimidated ALP was prepared and purified in the same manner as in Example 1 (1) except that CHM was used instead of BS.
実施例２
（１）ウサギ抗ＡＦＰＦａｂ’ の調製８抑のウサギ抗
ＡＦＰＦ（ａｂ’）ｔを含む０．１Ｍトリス−塩酸緩衝
液（ｐｉ４６．０） Ｌｘ（１１，＝７９メルカプトエ
チルアミンｌ 、 Ｉ ｘｇ及び５ｍＭＥＤＴＡを含む
０．１Ｍ）リス−塩酸緩衝液（ｐ Ｈ６０）５０μＱを
加え、３７℃で１．５時間撹拌した。Example 2 (1) Preparation of rabbit anti-AFPF Fab' 0.1M Tris-HCl buffer (pi 46.0) containing 80% rabbit anti-AFPF(ab')t Lx(11,=79 mercaptoethylamine l, Ixg 50 μQ of 0.1 M) Lis-HCl buffer (pH 60) containing 5 mM EDTA was added, and the mixture was stirred at 37° C. for 1.5 hours.
次に、その混合物をセファデックスＧ−２５（Ｓｅｐｈ
ａ−ｄｅｘＧ−２５）を充てんしたＰ。Next, the mixture was mixed with Sephadex G-25 (Seph
P filled with a-dexG-25).
ＩＯカラム（ファルマシア製）で脱塩、ａ縮した。Desalting and condensation were performed using an IO column (manufactured by Pharmacia).
（２）ＡＬＰ標識ウサつ抗ＡＦＰＦａｂ’の調製
実施例１（１）、（２）及び（３）で得られた３ｍｇの
マレイミド化ＡＬＰを含む０．１Ｍ）リス−塩酸緩衝液
（ｐＨ６，０）０．２ｚｆｆに実施例２で得られたウサ
ギ抗ＡＦＰＰａｂ’を含む０ 、１ Ｍ トリス−塩酸
緩衝液（ｐＨ６，０）０，５ｉ（２と０．１Ｍトリス−
塩酸緩衝液（ｐＨ６，０）３ｉ（！とを加え、３０℃で
１時間撹拌した。その混合物を４０℃で一昼夜静置した
後、３０００ ｒ ｐｍで１０分間遠心分離し、沈澱を
除去し、表−１に示す条件で高速液体クロマトグラフィ
ーにより精製した。(2) Preparation of ALP-labeled rabbit anti-AFPF Fab' in 0.1 M) Lis-HCl buffer (pH 6,0 ) 0.2zff containing rabbit anti-AFPPab' obtained in Example 2 in 0.5i (2 and 0.1M Tris-HCl buffer (pH 6.0)).
Hydrochloric acid buffer (pH 6,0) 3i (!) was added and stirred at 30°C for 1 hour. The mixture was allowed to stand overnight at 40°C, and then centrifuged at 3000 rpm for 10 minutes to remove the precipitate. It was purified by high performance liquid chromatography under the conditions shown in Table 1.
表−ｌ 高速液体クロマトグラフィーによる精製Ａは
、
Ｂは、
３００
Ｃは、
−３４
次に、
ず。Table 1 Purification by high performance liquid chromatography A is B is 300 C is -34 Next, Z.
ＴＳＫＧ３０００ＳＷ（東ソー製）、
セファクリル（Ｓｅｐｈａｃｒｙ Ｉ）Ｓ（ファルマシ
ア社製）、
アルトラゲル（Ｕｌ ｔ ｒａｇｅ Ｉ）Ａｃ（ＬＫＢ
社製）をそれぞれ示す。TSKG3000SW (manufactured by Tosoh), Sephacryl (Sephacry I) S (manufactured by Pharmacia), Ultragel (Ultrage I) Ac (LKB
(manufactured by the company) are shown respectively.
上記精製条件による再現性を表
２に示
表−２
この再現性（％）は精製剤の試料をカラムに添加してか
ら酵素標識抗体が溶出されるまでの液量を各場合につい
て求め、３回の標準蝙差を平均値で除して、１００を乗
じて算出した値である。The reproducibility under the above purification conditions is shown in Table 2. This reproducibility (%) is determined by calculating the volume of liquid from the time when the sample of the purification agent is added to the column until the enzyme-labeled antibody is eluted in each case. This is a value calculated by dividing the standard difference between times by the average value and multiplying by 100.
更に、上記精製条件で精製後、各フラクション（試験管
）の吸光度を測定した。その結果を第１図に試料添加後
の時間と吸光度の関係で示した。Furthermore, after purification under the above purification conditions, the absorbance of each fraction (test tube) was measured. The results are shown in Figure 1 as a relationship between absorbance and time after sample addition.
この図ではＡとＢ、Ｃとではカラムサイズ、流速が異な
るのでグラフの横軸が異なっている。また、Ａでは１０
〜２５分に酵素標識抗体が抗体数ごとに溶出されている
。更に２７分に未反応ＡＬＰか、３５分には未反応Ｆａ
ｂ′が溶出されている。In this figure, A, B, and C have different column sizes and flow rates, so the horizontal axes of the graphs are different. Also, in A, 10
Enzyme-labeled antibodies were eluted for each number of antibodies at ~25 minutes. Furthermore, unreacted ALP at 27 minutes or unreacted Fa at 35 minutes.
b' is eluted.
方、Ｂ、Ｃでは３００〜４００分に酵素標識抗体と未反
応のＡＬＰが、５００分付近に未反応のＦａｂ’が溶出
されている。従って、これらのことからＡはＢ及びＣに
比し分離能が大きく、また精製に要する時間も短いこと
がわかる。なお、架橋剤の種類の違いにかかわらす、吸
光度（よほぼ同一であったので、架橋剤としてＣＭＢＳ
を使用したしのについての測定結果を第１図に示した。On the other hand, in B and C, unreacted ALP with the enzyme-labeled antibody was eluted at 300 to 400 minutes, and unreacted Fab' was eluted around 500 minutes. Therefore, it can be seen from these facts that A has a higher separation ability than B and C, and also requires a shorter time for purification. Note that the absorbance was almost the same regardless of the type of crosslinking agent, so CMBS was used as the crosslinking agent.
