JPS59212768A - Reagent for measuring protein associated with basement membrane bmap originating in human placenta - Google Patents
Reagent for measuring protein associated with basement membrane bmap originating in human placentaInfo
- Publication number
- JPS59212768A JPS59212768A JP58088037A JP8803783A JPS59212768A JP S59212768 A JPS59212768 A JP S59212768A JP 58088037 A JP58088037 A JP 58088037A JP 8803783 A JP8803783 A JP 8803783A JP S59212768 A JPS59212768 A JP S59212768A
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- Prior art keywords
- bmap
- red blood
- human
- reagent
- antibody
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57449—Specifically defined cancers of ovaries
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- Hospice & Palliative Care (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
本発明は、ヒト胎盤由来基底映関連蛋白質ＢＭＡＰ測定
用試薬に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a reagent for measuring BMAP, a basal protein associated with human placenta.
ヒト胎盤由来の蛋白質は、水にｉ」溶性の抗ｊ足と不溶
性の抗原の２群に大別される。可溶性の抗原には１２種
が検索されており、血液凝固第」因子、フェリチア等が
これに端する。Proteins derived from human placenta are roughly divided into two groups: water-soluble anti-J antigens and insoluble antigens. Twelve types of soluble antigens have been discovered, including blood coagulation factor and ferritia.
ところで、このたび本発明者らはヒト胎盤力・ら回収し
え、ケル濾過法による分子量が約ｌＯ万、等重点がｐＨ
８，５、電気泳動による易ｌ！ｌ＆ｌＪ度がβ−グログ
リン領域、ａｔ含量がヘキソースとして１０％の蛋白質
であって、卵巣癌、胞状合胞に特異性の旨いヒト胎盤由
米基底映蛋白質を見出すと共に、これをｂＭＡＰと命名
した。By the way, the present inventors have recently recovered a human placenta, which has a molecular weight of about 10,000 by the Kell filtration method, and an emphasis on pH.
8,5, Easy by electrophoresis! We found a human placenta-based basal protein that is a protein with a β-globulin region and an AT content of 10% as hexose, and is highly specific to ovarian cancer and hydatid syncytia, and named it bMAP.
当、ｄＢＭＡＰは、上述の如く卵巣癌、胞状合胞に特異
性が高いので、ＢＭＡＰの血中濃度は卵巣癌、側状合胞
の指標となりうるものである。As mentioned above, dBMAP is highly specific for ovarian cancer and hydatid syncytia, so the blood concentration of BMAP can be an indicator of ovarian cancer and lateral syncytia.
従って、本発明は、多数の被検体について、迅速かつ正
確にＢＭＡＰを測定できる試薬を提供するものであり、
その目的自体がＷ１硯な発明である。Therefore, the present invention provides a reagent that can quickly and accurately measure BMAP for a large number of analytes.
The purpose itself is a unique invention.
即ち、木発＃ｇ４Ｉｒｉ特異抗ヒ）ＢＭＡＰ抗体を動物
赤血球に感作して得た抗ヒ）ＢＭＡＰ仇体感作赤皿球を
含む逆受身赤血球凝集反応用ヒトＢＭＡＰ測定用試楽に
関する。That is, the present invention relates to a sample for measuring human BMAP for reverse passive hemagglutination reaction, which includes red plate cells sensitized to anti-human BMAP obtained by sensitizing animal red blood cells with an anti-human BMAP antibody specific to Kihatsu #g4Iri.
本発明に係る測定試薬は、各試薬をキット化しておくこ
とが便宜的でめるので、以下キット化きれたものを例と
して本発明を説明する。Since it is convenient to prepare the measurement reagents according to the present invention in the form of a kit, the present invention will be explained below using the kits as an example.
木発８Ａ試楽をキット化した場合、次のごとき各試薬に
て構成される。即ち、餐）特異抗ヒｌ−ＢＭＡＰ仇体を
動物赤血球に感作して得た仇ヒトＨＭＡＰ感作赤血球（
以下、感作赤血球という。）、（ｏ）測定用緩衝液、虻
９標牛ヒト皿漿陽性コ／トロール（以下、陽性コノトロ
ールという）及び（に）標早ヒト血漿陰性コノトロール
（以下、陰性コノトロールという。）の絹合せにて当該
キットは溝底される。When Kibatsu 8A Shoraku is made into a kit, it is composed of the following reagents. That is, human HMAP-sensitized red blood cells obtained by sensitizing animal red blood cells with a specific anti-human HMAP antibody
Hereinafter referred to as sensitized red blood cells. ), (o) measurement buffer, silk combination of nine-labeled cow human plate plasma positive co/trol (hereinafter referred to as positive cootrol) and (to) pre-labeled human plasma negative cootrol (hereinafter referred to as negative cootrol). The kit will be discontinued.
