JP3496684B2 - Colorimetric analysis using colloidal gold particles - Google Patents
Colorimetric analysis using colloidal gold particlesInfo
- Publication number
- JP3496684B2 JP3496684B2 JP26667392A JP26667392A JP3496684B2 JP 3496684 B2 JP3496684 B2 JP 3496684B2 JP 26667392 A JP26667392 A JP 26667392A JP 26667392 A JP26667392 A JP 26667392A JP 3496684 B2 JP3496684 B2 JP 3496684B2
- Authority
- JP
- Japan
- Prior art keywords
- absorbance
- reaction
- gold
- solution
- hemoglobin
- 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
Description
【０００１】[0001]
【発明の利用分野】本発明は、金コロイド粒子を用いる
免疫分析法の改良法に関する。FIELD OF THE INVENTION The present invention relates to an improved method of immunoassay using colloidal gold particles.
【０００２】[0002]
【発明の背景】従来、抗原抗体反応の測定には、抗原
と抗体そのものの反応による濁度変化の測定、不溶化
担体を用いるラテックス法、寒天平板中のゲル内沈降
反応等があるが、いずれも溶媒中では濁りを生じる反応
であり、比色分析することは難である。また、凝集反応
は肉眼で判定するので、定性的或は半定量的にならざる
を得ず、また、個人により測定誤差が生じることも充分
考えられるので、凝集反応の判定には熟練が必要である
と共に問題点も多い。BACKGROUND OF THE INVENTION Conventionally, there have been known methods for measuring an antigen-antibody reaction, such as a change in turbidity due to a reaction between an antigen and an antibody itself, a latex method using an insolubilized carrier, and a precipitation reaction in a gel on an agar plate. It is a reaction that causes turbidity in a solvent, and colorimetric analysis is difficult. In addition, since the agglutination reaction is judged by the naked eye, it must be qualitative or semi-quantitative, and it is fully possible that an error in measurement will occur depending on the individual. There are many problems with it.
【０００３】また、免疫分析法においては、金コロイド
粒子を用い、この色調変化を利用するマンナンに対する
凝集反応分析や（Horisberger、Rossertら、J.Histoche
m.Cytochem.,25.295-305(1977)）、金コロイド粒子を担
体とした免疫分析法（特開昭57ー86051号公報）も知られ
ている。しかしながら、これらの金コロイド粒子を使用
した免疫分析法は、判定を目視により行っていたために
検出限界付近の陽性、陰性の判断は経験を積んでもなお
主観によって左右されやすいという短所があった。In the immunoassay method, gold colloidal particles are used, and agglutination reaction analysis for mannan utilizing this color change (Horisberger, Rossert et al., J. Histoche).
m.Cytochem., 25.295-305 (1977)), and an immunoassay method using gold colloidal particles as a carrier (JP-A-57-86051). However, the immunoassay method using these colloidal gold particles has a disadvantage that the judgment of positive and negative in the vicinity of the detection limit is apt to be influenced by the subjectivity even if it is experienced, because the judgment is performed visually.
【０００４】[0004]
【発明の目的】本発明は上記した如き問題点を解決する
ために成されたものであり、自動分析装置等を利用して
測定可能な、金コロイド粒子を用いた比色分析法を提供
することをその目的とする。SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and provides a colorimetric analysis method using colloidal gold particles, which can be measured by using an automatic analyzer or the like. That is the purpose.
【０００５】[0005]
【発明の構成】本発明は、ヘモグロビンに対する抗体が
結合した金コロイド粒子（以下、感作金コロイド粒子と
略記する。）と、糞便中に存在するヘモグロビンとを反
応させ、反応開始後の反応液の500〜550nmの吸光度変
化、より具体的には、反応開始後、反応液の吸光度を適
当な間隔で２回測定したものの差又は反応開始後の反応
液の吸光度変化率に基づいて、糞便中のヘモグロビンを
定量することを特徴とする比色分析法の発明である。BEST MODE FOR CARRYING OUT THE INVENTION According to the present invention, colloidal gold particles to which an antibody against hemoglobin is bound (hereinafter abbreviated as sensitized gold colloidal particles) and hemoglobin present in feces are reacted, and a reaction solution after the reaction is started. 500-550 nm absorbance change, more specifically, based on the difference between the absorbance of the reaction solution measured twice at an appropriate interval after the start of the reaction or the rate of change in the absorbance of the reaction solution after the start of the reaction, in feces Is an invention of a colorimetric analysis method characterized by quantifying hemoglobin of.