Figure 1 shows the results of the measurements made using this method.
次いで、第１図の溶出時間と吸光度のグラフ中、最も吸
光度の大きいフラクション（↓のとこる）の純度をＴＳ
ＫＧ３０００ＳＷｘＬカラム（分析用カラムで純度検定
等に使用されるゲル濾適用のカラム、東ソー製０．７８
Ｘ ３０ｃｍ、理論段数３０４４０）により０．２Ｍリ
ン酸緩衝液（ｐＨ６，９）、流速１０７分の溶出条件で
検定した。その結果を第２図に示す。Next, in the graph of elution time and absorbance in Figure 1, the purity of the fraction with the highest absorbance (↓) is determined by TS.
KG3000SWxL column (column for analysis and gel filtration used for purity testing, manufactured by Tosoh 0.78
The assay was performed using elution conditions of 0.2M phosphate buffer (pH 6,9) and a flow rate of 107 minutes using a x 30cm x 30440 theoretical plates. The results are shown in FIG.
第２図に示される溶出時間７．２分に見られるピークは
ＡＬＰ１分子に４分子の抗体が結合した酵素標識抗体で
ある。ＴＳＫＧ３０００ＳＷで精製した第２図（ａ）で
認められるピークはこの７．２分のピークのみで他には
認められないことからこのフラクションにはかなり純度
の高い抗体数５の酵素標識抗体が溶出されたことがわか
る。これに対し、セファクリルＳ−３００で精製した第
２図（ｂ）及びアルトラゲルＡｃＡ−３４で精製した第
２図（Ｃ）では溶出時間７．２分の他にもピークが認め
られる。即ち、７．６分、８．０分のピークはそれぞれ
抗体数が４．３の酵素標識抗体で、７．５分のピークは
未反応のＡＬＰであると考えられる。従ってこれらの使
用カラムではいずれも抗体数の異なる酵素標識抗体を抗
体数ごとに分取することはできず、未反応のＡＬＰも分
離できなかった。なお未反応の抗体は１０．５分にピー
クがあるが、第２図（ａ）、（ｂ）、（ｃ）のいずれら
の場合もこのピークは見られず未反応の抗体は分離でき
たと思われる。The peak seen at an elution time of 7.2 minutes shown in FIG. 2 is an enzyme-labeled antibody in which four molecules of antibody are bound to one ALP molecule. The only peak observed in Figure 2 (a) purified with TSKG3000SW is this 7.2 minute peak and no other peaks are observed, indicating that enzyme-labeled antibodies with a fairly high purity (number 5) were eluted in this fraction. I can see that. On the other hand, in FIG. 2(b) purified with Sephacryl S-300 and FIG. 2(C) purified with Altragel AcA-34, other peaks are observed in addition to the elution time of 7.2 minutes. That is, the peaks at 7.6 minutes and 8.0 minutes are thought to be enzyme-labeled antibodies with an antibody count of 4.3, and the peak at 7.5 minutes is considered to be unreacted ALP. Therefore, with any of these columns used, enzyme-labeled antibodies having different numbers of antibodies could not be fractionated by number of antibodies, and unreacted ALP could not be separated. Note that unreacted antibodies have a peak at 10.5 minutes, but this peak is not seen in any of the cases shown in Figure 2 (a), (b), and (c), indicating that unreacted antibodies were successfully separated. Seem.
第２図からＴＳＫＧ３０００ＳＷで精製した抗体数４の
酵素標識抗体が最も純度が高いことがわかる。従って、
このことは理論段数の大きいカラムを使用することで分
離能を上げることが可能であることを示している。From FIG. 2, it can be seen that the enzyme-labeled antibody with the number of antibodies purified using TSKG3000SW with the number of antibodies 4 has the highest purity. Therefore,
This shows that it is possible to increase the resolution by using a column with a large number of theoretical plates.
なお、純度検定の場合も架橋剤の相違にかかわらず、吸
光度の波形はほぼ同一であったので、架橋剤としてＧＭ
ＢＳを使用したものについての測定結果を第２図に示し
た。In addition, in the case of purity assay, the absorbance waveform was almost the same regardless of the difference in the crosslinking agent, so GM was used as the crosslinking agent.
The measurement results using BS are shown in FIG.
（３）ＡＦＰ−ＥＩＡの検量域
実施例２（２）で得られた第１図で示される溶出時間と
吸光度のグラフ中量も吸光度の大きいフラクション（↓
のとこる）の酵素標識抗体を用い、感度０，０．０１，
０．０５，０．１，０．５．１゜５ １０．２５ｎｇ
／ｍ（！のＡＦＩ”１’ＯＯμ（をプレートプラスデッ
ク製のウェルに入れ、０．１％ ＢＳＡ、５ｍＭ ＥＤ
ＴＡを含む０．１Ｍホウ酸緩衝に！７．（ｐＨ８，５）
（以下、ホウ酸−ＢＳＡと略す）２００μｆｆとヤギ抗
体ＡＲＰ Ｉ ｇＧ被覆ビーズｉｇを加え、室温にて６
時間静置した。次ぎに、固相を蒸留水にて３回洗浄し、
至適濃度に希釈した酵素標識抗体３００μＱを加え、室
温にて一晩静置した。更に、固相を蒸留水にて３回洗浄
後、試験管に移し、０．３ｍＭ４−メチルウンベリフェ
リルフォスフエイトを含む０．１Ｍ グリシン−ＮａＯ
Ｈ援衝液（ｐＨ９，５）３００μＱを加え、３０℃で１
００分反応させた。(3) Calibration range of AFP-EIA The amount in the graph of elution time and absorbance shown in Figure 1 obtained in Example 2 (2) is also the fraction with high absorbance (↓
Using the enzyme-labeled antibody of Notoru), the sensitivity was 0, 0.01,
0.05, 0.1, 0.5.1゜5 10.25ng
/m(!) of AFI"1'OOμ() was placed in a plate plus deck well, 0.1% BSA, 5mM ED
For 0.1M boric acid buffer containing TA! 7. (pH 8,5)
(hereinafter abbreviated as boric acid-BSA) and goat antibody ARP IgG coated beads ig were added, and at room temperature
Let it stand for a while. Next, the solid phase was washed three times with distilled water,
300 μQ of an enzyme-labeled antibody diluted to an optimal concentration was added, and the mixture was allowed to stand overnight at room temperature. Furthermore, after washing the solid phase three times with distilled water, it was transferred to a test tube and treated with 0.1M glycine-NaO containing 0.3mM 4-methylumbelliferyl phosphate.