感作赤血球は、特異抗ヒトＢＭＡＰ抗体を動物赤血球に
感作させた感作赤血球がその本体をなす。The main body of sensitized red blood cells is sensitized red blood cells obtained by sensitizing animal red blood cells with a specific anti-human BMAP antibody.
得ら７′する抗血清より精製することによってＳ！造さ
ねる。当該抗血清の製造は公知の方法にて行えばよく、
たとえばυ１度精製ヒｌ−Ｂ Ｍ Ａ Ｐとフロイ／ド
の完全アジュバントの混合乳液を作り、動物の皮内に５
〜６回注射し、数週後採血を行い室温でＭ＋６１せしめ
た後、４°Ｃで一夜放置後、Ｂ Ｏ００ｒｐｍ２０分向
の遠心分離により当該抗血清が得られる。By purifying the obtained antiserum, S! Create. The antiserum may be produced by a known method,
For example, make a mixed emulsion of υ1-degree purified Hi-BMAP and Freud's complete adjuvant, and inject it into the skin of an animal for 50 minutes.
After ~6 injections, blood was collected several weeks later to reach M+61 at room temperature, left overnight at 4°C, and centrifuged at BO00 rpm for 20 minutes to obtain the antiserum.
免疫にＩｆｌいる動物によって、ヒトＢＭＡＰに対する
抗体産生能力が異ることから、感受性のＩＱ Ｉ／−１
動物件、例えば、モルモット、ウサギ、ニワトリ、ウマ
等を選ぶことが経済的に有利である。当該ＤＦ。Since the ability to produce antibodies against human BMAP differs depending on the immunized animal, the susceptible IQ I/-1
It is economically advantageous to choose animals such as guinea pigs, rabbits, chickens, horses, etc. The DF.
血清の精製け、例えば、文献Ｊ、 Ａｍ、 Ｃｈｅｍ、
Ｓｏｃ、 。Purification of serum, e.g., Reference J, Am, Chem.
Soc.
６２．８８８６ （１９４０） ： Ｆｅｄ、 Ｐｒｏ
ｃ、 、１７ 。62.8886 (1940): Fed, Pro
c, ,17.
１１１６（１９５８）に記載の方法にて行われる。1116 (1958).
抗ヒトＢＭＡＰ抗体を感作させる赤租球は、特に動物を
選ぷ心易はないか、安定でしかも感度の商い試薬を作る
ために汀、ヒツジ、ニワトリ、ヒトの０型赤血球などが
望ましい。赤血球は生理食塩水で寸分洗浄した後、ゲル
タール・アルデヒド、ポルマリノなどの処理で安定化さ
せる。The red cells used to sensitize the anti-human BMAP antibody are not particularly sensitive to animals, and in order to produce stable and sensitive reagents, type 0 red blood cells from rats, sheep, chickens, and humans are preferable. After the red blood cells are thoroughly washed with physiological saline, they are stabilized by treatment with geltal aldehyde, polymerino, etc.
このような赤血球は５〜１５μｍ程度のものが良い。積
装抗体でこれらの赤血球を感作するのけ公知（医学のあ
ゆみ７８７５９（１９７０））の方法で￥施することが
出来る。それには、赤血球と抗体とを緩衝液中で感作さ
ぜるのがよく、一般にけ亦皿球浮遊准と抗悼合有ａを混
＠−することにより行う。Such red blood cells preferably have a diameter of about 5 to 15 μm. Sensitization of these red blood cells with loaded antibodies can be carried out by a known method (Igaku no Ayumi 78759 (1970)). For this purpose, it is best to sensitize the red blood cells and antibodies in a buffer solution, which is generally done by mixing a plate suspension with an anti-inflammatory agent.
本操作は、ｐ Ｈ５，０−８，５、温度２０−６０″Ｃ
Ｔ行うのがよく、感作赤血球は保結乾譲して、たとえば
バイアル中に、好ましくはナトリウムアンドなどの如き
保存剤を添加して、封入しておくことが好ましい、。This operation was carried out at pH 5.0-8.5 and temperature 20-60″C.
