JPS62218865A - Quantitative analysis of rheumatic factor - Google Patents
Quantitative analysis of rheumatic factorInfo
- Publication number
- JPS62218865A JPS62218865A JP6281886A JP6281886A JPS62218865A JP S62218865 A JPS62218865 A JP S62218865A JP 6281886 A JP6281886 A JP 6281886A JP 6281886 A JP6281886 A JP 6281886A JP S62218865 A JPS62218865 A JP S62218865A
- Authority
- JP
- Japan
- Prior art keywords
- reaction
- latex
- sensitized
- polyethylene glycol
- water
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 208000025747 Rheumatic disease Diseases 0.000 title abstract 2
- 230000000552 rheumatic effect Effects 0.000 title abstract 2
- 238000004445 quantitative analysis Methods 0.000 title 1
- 238000006243 chemical reaction Methods 0.000 claims abstract description 26
- 229920000126 latex Polymers 0.000 claims abstract description 22
- 239000004816 latex Substances 0.000 claims abstract description 22
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 18
- 230000003287 optical effect Effects 0.000 claims abstract description 18
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 18
- 150000001875 compounds Chemical class 0.000 claims abstract description 17
- 150000003839 salts Chemical class 0.000 claims abstract description 5
- 125000005270 trialkylamine group Chemical group 0.000 claims abstract description 5
- 108010074605 gamma-Globulins Proteins 0.000 claims abstract description 4
- 239000011259 mixed solution Substances 0.000 claims description 9
- 230000004520 agglutination Effects 0.000 claims description 6
- 150000003242 quaternary ammonium salts Chemical class 0.000 claims description 3
- 238000011002 quantification Methods 0.000 claims 1
- 238000005259 measurement Methods 0.000 abstract description 17
- 239000007788 liquid Substances 0.000 abstract description 8
- 239000000203 mixture Substances 0.000 abstract description 6
- 210000002966 serum Anatomy 0.000 abstract description 5
- 239000000969 carrier Substances 0.000 abstract description 2
- 230000004523 agglutinating effect Effects 0.000 abstract 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 abstract 1
- 239000003153 chemical reaction reagent Substances 0.000 description 32
- 238000000034 method Methods 0.000 description 18
- 238000011088 calibration curve Methods 0.000 description 12
- 238000002835 absorbance Methods 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 239000001763 2-hydroxyethyl(trimethyl)azanium Substances 0.000 description 5
- 235000019743 Choline chloride Nutrition 0.000 description 5
- SGMZJAMFUVOLNK-UHFFFAOYSA-M choline chloride Chemical compound [Cl-].C[N+](C)(C)CCO SGMZJAMFUVOLNK-UHFFFAOYSA-M 0.000 description 5
- 229960003178 choline chloride Drugs 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000000691 measurement method Methods 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 108010017384 Blood Proteins Proteins 0.000 description 3
- 102000004506 Blood Proteins Human genes 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 238000004220 aggregation Methods 0.000 description 3
- 230000002776 aggregation Effects 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 229920003008 liquid latex Polymers 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 206010039073 rheumatoid arthritis Diseases 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 108010044091 Globulins Proteins 0.000 description 2
- 102000006395 Globulins Human genes 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- JUGOREOARAHOCO-UHFFFAOYSA-M acetylcholine chloride Chemical compound [Cl-].CC(=O)OCC[N+](C)(C)C JUGOREOARAHOCO-UHFFFAOYSA-M 0.000 description 2
- 229960004266 acetylcholine chloride Drugs 0.000 description 2
- 229960003403 betaine hydrochloride Drugs 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- HOPSCVCBEOCPJZ-UHFFFAOYSA-N carboxymethyl(trimethyl)azanium;chloride Chemical compound [Cl-].C[N+](C)(C)CC(O)=O HOPSCVCBEOCPJZ-UHFFFAOYSA-N 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- 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 1
- JJCWKVUUIFLXNZ-UHFFFAOYSA-M 2-hydroxyethyl(trimethyl)azanium;bromide Chemical compound [Br-].C[N+](C)(C)CCO JJCWKVUUIFLXNZ-UHFFFAOYSA-M 0.000 description 1
- QUSNBJAOOMFDIB-UHFFFAOYSA-N Ethylamine Chemical compound CCN QUSNBJAOOMFDIB-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 238000011481 absorbance measurement Methods 0.000 description 1
- ZEHGKSPCAMLJDC-UHFFFAOYSA-M acetylcholine bromide Chemical compound [Br-].CC(=O)OCC[N+](C)(C)C ZEHGKSPCAMLJDC-UHFFFAOYSA-M 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- 238000003366 endpoint assay Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 230000035931 haemagglutination Effects 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 238000005375 photometry Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- ILWRPSCZWQJDMK-UHFFFAOYSA-N triethylazanium;chloride Chemical compound Cl.CCN(CC)CC ILWRPSCZWQJDMK-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
Description
【発明の詳細な説明】 (産業上の利用分野) 本発明は、リウマチ因子の定量法に関し、特に。[Detailed description of the invention] (Industrial application field) The present invention relates to a method for quantifying rheumatoid factor, and in particular to a method for quantifying rheumatoid factor.
