JPH0795067B2 - Immune complex assay - Google Patents
Immune complex assayInfo
- Publication number
- JPH0795067B2 JPH0795067B2 JP62088720A JP8872087A JPH0795067B2 JP H0795067 B2 JPH0795067 B2 JP H0795067B2 JP 62088720 A JP62088720 A JP 62088720A JP 8872087 A JP8872087 A JP 8872087A JP H0795067 B2 JPH0795067 B2 JP H0795067B2
- Authority
- JP
- Japan
- Prior art keywords
- igg
- mrf
- mouse
- rheumatoid factor
- type
- 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
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
【発明の詳細な説明】 （産業上の利用分野） 本発明は免疫複合体の測定法に係り、殊にモノクローナ
ルリウマトイド因子を用いる免疫複合体の測定法に係
る。TECHNICAL FIELD The present invention relates to a method for measuring an immune complex, and more particularly to a method for measuring an immune complex using a monoclonal rheumatoid factor.
免疫複合体は、全身性エリテマトーデス（SLE）を始め
として慢性関節リウマチ（RA）等の、膠原病、糸球体腎
炎、血管炎、感染症、肝疾患、肺疾患、ガン等の種々の
疾患に際して血清等の体液中に出現してくることが報告
されている。Immune complex is used in serum of various diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), collagen disease, glomerulonephritis, vasculitis, infectious disease, liver disease, lung disease, cancer, etc. It has been reported to appear in body fluids such as.
従って、本発明による測定法はこれら疾患についての臨
床診断検査法の一つとして利用することができる。Therefore, the assay method according to the present invention can be used as one of the clinical diagnostic test methods for these diseases.
（従来の技術及びその問題点） ヒト体液中、殊に血清中における免疫複合体の測定は従
来から種々の方法を用いて実施されており、これらの従
来法としては例えば ａ） 超遠沈法、 ｂ） ポチエリレングリコール（PEG）沈降法、 ｃ） 抗補体法、 ｄ） C1q法、 ｅ） ラジ（Raji）細胞法及び ｆ） リウマトイド因子（RF）による方法 等が汎用されている。(Prior art and its problems) The measurement of immune complexes in human body fluids, especially in serum, has been performed by various methods from the past. Examples of these conventional methods include a) ultracentrifugation method. The following methods are widely used: b) potierylene glycol (PEG) precipitation method, c) anti-complement method, d) C1q method, e) Raji cell method, and f) rheumatoid factor (RF) method.
これらの諸方法の内で、超遠心法は分析操作に長時間を
要するために多検体処理が困難である点に問題がある。
ポリエチレングリコール沈降法は操作が容易であると云
う利点を有しているが、免疫複合体以外の高分子蛋白も
測定してしまい、その結果として測定精度が低くなる点
に問題がある。抗補体法は、免疫複合体に結合した補体
の第３成分（C3）に対する抗体を利用して免疫複合体を
測定する方法であって、広範囲の免疫複合体の検出に応
用し得る利点を有しているが、測定において血中のC3の
影響を受け易い点に問題がある。C1q法は、補体の第１
成分であるC1qをヒト又は動物の血清から分離精製し、C
1qが免疫複合体と特異的に結合する性質を利用して免疫
複合体を測定する方法であって、C1qバインディングテ
スト、C1q固相ラジオイムノアッセイ（RIA）、C1q固相
エンザイムイムノアッセイ（EIA）等がある。これらのC
1q法は何れも特異性及び感度において優れているが、こ
の方法に用いるC1q自体の精製に手間を要し且つ多量の
血清を必要とするので、これらの入手の点に問題があ
り、又安定性が低い点に問題がある。尚、C1q法の実施
に際して、免疫複合体が血中で自己補体と結合している
場合には、測定に先立ちEDTA処理や非働化が必要とされ
る。又ラジ細胞法は、バーキットリンパ腫患者の腫瘍細
胞から株化されたラジ細胞を用いるものであり、このラ
ジ細胞がその表面に補体のC3b、C3dのリセプターを有し
ていることを利用し、免疫複合体と結合した補体を介し
て免疫複合体を測定する方法であるが、細胞を用いるた
めにその維持管理が必要であり、且つ細胞を培養するた
めの特別な設備を必要とする点に問題がある。Among these methods, the ultracentrifugation method has a problem in that it is difficult to process multiple samples because the analysis operation requires a long time.
