JPS6246263A - Method for quantifying prostate specific antigen and alpha1-antitrypsin composite - Google Patents
Method for quantifying prostate specific antigen and alpha1-antitrypsin compositeInfo
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- JPS6246263A JPS6246263A JP18523285A JP18523285A JPS6246263A JP S6246263 A JPS6246263 A JP S6246263A JP 18523285 A JP18523285 A JP 18523285A JP 18523285 A JP18523285 A JP 18523285A JP S6246263 A JPS6246263 A JP S6246263A
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- antibody
- antitrypsin
- gamma
- composite
- complex
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Abstract
Description
【発明の詳細な説明】
であるγ−セミノプロテイン（γ−Ｓｅｍｉｎｏｐｒｏ
ｔｅｉｎ）（以下［γ一ＳｍＪと略記する）とαｉ−ア
ンチトリプシンからなる複合体（以下「γーＳｍーαＩ
ＡＴ複合体」と略記する）の定１方法に関する。DETAILED DESCRIPTION OF THE INVENTION γ-Seminoprotein (γ-Seminopro
tein) (hereinafter abbreviated as γ-SmJ) and αi-antitrypsin (hereinafter referred to as γ-Sm-αI
This invention relates to a method for determining the composition of AT complex (hereinafter abbreviated as "AT complex").
本発明者は、血清中のγ−Ｓｍの性状を調べる目的で、
前立腺癌患者血清をゲルろ過し、γ−Ｓｍの酸素免疫測
定（ＥＩＡ）法でγ−Ｓｍを検出したところ、低分子量
（分子量約３万）と高分子１（分子量約９万）の分画に
γ−Ｓｍが検出された。The present inventor, for the purpose of investigating the properties of γ-Sm in serum,
Prostate cancer patient serum was gel-filtered and γ-Sm was detected using the γ-Sm oxygen immunoassay (EIA) method. As a result, low molecular weight (molecular weight approximately 30,000) and high molecular weight 1 (molecular weight approximately 90,000) were detected. γ-Sm was detected.
低分子量のγ一Ｓｍは精漿から単離されるγ−Ｓｍ（分
子量３３０００〜３４０００）と同じであるが、高分子
量のγ−Ｓｍは血清中に存在する蛋白と結合した複合体
ではないかと示唆された。そこで、γ−Ｓｍと複合体を
形成する血清中に存在する蛋白について検討した結果、
αｉ−アンチトリプシンがγ一Ｓｍと複合体を形成する
ことがわかった。すなわち、γ−Ｓｍは血清中において
遊離のγ一Ｓｍおよびγ一Ｓｍ一αＩＡＴＩ合体の状態
で存在している。Low molecular weight γ-Sm is the same as γ-Sm isolated from seminal plasma (molecular weight 33,000-34,000), but it is suggested that high molecular weight γ-Sm is a complex bound to proteins present in serum. It was done. Therefore, as a result of examining proteins present in serum that form complexes with γ-Sm,
It was found that αi-antitrypsin forms a complex with γ-Sm. That is, γ-Sm exists in serum in the form of free γ-Sm and γ-Sm-αIATI combination.
遊離のγ一Ｓｍおよびγ一Ｓｍ−αＩＡＴ複合体の総量
を測定するには、例えば抗γ−Ｓｍ抗体を用いたラジオ
イムノアッセイ（Ｒ Ｉ Ａ）法，ＥＩＡ法等で行うこ
とができる。この方法は、文献１日泌尿会誌、７４巻、
１３２０〜・１３２５頁、１９８３年（岡部勉他）、臨
床検査、２８巻、１７５５〜１７５８頁、１９８４年（
蒲池信−他）に報告されている。The total amount of free γ-Sm and γ-Sm-αIAT complex can be measured by, for example, a radioimmunoassay (RIA) method using an anti-γ-Sm antibody, an EIA method, or the like. This method is described in the literature 1-day Urology Journal, vol. 74,
pp. 1320-1325, 1983 (Tsutomu Okabe et al.), Clinical Examination, Vol. 28, pp. 1755-1758, 1984 (
It has been reported in Makoto Kamachi et al.
しかし、γ−３ｍ−αＩＡＴ複合体のみを測定すγ−３
ｍ抗体および抗αｉ−アンチトリプシン抗体を使用する
イムノアッセイ法を確立した。However, γ-3 which measures only the γ-3m-αIAT complex
An immunoassay method using m antibody and anti-αi-antitrypsin antibody was established.