Add 300 μQ of H buffer solution (pH 9,5) and incubate at 30°C for 1 hour.
The reaction was carried out for 00 minutes.
次に、０．ＩＭＢＤＴＡを含む０．５リン酸二水素カリ
ウム緩衝液（ｐＨ１０，４）３ｚ１２を加え、励起政長
３６０ｎｍ、蛍光波長４．５０ｎｍの蛍光を測定し、検
量線を作成した。Next, 0. A 0.5 potassium dihydrogen phosphate buffer (pH 10.4) 3z12 containing IMBDTA was added, and fluorescence at an excitation wavelength of 360 nm and a fluorescence wavelength of 4.50 nm was measured to create a calibration curve.
この検量線に基づいて検量下限から検出限界を、直線領
域の上限から検量上限をそれぞれ求め、検量域を定めた
。その結果を表−３に示す。表−３に示すように検量域
が最も広いのはＴＳＫＧ３０ｏｏｓｗで精製した酵素標
識抗体であり、これが検出限界も最も低かった。Based on this calibration curve, the detection limit was determined from the lower calibration limit, and the upper calibration limit was determined from the upper limit of the linear region, to define a calibration range. The results are shown in Table-3. As shown in Table 3, the enzyme-labeled antibody purified with TSKG30oosw had the widest calibration range, and it also had the lowest detection limit.
また、検量線の傾きも、ＴＳＫＧ３０００ＳＷの方が他
の２種のカラムを用いた場合よりも太きかった。Furthermore, the slope of the calibration curve was also thicker for TSKG3000SW than when the other two types of columns were used.
従って、このことからＴＳＫＧ３０００ＳＷにて精製を
行うことにより未反応ＡＬＰを取り除くことができ、良
い検量線が得られることがわかった。Therefore, it was found from this that unreacted ALP could be removed by purification using TSKG3000SW, and a good calibration curve could be obtained.
表
酵素標識抗体の検量域
実施例２（３）と同様の酵素標識抗体及び方法によりＦ
検定を行い検出限界を求めた。ＡＦＰｉ度ｉ：ｔ、０，
００１，０．０２５，０．０５．０．１ｎｇ／ｚＱ、と
しｎ＝７で行った。酵素標識抗体はＴＳＫＧ３０００Ｓ
Ｗで精製したものを２００倍、セファクリルＳ−３００
及びアルトラゲルＡｃＡ−３４で精製したものを５００
倍にそれぞれ希釈して使用した。なお、この測定は計３
回行ったが、いずれも再現性は良かった。その結果を表
−４に示す。表−４に示すように精製手段により検出限
界に差が見られ、最も低いのはＴＳＫＧ３０００ＳＷで
精製したもので０．０５ｎｇ／ｍＱであった。Calibration range of enzyme-labeled antibody using the same enzyme-labeled antibody and method as in Example 2 (3)
An assay was performed to determine the detection limit. AFPi degree i:t, 0,
001, 0.025, 0.05, 0.1 ng/zQ, and n=7. Enzyme labeled antibody is TSKG3000S
200 times purified with W, Sephacryl S-300
and purified with Altragel AcA-34.
Each was diluted twice and used. In addition, this measurement was performed for a total of 3
I tried it several times and the reproducibility was good. The results are shown in Table 4. As shown in Table 4, there were differences in the detection limit depending on the purification method, with the lowest detection limit being 0.05 ng/mQ for the one purified using TSKG3000SW.
これに対しセファクリルｌ−３００及びアルトラゲルＡ
ｃＡ−３４で精製したものはいずれも０．２５ｎｇ／ｘ
（であった。In contrast, Sephacryl l-300 and Altragel A
All products purified with cA-34 are 0.25ng/x
(Met.
以上の結果から、未反応ＡＬＰや種々の抗体数の酵素標
識抗体が混合しているセファクリル５３００及びアルト
ラゲルＡｃＡ−３４で精製した乙のよりも、同じ抗体数
の酵素標識抗体のみのＴＳＫＧ３０００ＳＷで精製した
ものの方が検出限界が低かった。再現性も良好で純度の
高い至適抗体数の酵素標識抗体の使用により検出限界を
改善することが可能である。From the above results, it was found that purification using TSKG3000SW with only enzyme-labeled antibodies of the same number of antibodies was superior to purification with Sephacryl 5300 and Altragel AcA-34, which contained unreacted ALP and various numbers of enzyme-labeled antibodies. The detection limit was lower. It is possible to improve the detection limit by using an optimal number of enzyme-labeled antibodies with good reproducibility and high purity.
表−４酵素標識抗体の検出限界
ウサギ抗ＡＦＰＩｇＧ被覆ポリスチレンボールに酵素標
識抗体を非特異的に吸着させ、固相に吸着した酵素標識
抗体と添加した酵素標識抗体のＡＬＰ活性を測定した。Table 4: Detection limits of enzyme-labeled antibodies Enzyme-labeled antibodies were non-specifically adsorbed onto rabbit anti-AFP IgG coated polystyrene balls, and the ALP activities of the enzyme-labeled antibodies adsorbed to the solid phase and the added enzyme-labeled antibodies were measured.