The polyethylene glycol precipitation method has an advantage that it is easy to operate, but it also has a problem in that high-molecular proteins other than the immune complex are also measured, and as a result, the measurement accuracy decreases. The anti-complement method is a method of measuring an immune complex by using an antibody against the third component (C3) of complement bound to the immune complex, and is advantageous in that it can be applied to the detection of a wide range of immune complexes. However, there is a problem in that it is easily affected by C3 in blood in the measurement. C1q method is the first of complement
The component C1q was separated and purified from human or animal serum,
It is a method to measure immune complex by utilizing the property that 1q specifically binds to the immune complex, such as C1q binding test, C1q solid phase radioimmunoassay (RIA), C1q solid phase enzyme immunoassay (EIA). is there. These c
Although the 1q method is excellent in specificity and sensitivity, purification of C1q itself used in this method requires labor and a large amount of serum is required.Therefore, there are problems in obtaining these and stable. There is a problem in that it is low in nature. When the immune complex is bound to self-complement in blood during the C1q method, EDTA treatment or inactivation is required prior to the measurement. In addition, the Raji cell method uses a Raji cell established from a tumor cell of a Burkitt lymphoma patient, and utilizes that the Raji cell has complement C3b and C3d receptors on its surface. , A method of measuring an immune complex through a complement bound to the immune complex, which requires maintenance of the cell and requires special equipment for culturing the cell. There is a problem with the point.
一方、本発明が関与する従来法であって、リウマトイド
因子による方法の内で慢性関節リウマチ患者の血清中に
存在するリウマトイド因子（RF）を利用する方法は、そ
のRFがポリクローナルRFであるために特異性が低く、こ
のRF含有血清では検出率が低い点に問題があった。尚、
ワルデンストローム・マクログブリネミア（Waldenstr
m macroglobulinemia）の患者血清中にはモノクロー
ナルリウマトイド因子（mRF）が存在しており、このmRF
を利用した免疫複合体の測定法は感度が高く且つ特異性
にも優れている点で有利であるが、使用すべきmRFの入
手自体が困難であり且つ又大量に得られない点に問題が
あった。更に、mRFに関しては、自己免疫疾患モデルマ
ウス（MRL/Mp−lpr/lpr）より調整されたmRF産生細胞を
用いてマウスミエローマ細胞とのハイブリドーマを作製
し、これからマウスmRFを得たことが報告され［「J.Ex
p.Med.」第158巻第901−919頁（1983年）、「ibid」第1
59巻第1429−1440頁（1984年）及び「第29回日本リウマ
チ学会会報」第174頁1985年）］、又免疫複合体測定系
へのその応用も報告されるに至っている（「第29回日本
リウマチ学会会報」第174頁（1985年）。On the other hand, in the conventional method involving the present invention, a method utilizing rheumatoid factor (RF) present in the serum of patients with rheumatoid arthritis among methods using rheumatoid factor, the RF is polyclonal RF because There was a problem in that the specificity was low and the detection rate was low in this RF-containing serum. still,
Waldenstr. McGrogbrinemia (Waldenstr.
Monoclonal rheumatoid factor (mRF) is present in the serum of patients with m macroglobulinemia.
The method for measuring an immune complex using the method is advantageous in that it has high sensitivity and excellent specificity, but there is a problem in that it is difficult to obtain the mRF to be used and it cannot be obtained in a large amount. there were. Furthermore, regarding mRF, it was reported that a hybridoma with mouse myeloma cells was prepared using mRF producing cells prepared from an autoimmune disease model mouse (MRL / Mp-lpr / lpr), from which mouse mRF was obtained. ["J.Ex
p.Med. "Vol. 158, pp. 901-919 (1983)," ibid "No. 1
Vol. 59, pp. 1429-1440 (1984) and "The 29th Annual Meeting of the Japanese Association of Rheumatology", p. 174, 1985)], and its application to an immune complex assay system has also been reported ("29. Annual Meeting of the Japanese Association of Rheumatology, p. 174 (1985).
しかしながら、従来報告されてきたモノクローナルリウ
マトイド因子（mRF）は全てIgM型のものであり、これを
免疫複合体の測定に利用するには難がある。即ちIgM型m
RFとは、長くて重いポリペプチド鎖（Ｈ鎖）２本と、短
くて軽いポリペプチド鎖（Ｌ鎖）２本とが結合して形成
されるＹ字状構造体が５つ複合したものであり、構造安
定性が低いからである。However, all of the previously reported monoclonal rheumatoid factors (mRF) are of the IgM type, and it is difficult to use them for the measurement of immune complexes. That is, IgM type m
RF is a composite of five Y-shaped structures formed by binding two long and heavy polypeptide chains (H chains) and two short and light polypeptide chains (L chains). This is because the structural stability is low.