【０００６】本発明の比色分析法の測定対象物質として
は、測定対象物質に対する抗体（或は抗原）が金コロイ
ド粒子に結合し得るものであれば特に限定されることな
く挙げられるが、より具体的には例えばアルブミン，ヘ
モグロビン，ミオグロビン，トランスフェリン，プロテ
インＡ，Ｃ反応性蛋白質（ＣＲＰ）等のタンパク質、例
えば高比重リポ蛋白質（ＨＬＤ），低比重リポ蛋白質
（ＬＤＬ），超低比重リポ蛋白質等の脂質蛋白質、例え
ばデオキシリボ核酸（ＤＮＡ），リボ核酸（ＲＮＡ）等
の核酸、例えばアルカリ性ホスファターゼ，乳酸脱水素
酵素，リパーゼ，アミラーゼ等の酵素、例えばＩgＧ，
ＩgＭ，ＩgＡ，ＩgＤ，ＩgＥ等の免疫グロブリン（或は
これらの、例えばＦc部，Ｆab部，Ｆ(ab)2部等の断
片）、例えばフィブリノーゲン，トロンビン等の血液凝
固因子、例えば抗ストレプトリジンＯ抗体，抗ウイルス
抗体，リュウマチ因子等の抗体等が挙げられる。The substance to be measured in the colorimetric method of the present invention is not particularly limited as long as the antibody (or antigen) to the substance to be measured can bind to the gold colloid particles, but Specifically, for example, proteins such as albumin, hemoglobin, myoglobin, transferrin, protein A, C-reactive protein (CRP), such as high-density lipoprotein (HLD), low-density lipoprotein (LDL), ultra-low density lipoprotein, etc. Nucleic acids such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), such as alkaline phosphatase, lactate dehydrogenase, lipase and amylase, such as IgG,
Immunoglobulins such as IgM, IgA, IgD and IgE (or fragments thereof such as Fc portion, Fab portion and F (ab) 2 portion), blood coagulation factors such as fibrinogen and thrombin, such as anti-streptolidine O Antibodies, antiviral antibodies, antibodies such as rheumatoid factor, etc. may be mentioned.
【０００７】本発明に用いられる金コロイド粒子は、市
販のものを用いればよいが、常法、例えば塩化金酸をク
エン酸ナトリウムで還元する方法（Nature Phys.Sci.,v
ol.241,20,1973等）により調製したものを用いてもよ
い。また、金コロイド粒子の粒径は特に限定されない
が、通常20nm〜90nm、好ましくは30nm〜70nmの範囲のも
のが挙げられる。As the gold colloidal particles used in the present invention, commercially available ones may be used, but a conventional method, for example, a method of reducing chloroauric acid with sodium citrate (Nature Phys. Sci., V
ol. 241, 20, 1973 etc.) may be used. Further, the particle size of the gold colloidal particles is not particularly limited, but examples thereof include those in the range of usually 20 nm to 90 nm, preferably 30 nm to 70 nm.
【０００８】本発明に於いて用いられる、感作金コロイ
ド粒子は、例えば以下の如くして容易に得ることができ
る。即ち、市販の金コロイド粒子、或は常法、例えば塩
化金酸をクエン酸ナトリウムで還元する方法（Nature P
hys.Sci.,vol.241,20,1973等）により調製された金コロ
イド粒子を常法（J.Histochem.Cytochem.,vol.25,1187
〜1200,1977、Experientia,vol.31,1147,1975等）によ
り処理することにより容易に調製することができる。よ
り具体的には、金コロイド粒子と、金コロイド粒子１mg
に対して通常0.5〜100μg、好ましくは１〜50μgの測定
対象物質に対する抗体（又は抗原）とを、適当な緩衝液
中で５〜30分間室温下に反応させた後、例えばカーボワ
ックス20Ｍ等の分散剤を添加して遠心分離等により目的
の感作金コロイド粒子を分取することにより容易に得ら
れる。尚、得られた感作金コロイド粒子は、例えばカー
ボワックス20Ｍ等の分散剤を含む溶液中に均一に分散さ
せて保存すればよい。尚、上記反応に於いて用いられる
緩衝液は、金コロイド粒子と測定対象物質に対する抗体
（又は抗原）との結合反応を阻害しないものであればそ
の種類、濃度、pＨ等は特に限定されない。The sensitized gold colloidal particles used in the present invention can be easily obtained, for example, as follows. That is, commercially available colloidal gold particles or a conventional method, for example, a method of reducing chloroauric acid with sodium citrate (Nature P
hys.Sci., vol. 241, 20, 1973, etc.) was used to prepare gold colloidal particles according to a conventional method (J. Histochem. Cytochem., vol. 25, 1187).
~ 1200, 1977, Experientia, vol. 31, 1147, 1975, etc.) can be easily prepared. More specifically, gold colloid particles and gold colloid particles 1 mg
On the other hand, usually 0.5 to 100 μg, preferably 1 to 50 μg of the antibody (or antigen) to the substance to be measured is reacted at room temperature for 5 to 30 minutes in an appropriate buffer, and then, for example, Carbowax 20M or the like. It can be easily obtained by adding a dispersant and collecting the target sensitized gold colloid particles by centrifugation or the like. The obtained sensitized gold colloid particles may be uniformly dispersed and stored in a solution containing a dispersant such as Carbowax 20M. The buffer solution used in the above reaction is not particularly limited in kind, concentration, pH and the like as long as it does not inhibit the binding reaction between the gold colloid particles and the antibody (or antigen) to the substance to be measured.