なお固相吸着量を添加量で除して、１００を乗じた値を
非特異的吸着率とした。この測定は３回行ったがいずれ
も再現性は良かった。その結果を表−５に示す、表−５
に示すようにＴＳＫＧ３０００ＳＷで精製した純度の高
い酵素標識抗体では、非特異的吸着率は約０．０１％で
あったが未反応ＡＬＰや種々の抗体数の酵素標識抗体が
混合しているセファクリルＳ−３００及びアルトラゲル
ＡｃＡ−３４により精製したものは、ＴＳＫＧ３０００
ＳＷにより精製したものに比し非特異的吸着か大きく、
また調製ロフトによる差も見られた。Note that the value obtained by dividing the solid phase adsorption amount by the addition amount and multiplying by 100 was defined as the nonspecific adsorption rate. This measurement was performed three times, and the reproducibility was good in all cases. The results are shown in Table-5.Table-5
As shown in Figure 3, the non-specific adsorption rate was approximately 0.01% for highly pure enzyme-labeled antibodies purified using TSKG3000SW, but Sephacryl S, which contains unreacted ALP and various numbers of enzyme-labeled antibodies, was -300 and those purified by Altragel AcA-34 are TSKG3000
The non-specific adsorption is greater than that purified by SW.
Differences were also observed depending on the preparation loft.
なお、ここでは結果を示さなかったがＴＳＫＧ３０００
ＳＷによる精製の際に抗体数４の酵素標識抗体と共に得
られた抗体数２および３の酵素標識抗体の非特異的吸着
率は抗体数３のそれとほぼ同じであった。Although the results are not shown here, TSKG3000
The nonspecific adsorption rates of the enzyme-labeled antibodies with 2 and 3 antibodies obtained together with the enzyme-labeled antibodies with 4 antibodies during purification by SW were almost the same as that with 3 antibodies.
以上の結果より、ＴＳＫＧ３０００ＳＷで精製した酵素
標識抗体の使用により非特異的吸着率が低減されたこと
がわかる。From the above results, it can be seen that the nonspecific adsorption rate was reduced by using the enzyme-labeled antibody purified with TSKG3000SW.
なお、未反応ＡＬＰ等を分離し、純度を高くすることで
非特異的吸着率を下げることら可能である。Note that it is possible to reduce the non-specific adsorption rate by separating unreacted ALP etc. and increasing the purity.
表
酵素標識抗体の非特異的吸着率
標識酵素としてＡＬＰ、抗体としてウサギ抗Ａ１”ｐＦ
（ａｂ′）ｔを用い、表−６に示す割合で混合して酵素
標識抗体を調製した。なお調製した試料１ｉＴｓＫＧ３
０００ＳＷｘＬカラムで分析した。Nonspecific adsorption rate of surface enzyme-labeled antibody ALP as labeled enzyme, rabbit anti-A1”pF as antibody
(ab')t was mixed in the ratio shown in Table 6 to prepare an enzyme-labeled antibody. In addition, prepared sample 1iTsKG3
Analysis was performed using a 000SWxL column.
その結果を第３図に示す。The results are shown in FIG.
第３図において、７．４分、７．７分、８．３分のピー
クはそれぞれ抗体数４．３．２の酵素標識抗体を、９．
６分、ＩＯ，４分のピークはそれぞれ未反応ＡＬＰ、未
反応Ｆａｂ’ を示す。抗体数４の酵素標識抗体は混合
モル比１：３．Ｉ：４．Ｉ：５で得られ、その中で最も
効率よく得られたのは混合モル比ｌ；４の場合であった
。また、抗体数３の酵素標識抗体は混合モル比１：１．
ｌ：２゜ｌ：３で得られ、特に効率よく得られたのは混
合モル比Ｉ：３の場合であった。更に抗体数２の酵素標
識抗体は混合モル比１：１．Ｉ：２．ｌ：３で得られ、
その中で最も効率よく得られたのはｌ：２の場合であっ
た。In FIG. 3, the peaks at 7.4 minutes, 7.7 minutes, and 8.3 minutes correspond to enzyme-labeled antibodies of 4.3, 2 and 9.9, respectively.
The peaks at 6 minutes, IO, and 4 minutes indicate unreacted ALP and unreacted Fab', respectively. Enzyme-labeled antibodies with 4 antibodies were mixed at a molar ratio of 1:3. I:4. The mixture was obtained at a mixing molar ratio of 1:5, and the most efficient one was obtained at a mixing molar ratio of 1:4. In addition, the enzyme-labeled antibodies with three antibodies were mixed at a molar ratio of 1:1.
It was obtained at a mixing molar ratio of 1:2 and 1:3, and the mixture with a molar ratio of 1:3 was particularly efficient. Furthermore, the enzyme-labeled antibodies with two antibodies were mixed at a molar ratio of 1:1. I:2. obtained with l:3,
Among them, the most efficient one was the case of 1:2.
以上のことから、 ■分子あたり３ ９〜４ ４分 子のマレイミ ド基をもつＡＬＰを用いた場合、 抗 体数と最適混合モル比は表 ７のようになる。From the above, ■3 per molecule 9-4 4 minutes child maleimi When ALP with a do group is used, anti The number of bodies and optimal mixing molar ratio are shown in the table. It will be like 7.
表−６
ＡＬＰとＦａｂ’の混合モル比
表−７
最適混合モル比
なお、
架橋剤の相違にかかわらず、
分析結果は
ほぼ同一であったので第３図には架橋剤としてＧＭ Ｂ
Ｓを使用したしのについての結果を示した。Table 6 Mixed molar ratio of ALP and Fab' Table 7 Optimum mixed molar ratio Note that regardless of the difference in crosslinking agent, the analysis results were almost the same, so GM B was used as the crosslinking agent in Figure 3.
The results for Shino using S were shown.