免疫ラテックス凝集法を利用するリウマチ因子の定量法
に関する。This invention relates to a method for quantifying rheumatoid factor using immunolatex agglutination method.
(従来の技術)
リウマチ因子(几heumatoid B’acto
r :以下几Fと略す)は、慢性リウマチ様関節炎患者
の血中に出現する物質で1分子量約100万の巨大蛋白
質といわれている。(Prior art) Rheumatoid factor
r: hereinafter abbreviated as F) is a substance that appears in the blood of patients with chronic rheumatoid arthritis and is said to be a large protein with a molecular weight of about 1 million.
RFの測定は、米国リウマチ協会の慢性関節リウマチ診
断の基準項目に指定されておシ、慢性関節リウマチ診断
の検査には臨床上不可欠とされている。Measurement of RF has been designated as a standard item for the diagnosis of rheumatoid arthritis by the American Rheumatology Association, and is considered clinically essential for testing for the diagnosis of rheumatoid arthritis.
現在、RFの測定には、ラテックス凝集反応を利用した
几A検査法やヒツジ赤血球を用いる受身血球凝集反応を
利用した几A−HA検査法が広く用いられている〔アク
タ・パス・マイクロビオルースカンディナビア7 (A
cta Path、 Microbiol。Currently, for RF measurement, the 几A test method that uses latex agglutination reaction and the 几A-HA test method that uses passive hemagglutination reaction using sheep red blood cells are widely used. Scandinavia 7 (A
cta Path, Microbiol.
5cand、)第17巻第172頁、1940年)。し
かしながら、これらの測定法は定性反応であることから
RF値の変動を時間の経過と共に把握するのが不可能で
あったシ、操作性の点でも欠陥があつ九。5cand, ) Vol. 17, p. 172, 1940). However, since these measurement methods are qualitative reactions, it is impossible to understand changes in RF values over time, and there are also flaws in terms of operability.
近年、検出法の進歩により血漿中の各種微量蛋白が定量
的に測定し得るようになりつつある。この検出法の一つ
が免疫比濁法である。免疫比濁法でば、血漿蛋白のうち
比較的血中濃度の高いIg G。In recent years, advances in detection methods have made it possible to quantitatively measure various trace proteins in plasma. One of these detection methods is immunoturbidimetry. In immunoturbidimetry, IgG has a relatively high blood concentration among plasma proteins.
IgA、 IgM、 C3,C4,CRPが測定される
が。IgA, IgM, C3, C4, and CRP are measured.
感度面から低濃度、血漿蛋白の測定には限界があった。In terms of sensitivity, there was a limit to the measurement of low concentrations and plasma proteins.
こういった動向から低濃度の各種血漿蛋白の測定には免
疫ラテックス凝集法が開発されてきている(特公昭58
−11575号公報、特開昭53−62826号公報)
。Due to these trends, the immunolatex agglutination method has been developed to measure various low-concentration plasma proteins (Special Publication No. 58).
-11575, Japanese Patent Application Laid-open No. 53-62826)
.