（発明の目的） 従って、本発明の目的は感度が高く且つ特異性に優れて
いるのみならず、測定試薬の供給性の面で満足でき、然
かも測定試薬の安定性において有利な免疫複合体の測定
法を提供することにある。(Object of the invention) Therefore, the object of the present invention is not only highly sensitive and excellent in specificity, but also satisfactory in terms of the supply of the measurement reagent, which is advantageous in the stability of the measurement reagent. To provide a measurement method of.
（目的を達成するための手段及び作用） 本発明によれば、上記の目的は、ヒト体液中の免疫複合
体の測定法であって、IgG型マウスモノクローナルリウ
マトイド因子を用いることを特徴とする、免疫複合体の
測定法により達成される。(Means and Actions for Achieving the Purpose) According to the present invention, the above-mentioned object is a method for measuring an immune complex in a human body fluid, characterized by using an IgG-type mouse monoclonal rheumatoid factor. It is achieved by a method of measuring immune complexes.
本発明による測定法に使用されるIgG型マウスモノクロ
ーナルリウマトイド因子は、自己免疫疾患モデルマウス
であるMRL/Mp−lpr/lprマウス由来のものであることが
好ましい。The IgG type mouse monoclonal rheumatoid factor used in the assay method of the present invention is preferably derived from MRL / Mp-lpr / lpr mouse which is an autoimmune disease model mouse.
本発明方法において「ヒト体液」としては血清、髄液、
尿等を用いることができ、殊に血清が用いられる。本発
明方法はヒト免疫複合体に対して特異的に反応する性質
を有するマウスmRF、殊にIgG型のマウスmRFを利用し、
これを抗体試薬として各種の抗原を免疫学的に測定しよ
うとするものであり、従ってこのIgG型マウスmRFを抗体
試薬として用い得る免疫学的測定法でだれば、どの測定
システムにも適用することができる。このような測定シ
ステム自体は既に種々開発実施されており、例えば凝集
反応法、凝集阻止法、免疫拡散法、免疫比漏法、免疫比
濁法、ラテックス比濁法、競合反応法（固相法、第二抗
体法等）、サンドイッチ法、イムノメトリック法、リポ
ソームイムノライシスアッセイ（LILA）があり、又標識
物質を用いる測定システムとしてはラジオイムノアッセ
イ（RIA）、エンザイムイムノアッセイ（EIA）、蛍光イ
ムノアッセイ（FIA）、化学発光イムノアッセイ、金属
イムノアッセイ、スピンイムノアッセイ（SIA）等があ
る。In the method of the present invention, "human body fluid" includes serum, spinal fluid,
Urine or the like can be used, and particularly serum is used. The method of the present invention utilizes mouse mRF having the property of reacting specifically with human immune complexes, in particular IgG type mouse mRF,
It is intended to immunologically measure various antigens using this as an antibody reagent. Therefore, any immunological assay that can use this IgG mouse mRF as an antibody reagent should be applied to any assay system. You can A variety of such measurement systems have already been developed and implemented. For example, agglutination reaction method, agglutination inhibition method, immunodiffusion method, immunospecific leakage method, immunoturbidimetric method, latex nephelometric method, competitive reaction method (solid phase method). , The second antibody method, etc.), the sandwich method, the immunometric method, and the liposome immunolysis assay (LILA), and the measurement system using a labeling substance is radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescent immunoassay (FIA). ), Chemiluminescence immunoassay, metal immunoassay, spin immunoassay (SIA) and the like.
例えば、本発明方法は、IgG型マウスmRFを固相化し、試
料と反応させた後、標識化した抗ヒトIgG抗体類又は上
記のIgG型マウスmRFを添加して結合させ、その結果標識
量から免疫複合体を測定したり、微粒子例えばラテック
ス粒子や赤血球等にIgG型マウスmRFを吸着させ、これと
試料とを反応させることにより生じる凝集の度合から免
疫複合体を測定したり、或いはIgG型マウスmRFと試料と
を液相中で反応させ、生じた濁度から免疫複合体を測定
することにより実施することができる。For example, the method of the present invention comprises immobilizing IgG type mouse mRF, reacting it with a sample, adding labeled anti-human IgG antibodies or the above IgG type mouse mRF and binding them, and as a result, from the labeled amount. The immune complex is measured, or the immune complex is measured from the degree of aggregation generated by adsorbing IgG mouse mRF to microparticles such as latex particles or erythrocytes and reacting it with a sample, or IgG mouse It can be carried out by reacting mRF with a sample in a liquid phase and measuring the immune complex from the turbidity generated.