【０００９】本発明は、上記の如くして得られた感作金
コロイド粒子と、測定対象物質を含む試料、例えば血
液、血漿、血清、髄液、尿、糞便等の生体由来の試料や
これらを適宜緩衝液等で希釈したもの等とを、適宜混合
し、その結果生ずる抗原抗体反応に起因する金コロイド
粒子の吸光度変化を測定し、その結果を、予め作成して
おいた金コロイド粒子の吸光度変化と測定対象物質量と
の関係を表わす検量線に当てはめる等することにより、
容易に実施し得る。尚、吸光度変化が一定値以下であれ
ば陰性、一定値以上であれば陽性としておけば、本発明
の方法により測定対象物質の半定量も可能である。The present invention is directed to a sample containing the sensitized gold colloid particles obtained as described above and a substance to be measured, for example, a sample derived from a living body such as blood, plasma, serum, cerebrospinal fluid, urine, feces, or the like. Is appropriately diluted with a buffer or the like, and mixed appropriately, and the change in absorbance of the gold colloid particles resulting from the antigen-antibody reaction that occurs as a result is measured, and the result is calculated for the gold colloid particles prepared in advance. By applying it to a calibration curve that shows the relationship between the change in absorbance and the amount of the substance to be measured,
It can be easily implemented. In addition, if the change in absorbance is a certain value or less, it is negative, and if it is more than a certain value, it is positive, and the semi-quantitative determination of the substance to be measured is possible by the method of the present invention.
【００１０】本発明に於ける吸光度変化は、例えば以下
の如くして求めればよい。
(1)感作金コロイド粒子と測定対象物質であるヘモグロ
ビンとの反応開始後、反応液の吸光度を適当な間隔で２
回測定し、その差を吸光度変化とする。
(2)感作金コロイド粒子と測定対象物質であるヘモグロ
ビンとの反応開始後の反応液の吸光度変化率（特に、そ
の最大変化率）を吸光度変化とする。The change in absorbance in the present invention may be obtained as follows, for example. (1) After the reaction between the sensitized gold colloid particles and hemoglobin, which is the substance to be measured, is started, the absorbance of the reaction solution is adjusted to 2 at appropriate intervals.
Measure the number of times and use the difference as the change in absorbance. (2) The rate of change in absorbance (particularly the maximum rate of change) of the reaction solution after the reaction between the sensitized gold colloid particles and hemoglobin, which is the substance to be measured, is defined as the change in absorbance.
【００１１】本発明に於いて、吸光度変化を測定するた
めの波長としては、吸光度変化を測定可能な波長であれ
ば、特に限定されないが、金コロイド粒子の極大吸収波
長付近の500〜550nm付近が測定感度を高くすることがで
きるので望ましい。また、吸光度変化は、主波長と副波
長を使用する２波長測光により求めてもよいことは言う
までもない。In the present invention, the wavelength for measuring the change in absorbance is not particularly limited as long as it is a wavelength capable of measuring the change in absorbance, but is in the vicinity of 500 to 550 nm, which is the maximum absorption wavelength of the gold colloid particles. It is desirable because the measurement sensitivity can be increased. Also, it goes without saying that the change in absorbance may be obtained by two-wavelength photometry using the main wavelength and the sub wavelength.
【００１２】本発明によれば、抗原抗体反応に起因する
変化を吸光度変化として測定することができるので、抗
原抗体反応を利用する測定法を例えば比色計、分光光度
計、マイクロプレ−トリ−ダ−、生化学自動分析機等で
測定することが可能である。また、上記した如き装置で
測定が可能であるため、従来法の目視による判定で生じ
ていた、例えば測定場所の照度、照明に用いる光源の種
類等の外的因子や例えば判定者の経験度や習熟度或は体
調等の人的因子に起因する判定の不一致を回避すること
ができる。また、本発明を生化学自動分析機に応用する
ことにより、多数の試料を短時間に処理することも可能
である。According to the present invention, since a change caused by an antigen-antibody reaction can be measured as a change in absorbance, a measuring method utilizing the antigen-antibody reaction can be, for example, a colorimeter, a spectrophotometer, a microplate. It is possible to measure with an automatic analyzer or biochemical analyzer. Further, since it is possible to measure with the device as described above, the external factors such as the illuminance at the measurement location, the type of the light source used for illumination, and the like, which are caused by the conventional visual determination, and the experience of the determiner, It is possible to avoid discrepancies in judgments due to human factors such as proficiency or physical condition. Moreover, by applying the present invention to an automatic biochemical analyzer, it is possible to process a large number of samples in a short time.
【００１３】以下に本発明の実施例を挙げ、本発明をよ
り具体的に説明するが、本発明はこれによって何等限定
されるものではない。Hereinafter, the present invention will be described in more detail with reference to examples of the present invention, but the present invention is not limited thereto.