すなわち、本発明は抗γ−３ｍ抗体ならびに抗αｉ−ア
ンチトリプシン抗体を用いることからなるγ−３ｍとα
ｉ−アンチトリプ／ンの複合体（γ−Ｓｍ−α＋ＡＴｉ
合体）の測定方法および少なくとも当該２種の抗体を含
むγ−Ｓｍ−αｔＡＴｍ合体測定用組成物である。That is, the present invention provides γ-3m and α
i-antitrypone complex (γ-Sm-α+ATi
The present invention provides a method for measuring γ-Sm-αtATm combination and a composition for measuring γ-Sm-αtATm combination containing at least the two types of antibodies.
本発明はこの分野で一般に実施されているサンドインチ
型のイムノアッセイ法によって行うことた抗αｉ−アン
チトリプシン抗体に酵素、放射性同位元素あるいは蛍光
物質の如き５″Ｈ剤で標識することにより標識抗体とし
、この２つの抗体を被検試料中のγ−３ｍ−α（ＡＴ複
合体と抗原抗体反応させ、結合した標識剤の量から当該
複合体の量を求めることができる。また、上記とは逆に
抗αｉ−アンチトリプシン抗体を不溶化抗体とし、一方
抗γ−３ｍ抗体を標識抗体としてもよい。The present invention is a labeled antibody by labeling an anti-αi-antitrypsin antibody with a 5"H reagent such as an enzyme, a radioisotope, or a fluorescent substance, which is carried out by a sandwich-type immunoassay method commonly practiced in this field. , these two antibodies are allowed to react with the γ-3m-α (AT complex) in the test sample and the antigen-antibody reaction, and the amount of the complex can be determined from the amount of bound labeling agent. Alternatively, the anti-αi-antitrypsin antibody may be used as an insolubilized antibody, while the anti-γ-3m antibody may be used as a labeled antibody.
本発明における抗γ−Ｓｍ抗体および抗αｉ−アンチト
リプシン抗体はポリクローナル抗体またはモノクローナ
ル抗体のいずれであってもよく、それぞれγ−３ｍある
いは、αｉ−アンチトリブ／ンを抗原として宿主動物（
マウス、ラット、ウサギ。The anti-γ-Sm antibody and anti-αi-antitrypsin antibody in the present invention may be either polyclonal antibodies or monoclonal antibodies, and each uses γ-3m or αi-antitrib/n as an antigen in a host animal.
Mouse, rat, rabbit.
ヤギ等）に免疫して、該動物の血清中からポリクローナ
ル抗体として得るとか、あるいは該宿主動物の膵臓細胞
とミエローマ細胞との融合細胞からモノクローナル抗体
として得る等、いずれも当該分野における既知の技術に
よって得ることができる。goat, etc.) and obtain it as a polyclonal antibody from the serum of the animal, or obtain it as a monoclonal antibody from a fused cell of pancreatic cells and myeloma cells of the host animal, either by techniques known in the art. Obtainable.
不溶化抗体を作製する際の不溶化担体は、一般のイムノ
アッセイ用の担体でよ（、任意であってよい。担体の形
状はボール吠、プレート状、 Ｉａ状。The insolubilizing carrier used to produce the insolubilized antibody may be a general immunoassay carrier (it may be of any type. The shape of the carrier may be a ball shape, a plate shape, or a Ia shape.
角状その他任意であってよく、ゲルろ過に用いる担体、
例えばセファロース４Ｂ等でもよい。担体の材質は、ポ
リスチレン、ポリアセテート、ポリカーボネート、アク
リルニトリルーブタノエンースチレン共重合体等のプラ
スチノ・り、セルロース等の紙類、その他イムノア、セ
イに従来使用されている任αの材料でよい。また、当該
担体に抗体を結合させるには、物理化学的な吸胃あるい
は化学的に結合させる等の既知の技術によって達成でき
る。A carrier used for gel filtration, which may be angular or other arbitrary shape;
For example, Sepharose 4B or the like may be used. The material of the carrier may be polystyrene, polyacetate, polycarbonate, plastino resin such as acrylonitrile-butanoene-styrene copolymer, paper such as cellulose, or any other material conventionally used for immunotherapy. . Furthermore, binding of the antibody to the carrier can be achieved by known techniques such as physicochemical absorption or chemical binding.