本発明方法において使用されるIgG型マウスモノクロー
ナルリウマトイド因子（mRF）、殊に自己免疫疾患モデ
ルマウスであるMRL/Mp−lpr/lprマウス由来のIgG型マウ
スモノクローナルリウマトイド因子は特願昭62−42747
号明細書（特開昭63−209598号公報）に開示のものであ
って、同明細書に開示の方法によって得られる。即ち、
このIgG型マウスmRFはMRL/Mp−lpr/lprマウスの脾細胞
とミエローマ細胞とを細胞融合させてハイブリドーマを
作製し、各ハイブリドーマを選択培養し、培養により生
育した各ハイブリドーマについて変性ヒトIgG及び免疫
複合体に対する反応性を調べることによりリウマトイド
因子産生ハイブリドーマを特定しクローン化してモノク
ローナルリウマトイド因子を安定に産生する細胞株を取
得し、この細胞株を培地で培養するか、又はマウスの腹
腔内に移植し増殖させてモノクローナルリウマトイド因
子を産生させ、次いで培養物又は腹水を採取し分離精製
することにより得ることができる。The IgG-type mouse monoclonal rheumatoid factor (mRF) used in the method of the present invention, particularly the IgG-type mouse monoclonal rheumatoid factor derived from MRL / Mp-lpr / lpr mouse, which is an autoimmune disease model mouse, is disclosed in Japanese Patent Application No. 62-42747.
JP-A-63-209598, which can be obtained by the method disclosed in the specification. That is,
This IgG-type mouse mRF is a hybridoma prepared by cell fusion of splenocytes and myeloma cells of MRL / Mp-lpr / lpr mice, each hybridoma is selectively cultured, and each hybridoma grown by the culture is denatured human IgG and immunized. A rheumatoid factor-producing hybridoma was identified and cloned by examining its reactivity to the complex to obtain a cell line that stably produces the monoclonal rheumatoid factor, and this cell line was cultured in a medium or transplanted into the abdominal cavity of mice. It can be obtained by subjecting the culture or ascites fluid to collection and separation purification in order to produce a monoclonal rheumatoid factor.
本発明方法の実施において、モノクローナルリウマトイ
ド因子として、このようにモノクローナルリウマトイド
因子産生細胞株から得られたものを用いれば、その性質
は常に一定であり且つその入手性も安定しており、又モ
ノクローナルリウマトイド因子であるが故に測定感度や
反応特異性に優れている。更に、本発明方法に使用され
るモノクローナルリウマトイド因子はIgG型mRFであり、
従って単一のＹ字状構造体［長くて重いポリペプチド鎖
（Ｈ鎖）２本と、短くて軽いポリペプチド鎖（Ｌ鎖）２
本とから形成されている］からなっているために構造安
定性が従来発表されたIgM型mRFと比較して高く、その結
果としてIgG型モノクローナルリウマトイド因子は試薬
化されても安定性に優れているのである。In the practice of the method of the present invention, when the monoclonal rheumatoid factor thus obtained is used from the monoclonal rheumatoid factor-producing cell line, its properties are always constant and its availability is stable, and the monoclonal rheumatoid factor is also stable. Since it is a factor, it has excellent measurement sensitivity and reaction specificity. Further, the monoclonal rheumatoid factor used in the method of the present invention is IgG type mRF,
Therefore, a single Y-shaped structure [two long and heavy polypeptide chains (H chains) and two short and light polypeptide chains (L chains) 2]
The structure stability is higher than that of previously published IgM-type mRF, and as a result, the IgG-type monoclonal rheumatoid factor has excellent stability even when it is made into a reagent. Is there.
（実施例等） 次に、参考例及び実施例に関連して本発明を更に詳細に
説明する。(Examples, etc.) Next, the present invention will be described in more detail with reference to Reference Examples and Examples.