(発明が解決しようとする問題点)
免疫ラテックス凝集法はレーザー光を用いるので高感度
ではあるが、RFをこの方法を利用して定量する場合、
免疫反応(抗原−抗体反応)を速やかに行なわせるため
にポリエチレングリコールの存在下に該反応を行なわせ
るのが好ましい。しかし、この場合、良好な直線性を有
する検fk#Jが得られず、従って、定量に際し、検量
線の作成が煩雑になり、また、定量の精度が低下する問
題がある。(Problems to be Solved by the Invention) Although the immunolatex agglutination method uses laser light and is highly sensitive, when quantifying RF using this method,
In order to rapidly carry out the immune reaction (antigen-antibody reaction), it is preferable to carry out the reaction in the presence of polyethylene glycol. However, in this case, a test fk#J having good linearity cannot be obtained, and therefore, there are problems in that the preparation of a calibration curve is complicated during quantitative determination, and the accuracy of quantitative determination is reduced.
(問題点を解決するための手段)
本発明は、試料及びヒトr−グロブリンを感作した不溶
性担体を混合して混合液とし、該混合液中にポリエチレ
ングリコール並びにトリアルキルアミン、その塩及び第
4級アンモニウム塩からなる群から選ばれる少なくとも
一種の水浴性化合物を共存させて抗原−抗体反応による
ラテックス凝集反応を起こさせて、光学的強度を測定し
、この測定値から試料中のリウマチ因子を定量すること
を特徴とするリウマチ因子の定蓋法に関する。(Means for Solving the Problems) The present invention involves mixing a sample and an insoluble carrier sensitized with human r-globulin to form a mixed solution, and adding polyethylene glycol, trialkylamine, a salt thereof, and At least one type of water-based compound selected from the group consisting of quaternary ammonium salts is allowed to coexist to cause a latex agglutination reaction by an antigen-antibody reaction, and the optical intensity is measured. From this measurement value, the rheumatoid factor in the sample is determined. This invention relates to a constant lid method for quantifying rheumatoid factors.
上記試料としては、血清等がある。The above-mentioned sample includes serum and the like.
上記の不溶性担体としては2診断用ポリスチレン系ラテ
ックス粒子等公知のものが使用でき、該担体にヒトーr
グロブリンを物理的又は化学的に吸着させてヒトーrグ
ロブリンが感作された不溶性担体とされる。不溶性担体
は、測定感度向上の点から9粒径が0.05〜0.2μ
mであるのが好ましい。As the above-mentioned insoluble carrier, known carriers such as polystyrene latex particles for diagnosis can be used.
The insoluble carrier is sensitized with human globulin by adsorbing globulin physically or chemically. The particle size of the insoluble carrier is 0.05 to 0.2μ to improve measurement sensitivity.
Preferably, it is m.
この感作された不溶性担体は、上記混合液中に適宜の濃
度で使用されるが、吸光度の測定の容易さから0,05
5重量以下になるように使用されるのが好ましく、十分
に反応させる点からは0,01重量%以上が好ましい。This sensitized insoluble carrier is used in the above mixed solution at an appropriate concentration, but from the viewpoint of ease of absorbance measurement, 0.05
It is preferably used in an amount of 5% by weight or less, and from the viewpoint of sufficient reaction, 0.01% by weight or more is preferable.
前記ポリエチレングリコールとしては2通常。The polyethylene glycol is usually 2.
平均分子量が3.000以上のものが好ましい。分子量
が大きくなるとラテックス凝集による光学的強度が大き
くなる。ポリエチレングリコールハ前8ピ混合液中に、
り重量%以下で存在させるのが好ましい。ポリエチレン
グリコールの濃度が高くなりすぎると感作された不溶性
担体の非特異的な凝集が起こりやすくなる。ポリエチレ
ングリコールは、前記混合液中に、0.5重量%以上の
濃度で存在するのが好ましい。少なすぎると反応促進の
効果が小さい。Those having an average molecular weight of 3.000 or more are preferred. As the molecular weight increases, the optical intensity due to latex aggregation increases. Add polyethylene glycol to the 8-pi mixture,
Preferably, it is present in an amount less than or equal to % by weight. If the concentration of polyethylene glycol becomes too high, nonspecific aggregation of the sensitized insoluble carrier tends to occur. Preferably, polyethylene glycol is present in the mixed liquid at a concentration of 0.5% by weight or more. If the amount is too small, the effect of promoting the reaction will be small.