【００１４】実施例１．
(1)抗ヒトＩｇＧモノクローナル抗体感作金コロイト゛試液の
調製
抗ヒトＩｇＧモノクローナル抗体溶液（１mg/ml、和光
純薬工業(株)製）１７μｌに、0.2M Ｋ2ＣＯ3水溶液でp
Ｈ=6.0に調整した金コロイト゛ソ゛ル溶液（ジャンセン社製）１
０ｍｌをすばやく混合し、そのまま１０分間室温放置し
た。その後、反応液に１％(w/w)カーボワックス20Ｍ溶
液500μｌを加えて均一になるように混合し、7000ｇで
２０分間遠心分離し、その沈澱物に分散液（50mMリン酸
ナトリウム、0.0005%セ゛ラチン、0.02%カーホ゛ワックス20Ｍ pH6.5）を加
えてＯＤ540=5.0となるようにしたものを、抗ヒトＩｇ
Ｇモノクローナル抗体感作金コロイト゛試液とした。
(2)金コロイト゛試液を用いた検量線の作成
96穴プレートの各ウェルに濃度既知のヒトIgＧ溶液４０
μｌを入れ、さらに上記(1)で得た抗ヒトＩｇＧモノク
ローナル抗体感作金コロイト゛試液100μｌを添加し、ＳＯＦ
ＴmaxーＪ（Ver.2.01J、和光純薬工業(株)製）によりλ1
=540nm、λ2=405nm、オートミックス一回、測定時間１０min、
測定インターハ゛ル12sec.に条件設定したマイクロプレートリ
ーダーＵＶmax（モレキュラーデバイス社製）で二波長ネ
カ゛ティフ゛カイネティック測定した。得られた測定結果を、更にＳ
ＯＦＴmaxーＪを使用して、ラク゛タイム１２sec,Vmaxホ゜イント=２
の条件で処理して最大反応速度Ｖmaxを求めた。得られ
たＶmaxとIgＧ濃度（μg/ml）との関係を表わす検量線
を図１に示す。図１から明らかな如く、良好な直線性を
有する検量線が得られることが判る。Example 1. (1) Preparation of anti-human IgG monoclonal antibody sensitized colony test solution 17 μl of anti-human IgG monoclonal antibody solution (1 mg / ml, manufactured by Wako Pure Chemical Industries, Ltd.) was added with 0.2 M K 2 CO 3 aqueous solution.
Gold Korodesol solution adjusted to H = 6.0 (manufactured by Jansen) 1
0 ml was mixed rapidly and left at room temperature for 10 minutes. Then, 500 μl of 1% (w / w) Carbowax 20M solution was added to the reaction mixture and mixed to homogeneity, followed by centrifugation at 7,000 g for 20 minutes, and the precipitate was dispersed (50 mM sodium phosphate, 0.0005% Anti-human Ig was prepared by adding keratin, 0.02% carbowax 20M pH6.5) so that OD 540 = 5.0.
It was used as a G monoclonal antibody sensitized gold colloid test solution. (2) Preparation of calibration curve using gold colloid reagent 40 Human IgG solution of known concentration in each well of 96-well plate
Then, add 100 μl of the anti-human IgG monoclonal antibody-sensitized gold colloid reagent solution obtained in (1) above, and add SOF.
Λ 1 by Tmax-J (Ver.2.01J, Wako Pure Chemical Industries, Ltd.)
= 540nm, λ 2 = 405nm, once automix, measurement time 10min,
Two-wavelength negative kinetic measurement was carried out with a microplate reader UVmax (manufactured by Molecular Devices Co., Ltd.) under the condition that the measurement interval was set to 12 sec. The obtained measurement result is further S
Using OFTmax-J, lag time 12sec, Vmax point = 2
The maximum reaction rate Vmax was obtained by treating under the conditions of. A calibration curve showing the relationship between the obtained Vmax and the IgG concentration (μg / ml) is shown in FIG. As is clear from FIG. 1, it can be seen that a calibration curve having good linearity can be obtained.
【００１５】実施例２．
(1)抗ＣＲＰポリクローナル抗体感作金コロイト゛試液の調製
抗ＣＲＰポリクローナル抗体（ウサギ）溶液（41μg/m
l、和光純薬工業(株)製）65μｌに、0.1Ｍリン酸二水素
カリウムー0.05Ｍ四ホウ酸ナトリウム緩衝液でpＨを6.8
に調整した金コロイト゛ソ゛ル溶液（ジャンセン社製）１０ｍｌ
をすばやく混合し、そのまま１０分間室温放置した。そ
の後、反応液に１％(w/w)カーボワックス20Ｍ溶液500μ
ｌを加えて均一になるように混合し、7000ｇで２０分間
遠心分離し,得られた沈澱物を分散液（50mMリン酸ナトリウ
ム、0.02%カーホ゛ワックス20Ｍ、pH7.4）でＯＤ540=5.0となるよ
うに調整したものを、抗ＣＲＰポリクローナル抗体感作
金コロイト゛試液とした。
(2)金コロイト゛試液を用いた検量線の作成
96穴プレートの各ウェルに濃度既知のＣＲＰ溶液４０μ
ｌを入れ、さらに上記抗ＣＲＰポリクローナル抗体感作
金コロイト゛試液100μｌを添加し、ＳＯＦＴmaxーＪ（Ver.2.