標識抗体は、抗体を３Ｈ９１４ｃ、 ｌ！５■、
１３１ ■等の放射性同位元素、ベルオキンダーゼ、ア
ルカリ性フォスファターゼ、β−ガラクト／ダーゼ等の
酵素。The labeled antibody is 3H914c, l! 5■,
Radioactive isotopes such as 131 ■, enzymes such as verokindase, alkaline phosphatase, β-galacto/dase, etc.
フルオレノセイン、ローダミンＢ等の蛍光物質。Fluorescent substances such as fluorenocein and rhodamine B.
イソルミノール誘導体等の発光物質等当該分野で一般的
に用いられている標識剤で常法により標識することによ
り得られる。It can be obtained by labeling with a labeling agent commonly used in the field, such as a luminescent substance such as an isoluminol derivative, by a conventional method.
次に、不溶化抗体および標識抗体を用いた本発明の実施
方法を簡単に説明する。Next, a method of carrying out the present invention using an insolubilized antibody and a labeled antibody will be briefly explained.
先ず、測定すべき試料を一定量、反応容器にとり、ラン
血清アルブミン（ＢＳＡ）、 ウンγ−グロブリンま
たは動物血清を０．１〜５％好ましくは０．５〜２％Ｂ
ＳＡを含む緩衝液を一定量加えて混和する。この場合の
緩衝液は、０．０１〜０．５Ｍのホウ酸塩緩衝液（ｐＨ
７，２〜９．０）、Ｏ，Ｏｆ〜０．５Ｍのリン酸塩緩衝
液（ｐＨ５，２〜８．５）、 ０．０１〜０．５Ｍのベ
ロナール緩衝液（ｐＨ６，８〜９．６）または０．０１
〜０．５Ｍのトリス−塩酸緩衝液（ｐ Ｈ７，２〜９．
１）等の緩衝液、あるいは０．０５〜０．５Ｍの塩化ナ
トリウム、０．０５〜０．５ Ｍの塩化カリウム等の塩
を含む上記緩衝液を使用する。次に、試料と緩衝液の混
合液に不溶化抗体（抗γ−Ｓｍ抗体または抗αｉ−アン
チトリプシン抗体を結合した担体）を加え、一定時間反
応させる。反応後、担体を場合により洗浄してから、上
記の緩衝液に溶解した標識抗体（不溶化抗体が抗γ−Ｓ
ｍ抗体の場合は、標識剤で標識した抗αｉ−アンチトリ
プシン抗体、不溶化抗体が抗αｉ−アンチトリプシン抗
体の場合は標識剤で標識した抗γ−３ｍ抗体を用いる）
と担体を一定時間反応させる。反応後、担体を洗浄して
から、担体に結合している標識抗体の標識剤の量を検出
することにより、γ−３ｍ−αＩＡＴ？３Ｊ合体の量を
知ることができる。First, a certain amount of the sample to be measured is placed in a reaction container, and 0.1 to 5% of BSA, unγ-globulin, or animal serum is added to the reaction vessel, preferably 0.5 to 2% of BSA.
Add a certain amount of a buffer containing SA and mix. The buffer in this case is a 0.01-0.5M borate buffer (pH
7,2-9.0), O,Of~0.5M phosphate buffer (pH 5,2-8.5), 0.01-0.5M veronal buffer (pH 6,8-9. 6) or 0.01
~0.5M Tris-HCl buffer (pH 7,2-9.
A buffer such as 1) or the above buffer containing a salt such as 0.05 to 0.5 M sodium chloride or 0.05 to 0.5 M potassium chloride is used. Next, an insolubilized antibody (a carrier bound to an anti-γ-Sm antibody or an anti-αi-antitrypsin antibody) is added to the sample and buffer mixture, and the mixture is allowed to react for a certain period of time. After the reaction, the carrier is optionally washed, and then the labeled antibody (insolubilized antibody is anti-γ-S) dissolved in the above buffer is added.
In the case of the m antibody, use an anti-αi-antitrypsin antibody labeled with a labeling agent, and if the insolubilized antibody is an anti-αi-antitrypsin antibody, use an anti-γ-3m antibody labeled with a labeling agent)
and the carrier for a certain period of time. After the reaction, the carrier is washed and the amount of the labeling agent of the labeled antibody bound to the carrier is detected to determine whether γ-3m-αIAT? You can know the amount of 3J coalescence.