前記水溶性化合物としては、トリアルキルアミンとして
トリエチルアミン等、トリアルキルアミンの塩としてト
リエチルアミンの塩酸塩等及び第4級アンモニウム塩と
して塩化コリン、臭化コリン、塩化アセチルコリン、臭
化アセチルコリン。Examples of the water-soluble compounds include triethylamine as a trialkylamine, triethylamine hydrochloride as a salt of trialkylamine, and choline chloride, choline bromide, acetylcholine chloride, and acetylcholine bromide as quaternary ammonium salts.
塩酸ベタイン等がある。これらの化合物は、一種又は二
種以上使用される。この水溶性化合物は。Examples include betaine hydrochloride. These compounds may be used alone or in combination. This water-soluble compound is.
前記混合液中に、適宜の濃度になるように使用されるが
、好ましくは0.1〜10i1i%になるように使用さ
れる。該水溶性化合物が少なすぎると添加することによ
る効果が小さく、多すぎると測定感度が低下しやすくな
る。It is used in the mixed solution at an appropriate concentration, preferably 0.1 to 10i1i%. If the amount of the water-soluble compound is too small, the effect of adding it will be small, and if it is too large, the measurement sensitivity will tend to decrease.
前記の感作された不溶性担体・ポリエチレングリコール
及び水溶性化合物は、適当な媒体に分散及び溶解して試
薬として使用するのが好ましい。The sensitized insoluble carrier, polyethylene glycol, and water-soluble compound described above are preferably used as a reagent after being dispersed and dissolved in a suitable medium.
この場合、試薬としては1次の形態がある。In this case, the reagent has a primary form.
(1)感作された不溶性担体、ポリエチレングリコール
及び水溶性化合物を共に同一の媒体に分散又は溶解させ
た試薬(1液のラテックス試薬)。(1) A reagent (one-liquid latex reagent) in which a sensitized insoluble carrier, polyethylene glycol, and a water-soluble compound are both dispersed or dissolved in the same medium.
(2)感作された不溶性担体を媒体に分散させた試薬(
ラテックス試薬)
と
ポリエチレングリコールと水溶性化合物を同じ媒体に溶
解した試薬
からなる2液型の試薬。(2) A reagent in which a sensitized insoluble carrier is dispersed in a medium (
A two-component reagent consisting of a latex reagent), polyethylene glycol, and a water-soluble compound dissolved in the same medium.
(3)感作された否溶性担体と水溶性化合物を共に同一
の媒体に分散又は溶解した試薬(ラテックス試薬)
ポリエチレングリコールを溶解した試薬からなる2液型
の試薬。(3) A reagent in which a sensitized insoluble carrier and a water-soluble compound are both dispersed or dissolved in the same medium (latex reagent) A two-component reagent consisting of a reagent in which polyethylene glycol is dissolved.
(4)感作された不溶性担体を媒体に分散した試薬(ラ
テックス試薬)。(4) A reagent (latex reagent) in which a sensitized insoluble carrier is dispersed in a medium.
ポリエチレングリコールを媒体に溶解した試薬と 水溶性化合物を媒体に溶解した試薬 からなる3液型の試薬。A reagent in which polyethylene glycol is dissolved in a medium and A reagent containing a water-soluble compound dissolved in a medium A three-component reagent consisting of:
これらのうち、(2)及び(3)の形態が最も好ましい
。Among these, forms (2) and (3) are most preferred.
前記において、媒体としては、リン酸緩衝液。In the above, the medium is a phosphate buffer.
グリシン緩衝液、トリス塩酸緩衝液、グツド緩衝液等が
好ましく、pHを6〜10に調整したものが好ましい。Glycine buffer, Tris-HCl buffer, Gud buffer, etc. are preferred, and those whose pH is adjusted to 6 to 10 are preferred.