01J、和光純薬工業(株)製）によりλ1=540nm、λ2=650n
m、オートミックス一回、測定時間１０min、測定インターハ゛ル20sec.
に条件設定したマイクロプレートリーダーＵＶmax（モ
レキュラーデバイス社製）で二波長ネカ゛ティフ゛カイネティック測定
した。得られた吸光度とＣＲＰ濃度（μg/ml）との関係
を表わす検量線を図２に示す。図２から明らかな如く、
良好な直線性を有する検量線が得られることが判る。Example 2. (1) Preparation of anti-CRP polyclonal antibody-sensitized colony test solution Anti-CRP polyclonal antibody (rabbit) solution (41 μg / m
l, Wako Pure Chemical Industries, Ltd.) 65 μl with 0.1 M potassium dihydrogen phosphate-0.05 M sodium tetraborate buffer to a pH of 6.8
10 ml of gold corodiesol solution (manufactured by Jansen) adjusted to
Were mixed rapidly and left at room temperature for 10 minutes. Then 500μ of 1% (w / w) Carbowax 20M solution in the reaction mixture
1) and mixed to homogeneity, followed by centrifugation at 7,000 g for 20 minutes, and the resulting precipitate was treated with a dispersion (50 mM sodium phosphate, 0.02% carbowax 20M, pH 7.4) to give OD 540 = 5.0. The solution prepared as described above was used as an anti-CRP polyclonal antibody-sensitized gold reagent solution. (2) Preparation of calibration curve using gold colloid reagent 40 μl of known CRP solution in each well of 96-well plate
Then, add 100 μl of the anti-CRP polyclonal antibody-sensitized gold color reagent solution described above, and add SOFTmax-J (Ver.2.
01J, Wako Pure Chemical Industries, Ltd.) λ 1 = 540nm, λ 2 = 650n
m, auto mix once, measurement time 10 min, measurement interval 20 sec.
Two-wavelength negative kinetic measurement was performed with a microplate reader UVmax (manufactured by Molecular Devices Co., Ltd.) set to the above conditions. A calibration curve showing the relationship between the obtained absorbance and the CRP concentration (μg / ml) is shown in FIG. As is clear from FIG.
It can be seen that a calibration curve having good linearity can be obtained.
【００１６】実施例３．
(1)抗ヒトフィブリノーゲンポリクローナル抗体感作金コ
ロイト゛試液の調製
抗ヒトフィブリノーゲンポリクローナル抗体溶液（１mg
/ml、和光純薬工業(株)製）３２μｌに、０．２Ｍ 3-(N
-モルホリノ)プロパンスルホン酸（ＭＯＰＳ）緩衝液で
pH=6.6に調整した金コロイト゛ソ゛ル溶液（ジャンセン社製）１
０ｍｌをすばやく混合し、そのまま１０分間室温放置し
た。その後、反応液に１％(w/w)カーボワックス２０Ｍ
溶液500μｌを加えて均一になるように混合し、7000ｇ
で２０分間遠心分離し、得られた沈澱物を分散液（0.2M
ＭＯＰＳ、0.02%カーホ゛ワックス20Ｍ、0.1%窒化ソータ゛、pH7.4）
でＯＤ540=10.0となるように調整したものを抗ヒトフィ
ブリノーゲンポリクローナル抗体感作金コロイト゛試液とし
た。
(2)金コロイト゛試液を用いた検量線の作成
96穴プレートの各ウェルに濃度既知のヒトフィブリノー
ゲン溶液４０μｌを入れ、さらに上記抗ヒトフィブリノ
ーゲンポリクローナル抗体感作金コロイト゛試液100μｌを添
加し、ＳＯＦＴmaxーＪ（Ver.2.01J、和光純薬工業(株)
製）によりλ1=540nm、λ2=650nm、オートミックス一回、測定
時間１０min、測定インターハ゛ル30sec.に条件設定したマイク
ロプレートリーダーＵＶmax（モレキュラーデバイス社
製）で二波長ネカ゛ティフ゛カイネティック測定した。得られた測定結
果を、更にＳＯＦＴmaxーＪにより解析して、測定開始後
２分後の吸光度と１０分後の吸光度の差を求めた。この
吸光度差とフィブリノーゲン（Ｆgn）濃度（μg/ml）と
の関係を表わす検量線を図３に示す。図３から明らかな
如く、良好な直線性を有する検量線が得られることが判
る。Embodiment 3. (1) Preparation of anti-human fibrinogen polyclonal antibody sensitized gold reagent solution Anti-human fibrinogen polyclonal antibody solution (1 mg
/ ml, Wako Pure Chemical Industries, Ltd.) 32 μl, 0.2M 3- (N
-With morpholino) propanesulfonic acid (MOPS) buffer
Gold Corodiesol solution (manufactured by Jansen) adjusted to pH = 6.6 1
0 ml was mixed rapidly and left at room temperature for 10 minutes. Then, 1% (w / w) Carbowax 20M in the reaction solution
Add 500 μl of solution and mix evenly, 7,000 g
After centrifuging for 20 minutes, the resulting precipitate is dispersed (0.2M
MOPS, 0.02% carbowax 20M, 0.1% sodium nitride, pH 7.4)
The anti-human fibrinogen polyclonal antibody-sensitized gold colony test solution was prepared so that OD 540 = 10.0. (2) Preparation of calibration curve using gold corolide test solution 40 μl of human fibrinogen solution of known concentration was placed in each well of a 96-well plate, and 100 μl of the anti-human fibrinogen polyclonal antibody-sensitized gold corolide test solution was added to the solution to obtain SOFTmax-J (Ver.2.01J, Wako Pure Chemical Industries, Ltd.