The sensitized red blood cells are preferably dried and sealed in a vial, preferably with the addition of a preservative such as sodium chloride.
本発り」に関する１′−Ｊｈ性コントロールは、標準倹
は緑を作１祝するために用いられるものであり、連成は
ヒトＢＭＡＰ含有の凍結乾祿品よりなるものである。The 1'-Jh sex control for the present invention was a standard one used for cultivating green plants, and the coupling consisted of a freeze-dried product containing human BMAP.
ヒトＢ Ｍ Ａ Ｐは緩衝液に雛解して使用され、この
際例えは５〜５０ ｎＶ／ｍｔとなるように４〜６ｍ砲
Ｊ’ｌ；一段階まで小力けしで標キ快量椋の４′［戚に
使用される。Human BMAP is used by dissolving it in a buffer solution, and in this case, for example, it is 4 to 6 m gun J'l;4' [Used for relatives.
陽性コノトロールに１．たとえば／くイアル中に、好ま
しくはナトリウムアンドなとの閑存ｉｌＪを添〃１１し
−Ｃ刺入しておく。1 for positive conotrols. For example, in a vial, preferably add some sodium chloride and insert it into the vial.
陰性コノトロールは、ヒト仇ＢＭＡＰ抗体及びヒ）ＨＭ
Ａ）’が共に陽性の成体（たとえば、生理穴場ｅ、なと
）よりなり、たとえば、ノくイアル中に。Negative conotrols include human anti-BMAP antibodies and human) HM
A)' consists of both positive adults (for example, menstruation secret spot e, nato), for example, in nokuial.
好ｆ Ｌ ＜ ｉｔナトリウムアシドなどの呆仔＝ｌＪ
ｋ ｔＡ＝加して、封入しておく。Good f L <it sodium acid etc. = lJ
Add k tA= and seal.
測定用緩悔＊ば、感作赤血球、陽性コノトロール、陰性
コノトロールを重訳するために用いられるものであり、
たとえばリン酸緩衝漱よりなり、非特異的反応の生じる
のを防止するために、たとえばストローマ、動物血清な
どを添加してもよい。Measuring remission* is used to retranslate sensitized red blood cells, positive conotrols, and negative conotrols,
For example, it is made of a phosphate buffer, and to prevent non-specific reactions, stroma, animal serum, etc. may be added.
また、たとえばナトリウムアジドなどの保存剤を添加し
ておくことが好ましい。It is also preferable to add a preservative such as sodium azide.
実施例
５〜蛋白の精製ヒトＢＭＡＰを馬に免疫して抗ヒトＢＭ
ＡＰｍ消を得た。この抗血清を硫安分画、陰イオ／文換
樹脂（Ｄ Ｅ Ａ Ｅ −ｃｅｌｌｕｌｏｓｅ ）、
ケルｐ過方法等によシ精製ヒ）ＢＭＡＰ抗俸の純度を上
げた。即ち、この抗血清を生理良塩歇で２倍布釈し、硫
酸アンモニウム飽和に液の同等量を混合し、３０分間攪
拌して３０が開静置後遠心分離により沈澱を回収した。Example 5 - Purification of protein Immunization of horses with human BMAP to produce anti-human BM
Obtained AP m erasure. This antiserum was subjected to ammonium sulfate fractionation, anion/cellulose,
Purification by Kelp filtration method etc.) The purity of BMAP anti-salt was increased. That is, this antiserum was diluted twice with physiological saline, an equal amount of the solution was mixed with saturated ammonium sulfate, stirred for 30 minutes, left to stand for 30 minutes, and then centrifuged to collect the precipitate.
この沈澱をｐＨ６，８の０．０１７５ＭＩＪン酸緩衝液
に溶解して、同緩衝液で平衡化したＤｂＡＥ−セルロー
スに通して、同緩衝液でセルロース内の残准を浴出しつ
くして精製した。この射出液を減圧透析法により透析と
澱縮を行ったのち、生理賞塩水＃に度の食塩を同上緩衝
液に加えたもので、Ｇ−２００セフアデツクスを膨潤し
たり°ル全用いてゲル濾過を行い、その最初に分収した
ヒーク部分を精製ヒ）ＢＭＡＰ抗体として感作用に使用
した。This precipitate was dissolved in a 0.0175 MIJ acid buffer of pH 6.8, passed through DbAE-cellulose equilibrated with the same buffer, and the residue in the cellulose was washed out with the same buffer for purification. After dialysis and starvation of this injection solution using the vacuum dialysis method, gel filtration was performed using G-200 Sephadex, which was swollen with saline solution containing the same amount of saline added to the same buffer solution. The first fraction of Heak was used as a purified human BMAP antibody for sensitization.