次に、試験例によって前立腺癌患者の血清中のγ−３ｍ
−α７ＡＴ複合体の検出と、該複合体のみの測定によっ
ても前立腺癌の診断が可能であることを説明する。尚、
試験例ならびに後述する実施例において用いた抗γ−Ｓ
ｍモノクローナル抗体は文献、臨床検査、２８巻、１７
５５〜１７５８頁、１９８４年（蒲池信−他）に従って
作製したものを用いた。Next, according to a test example, γ-3m in the serum of prostate cancer patients
It will be explained that prostate cancer can be diagnosed by detecting -α7AT complex and measuring only this complex. still,
Anti-γ-S used in test examples and examples described below
m monoclonal antibodies in literature, clinical examination, vol. 28, 17
55-1758, 1984 (Nobu Kamachi et al.) was used.
試験例１前立腺題意者血清中のγ−Ｓｍ−αｉＡＴ複合
体の検出
前立腺癌患者血清３０ｍ１をセファデックスＧ−１００
カラム（５Ｘ９０ｃｍ）に添加後、０，９％塩化ナトリ
ウムを含む０．１ Ｍホウ酸塩緩衝液（ｐＨ８，０）で
溶出し、１０ｍ１ずつ分取した。各フラクンヨン１００
μｍを用いて、前出の文献、臨床検査、２８巻、１７５
５〜ＩＴ′７５８頁、１９８４年（蒲池信−他）の測定
操作法に従って抗γ−Ｓｍモノクローナル抗体結合ＡＢ
Ｓビーズとペルオキシダーゼ標識抗γ−３ｍ抗体で遊離
のγ−Ｓｍとγ−Ｓｍ−α７ＡＴ？！［合体の両方を合
わせて測定し、一方抗γ−Ｓｍモノクローナル抗体結合
ＡＢＳビーズとペルオキシダーゼ標識抗αｉ−アンチト
リプシン抗体を用いて後述する実施例（４）に従って、
γ−Ｓｍ−αＩＡＴ複合体のみを測定した。結果を第１
図に示した。Test Example 1 Detection of γ-Sm-αiAT complex in the serum of a prostate patient
After loading onto a column (5 x 90 cm), it was eluted with 0.1 M borate buffer (pH 8,0) containing 0.9% sodium chloride, and 10 ml aliquots were collected. 100 pieces each
Using μm, the above-mentioned literature, Clinical Examination, Vol. 28, 175
Anti-γ-Sm monoclonal antibody binding AB according to the measurement procedure of 5-IT'758, 1984 (Shin Kamachi et al.)
Free γ-Sm and γ-Sm-α7AT using S beads and peroxidase-labeled anti-γ-3m antibody? ! [Both the coalescence was measured together, and on the other hand, according to Example (4) described below using anti-γ-Sm monoclonal antibody-bound ABS beads and peroxidase-labeled anti-αi-antitrypsin antibody,
Only the γ-Sm-αIAT complex was measured. Results first
Shown in the figure.
この結果より、前出の文献、臨床検査、２８巻。Based on this result, the above-mentioned document, Clinical Examination, Volume 28.
１７５５〜１７５８頁、１９８４年（蒲池信−他）に従
って不溶化抗体と酵素標識抗体の両名とも抗γ−３ｍ抗
体を使用すると、遊離のγ−３ｍとγ−３ｍ−α＋Ａ
Ｔ ？Ｓ１合体の両者が検出され、一方不溶化抗体と酵
素標識抗体に、異なる抗体（抗γ−３ｍ抗体およびαｉ
−アンチトリプシン抗体）を使用することによりγ−Ｓ
ｍ−αＩＡ Ｔ 複合体のみを検出することができる。When anti-γ-3m antibodies are used for both insolubilized antibodies and enzyme-labeled antibodies according to Nobu Kamachi et al., 1984, pp. 1755-1758, free γ-3m and γ-3m-α+A
T? Both S1 conjugates were detected, while the insolubilized antibody and the enzyme-labeled antibody were combined with different antibodies (anti-γ-3m antibody and αi
-antitrypsin antibody) by using γ-S
Only the m-αIAT complex can be detected.