Λ 1 = 540 nm, λ 2 = 650 nm, automix once, measurement time 10 min, measurement interval 30 sec., And a two-wavelength negative kinetic measurement was carried out with a microplate reader UVmax (manufactured by Molecular Devices). The obtained measurement results were further analyzed by SOFTmax-J, and the difference between the absorbance 2 minutes after the start of the measurement and the absorbance 10 minutes after the measurement was started. A calibration curve showing the relationship between this absorbance difference and the fibrinogen (Fgn) concentration (μg / ml) is shown in FIG. As is clear from FIG. 3, it can be seen that a calibration curve having good linearity can be obtained.
【００１７】実施例４．市販の金コロイド凝集反応法用
試薬（イムノゴールド Ｈｅｍ：和光純薬工業(株)製）
を用いて、ヘモグロビンの比色分析を行った。
（金コロイド試液）イムノゴールド Ｈｅｍの金コロイト゛試
薬（2.5ml用）１瓶を、同キットの金コロイト゛試液溶解液5.
0mlで溶解したものを金コロイト゛試液とした。
（ヘモグロビン標準液）所定濃度のヘモグロビンを含む
50mM リン酸緩衝液（pＨ7.4）溶液をヘモグロビン標準
液とした。
（使用装置）自動分析装置スーパーＺ８２８（ニッテク
社製）を一部改良して用いた。
（操作法）上記自動分析装置を下記の測定条件に設定し
て測定を行った。
・サンプル採取量 83μl。
・金コロイト゛試液採取量 200μl。
・測定波長 542/659nm。
・測定方法 ２ポイントエンドアッセイ（反応
開始後２分後の吸光度と９分後の吸光度との差を測
定。）
（結果）得られた吸光度差とヘモグロビン濃度との関係
を表わす検量線を図４に示す。図４から明らかな如く、
良好な検量線が得られることが判る。Embodiment 4. Commercially available gold colloid agglutination reaction reagent (ImmunoGold Hem: Wako Pure Chemical Industries, Ltd.)
Was used for colorimetric analysis of hemoglobin. (Gold colloidal test solution) 1 bottle of Immunogold Hem's gold colloid reagent (for 2.5 ml) was added to the same solution of the gold colloid reagent of the kit 5.
What was melt | dissolved in 0 ml was made into the gold-colloid test liquid. (Hemoglobin standard solution) Contains a predetermined concentration of hemoglobin
A 50 mM phosphate buffer solution (pH 7.4) was used as a hemoglobin standard solution. (Apparatus to be used) An automatic analyzer Super Z828 (manufactured by Nitteku Co., Ltd.) was partially modified and used. (Operation method) The above-mentioned automatic analyzer was set under the following measurement conditions to perform measurement.・ Sample volume 83 μl.・ Amount of gold test solution collected is 200 μl.・ Measurement wavelength 542/659 nm. -Measurement method 2-point end assay (measurement of the difference between the absorbance 2 minutes after the start of the reaction and the absorbance 9 minutes after the reaction was started.) (Results) A calibration curve showing the relationship between the obtained absorbance difference and the hemoglobin concentration is shown in FIG. Shown in. As is clear from FIG.
It can be seen that a good calibration curve can be obtained.
【００１８】実施例５．ヒト糞便中のヘモグロビンの検
出
（金コロイド試液）実施例４と同じ。
（サンプル）新鮮ヒト糞便67検体についてイムノゴール
ド Ｈｅｍに添付の現品説明書に従って調製された検体
抽出液をサンプルとした。
（使用装置及び操作法）実施例４と同じ。
（結果）得られた吸光度差が0.06以上のものを陽性、0.
06未満のものを陰性として判定を行った結果を表１及び
表２に示す。Example 5. Detection of hemoglobin in human feces (gold colloidal test solution) Same as in Example 4. (Sample) For 67 samples of fresh human feces, a sample extract prepared according to the instruction manual attached to Immunogold Hem was used. (Apparatus used and operation method) The same as in Example 4. (Result) Positive results with an absorbance difference of 0.06 or more, 0.
Tables 1 and 2 show the results of the judgment with negative values less than 06.