実施例３ ＡＬＰの活性測定
実施例２（６）で得られた抗体数４．３．２のＭ素標識
抗体及びそれらの酵素標識抗体と未反応のＡＬＰとの混
合物のＡＬＰ活性を次の方法により測定した。Example 3 Measurement of ALP activity The ALP activity of the 4.3.2 M-labeled antibodies obtained in Example 2 (6) and a mixture of those enzyme-labeled antibodies and unreacted ALP was measured by the following method. It was measured by
まず、各酵素標識抗体を１ ｍＭ Ｍｇ ｃ ｌ ｔ。First, each enzyme-labeled antibody was added to 1mM Mgclt.
０、ＩｍＭ Ｚｎｃ Ｉｚ、０．２５９／Ｑ卵アルブ
ミンを含む０．１Ｍ グリシン−ＮａＯＨ緩衝液（ｐ
Ｈ９，５）（以下、酵素希釈液と略す）で１０万倍に希
釈し、１００μｇを試験管に加えた。0.1M glycine-NaOH buffer (p
H9,5) (hereinafter abbreviated as enzyme diluent) to 100,000 times, and 100 μg was added to a test tube.
次に、ＡＬＰ１００μｇを含む酵素希釈液！００μＱを
別の試験管に加えた。Next, enzyme dilution containing 100 μg of ALP! 00 μQ was added to another tube.
更に、０．３ｍＭ４−メチルウンベリフェリルフォスフ
エイトを含む酵素希釈液３００μＱをそれぞれの試験管
に加え、３０℃で６０分間インキュベートした。次にＩ
ＯｍＭＥＤＴＡを含む０．５Ｍリン酸カリウム−Ｋ Ｏ
１−Ｉ緩衝液（ｐＨ１０，４）２．５村をそれぞれの試
験管に加え、励起波長３６０ｎｍ、蛍光波長４５０ ｎ
ｍで蛍光を測定した。Furthermore, 300 μQ of an enzyme dilution solution containing 0.3 mM 4-methylumbelliferyl phosphate was added to each test tube and incubated at 30° C. for 60 minutes. Next I
0.5M Potassium Phosphate-KO with OmMEDTA
Add 2.5 μl of 1-I buffer (pH 10,4) to each test tube, set the excitation wavelength to 360 nm and the fluorescence wavelength to 450 nm.
Fluorescence was measured at m.
このようにしてＡＬＰ活性をそれぞれ測定し、標識して
いない未処理のＡＬＰ活性を１として、各々の酵素標識
抗体の比活性を求めた。その結果を表−８に示す。表−
８に示すように抗体数の違いによるＡＬＰ活性の差は見
られず、またセファクリルＳ−３００により精製した各
種抗体数を含む場合のものとの差も見られなかった。従
って、この結果から抗体数増大に伴う酵素活性の低下は
見られず、抗体数が違っても酵素活性に差かないことか
わかった。The ALP activity was measured in this way, and the specific activity of each enzyme-labeled antibody was determined, setting the unlabeled, untreated ALP activity as 1. The results are shown in Table-8. Table -
As shown in Fig. 8, no difference in ALP activity was observed depending on the number of antibodies, and no difference was observed between the ALP activity and the case containing the number of various antibodies purified by Sephacryl S-300. Therefore, from this result, no decrease in enzyme activity was observed as the number of antibodies increased, and it was found that there was no difference in enzyme activity even if the number of antibodies was different.
表
ＡＬＰ活性
実施例３と同一の試料を用いて二抗体法による力価検定
を次の方法により行った。Table ALP Activity Using the same sample as in Example 3, titer assay using the two-antibody method was carried out in the following manner.
まず、１％正常ウサギ血清、０．０５Ｍエチレンジアミ
ン四酢酸四ナトリウム０．１５Ｍナトリウムを含む０．
０１Ｍリン酸緩衝液（ｐＨ７，５）で酵素標識抗体を１
０〜ｔｏｏｏ倍に希釈した。First, 1% normal rabbit serum, 0.05M tetrasodium ethylenediaminetetraacetic acid, and 0.15M sodium were used.
0.01M phosphate buffer (pH 7.5) with enzyme-labeled antibody.
Diluted 0 to too many times.
次に、上記抗体希釈液０ 、１ ｘＱ、、 ′２Ｊ標識
ＡＦＰ０．１ｍ１２（１，５Ｘ １０’ｃ ｐｍに相当
）と、！％ＢＳＡ、０．１５Ｍナトリウムを含む０．１
Ｍリン酸緩衝液（ｐＨ７，５）０．２ｘＱを試験管に加
え、４℃で一晩放置した。Next, the above antibody dilution solution 0. % BSA, 0.1 with 0.15M sodium
M phosphate buffer (pH 7,5) 0.2xQ was added to the test tube and left overnight at 4°C.
更に、ヤギ抗つサギγ−グロブリン血清を０．０５Ｍエ
チレンジアミン四酢酸四ナトリウム０．１５Ｍナトリウ
ムを含む０．０１Ｍリン酸緩衝液（ｐＨ７，５）で２倍
に希釈し、その０ 、 Ｉ ｘ（ｌを前記試験管に加え
、４℃で一晩放置した。次に３０００ｒｐｍでＩ５分間
遠心分離し、沈澱物の放射能を測定した。この際、最大
カウントをＢｏ、各濃度のカウントをＢとし、Ｂ ／
Ｂ ｏと酵素標識抗体の濃度との関係をグラフに描き、
Ｂ ／ Ｂ 。Furthermore, the goat anti-heron γ-globulin serum was diluted 2 times with 0.01 M phosphate buffer (pH 7.5) containing 0.05 M tetrasodium ethylenediaminetetraacetate and 0.15 M sodium, and its 0, I x ( 1 was added to the test tube and left at 4°C overnight.Next, it was centrifuged at 3000 rpm for 5 minutes, and the radioactivity of the precipitate was measured.At this time, the maximum count was Bo, and the count at each concentration was B. ,B/
Draw a graph of the relationship between Bo and the concentration of enzyme-labeled antibody,
B/B.
０．５ となる酵素標識抗体濃度を求めた。この濃度
か抗体力価である。その結果を表−９に示す。The enzyme-labeled antibody concentration was determined to be 0.5. This concentration or antibody titer. The results are shown in Table-9.