参考例１（IgG型マウスmRFの調製） 自己免疫疾患モデル動物であるMRL/Mp−lpr/lprマウス
であって血中にリウマトイド因子を産生しているマウス
を選択し、これらのマウスから脾臓を摘出し、常法によ
り脾細胞懸濁液を調製した。この脾細胞懸濁液と、マウ
スミエローマ細胞（P3−NS1−１−Ag4−１）の懸濁液と
を10対１の細胞比となるような割合で混合し、遠心処理
した後に50％ポリエチレングリコール（メルク社製、分
子量4000）を滴下し、２分間反応させて上記の両細胞を
融合させた。Reference Example 1 (Preparation of IgG type mouse mRF) MRL / Mp-lpr / lpr mice which are autoimmune disease model animals and which produce rheumatoid factor in blood are selected, and spleens are extracted from these mice. It was extracted and a splenocyte suspension was prepared by a conventional method. This spleen cell suspension was mixed with a suspension of mouse myeloma cells (P 3 -NS1-1-Ag4-1) at a ratio such that the cell ratio was 10: 1 and after centrifugation 50% Polyethylene glycol (Merck, molecular weight 4000) was added dropwise and reacted for 2 minutes to fuse both cells.
得られた細胞を無血清培地（日水製薬株式会社製のSFM
−101）に懸濁させて培養を行うことにより融合細胞の
選択を行った（この融合細胞の選択は自体公知のHAT選
択法により実施することもできる）。融合細胞の培養14
日目位に培養上清を用い変性ヒトIgG及び免疫複合体に
対する反応性についてラテックス凝集法及びEIA法によ
り検討し、リウマトイド因子産生の有無を調べた。これ
により判明したリウマトイド因子産生融合細胞につい
て、限界稀釈法によるクローニングを行って単個細胞由
来のクローンとなし、このクローニングによりモノクロ
ーナルリウマトイド因子を安定に産生する細胞株を得、
これを「IV3−５株」と命名した。The cells obtained were serum-free medium (SFM manufactured by Nissui Pharmaceutical Co., Ltd.
The fused cells were selected by suspending the cells in (-101) and culturing (the selection of the fused cells can also be performed by a HAT selection method known per se). Culture of fused cells 14
The reactivity to denatured human IgG and immune complexes was examined by using the culture supernatant at the day position by the latex agglutination method and the EIA method to examine the presence or absence of rheumatoid factor production. Regarding the rheumatoid factor-producing fused cells found out by this, cloning by the limiting dilution method to obtain a clone derived from a single cell, and by this cloning, a cell line stably producing a monoclonal rheumatoid factor was obtained,
This was named "IV3-5 strain".
参考例２（IgG型マウスmRFの産生と精製） 参考例１で得たIV3−５株を無血清培地（上記のSFM−10
1）を用い培養してモノクローナルリウマトイド因子を
産生させた。次いで培養上清を採取して濃縮し、50％飽
和硫安を用いて分画することにより所望の精製IgG型マ
ウスモノクローナルリウマトイド因子を得た。Reference Example 2 (Production and purification of IgG-type mouse mRF) IV3-5 strain obtained in Reference Example 1 was treated with serum-free medium (SFM-10 described above).
1) was cultured to produce monoclonal rheumatoid factor. Then, the culture supernatant was collected, concentrated, and fractionated with 50% saturated ammonium sulfate to obtain the desired purified IgG mouse murine monoclonal rheumatoid factor.
尚、モノクローナルリウマトイド因子の産生は上記の細
胞株をマウスの腹腔内に投与することによっても行うこ
とができ、この場合には腹水を採取し、これをプロテイ
ンＡカラムクロマトグラフィーにかけることにより精製
を行うことができる。The production of the monoclonal rheumatoid factor can also be carried out by intraperitoneally administering the above cell line to the mouse. In this case, ascites is collected and subjected to protein A column chromatography for purification. It can be carried out.
上記の精製モノクローナルリウマトイド因子の免疫グロ
ブリンクラスのサブタイプを、抗マウスイムノグロブリ
ンサブタイプ血清（マイルス社製及びシグマ社製）を用
いたオクタロニー法により、並びに酵素標識抗マウスイ
ムノグロブリンサブタイプ血清（大日本製薬株式会社
製）を用いたEIA法により調べた結果、IgGタイプ（IgG
1）のモノクローナルリウマトイド因子であった。Immunoglobulin class subtypes of the above purified monoclonal rheumatoid factor, by the Ouchterlony method using anti-mouse immunoglobulin subtype serum (manufactured by Miles and Sigma), and enzyme-labeled anti-mouse immunoglobulin subtype serum (large As a result of examination by the EIA method using Nippon Pharmaceutical Co., Ltd., IgG type (IgG
It was the rheumatoid factor of 1).