【００１９】参考例１．実施例５で使用した新鮮ヒト糞
便67検体について、イムノゴールド Ｈｅｍを使用して
ヘモグロビンの検出を行った。尚、測定操作は、該キッ
トに添付の現品説明書に記載の標準操作法により行い、
判定は習熟した検査員により目視で行われたものであ
る。得られた結果を表１に併せて示す。Reference Example 1. Hemoglobin was detected in 67 samples of the fresh human feces used in Example 5 by using Immunogold Hem. In addition, the measurement operation is performed by the standard operation method described in the item description attached to the kit,
The judgment was made visually by a trained inspector. The obtained results are also shown in Table 1.
【００２０】参考例２．実施例５で使用した新鮮ヒト糞
便67検体について、ＯＣ-ヘモディア‘栄研’（栄研化
学(株)登録商標）を使用してヘモグロビンの検出を行っ
た。尚、測定操作は、該キットに添付の現品説明書に記
載の標準操作法により行い、判定は習熟した検査員によ
り目視で行われたものである。得られた結果を表２に併
せて示す。Reference Example 2. The 67 samples of fresh human feces used in Example 5 were subjected to detection of hemoglobin using OC-hemodia'Eiken '(registered trademark of Eiken Chemical Co., Ltd.). The measurement operation was performed by the standard operation method described in the instruction manual attached to the kit, and the determination was visually performed by a trained inspector. The obtained results are also shown in Table 2.
【００２１】[0021]
【表１】 [Table 1]
【００２２】[0022]
【表２】
表１及び表２の結果から明らかな如く、本発明の方法に
より得られる判定結果は、習熟した検査員により目視判
定されたものと良く一致することが判る。[Table 2] As is clear from the results of Tables 1 and 2, it can be seen that the judgment results obtained by the method of the present invention are in good agreement with those visually judged by a trained inspector.
【００２３】[0023]
【発明の効果】以上述べた如く、本発明は、自動分析装
置等への応用が可能な金コロイド粒子を用いた比色分析
法を提供するものであり、従来法に於ける問題点、例え
ば目視による判定で生じていた、例えば測定場所の照
度、照明に用いる光源の種類等の外的因子や例えば判定
者の経験度や習熟度或は体調等の人的因子に起因する判
定の不一致等を回避することができると共に、測定対象
物質を定量する（半定量ではなく）ことを可能にした点
に顕著な効果を奏する発明であり、斯業に貢献するとこ
ろ大なる発明である。INDUSTRIAL APPLICABILITY As described above, the present invention provides a colorimetric analysis method using colloidal gold particles which can be applied to an automatic analyzer and the like. Discrepancies in judgment caused by external factors such as the illuminance at the measurement location, the type of light source used for illumination, etc. and human factors such as the experience and proficiency of the judge or physical condition, etc. It is an invention that has a remarkable effect in that it is possible to avoid the above, and that it is possible to quantify (not semi-quantitatively) the substance to be measured, and it is a great invention that contributes to the art.
【図１】実施例１に於いて得られた、ＶmaxとIgＧ濃度
との関係を表わす検量線を示す。FIG. 1 shows a calibration curve showing the relationship between Vmax and IgG concentration, which was obtained in Example 1.
【図２】実施例２に於いて得られた、吸光度とＣＲＰ濃
度との関係を表わす検量線を示す。FIG. 2 shows a calibration curve showing the relationship between absorbance and CRP concentration obtained in Example 2.
【図３】実施例３に於いて得られた、吸光度差とフィブ
リノーゲン濃度との関係を表わす検量線を示す。FIG. 3 shows a calibration curve showing the relationship between the difference in absorbance and the concentration of fibrinogen, which was obtained in Example 3.
【図４】実施例４に於いて得られた、吸光度差とヘモグ
ロビン濃度との関係を表わす検量線を示す。FIG. 4 shows a calibration curve showing the relationship between the difference in absorbance and the hemoglobin concentration, which was obtained in Example 4.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 佐 藤 淳 一 宝塚市仁川北１−１−11 (72)発明者 熊 井 恵 尼崎市東園田町６丁目67−３ (56)参考文献 特開 昭58−211659（ＪＰ，Ａ) 「臨床病理」Ｖｏｌ．39，Ｎｏ．11, ｐｐ．1184〜1190（1991) 「臨床検査 機器・試薬」Ｖｏｌ. 13，Ｎｏ．６，ｐｐ．1097〜1104 （1990) 「日本臨床検査自動化学会会誌」Ｖｏ ｌ．13，Ｎｏ．６，ｐｐ．736〜740 （1988) ─────────────────────────────────────────────────── ─── Continued front page (72) Inventor Junichi Sato 1-1-11 Incheon North, Takarazuka (72) Inventor Megumi Kumai 67-3 Higashi Sonoda-cho, Amagasaki-shi (56) References JP-A-58-211659 (JP, A) "Clinical Pathology" Vol. 39, No. 11, pp. 1184 ~ 1190 (1991) "Clinical test equipment and reagents" Vol. 13, No. 6, pp. 1097 ~ 1104 (1990) "Journal of Japan Society for Clinical Laboratory Automation" Vo l. 13, No. 6, pp. 736-740 (1988)
Claims (2)
ロイド粒子（以下、感作金コロイド粒子と略記する。）
と、糞便中に存在するヘモグロビンとを反応させ、反応
開始後の反応液の500〜550nmの吸光度変化に基づいてヘ
モグロビンを定量することを特徴とする、糞便中のヘモ
グロビンの比色分析法。1. Colloidal gold particles to which an antibody against hemoglobin is bound (hereinafter abbreviated as sensitized gold colloidal particles).