表−９に示すように抗体数の相違から抗体力価に差が見
られ、抗体数３．４の酵素標識抗体の力価は抗体数２の
それの力価のそれぞれ約２倍、約３倍であった。なおセ
ファクリルＳ−３００で精製した混合物の力価は抗体数
２の力価とほぼ同一であった。これらのことから、抗体
数により抗体力価に差か見られ、抗体数が多いほど力価
も大きかった。As shown in Table 9, there are differences in antibody titer due to the difference in the number of antibodies, and the titer of the enzyme-labeled antibody with 3.4 antibodies is about twice that of the titer with 2 antibodies, and about 3 times that of the enzyme-labeled antibody with 2 antibodies. It was double that. Note that the titer of the mixture purified with Sephacryl S-300 was almost the same as the titer of 2 antibodies. From these results, it was seen that there were differences in antibody titer depending on the number of antibodies, and the greater the number of antibodies, the greater the titer.
（以下余白）
表
二抗体法による抗体力価の評価
実施例３と同一の試料を用いて抗原抗体反応による特異
的結合の評価を次のように行った。(The following is a blank space) Table 2 Evaluation of antibody titer by antibody method Using the same sample as in Example 3, evaluation of specific binding by antigen-antibody reaction was performed as follows.
まず０，１，１０，２５ｎｇ／ｙｆｆのＡＦＰ ｌ ０
０μＱをプラスチック製のプレートのウェルに入れ、０
．１％Ｂ Ｓ Ａ 、 ５ ｍ Ｍ Ｅ Ｄ Ｔ Ａを
含むＯ，ＩＭホウ酸緩衝液（ｐＨ８，５）（以下、ホウ
酸−ＢＳＡと略す）３００μＱとヤギ抗ＡＦＰＩｇＧ被
覆ビーズ１個とを加え、室温で６時間インキユベーソヨ
ンした。First, AFP l 0 of 0, 1, 10, 25 ng/yff
Place 0μQ into the wells of a plastic plate and
．． Add 300 μQ of O, IM borate buffer (pH 8.5) (hereinafter abbreviated as boric acid-BSA) containing 1% BSA, 5 mM EDT A and one goat anti-AFP IgG coated bead, The ink was incubated at room temperature for 6 hours.
次に得られた固相を蒸留水で３回洗浄した。更にこれに
至適濃度に希釈したＡＬＰ標識ウザつ抗ＡＦＰＦ（ａｂ
’）＊ ３００ｕ（ｌを加え、室温にて一晩インキユベ
ートした。次に固相を蒸留水で３回洗浄し、試験管に移
した。この試験管に０．３ｍＭ４−メチルウンベリフェ
リル−フォスフエイトを含むＯ，１Ｍグリシン−ＮａＯ
Ｈ３００μＲを加え、室温で一晩インキユベーンヨンし
た。The resulting solid phase was then washed three times with distilled water. Furthermore, ALP-labeled annoying anti-AFPF (ab
') * 300 u(l) was added and incubated overnight at room temperature. The solid phase was then washed three times with distilled water and transferred to a test tube. To this test tube was added 0.3 mM 4-methylumbelliferyl-phosphate. containing O, 1M glycine-NaO
300 μR of H was added and incubation was carried out overnight at room temperature.
次に、１０ｍＭＥＤＴＡを含む０．５Ｍ リン酸カリウ
ム−ＫＯＨ緩衝液（ｐＨ１Ｏ，４）２．５岬を加え、励
起波長３６０ｎｍ、蛍光波長４５０ｎｍで蛍光を測定し
た。その結果を表−ＩＯに示す。Next, 2.5 caps of 0.5M potassium phosphate-KOH buffer (pH 1O, 4) containing 10mM EDTA was added, and fluorescence was measured at an excitation wavelength of 360 nm and a fluorescence wavelength of 450 nm. The results are shown in Table-IO.
表−ＩＯに示すように３ＡＦＰ農度におけろＳ／Ｎは抗
体数により異なり抗体数が多いほど大きかった。また、
セファクリルｌ−３００で精製した各種抗体数を含む混
合物は比較例５で示す抗体数２のらのと同様な値を示し
た。なお、口内再現性は抗体数による差は見られず、い
ずれも１０％以下であった。As shown in Table IO, the S/N in the 3AFP crop varied depending on the number of antibodies, and the higher the number of antibodies, the higher the S/N. Also,
A mixture containing various antibodies purified with Sephacryl 1-300 showed a value similar to that of Comparative Example 5 with 2 antibodies. In addition, no difference was observed in the intraoral reproducibility depending on the number of antibodies, and all were below 10%.
これらのことから特異的結合は抗体数によって異なり、
抗体数が多いほど大きいことがわかる。From these facts, specific binding varies depending on the number of antibodies,
It can be seen that the larger the number of antibodies, the larger the number.
また、至適抗体数の酵素標識抗体の使用により特異的結
合を増大させることが可能である。Further, specific binding can be increased by using an optimal number of enzyme-labeled antibodies.
（以下余白）
表
０
特異的結合
※ Ｓ／ＮはＯｎｇ／ｙｔｔ（ｌの化学発光量をノイズ
（Ｎ）、■、 １０ 、 ２５ｎｇ／ｍ（ｌの化学発
光量をシグナル（Ｓ）とし、各々のシグナル（Ｓ）をノ
イズ（Ｎ）で除して求めた。また日内再現性は各測定点
（ｎ＝５）の標準偏差を平均値で除して１０Ｏを乗じた
値とした。(Leaving space below) Table 0 Specific binding *S/N is Ong/ytt (the amount of chemiluminescence in 1 is taken as noise (N), ■, 10, 25 ng/m (the amount of chemiluminescence in 1 is taken as signal (S), respectively. It was calculated by dividing the signal (S) by the noise (N).The intraday reproducibility was calculated by dividing the standard deviation of each measurement point (n=5) by the average value and multiplying it by 10O.