参考例３（IgG型マウスmRFの性質） 参考例２で得た精製IgG型マウスmRFがヒトIgG（熱変性I
gG及び無処理IgG）に対して示す反応性をEIA法により検
討した。Reference Example 3 (Properties of IgG-type mouse mRF) The purified IgG-type mouse mRF obtained in Reference Example 2 was human IgG (heat-denatured I
The reactivity against gG and untreated IgG) was examined by the EIA method.
即ち、精製IgG型mRFを96穴マイクロプレート（EIA用）
に４℃で24時間放置し、次いで洗浄した後に0.1％BSA/P
BS（−）によりブロッキングしてIgG型mRF感作プレート
を作製した。That is, purified IgG-type mRF is a 96-well microplate (for EIA)
After standing at 4 ℃ for 24 hours and then washing, 0.1% BSA / P
An IgG type mRF sensitized plate was prepared by blocking with BS (-).
この感作プレートに各濃度の無処理ヒトIgG及び熱変性
ヒトIgGを添加して２時間放置した後に洗浄し、それら
が反応したか否かをアルカリフォスファターゼ標識抗ヒ
トIgGにより調べた。Untreated human IgG and heat-denatured human IgG at respective concentrations were added to this sensitized plate, left for 2 hours and then washed, and whether or not they reacted was examined by alkaline phosphatase-labeled anti-human IgG.
結果は第１図に示されている通りであり、このIgG型mRF
は変性ヒトIgGに対しては強い反応性を示すが、無処理
ヒトIgGに対しては反応性が殆ど認められなかった。The results are shown in Fig. 1, and this IgG type mRF
Shows a strong reactivity to denatured human IgG, but hardly any reactivity to untreated human IgG.
更に、熱変性ヒトIgGをウルトラゲルAcA22によりポリマ
ーIgGとモノマーIgGとに分画し、これらの画分につき上
記と同様にしてEIA法により反応性を検討した。結果は
第２図に示される通りであり、ポリマーIgGに関してはI
gG濃度0.05μg/ml以上で反応が認められたが、モノマー
IgGに関しては反応性が殆ど認められなかった。Furthermore, heat-denatured human IgG was fractionated into polymer IgG and monomer IgG by Ultragel AcA22, and the reactivity of these fractions was examined by the EIA method in the same manner as above. The results are shown in Figure 2 and for polymer IgG I
A reaction was observed at a gG concentration of 0.05 μg / ml or more, but the monomer
Almost no reactivity was observed for IgG.
上記の結果から、参考例２で得られたIgG型mRF、即ちMR
L/Mp−lpr/lprマウス由来のIgG型mRFは、熱変性ヒトIg
G、殊にポリマーIgGに対して強く反応し、無処理IgG及
びモノマーIgGに対しては殆ど反応しないと云うヒトRF
の活性と同様の活性を有していることが判る。From the above results, IgG type mRF obtained in Reference Example 2, namely MR
IgG type mRF derived from L / Mp-lpr / lpr mouse is heat-denatured human Ig.
Human RF that reacts strongly with G, especially with polymer IgG, but hardly with untreated IgG and monomer IgG
It is found that it has an activity similar to that of.
参考例４（IgG型マウスmRFと各種動物の免疫グロブリン
との反応性） 参考例２で得た精製IgG型マウスmRFと各種動物の免疫グ
ロブリン（ヒト、家兎、山羊、馬及びマウス）との反応
性をEIA法により検討した。Reference Example 4 (Reactivity between IgG mouse mRF and immunoglobulin of various animals) Purified IgG mouse mRF obtained in Reference Example 2 and immunoglobulin of various animals (human, rabbit, goat, horse and mouse) The reactivity was examined by the EIA method.
即ち、参考例３におけると同様にIgG型mRF感作プレート
を作製し、これに各種動物の変性IgGを反応させ、反応
の有無をアルカリフォスファターゼ標識抗ヒトIgG、抗
ヒトIgA、抗ヒトIgM、抗家兎IgG、抗山羊IgG、抗馬IgG
及び抗マウスIgGにより調べた。尚、対照としてMRL/Mp
−lpr/lprマウス脾細胞から得られたIgM型のmRFについ
ても同様の検討を行った。That is, an IgG-type mRF sensitized plate was prepared in the same manner as in Reference Example 3 and reacted with denatured IgG of various animals, and the presence or absence of reaction was checked for alkaline phosphatase-labeled anti-human IgG, anti-human IgA, anti-human IgM, and anti-human IgM. Rabbit IgG, anti-goat IgG, anti-horse IgG
And anti-mouse IgG. As a control, MRL / Mp
The same study was performed for IgM type mRF obtained from −lpr / lpr mouse splenocytes.