試験例２血清中のγ−３ｍ−αＩＡＴ１５１合体と前立
腺癌との関連の検討
正常者１９例、前立腺肥大症患者１９例および前立腺癌
患者（未治療）１９例の血清中のγ−３ｍ−αＩＡＴ複
合体を後述する実施例（４）に従って測定した。この結
果を第２図に示す。結果から明らかな如く、前立腺癌患
者血清中のγ−Ｓｍ−αＩＡＴ複合体の濃度は正常者お
よび前立腺肥大症…者よりも高ｌ！１度であった。以上
の試験例から明らかな如く、本発明のγ−Ｓｍ−αＬＡ
Ｔ複合体の測定は前立腺癌の診断に打用なものである。Test Example 2 Examination of the relationship between γ-3m-αIAT151 combination in serum and prostate cancer γ-3m-αIAT in serum of 19 normal subjects, 19 patients with benign prostatic hyperplasia, and 19 patients with prostate cancer (untreated) The composite was measured according to Example (4) described below. The results are shown in FIG. As is clear from the results, the concentration of γ-Sm-αIAT complex in the serum of prostate cancer patients is higher than that of normal subjects and subjects with benign prostatic hyperplasia! It was once. As is clear from the above test examples, the γ-Sm-αLA of the present invention
Measurement of T complex is useful in the diagnosis of prostate cancer.
次に、実施例によって本発明を説明する。Next, the present invention will be explained by examples.
実施例
（１）不溶化抗体の調整
抗γ−Ｓｍモノクローナル抗体または抗αｉ−アンチト
リプシン抗体を６・％塩化ナトリウムを含む０．１Ｍホ
ウ酸塩緩衝液（ｐＨ８，０）で１５μｇ／ｍｌとした。Example (1) Preparation of insolubilized antibody Anti-γ-Sm monoclonal antibody or anti-αi-antitrypsin antibody was adjusted to 15 μg/ml in 0.1 M borate buffer (pH 8.0) containing 6% sodium chloride.
球状（直径６．３Ｓｍｍ）のアクリルニトリル−ブタジ
ェン−スチレン（ＡＢＳ）ビーズを洗剤と蒸留水にてよ
く洗浄し、各々の抗体溶液２０ｍ１にＡＢＳビーズ１０
０個を浸した。３７°Ｃで２４時間放置後、０．９％塩
化ナトリウム液でＡＢＳビーズを洗浄し、１％タウン清
アルブミン、０．１％アジ化ナトリウムおよび０．９％
塩化ナトリウムを含む０．１Ｍホウ酸塩緩衝液（ｐＨ７
，４）に浸し４°Ｃで保存した。Wash spherical (6.3 S mm diameter) acrylonitrile-butadiene-styrene (ABS) beads thoroughly with detergent and distilled water, and add 10 ABS beads to 20 ml of each antibody solution.
0 pieces were soaked. After standing at 37°C for 24 hours, the ABS beads were washed with 0.9% sodium chloride solution, 1% town clear albumin, 0.1% sodium azide and 0.9% sodium chloride solution.
0.1 M borate buffer (pH 7) containing sodium chloride
, 4) and stored at 4°C.
（２）酵素標識抗体の調整
１）ペルオキシダーゼ標識抗γ−３ｍ抗体抗γ−Ｓｍウ
サギ血清から常法によりＦ（ａｂ’）２をＦＲＭし、２
−メルカプトエチルアミン・塩酸で還元してＦａｂ’を
得た。次にペルオキシダーゼにＮ−（４−カルボキシシ
クロヘキシルメチル）マレイミドのＮ−ハイドロキシサ
クシニミドエステルを用いてマレイミド基を導入し、Ｆ
ａｂ’のチオール基と反応させ、ペルオキシダーゼ標識
抗γ−Ｓｍ抗体を調製した。(2) Preparation of enzyme-labeled antibody 1) Peroxidase-labeled anti-γ-3m antibody Anti-γ-Sm FRM of F(ab')2 from anti-γ-Sm rabbit serum by a conventional method,
-Reduction with mercaptoethylamine/hydrochloric acid to obtain Fab'. Next, a maleimide group was introduced into peroxidase using N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl)maleimide, and F
A peroxidase-labeled anti-γ-Sm antibody was prepared by reacting with the thiol group of ab'.