また、試薬には、適宜、牛血清アルブミン、塩濃度n1
″1整のためのNa C1等を溶解させてもよい。In addition, the reagents include bovine serum albumin and salt concentration n1, as appropriate.
You may dissolve NaCl etc. for ``1 adjustment''.
前記1液のラテックス試薬及び多液型のラテックス試薬
において、感作された不溶性担体は、前記混合液中の濃
度が調整しやすくなるよう適宜の濃度で使用されるが、
0.1〜0.5重量%の濃度になるようにするのが、一
般に使用しやすい。前記のポリエチレングリコール及び
水溶性化合物は。In the one-liquid latex reagent and multi-liquid latex reagent, the sensitized insoluble carrier is used at an appropriate concentration so that the concentration in the mixed liquid can be easily adjusted.
A concentration of 0.1 to 0.5% by weight is generally easy to use. The polyethylene glycol and water-soluble compound mentioned above.
1液のラテックス試薬及び多液型の試薬において。In one-part latex reagents and multi-part reagents.
前記混合液中での濃度が調整しやすくなるように適宜の
濃度で溶解される。It is dissolved at an appropriate concentration so that the concentration in the liquid mixture can be easily adjusted.
マ九、前記の媒体に、適宜、牛血清アルブミン。Add bovine serum albumin to the above medium as appropriate.
NaC1等を溶解させたものを、前記試薬と共に。Dissolve NaCl etc. together with the above reagent.
液fl−調整のために使用してもよい。It may also be used for liquid fl-regulation.
本発明において、試料、前記の感作された不溶性担体、
ポリエチレングリコール及び前記水溶性化合物は適宜の
順序で混合され、混合液とされる。In the present invention, a sample, the sensitized insoluble carrier,
Polyethylene glycol and the water-soluble compound are mixed in an appropriate order to form a mixed solution.
上記抗原−抗体反応は、25〜37℃で行なうのが好ま
しく1反応中は恒温にするのが好ましい。The above antigen-antibody reaction is preferably carried out at a temperature of 25 to 37°C, preferably at a constant temperature during one reaction.
この範囲をはずれると抗原−抗体反応が不安定になりや
すい。さらに、この反応は、30秒〜15分間行なわれ
るのが好ましい。30秒未満では。Outside this range, the antigen-antibody reaction tends to become unstable. Furthermore, this reaction is preferably carried out for 30 seconds to 15 minutes. In less than 30 seconds.
上記反応が不充分であり、吸光度から几Fを定量するの
が困難になり、15分を越えると短時間測定の長所が減
じる。The above reaction is insufficient, making it difficult to quantify F from absorbance, and if the time exceeds 15 minutes, the advantage of short-time measurement is diminished.
上記ラテックス凝集反応開始後、混合液の光学的強度が
適尚な波長を選択して測定される。After the latex aggregation reaction starts, the optical intensity of the mixed liquid is measured by selecting an appropriate wavelength.
ここで、光学的強度とは吸光度又は散乱光強度を意味す
る。波長は2通常500〜1,000nmの範囲から選
択される。Here, optical intensity means absorbance or scattered light intensity. The wavelength is usually selected from the range of 500 to 1,000 nm.
光学的強度の測定は1反応開始後1回測定する方法〔終
末点測定(エンドポイントアッセイ)。The optical intensity is measured once after the start of one reaction [end point assay].
反応開始後の光学的強度の増加を測定する方法〕及び反
応開始後2回以上測定し、その間の光学的強度の増加分
又は単位時間当りの増加分を求める方法並びに−足元学
的強度に達するまでの時間を測定する方法のいずれfも
採用することができる。A method of measuring the increase in optical intensity after the start of the reaction] and a method of measuring at least two times after the start of the reaction and determining the increase in optical intensity or the increase per unit time during that time, and - reaching the optical intensity Any method f for measuring the time up to this point can be adopted.
定量は、几F既知量の試料(例えばBP標準血清とその
希釈系列)について、前記の測定を行ない、その測定値
とRF量とから検量線を作成しておき、几F未知量の試
料について同一条件で測定した測定値から該検量線によ
って対応するIt ル”量を求めることによって行なう
ことができる。For quantitative determination, perform the above measurements on a sample with a known amount of F (for example, BP standard serum and its dilution series), create a calibration curve from the measured values and the RF amount, and then perform the measurement on a sample with an unknown amount of F. This can be done by determining the corresponding Itle amount from the measured values measured under the same conditions using the calibration curve.