結果は下記の表に示されている通りであり、IgG型mRFは
ヒトIgG、家兎IgG及び馬IgGに対して反応を示したが、
ヒトIgA、ヒトIgM、山羊IgG及びマウスIgGに対しては反
応を示さなかった。一方、IgM型mRFはヒトIgG、山羊Ig
G、馬IgG及びマウスIgGに対して反応を示し、ヒトIgA、
ヒトIgM及び家兎IgGに対しては反応を示さなかった。従
って、MRL/Mp−lpr/lpr由来のmRFであっても、両者の反
応性は異なることが判る 測定実施例１ 参考例２で得た精製IgG型マウスmRFがヒトの免疫複合体
に対して示す反応性をEIA法により検討した。The results are as shown in the table below, IgG type mRF showed a reaction to human IgG, rabbit IgG and horse IgG,
It showed no reaction to human IgA, human IgM, goat IgG and mouse IgG. On the other hand, IgM type mRF is human IgG, goat Ig
Reacts with G, horse IgG and mouse IgG, human IgA,
It showed no reaction to human IgM and rabbit IgG. Therefore, it can be seen that the reactivity of both is different even for mRF derived from MRL / Mp-lpr / lpr. Measurement Example 1 The reactivity of the purified IgG mouse mRF obtained in Reference Example 2 to human immune complexes was examined by the EIA method.
即ち、参考例３におけると同様にIgG型mRF感作プレート
を作製し、各濃度の免疫複合体（三光純薬株式会社製の
「Immuno Complex検出用試薬」）を添加して２時間反応
させた後に洗浄し、反応の有無をアルカリフォスファタ
ーゼ標識を施した抗ヒトIgG（山用IgG）、抗ヒトIgG
［山用IgG−Ｆ（ab′）２］、抗ヒトIgG（マウスモノク
ローナル）、IgG型mRF（マウスモノクロナール）及びIg
M型mRF（マウスモノクロナール）を用いて比較検討し
た。結果は第3A及び3B図に示される通りであった。That is, an IgG-type mRF sensitized plate was prepared in the same manner as in Reference Example 3, an immune complex (“Immuno Complex detection reagent” manufactured by Sanko Junyaku Co., Ltd.) at each concentration was added, and reacted for 2 hours. After washing, anti-human IgG (mountain IgG) and anti-human IgG labeled with alkaline phosphatase for reaction
[Mountain IgG-F (ab ') 2 ], anti-human IgG (mouse monoclonal), IgG-type mRF (mouse monoclonal) and Ig
A comparative study was carried out using M-type mRF (mouse monoclonal). The results were as shown in Figures 3A and 3B.
尚、対照としてIgM型mRFを感作させた場合についても同
様の検討を行った。結果は第4A及び4B図に示される通り
であった。As a control, the same examination was performed also in the case of sensitizing IgM type mRF. The results were as shown in Figures 4A and 4B.
これらの結果から、何れの型のmRFを用いても上記のよ
うな標識抗体により免疫複合体の測定が可能であるこ
と、又固相化抗体としてIgG型mRFを用いることによりIg
M型mRFを用いる場合よりも高感度な測定系を組めること
が判明した。From these results, it is possible to measure the immune complex with the labeled antibody as described above using any type of mRF, and by using IgG type mRF as the immobilized antibody, Ig
It was found that a measurement system with higher sensitivity than in the case of using M type mRF can be set up.
測定実施例２（C1q法との比較） 測定実施例１と同様にして、但しプレートにIgG型mRF
（参考例２）及びC1qをそれぞれ感作させ、EIA法により
免疫複合体を測定して両者を比較検討した。結果は第５
図に示される通りであり、IgG型mRFを用いる方が高感度
で測定し得ることが判明した。Measurement Example 2 (comparison with C1q method) In the same manner as in Measurement Example 1, except that IgG type mRF was added to the plate.
(Reference Example 2) and C1q were sensitized respectively, and immune complexes were measured by the EIA method to compare and examine them. Result is fifth
As shown in the figure, it was found that the measurement using IgG type mRF can be performed with higher sensitivity.