＋１） ペルオキシダーゼ標識抗αＩ−アンチトリブ
ンン抗体
ベルオキンダーゼ標識抗αｉ−アンチトリプ／ン抗体（
ヤギ、カッペル社製）を適量に希釈して調整した。 ′
（３） ７− Ｓ ｍ −αＩＡＴｉ合体の調製ヒト
血清６ｍｌにｒ−Ｓｍ溶液（２，６ｍｇ／ｍｌ）２ｍｌ
を加え、３７°Ｃで４８時間放置後、混合液をセファデ
ックスＧ−１００カラム（５Ｘ９０ｃｍ）に添加し、０
．９％塩化ナトリウムを含む０．１Ｍホウ酸緩衝、夜（
ｐＨ８，０）で溶出し、Ｓｍｌずつ分取した。γ−Ｓｍ
−αＩＡ Ｔ ｉ合体を含む分画を集め、抗γ−Ｓｍモ
ノクローナル抗体を結合したセファロース４Ｂカラム（
ＩＸｌｏｃｍ）に添加した。カラムを０．５％塩化ナト
リウムを含む０．１Ｍホウ酸塩緩衝液（ｐＨ８，０）約
５０ｍ１で洗った後、３．５Ｍ千オシアン酸カリウムを
含む０．１Ｍホウ酸塩緩衝液（ｐＨ８，０）で溶出し、
３ｍｌずつ分取した。γ−Ｓｍ−αＩＡ Ｔ 複合体を
含む分画を集めてＩＱ縮しく約Ｓｍ１）、０．９％塩化
ナトリウムを含む０．１Ｍホウ酸塩緩衝液（ｐＨ８，０
）５１に対して透析した。透析後、セフアゾ、クスＧ−
１００カラム（２％９０ｃｍ）に添加し、０．９％塩化
す） ＩＪウムを含む０．・１Ｍホウ酸塩緩衝液（ｐＨ
８，０）で溶出し、３ｍｌずつ分取した。+1) peroxidase-labeled anti-αI-antitrypton antibody peroxidase-labeled anti-αi-antitrypton antibody (
Goat (manufactured by Kappel) was diluted to an appropriate amount. ' (3) Preparation of 7-S m -αIATi combination 2 ml of r-Sm solution (2.6 mg/ml) in 6 ml of human serum
was added and left at 37°C for 48 hours, the mixture was added to a Sephadex G-100 column (5 x 90 cm) and 0.
．． 0.1 M borate buffer containing 9% sodium chloride, overnight (
It was eluted at pH 8.0) and fractionated into Sml portions. γ-Sm
- The fractions containing the αIA Ti complex were collected and applied to a Sepharose 4B column bound with an anti-γ-Sm monoclonal antibody (
IXlocm). After washing the column with approximately 50 ml of 0.1 M borate buffer (pH 8,0) containing 0.5% sodium chloride, it was washed with approximately 50 ml of 0.1 M borate buffer (pH 8,0) containing 3.5 M potassium thousandocyanate. 0),
Aliquots of 3 ml were taken. Fractions containing the γ-Sm-αIAT complex were collected, IQ approximately Sm1), and 0.1M borate buffer containing 0.9% sodium chloride (pH 8,0
) 51. After dialysis, Cefazo, Cus G-
100 column (2% 90 cm) containing 0.9% chloride).・1M borate buffer (pH
8,0) and aliquots of 3 ml were collected.
γ−３ｍ−αｉＡＴ？１合体を含む分画を集めて濃縮す
ることによりγ−Ｓｍ−α、ＡＴ複合体を得た。γ-3m-αiAT? The γ-Sm-α, AT complex was obtained by collecting and concentrating the fractions containing 1 complex.
なお、２８０ｎｍにおける吸光度を測定し、吸光度ｉ、
ｏｏｏを示す濃度を１単位／ｍｌのγ−Ｓｍ−α、Ａ
Ｔ １合体と規定した。これを基に実施例（４）におけ
るγ−Ｓｍ−αＩＡ Ｔ ？１合体の標準液を調製した
。In addition, the absorbance at 280 nm was measured, and the absorbance i,
The concentration showing ooo is 1 unit/ml γ-Sm-α, A
It was defined as T1 coalescence. Based on this, γ-Sm-αIAT? in Example (4)? A standard solution of one combination was prepared.