感作のための動物赤血球は、ヒツジ赤血球をリン酸緩衝
液で充分に洗浄後、ゲルタールアルｔヒトを終一度０．
５％ｗ／ｖＫ添力口して約１時間室温処理し、上記緩衝
液でグルクールアルデヒドを除いて同定化赤血球を得た
。To prepare animal red blood cells for sensitization, sheep red blood cells were thoroughly washed with phosphate buffer, and then geltal alt-human was added to 0.000ml for a final time.
After adding 5% w/v K, the mixture was treated at room temperature for about 1 hour, and the glucuraldehyde was removed with the above buffer to obtain identified red blood cells.
５％ｗ／ｖ固定化赤固定化赤肉准と等旬の上、妃の精製
ヒトＢＭＡＰｈ俸（Ｅ２８ｏｎｍで０．Ｏ１１柿）全混
合し、ｐ Ｈ７，２でタノニ／酸５〜３００〜／ｄｔを
添加・攪拌後、４°Ｃ″″ｃｌ夜放置して感作し仇ヒ）
ＢＭＡＰ抗体感作赤血球を得た。そして充分に同上り／
酸緩衝欣で洗浄した。この感作血球を分注してから凍結
乾燥して試薬を得た。5% w/v immobilized red immobilized red meat and isojun, purified human BMAPH (0.011 persimmon at E28onm) were mixed completely, and at pH 7.2, tanoni/acid 5-300-/ After adding dt and stirring, leave it at 4°C for sensitization.
BMAP antibody-sensitized red blood cells were obtained. And quite the same /
Washed with acid buffer. The sensitized blood cells were dispensed and freeze-dried to obtain a reagent.
凍結乾燥後の仇ヒトＨＭＡ）’抗体感作赤血球を動物血
＠を含有する塩化ナトリウム含有等張化り／酸ｇ、備赦
（ｐＨ７，２）に２≠以上のヒツジ赤血球ストローマを
含むリン酸緩１藺故で０６５襲に調整、溶解し、ヒトＢ
ＭＡＰＶｃ対する凝果反応をマイクロプレート法で測定
した。検体中に台上−ＷるヒトＢＭＡＰ量の５ ｎｆ７
ｍｌまでの測定（仕司能でありた。After lyophilization, the enemy human HMA)' antibody-sensitized red blood cells are made isotonic with sodium chloride containing animal blood/acid (pH 7,2) and phosphoric acid containing 2≠ or more sheep red blood cell stroma. Adjusted to 065 attack due to slow 1st phase, dissolved, human B
The fruit set reaction to MAPVc was measured by a microplate method. The amount of human BMAP present in the sample on the bench was 5 nf7.
Measurement to ml
実嵐例２ キットの構成
不発り」のキットに含まれているル（薬類１ｄ、次の５
柿である。Actual Arashi Example 2: Kit configuration failure (drugs 1d, following 5) included in the kit.
It's a persimmon.
■ 抗ヒトＢＭＡＰ感作ヒツジ赤血球。■ Anti-human BMAP sensitized sheep red blood cells.
ｌバイアルの内容物は、乾燥感作ヒツジ赤血球および作
＃背りで■のリン酸緩価ｇ（ＰＢＳ）の５−をこれに加
えて懸ｌ蜀させるとき、０．５％（ｎ／ｖ ）赤血球浮
遊液が得られる。保存剤としてナトリウムアジドｌ＋η
が添加されている。The contents of the vial are 0.5% (n/v) when dried sensitized sheep red blood cells and 5-g of phosphoric acid (PBS) are added to it and suspended. ) A red blood cell suspension is obtained. Sodium azide l+η as a preservative
is added.
■ ヒトＢＭＡＰ陽性コ／トロール
ｌバイアル中、除菌濾過したヒトＨＭＡＰ ｌｄを台上
する。本溶畝のＲＰＨＡ法によるヒトＢＭＡＰ魚はｌ・
６４以上である。保存剤としてナトリウムアンドｌ〜（
０，１％）が添加ごれている。■ Place sterile-filtered human HMAP ld in a human BMAP-positive co/trol vial. The human BMAP fish obtained by the RPHA method of the main melted ridge is l.
64 or more. Sodium and l~(
0.1%) is added and contaminated.