（発明の効果） 本発明方法に使用されるモノクローナルリウマトイド因
子はその産生細胞株から得られたものであることができ
るので、性質が常に一定であり且つその入手性も安定し
ており、又モノクローナルリウマトイド因子であるが故
に測定感度や反応特異性に優れている。更に、本発明方
法に使用されるモノクローナルリウマトイド因子はIgG
型のものであり、従来発表されてきたIgM型のものと比
較して構造が単純であり、安定性において優れている。(Effects of the Invention) Since the monoclonal rheumatoid factor used in the method of the present invention can be obtained from its producing cell line, its properties are always constant and its availability is stable. Since it is a rheumatoid factor, it has excellent measurement sensitivity and reaction specificity. Further, the monoclonal rheumatoid factor used in the method of the present invention is IgG
It has a simple structure and is excellent in stability as compared with the previously announced IgM type.
第１図は本発明方法において使用されるIgG型マウスモ
ノクローナルリウマトイド因子と無処理及び熱変性ヒト
IgGとの反応性を示すグラフ、第２図は第１図と同様
の、但し熱変性ヒトIgG中のポリマーIgG及びモノマーIg
Gとの反応性を示すグラフ、第3A図及び3B図はIgG型マウ
スモノクローナルリウマトイド因子をプレートに感作さ
せることにより固相化させ、これを患者の血清と接触さ
せて反応の有無を酵素標識した各種の抗体で調べた結果
を示すグラフ、第4A及び4B図は第3A及び3B図と同様な、
但しIgM型マウスモノクローナルリウマトイド因子をプ
レートに感作させた場合の結果を示すグラフ、第５図は
IgG型マウスモノクローナルリウマトイド因子とC1qとを
それぞれ用いて免疫複合体を測定し、両者の測定結果を
比較して示したグラフである。FIG. 1 shows IgG type mouse monoclonal rheumatoid factor used in the method of the present invention and untreated and heat-denatured human.
Graph showing reactivity with IgG, Fig. 2 is similar to Fig. 1, except that polymer IgG and monomer Ig in heat-denatured human IgG are
Graphs showing reactivity with G, FIGS. 3A and 3B are solid-phase immobilized by sensitizing IgG type mouse monoclonal rheumatoid factor to a plate, and contacted with serum of a patient to detect the presence or absence of reaction with an enzyme label. Graph showing the results of examining various antibodies, 4A and 4B are similar to FIGS. 3A and 3B,
However, a graph showing the results of sensitizing plates with IgM mouse monoclonal rheumatoid factor, Fig. 5
5 is a graph showing the results of measuring immune complexes using IgG type mouse monoclonal rheumatoid factor and C1q, respectively, and comparing the measurement results of both.
───────────────────────────────────────────────────── フロントページの続き (56)参考文献 特開 昭61−114165（ＪＰ，Ａ) ─────────────────────────────────────────────────── ─── Continuation of front page (56) References JP-A-61-114165 (JP, A)
Claims (2)
て、IgG型マウスモノクローナルリウマトイド因子を用
いることを特徴とする免疫複合体の測定法。1. A method for measuring an immune complex in a human body fluid, which comprises using an IgG-type mouse monoclonal rheumatoid factor.
因子が自己免疫疾患モデルマウスであるMRL/Mp−lpr/lp
rマウス由来のものであることを特徴とする、特許請求
の範囲第１項に記載の免疫複合体の測定法。2. An IgG type mouse monoclonal rheumatoid factor, MRL / Mp-lpr / lp, which is a model mouse for autoimmune disease.
The method for measuring an immune complex according to claim 1, which is derived from r mouse.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62088720A JPH0795067B2 (en) | 1987-04-13 | 1987-04-13 | Immune complex assay |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62088720A JPH0795067B2 (en) | 1987-04-13 | 1987-04-13 | Immune complex assay |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63255662A JPS63255662A (en) | 1988-10-21 |
| JPH0795067B2 true JPH0795067B2 (en) | 1995-10-11 |
Family
ID=13950739
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62088720A Expired - Lifetime JPH0795067B2 (en) | 1987-04-13 | 1987-04-13 | Immune complex assay |
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| Country | Link |
|---|---|
| JP (1) | JPH0795067B2 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001055724A1 (en) * | 2000-01-27 | 2001-08-02 | Shibayagi Co., Ltd | Method for quantitating autoimmune disease antibody and quantification reagent and quantification kit therefor |
| WO2007113211A1 (en) * | 2006-03-30 | 2007-10-11 | Gyros Patent Ab | Ig-assay |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61114165A (en) * | 1984-11-09 | 1986-05-31 | Yamasa Shoyu Co Ltd | Measuring method of immune complex |
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1987
- 1987-04-13 JP JP62088720A patent/JPH0795067B2/en not_active Expired - Lifetime
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| Publication number | Publication date |
|---|---|
| JPS63255662A (en) | 1988-10-21 |
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