（４）γ−Ｓｍ−αＩＡＴ複合体の測定検体または標準
液１００μｌを試験管に取り、１％ウシ血清アルブミン
、０．３Ｍ塩化ナトリウムを含む０．１Ｍホウ酸塩緩衝
液（ｐＨ７，４）２００μｍを加え攪拌した。これに抗
γ−Ｓｍモノクローナル抗体結合ＡＢＳビーズ１個を入
れ、４°Ｃで１５時間放置した。０．０５％ツイーン２
０を含む生理食塩水４ｍｌでＡＢＳビーズを２回洗浄し
た。(4) Measurement of γ-Sm-αIAT complex Transfer 100 μl of the sample or standard solution to a test tube and add 200 μl of 0.1 M borate buffer (pH 7.4) containing 1% bovine serum albumin and 0.3 M sodium chloride. was added and stirred. One anti-γ-Sm monoclonal antibody-conjugated ABS bead was added to this, and the mixture was left at 4°C for 15 hours. 0.05% Tween 2
The ABS beads were washed twice with 4 ml of physiological saline containing 0.
ペルオキ／ダーゼｆｆ１ｍ抗αｉ−アンチトリプシン抗
体液（１％ウソ血清アルブミン、０．３Ｍ塩化ナトリウ
ムを含む０．１Ｍホウ酸塩緩衝液、ｐＨ７，４で希釈し
て調製）３００μｍを加え、室温で２時間放置した。０
．０５％ツイーン２０を含む生理食塩水４ｍｌでＡＢＳ
ビーズを洗浄した。新しい試験管に０．２％０−フェニ
レンンアミンニ塩酸塩と０．０１５％過酸化水素水を含
むクエン酸−リン酸塩緩衝液（ｐＨ６，０）ｏ、Ｓｍｌ
を取り、これに洗浄したＡＢＳビーズを入れ、室温で１
時間放置した。２Ｎ硫酸２．０ｍｌを加えて攪拌し、４
９２ｎｍにおける吸光度を測定して、標準曲線から検体
中のγ−Ｓｍ−αＩＡＴｆｆ１合体の７８度を求めた。Add 300 μm of peroxidase ff1m anti-αi-antitrypsin antibody solution (prepared by diluting with 0.1 M borate buffer containing 1% bovine serum albumin and 0.3 M sodium chloride, pH 7.4), and incubate at room temperature for 2 hours. I left it for a while. 0
．． ABS with 4 ml of physiological saline containing 05% Tween 20
Beads were washed. Citric acid-phosphate buffer (pH 6,0) containing 0.2% 0-phenyleneamine dihydrochloride and 0.015% hydrogen peroxide in a new test tube, Sml
Put the washed ABS beads into it and let it cool for 1 hour at room temperature.
I left it for a while. Add 2.0 ml of 2N sulfuric acid and stir.
The absorbance at 92 nm was measured, and 78 degrees of the γ-Sm-αIATff1 combination in the sample was determined from the standard curve.
第１図は、試験例１における前立腺癌患者面７Ｎ中の遊
離γ−３ｍとγ−Ｓｍ−αｉＡ Ｔ １合体を分離した
、クロマトグラムである。実線は、不溶化ｂｔ体として
抗γ−Ｓｍモノクローナル抗体、酵素標識抗体として抗
αｉ−アンチトリプシン抗体を用いて検出した結果を示
す。破線は、不溶化抗体、酵素標識抗体の両方に抗γ−
３ｍ抗体を用いて検出した結果を示す。
第２図は、正常者、前立腺肥大症（ＢＰＨ）患者および
前立腺癌（ＰＣａ）患者血清中のγ−Ｓｍ−αＩＡＴｉ
合体１４度（μＵｎ ｉ ｔ／ｒａりを示すグラフであ
る。
オフ図
！
す
θ ｌ−０／１０ １０ Ｊ？Ｄ
／ρＯ試、駿で６′；？
才り図
へFIG. 1 is a chromatogram showing separation of free γ-3m and γ-Sm-αiA T 1 combination in prostate cancer patient surface 7N in Test Example 1. The solid line shows the results of detection using an anti-γ-Sm monoclonal antibody as the insolubilized bt body and an anti-αi-antitrypsin antibody as the enzyme-labeled antibody. The dashed line indicates anti-γ-
The results of detection using the 3m antibody are shown. Figure 2 shows γ-Sm-αIATi in the serum of normal subjects, benign prostatic hyperplasia (BPH) patients, and prostate cancer (PCa) patients.