反応開始後、1回測定する方法で前記の2液型の試薬を
用いる場合には、さらに2次のようにして定量すること
ができる。When the above-mentioned two-component reagent is used in a method in which measurement is performed once after the start of the reaction, quantitative determination can be further carried out in the following manner.
すなわち、試料について光学的強度(ET)を測定し、
この値から試料に起因する吸光度(EBB)とラテック
ス試薬に起因する吸光度を差し引き、算出強度(E R
F)を求める。That is, measuring the optical intensity (ET) of the sample,
From this value, subtract the absorbance attributable to the sample (EBB) and the absorbance attributable to the latex reagent to obtain the calculated intensity (ER
Find F).
ここで、試料に起因する光学的強度とは9例えば、上記
混合液の調整において、ラテックス試薬の代わりに生理
食塩水を使用して得た液の光学的強度である。Here, the optical intensity caused by the sample is the optical intensity of a solution obtained by using physiological saline instead of the latex reagent in the preparation of the above-mentioned mixed solution, for example.
ラテックス試薬に起因する光学的強度とは1例えば上記
した混合液の調整において、試料の代わりに生理食塩水
を使用して得た液の光学的強度(E an)から、上記
した混合液において試料及びラテックス試薬の代わりに
生理食塩水を使用して得た光学的強度(EBB)を差し
引いた値である。What is the optical intensity caused by the latex reagent?1 For example, in preparing the above-mentioned mixed solution, from the optical intensity (E an) of the solution obtained by using physiological saline instead of the sample, the optical intensity of the sample in the above-mentioned mixed solution is calculated. and minus the optical intensity (EBB) obtained using saline instead of the latex reagent.
上記ET、 Esa* Eia、 EaaからRFに関
する光学的強度Earが次の式により求められる。The optical intensity Ear regarding RF is determined from the above ET, Esa*Eia, and Eaa using the following formula.
Enp= ET −Ess (ERB E
BBンEarからRFを定量する仕方は、前記の定量の
仕方と同様である。Enp= ET −Ess (ERB E
The method of quantifying RF from BBB and Ear is the same as the method of quantitative determination described above.
(実施例)
次に試薬、測定方法、実測結果などに関連して本発明方
法を詳細に説明する。以下1%はM量チを意味する。(Example) Next, the method of the present invention will be explained in detail in relation to reagents, measurement methods, actual measurement results, etc. The following 1% means the amount of M.
実施例1
1)試薬
り希釈液
0.9%ポリエチレングリコール(平均分子量7500
)、 0.15M NaC1、及び0.1%牛血
清アルブミン含有0.05 Mリン酸緩衝液11)
ラテックス試薬
上記1)の希釈液からポリエチレングリコールを除き、
0.1M塩化コリンを溶解し、ヒトγ−グロブリンを感
作した粒径0.2μm以下のポリスチレン系ラテックス
を分散させた試液(ラテックス濃度0.4重量%)。こ
のラテックス試薬は0〜4℃で保存した場合には少なく
とも12力月間安定である。Example 1 1) Reagent diluent 0.9% polyethylene glycol (average molecular weight 7500
), 0.05 M phosphate buffer containing 0.15 M NaCl, and 0.1% bovine serum albumin 11)
Latex reagent Remove polyethylene glycol from the diluted solution in 1) above,
A test solution in which 0.1M choline chloride is dissolved and polystyrene latex sensitized with human γ-globulin and having a particle size of 0.2 μm or less is dispersed (latex concentration 0.4% by weight). This latex reagent is stable for at least 12 months when stored at 0-4°C.