And a hemoglobin present in feces, and quantifying hemoglobin based on the change in absorbance at 500 to 550 nm of the reaction solution after the start of the reaction, which is a colorimetric analysis method of hemoglobin in feces.
グロビンとの反応開始後、反応液の吸光度を適当な間隔
で２回測定したものの差又は反応開始後の反応液の吸光
度変化率である、請求項１に記載の分析法。2. The change in absorbance is the difference between the absorbance of the reaction solution measured twice at appropriate intervals after the reaction between the sensitized gold colloid particles and hemoglobin, or the rate of change in the absorbance of the reaction solution after the initiation of the reaction. The analysis method according to claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP26667392A JP3496684B2 (en) | 1992-09-09 | 1992-09-09 | Colorimetric analysis using colloidal gold particles |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP26667392A JP3496684B2 (en) | 1992-09-09 | 1992-09-09 | Colorimetric analysis using colloidal gold particles |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0694719A JPH0694719A (en) | 1994-04-08 |
| JP3496684B2 true JP3496684B2 (en) | 2004-02-16 |
Family
ID=17434107
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP26667392A Expired - Lifetime JP3496684B2 (en) | 1992-09-09 | 1992-09-09 | Colorimetric analysis using colloidal gold particles |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3496684B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009117510A2 (en) * | 2008-03-20 | 2009-09-24 | Abaxis, Inc. | Multi-wavelength analyses of sol-particle specific binding assays |
| CN110940805B (en) * | 2020-02-14 | 2022-11-29 | 北京纳百生物科技有限公司 | Immune colloidal gold homogeneous phase labeling method |
-
1992
- 1992-09-09 JP JP26667392A patent/JP3496684B2/en not_active Expired - Lifetime
Non-Patent Citations (3)
| Title |
|---|
| 「日本臨床検査自動化学会会誌」Ｖｏｌ．１３，Ｎｏ．６，ｐｐ．７３６〜７４０（１９８８） |
| 「臨床検査 機器・試薬」Ｖｏｌ．１３，Ｎｏ．６，ｐｐ．１０９７〜１１０４（１９９０） |
| 「臨床病理」Ｖｏｌ．３９，Ｎｏ．１１，ｐｐ．１１８４〜１１９０（１９９１） |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0694719A (en) | 1994-04-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP3033620B1 (en) | Method for the detection of the prozone effect of photometric assays | |
| US6087188A (en) | Two-site immunoassay for an antibody with chemiluminescent label and biotin bound ligand | |
| US20070243559A1 (en) | Immunoassay method | |
| US20060089810A1 (en) | Biological sample analyzing apparatus and biological sample analyzing method | |
| WO2002039114A2 (en) | Improved assay and reagents or immunological determination of analyte concentration | |
| CN108152512A (en) | Heparin-binding protein detection kit and preparation method thereof | |
| EP1801590B1 (en) | Method of assaying antigen and reagent therefor | |
| JP3194585B2 (en) | Two-site immunoassay for antibodies with chemiluminescent label and biotin-binding ligand | |
| JP3266960B2 (en) | Immunoassay using colloidal gold particles | |
| WO2009054538A1 (en) | Method and kit for measurement of acrolein adduct in sample utilizing agglutination reaction of immunological microparticle | |
| JP3496684B2 (en) | Colorimetric analysis using colloidal gold particles | |
| JP2618629B2 (en) | Specific immunological quantification method | |
| JP5294257B2 (en) | Hemoglobin measurement method and measurement kit | |
| JP7438910B2 (en) | Ferritin measurement reagent | |
| JP7483109B2 (en) | Ferritin measurement reagent | |
| JP3328053B2 (en) | Determination of antibody or antigen concentration by immunoagglutination | |
| JP3828614B2 (en) | Protein quantification by turbidimetry and turbidity analysis unaffected by excess antigen | |
| JP7483168B2 (en) | Ferritin measurement reagent | |
| Xiang et al. | Adapting dried blood spot sampling for an anti-therapeutic antibody immunogenicity assay | |
| Sheet et al. | HUMAN CD14ELISA | |
| CN117890578A (en) | Single reagent for detecting trypsinogen 2 (Try-2) and preparation method thereof | |
| Specifications | Human IL-1β ELISA Kit | |
| WO2022050264A1 (en) | Immunological measurement method | |
| Specifications | Human sCD141 ELISA Kit | |
| TW202040132A (en) | Methods for suppressing abnormal detections in immunoassays conducted with an automatic analyzer, detection methods, immunoassay reagents, and immunoassay reagent kits |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20010703 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20121128 Year of fee payment: 9 |
|
| EXPY | Cancellation because of completion of term | ||
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20121128 Year of fee payment: 9 |