This is a graph showing the combined 14 degrees (μUnit/ra. Off diagram! S θ l-0/10 10 J?D
/ρO test, 6' with Shun;? To the talent map
Claims (4)
ン抗体を用いることを特徴とするγ−Ｓｍ−α＿ｉ−ア
ンチトリプシン複合体の定量方法。(1) A method for quantifying a γ-Sm-α_i-antitrypsin complex, characterized by using an anti-γ-Sm antibody and an anti-α_i-antitrypsin antibody.
ン抗体のいずれか一方が不溶化され、他方が標識化され
た抗体である特許請求の範囲第１項記載の方法。(2) The method according to claim 1, wherein either the anti-γ-Sm antibody or the anti-α_i-antitrypsin antibody is insolubilized, and the other is a labeled antibody.
チトリプシン抗体の２つを含むことを特徴とするγ−Ｓ
ｍ−α＿ｉ−アンチトリプシン複合体の定量用試薬。(3) γ-S comprising at least two anti-γ-Sm antibodies and anti-α_i-antitrypsin antibodies
A reagent for quantifying m-α_i-antitrypsin complex.
ン抗体のいずれか一方が不溶化され、他方が標識化され
た抗体である特許請求の範囲第３項記載の定量試薬。(4) The quantitative reagent according to claim 3, wherein either the anti-γ-Sm antibody or the anti-α_i-antitrypsin antibody is insolubilized and the other is a labeled antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP18523285A JPH0690203B2 (en) | 1985-08-23 | 1985-08-23 | Method for quantifying prostate-specific antigen and α-lower 1-antitrypsin complex |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP18523285A JPH0690203B2 (en) | 1985-08-23 | 1985-08-23 | Method for quantifying prostate-specific antigen and α-lower 1-antitrypsin complex |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6246263A true JPS6246263A (en) | 1987-02-28 |
| JPH0690203B2 JPH0690203B2 (en) | 1994-11-14 |
Family
ID=16167185
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP18523285A Expired - Lifetime JPH0690203B2 (en) | 1985-08-23 | 1985-08-23 | Method for quantifying prostate-specific antigen and α-lower 1-antitrypsin complex |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0690203B2 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0313863A (en) * | 1989-06-08 | 1991-01-22 | Eiken Chem Co Ltd | Method for measuring alpha1-antitrypsin in excrement using immunological latex agglutination reaction and reagent used for the method |
| US5501983A (en) * | 1990-07-23 | 1996-03-26 | Lilja; Hans | Assay of free and complexed prostate-specific antigen |
| US5614372A (en) * | 1995-02-24 | 1997-03-25 | Lilja; Hans | Early detection of prostate cancer (CAP) by employing prostate specific antigen (PSA) and human glandular kallikrein (hGK-1) |
| US5840501A (en) * | 1996-10-25 | 1998-11-24 | Bayer Corporation | Determination of cPSA |
| US5928878A (en) * | 1996-10-25 | 1999-07-27 | Bayer Corporation | Differentiation of prostate cancer from BPH by assaying PSA-ACT |
-
1985
- 1985-08-23 JP JP18523285A patent/JPH0690203B2/en not_active Expired - Lifetime
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0313863A (en) * | 1989-06-08 | 1991-01-22 | Eiken Chem Co Ltd | Method for measuring alpha1-antitrypsin in excrement using immunological latex agglutination reaction and reagent used for the method |
| US5501983A (en) * | 1990-07-23 | 1996-03-26 | Lilja; Hans | Assay of free and complexed prostate-specific antigen |
| US5912158A (en) * | 1990-07-23 | 1999-06-15 | Lilja; Hans | Prostate specific antigen (PSA)-proteinase inhibitor complexes |
| US5939533A (en) * | 1990-07-23 | 1999-08-17 | Lilja; Hans | Assay of free and complexed prostate-specific antigen (PSA) |
| US5614372A (en) * | 1995-02-24 | 1997-03-25 | Lilja; Hans | Early detection of prostate cancer (CAP) by employing prostate specific antigen (PSA) and human glandular kallikrein (hGK-1) |
| US5840501A (en) * | 1996-10-25 | 1998-11-24 | Bayer Corporation | Determination of cPSA |
| US5928878A (en) * | 1996-10-25 | 1999-07-27 | Bayer Corporation | Differentiation of prostate cancer from BPH by assaying PSA-ACT |
| US6107049A (en) * | 1996-10-25 | 2000-08-22 | Bayer Corporation | Sandwich immunoassay determination of cPSA |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0690203B2 (en) | 1994-11-14 |
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