2)測定方法
以下全白
表 組成及び吸光度
上記の表の4糧の液を調製し、各液を37℃で10分間
恒温反応した後、570nmの波長吸光度を測定し、検
体血清(試料)の吸光度(ARF)を式%式%)
から算出する。検体血清としてリウマチ因子陽性血清(
上記几F二次標準血−ff)O115(24IU/di
)から515 (120IU/dIりまで5個の希釈系
列を用いて吸光度と几F値(IU/mIりの検量線を作
成した。測定は1日立自動分析装置705型(以下日立
705形と略す)を用い、上記測定原理を適用した。2) Measurement method: Full white table below Composition and absorbance Prepare the four solutions shown in the table above, and after reacting each solution at 37°C for 10 minutes, measure the absorbance at a wavelength of 570 nm, and determine whether the sample serum (sample) Absorbance (ARF) is calculated from the formula %. Rheumatoid factor positive serum (
The above ⇠F secondary standard blood-ff) O115 (24IU/di
) to 515 (120 IU/dI) to create a calibration curve of absorbance and F value (IU/ml). ), and the above measurement principle was applied.
日立705形では分析法プログラムの2ポイントエンド
法を使用すると自動的に装置が上記測定法にもとづいて
演算し、測定結果を算出する。反応温度は25℃〜37
℃、測定波長は57 Onm学轡q寄eを選択し、−波
長測光を採用した。In the Hitachi Model 705, when the 2-point end method of the analysis method program is used, the device automatically performs calculations based on the above measurement method and calculates the measurement results. Reaction temperature is 25℃~37℃
℃, the measurement wavelength was 57 Onm, and -wavelength photometry was adopted.
3)実測結果 上記で得られた検量線を第1図にグラフ1として示す。3) Actual measurement results The calibration curve obtained above is shown in FIG. 1 as Graph 1.
実施例2
実施例1において、ラテックス試薬中の0.1M塩化コ
リンを、0.12M塩化アセチルコリン。Example 2 In Example 1, 0.1M choline chloride in the latex reagent was replaced with 0.12M acetylcholine chloride.
0、08 M塩酸ベタイン及び0.13M1−リエチル
アミンに換えたこと以外は、実施例1と同様に行なった
。それぞれの場合に、第1図のグラフ1と同様の直線性
に優れた検量線が得られた。The same procedure as in Example 1 was carried out except that 0.08M betaine hydrochloride and 0.13M 1-ethylamine were used. In each case, a calibration curve with excellent linearity similar to Graph 1 in FIG. 1 was obtained.
比較例1
実施例1における希釈液に、塩化コリンを含有させない
こと以外は、実施例1と同様に行なった。Comparative Example 1 The same procedure as in Example 1 was conducted except that the diluent in Example 1 did not contain choline chloride.
得られた検量線を第1図に、グラフ2として示す。The obtained calibration curve is shown in FIG. 1 as Graph 2.
第1図から明らかなように、ポリエチレングリコールを
使用した場合に、検量線が直線性を示さない場合(グラ
フ2)でも、水溶性化合物(塩化コリン等)を使用する
ことによシ、良好な直線性を有する検量線とすることが
できる。As is clear from Figure 1, even if the calibration curve does not show linearity when polyethylene glycol is used (Graph 2), a good result can be obtained by using a water-soluble compound (such as choline chloride). A calibration curve with linearity can be obtained.
(発明の効果)
本発明によれば、几Fの測定において良好な直線を有す
る検量線が得られるため、検量線の作成が容易になり、
定量精度も向上する。(Effects of the Invention) According to the present invention, a calibration curve having a good straight line can be obtained in the measurement of F, making it easy to create a calibration curve.
Quantitative accuracy also improves.