実施例６ 非特異的吸着率の評価
実施例３と同一の試料を用いて次の方法により非特異的
吸着率を評価した。Example 6 Evaluation of non-specific adsorption rate Using the same sample as in Example 3, the non-specific adsorption rate was evaluated by the following method.
まず、０．１ＭグリンンーＮａＯＨ緩衝液（ｐ■−（９
，５）で １０万倍あるいは１万倍に希釈した酵素標識
抗体１００μｅを試験管に入れた。次に実施例３と同様
な方法でＡＬＰ活性を求めた。更に、固相に吸着した抗
体標識抗体のＡＬＰ活性を添加した酵素標識抗体のＡＬ
Ｐ活性で除して、非特異的吸着率を求めた。その結果を
表−２に示す。First, 0.1M green-NaOH buffer (p■-(9
, 5), 100 μe of the enzyme-labeled antibody diluted 100,000 times or 10,000 times was placed in a test tube. Next, ALP activity was determined in the same manner as in Example 3. Furthermore, AL of the enzyme-labeled antibody to which the ALP activity of the antibody-labeled antibody adsorbed to the solid phase was added.
The nonspecific adsorption rate was determined by dividing by the P activity. The results are shown in Table-2.
表−１１に示すように抗体数が増加すると非特異的吸着
がわずかながら上昇した。またセファクリルＳ−３００
で精製した各種抗体数を含むものは、ＴＳＫＧ３０００
ＳＷで精製した抗体数３及び２のものと同程度の非特異
性を示した。このことから、抗体数により非特異的吸着
にわずかな差は見られるがいずれらＥＩＡに大きく影響
を与えるほど大きな値ではなく、問題にはならないこと
がわかる。As shown in Table 11, as the number of antibodies increased, nonspecific adsorption increased slightly. Also Sephacryl S-300
Those containing various antibodies purified with TSKG3000
It showed the same level of non-specificity as those of antibodies 3 and 2 purified by SW. From this, it can be seen that although there are slight differences in nonspecific adsorption depending on the number of antibodies, the values are not large enough to significantly affect EIA and do not pose a problem.
表−１１非特異的吸着
実施例７
化学発光法による検出限界
実施例３と同一の試料を用いて実施例５と同様の方法に
より化学発光法による検出限界を評価した。Table 11 Non-Specific Adsorption Example 7 Detection Limit by Chemiluminescence Method Using the same sample as in Example 3, the detection limit by chemiluminescence method was evaluated in the same manner as in Example 5.
０．０．１，０．２５，０．５ｎｇ／ｘ（のＡＦＰ（実
施例７）及び０，０．１，０．２５，０．５ｎｇ／ｆｆ
１２のＡＦＰ （比較例）を用いた。また、酵素標識抗
体は実施例７．比較例２の０．０２５，０．１゜ｎｇ／
ｘＩ２は１０００倍希釈し、比較例５の０．２ｎｇ／ｌ
１２は６０００倍に希釈して使用した。その結果を表−
１２に示す。表−１２に示すように抗体数により検出限
界に差が見られ、抗体数４が最も低く、次いで抗体数３
．２の順であった。また抗体数４の酵素標識抗体を使用
した実施例７の検出限界は０．１ｎｇ／ｍＱで、セファ
クリルＳ−３００で精製した混合物（比較例）の検出限
界０．５ｎｇ／ｆｆ（よりし低濃度であった。0.0.1, 0.25, 0.5 ng/x (AFP (Example 7) and 0, 0.1, 0.25, 0.5 ng/ff
No. 12 AFP (comparative example) was used. In addition, the enzyme-labeled antibody was prepared in Example 7. 0.025, 0.1゜ng/ of Comparative Example 2
xI2 was diluted 1000 times and was 0.2 ng/l of Comparative Example 5.
No. 12 was used after being diluted 6000 times. Table the results.
12. As shown in Table 12, there are differences in the detection limit depending on the number of antibodies, with 4 antibodies being the lowest, followed by 3 antibodies.
．． The order was 2. In addition, the detection limit of Example 7 using 4 enzyme-labeled antibodies was 0.1 ng/mQ, and the detection limit of the mixture purified with Sephacryl S-300 (comparative example) was 0.5 ng/ff (low concentration of Met.
これらのことから、抗体数が多いほど検出限界は低いこ
とがわかる。即ち、抗体数４の酵素標識抗体の使用によ
り検出限界は従来の０ 、５ ｎｇ／ ｍ（１（比較例
）から０．１ｎｇ／ｉｉ２となり、ＥＩＡの感度が著し
く上昇した。また、至適な抗体数の酵素標識抗体の使用
により検出限界を改善することができる。なお、架橋剤
の種類の違いにかかわらず、同一の結果を得たので、架
橋剤としてＧＭＢＳを使用したものについての測定結果
を表−１２に示した。From these facts, it can be seen that the larger the number of antibodies, the lower the detection limit. That is, by using enzyme-labeled antibodies with 4 antibodies, the detection limit became 0.1 ng/ii2 from the conventional 0.5 ng/m (1 (comparative example)), and the sensitivity of EIA increased significantly. The detection limit can be improved by using enzyme-labeled antibodies of the same number of antibodies.The same results were obtained regardless of the type of cross-linking agent, so the measurement results using GMBS as the cross-linking agent are shown below. are shown in Table-12.
（以下余白）
表−１２
検出限界
Ｇ１発明の効果
本発明の酵素免疫測定用試薬は、酵素に至適抗体数があ
り、至適抗体数の酵素標識体を酵素免疫測定用試薬とし
て使用することにより、酵素免疫測定法の感度及び再現
性を向上させることができ、これにより臨床検査部門に
おけるガンマ−カーなどを高感度に微量分析できガンの
早期発見、早期治療に役立つ。(Left below) Table 12 Detection limit G1 Effect of the invention In the enzyme immunoassay reagent of the present invention, the enzyme has an optimum number of antibodies, and the enzyme labeled with the optimum number of antibodies can be used as the enzyme immunoassay reagent. This makes it possible to improve the sensitivity and reproducibility of enzyme-linked immunosorbent assays, which enables highly sensitive microanalysis of gamma markers and the like in clinical testing departments, which is useful for early detection and early treatment of cancer.