第1図は、実施例1及び比較例1で得られた検量線を示
す。
符号の説明FIG. 1 shows the calibration curves obtained in Example 1 and Comparative Example 1. Explanation of symbols
Claims (1)
体を混合して混合液とし、該混合液中にポリエチレング
リコール並びにトリアルキルアミン、その塩及び第4級
アンモニウム塩からなる群から選ばれる少なくとも一種
の水溶性化合物を共存させて抗原−抗体反応によるラテ
ックス凝集反応を起こさせて、光学的強度を測定し、こ
の測定値から試料中のリウマチ因子を定量することを特
徴とするリウマチ因子の定量法。1. A sample and a human insoluble carrier sensitized with γ-globulin are mixed to form a mixed solution, and in the mixed solution at least one selected from the group consisting of polyethylene glycol, trialkylamine, its salt, and quaternary ammonium salt is added. Quantification of rheumatoid factor, which involves causing a latex agglutination reaction by an antigen-antibody reaction in the coexistence of a type of water-soluble compound, measuring optical intensity, and quantifying rheumatoid factor in a sample from this measured value. Law.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP61062818A JPH0668492B2 (en) | 1986-03-20 | 1986-03-20 | Method for quantifying rheumatoid factor |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP61062818A JPH0668492B2 (en) | 1986-03-20 | 1986-03-20 | Method for quantifying rheumatoid factor |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS62218865A true JPS62218865A (en) | 1987-09-26 |
JPH0668492B2 JPH0668492B2 (en) | 1994-08-31 |
Family
ID=13211291
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP61062818A Expired - Fee Related JPH0668492B2 (en) | 1986-03-20 | 1986-03-20 | Method for quantifying rheumatoid factor |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH0668492B2 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002018953A1 (en) * | 2000-08-29 | 2002-03-07 | Kyowa Medex Co.,Ltd | Highly reproducible agglutination immunoassay method and reagents |
JP2011514517A (en) * | 2008-02-20 | 2011-05-06 | アクシス−シールド ダイアグノスティックス リミテッド | Method for assaying antibodies against cyclic citrullinated peptides |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5426327A (en) * | 1977-07-27 | 1979-02-27 | Eiken Chemical | Reagent for detecting rheumatism factor |
JPS5610254A (en) * | 1979-07-07 | 1981-02-02 | Wako Pure Chem Ind Ltd | New rheumatism factor measuring reagent |
JPS5631647A (en) * | 1979-08-24 | 1981-03-31 | Yatoron:Kk | Stabilizing method of reagent for detecting rheumatoid factor |
JPS5847256A (en) * | 1981-09-14 | 1983-03-18 | Mitsubishi Chem Ind Ltd | Measuring method for antigen-antibody reaction |
JPS5924387A (en) * | 1982-07-30 | 1984-02-08 | Sharp Corp | Electronic memo |
-
1986
- 1986-03-20 JP JP61062818A patent/JPH0668492B2/en not_active Expired - Fee Related
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5426327A (en) * | 1977-07-27 | 1979-02-27 | Eiken Chemical | Reagent for detecting rheumatism factor |
JPS5610254A (en) * | 1979-07-07 | 1981-02-02 | Wako Pure Chem Ind Ltd | New rheumatism factor measuring reagent |
JPS5631647A (en) * | 1979-08-24 | 1981-03-31 | Yatoron:Kk | Stabilizing method of reagent for detecting rheumatoid factor |
JPS5847256A (en) * | 1981-09-14 | 1983-03-18 | Mitsubishi Chem Ind Ltd | Measuring method for antigen-antibody reaction |
JPS5924387A (en) * | 1982-07-30 | 1984-02-08 | Sharp Corp | Electronic memo |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002018953A1 (en) * | 2000-08-29 | 2002-03-07 | Kyowa Medex Co.,Ltd | Highly reproducible agglutination immunoassay method and reagents |
US7166476B2 (en) | 2000-08-29 | 2007-01-23 | Kyowa Medex Co., Ltd. | Highly reproducible agglutination immunoassay method and reagents |
KR100778102B1 (en) * | 2000-08-29 | 2007-11-27 | 가부시키가이샤 티에프비 | Highly reproducible agglutination immunoassay method and reagents |
JP4733335B2 (en) * | 2000-08-29 | 2011-07-27 | 協和メデックス株式会社 | Aggregation immunoassay and reagent with good reproducibility |
JP2011514517A (en) * | 2008-02-20 | 2011-05-06 | アクシス−シールド ダイアグノスティックス リミテッド | Method for assaying antibodies against cyclic citrullinated peptides |
EP2255191B1 (en) | 2008-02-20 | 2016-01-06 | Axis-Shield Diagnostics Ltd. | Assay method for antibodies against cyclic citrullinated peptide |
Also Published As
Publication number | Publication date |
---|---|
JPH0668492B2 (en) | 1994-08-31 |
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