また、本発明は上記酵素標識体を調製する際、酵素１モ
ルに対して、抗原又は抗体を酵素標識抗体中の抗体数と
同じ割合で混合することにより、酵素標識中の抗原又は
抗体数を制御することかでき、これにより所望の抗原又
は抗体数をもつ酵素標識抗体を収率よく調製することが
できる。Furthermore, when preparing the enzyme-labeled product of the present invention, the number of antigens or antibodies in the enzyme-labeled antibody can be reduced by mixing the antigen or antibody in the same ratio as the number of antibodies in the enzyme-labeled antibody per mole of enzyme. This allows enzyme-labeled antibodies with a desired number of antigens or antibodies to be prepared with good yield.
更に本発明の綱要方法で得られる酵素標識体は、ＡＬＰ
、Ｆａｂ’の分子量がそれぞれ１０万、４，５万である
ことから、抗体数４．３．Ｉの酵素標識抗体の分子量は
それぞれ２５万、２０万、１５万となり、分子量マーカ
ーとしてゲル濾過、電気泳動等に使用することもできる
。Furthermore, the enzyme labeled product obtained by the method of the present invention is ALP.
, Fab' have a molecular weight of 100,000, 4,500,000, respectively, so the number of antibodies is 4.3. The molecular weights of the enzyme-labeled antibodies I are 250,000, 200,000, and 150,000, respectively, and can be used as molecular weight markers in gel filtration, electrophoresis, etc.
第１図は精製された酵素標識抗体の吸光度を示すグラフ
、第２図は精製された酵素標識抗体の純度を示すグラフ
、第３図は抗体数の異なる酵素標識抗体を示すグラフで
ある。
（イ）
時間（分）□
（口〉
時間（分）□
（ハ）
時間（分＞＝
第３図
時間（分）FIG. 1 is a graph showing the absorbance of purified enzyme-labeled antibodies, FIG. 2 is a graph showing the purity of purified enzyme-labeled antibodies, and FIG. 3 is a graph showing enzyme-labeled antibodies with different numbers of antibodies. (B) Time (minutes)□ (mouth) Time (minutes)□ (C) Time (minutes>= Figure 3 Time (minutes)
Claims (2)
した抗原抗体反応の定量的測定法に用いられる酵素免疫
測定用試薬において、 前記酵素標識体がアルカリフォスファターゼ１分子に抗
原又は抗体が４分子結合していることを特徴とする前記
酵素免疫測定用試薬。(1) In an enzyme immunoassay reagent used for quantitative measurement of antigen-antibody reactions using an enzyme-labeled antigen or antibody labeled with an enzyme, the enzyme-labeled enzyme contains one molecule of alkaline phosphatase and four antigens or antibodies. The enzyme immunoassay reagent characterized in that it is molecularly bonded.
抗原又は抗体のもつチオール基とを反応させて、抗原又
は抗体に酵素を標識した酵素標識体を利用した抗原抗体
反応の定量的測定法に用いる酵素免疫測定用試薬の調製
方法において、 前記酵素がアルカリフォスファターゼであり、該アルカ
リフォスファターゼ１モルに対して、ｎ倍（但し、ｎは
２以上の整数である）の割合で抗原又は抗体を混合する
ことを特徴とする前記酵素免疫測定用試薬の調製方法。(2) Quantitative measurement method for antigen-antibody reactions using an enzyme-labeled enzyme labeled antigen or antibody by introducing a maleimide group into an enzyme and reacting the maleimide group with a thiol group of an antigen or antibody. The enzyme is alkaline phosphatase, and the antigen or antibody is added at a ratio of n times (where n is an integer of 2 or more) to 1 mole of the alkaline phosphatase. A method for preparing the reagent for enzyme immunoassay, which comprises mixing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP22552389A JPH0389164A (en) | 1989-08-31 | 1989-08-31 | Reagent for measurement of enzyme immunity and preparation thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP22552389A JPH0389164A (en) | 1989-08-31 | 1989-08-31 | Reagent for measurement of enzyme immunity and preparation thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0389164A true JPH0389164A (en) | 1991-04-15 |
Family
ID=16830640
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP22552389A Pending JPH0389164A (en) | 1989-08-31 | 1989-08-31 | Reagent for measurement of enzyme immunity and preparation thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0389164A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6252053B1 (en) | 1998-09-16 | 2001-06-26 | Nichirei Corporation | Enzyme-antibody complex and a method for manufacturing the same |
| US7629295B2 (en) | 1994-07-25 | 2009-12-08 | Roche Diagnostics Gmbh | Determination of a specific immunoglobulin using multiple antigens |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59138957A (en) * | 1983-01-25 | 1984-08-09 | アボツト ラボラトリ−ズ | Alkaline phosphatase mark steroid hormone glucuronide |
| JPS6113156A (en) * | 1984-06-28 | 1986-01-21 | Sumitomo Chem Co Ltd | Reagent for quantitative analysis of human interferon-alpha and determination method thereof |
-
1989
- 1989-08-31 JP JP22552389A patent/JPH0389164A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59138957A (en) * | 1983-01-25 | 1984-08-09 | アボツト ラボラトリ−ズ | Alkaline phosphatase mark steroid hormone glucuronide |
| JPS6113156A (en) * | 1984-06-28 | 1986-01-21 | Sumitomo Chem Co Ltd | Reagent for quantitative analysis of human interferon-alpha and determination method thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7629295B2 (en) | 1994-07-25 | 2009-12-08 | Roche Diagnostics Gmbh | Determination of a specific immunoglobulin using multiple antigens |
| US6252053B1 (en) | 1998-09-16 | 2001-06-26 | Nichirei Corporation | Enzyme-antibody complex and a method for manufacturing the same |
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