■ 陰性コントロール・
■／９イアル中、除菌濾過した仇ヒ）ＢＭＡＩ’抗体な
らびにヒトＢＭＡＰｉ原が共に陰性の生理食塩液１ｒｎ
ｌを含有する。保存剤としてナトリウムアジドＩＩｎｇ
（０，１％）が添加されてい乙。■ Negative control - 1rn physiological saline solution that is negative for both BMAI' antibody and human BMAPi antigen after sterilization and filtration in /9 bottles
Contains l. Sodium azide IIng as preservative
(0.1%) is added.
（４） リン酸ｃ！ｉ価液・
１ノくイアル中、除菌濾過した塩化ナトリウム加等張す
ン酸緩働柩、ｐＨ７，２（ＰＢＳ ）５０−を含有する
。ｌ） Ｂ Ｓには、試験に除して丼＠異反応の生じる
の全防止するため、可溶化ストローマおよび動物血清を
配合している。(4) Phosphoric acid c! One bottle of i-valent solution contains sterile-filtered sodium chloride, isotonic acid slowing coffin, pH 7.2 (PBS) 50-. l) BS contains solubilized stroma and animal serum to completely prevent the occurrence of different reactions during the test.
組成（５０ｒｎｌ中）
リン、吸ニナトリウム（，１ｉｔｕ水） ３９Ｅ＋ｆ
ｆ＋９９／畝−カリウム １５５ｆｆ＋７
塩化ナトリウム ２２５すｎノストローマ
３ ％動物血清
１ ％
ナトリウムアシド ５０ｆｆ＋７−−リ□
−喝一一一、１．、− ＾÷−□ □−−−−−
−−−ｐＨ７，２
リノ敵級ｔｈ柩のＩＯ−は仇ヒトＢＭＡＰ仇体感件ヒツ
ジ赤血球の懸濁用に用いられ、伐りは被験液のイ】釈に
用いる。保存剤としてナトリウムアシ）’ ｌ ｎＪ（
ｔｌ、１％）を加えておくことが好ましい。Composition (in 50 rnl) Phosphorus, absorbent sodium (, 1 itu water) 39E+f
f+99/ridge-potassium 155ff+7
Sodium chloride 225n nostroma 3% animal serum
1% Sodium acid 50ff+7--li□
-Keiichi, 1. , − ＾÷−□ □−−−−−
---pH 7.2 The IO- of the Reno grade th coffin is used for suspending sheep red blood cells that are susceptible to human BMAP, and the cut is used for diluting the test solution. Sodium acid as a preservative)' l nJ(
tl, 1%) is preferably added.
■ 特異性判定のための侮認試楽：
■バイアル中、除菌濾過したｓｊ製仇ヒトＢＡｉＡＰ仇
体（ＰＨＡＩ′１ＩＪＪでｌ：５１２以上）を含む凍結
乾燥品である。これを生理食塩水の５ｍｅ′″Ｃ溶解す
る。■ Test sample for determining specificity: ■ The vial is a lyophilized product containing a sterilized and filtered human BAiAP body manufactured by SJ (PHAI'1IJJ: 1:512 or more). This is dissolved in 5 me'''C of physiological saline.
失施例３ 失施例２に示した試薬を用いて定量試断を次
の透引実施した。Missing Example 3 Using the reagent shown in Missing Example 2, the following quantitative test was carried out by diafiltration.
■ 仇ヒトＢＭＡＰ抗体感作ヒツジ赤血球を、ｌバイア
ル当たりリン酸緩衝敵５艷に懸濁する。■ Suspend human BMAP antibody-sensitized sheep red blood cells in 5 liters of phosphate buffered vial.
■ （」−プレートの各穴に、ｄｒｏｐｐｅｒ でり
／Ｄ緩衝液をそれぞれ２５μｔずつ入れる。■ (''-Pour 25 μt of dropper Deli/D buffer into each hole of the plate.
■ ｄｒｏｐｐｅｒ により、Ａ列の穴に被験液各２
５μｔずつを入れる。Ａ列の混液をｄｉｌｕｔｅｒでＢ
列＼移し、更にＢ列より０列へ移し、次々と順次移して
１列すでこれを行う。これにより被験ｇ、は、２倍段階
希釈されて診す、１列の希Ｗ＜倍数け１０２４倍となり
ている。■ Using a dropper, drop 2 test liquids into each hole in row A.
Add 5μt each. Mixed liquid in row A with diluter B
Move to column \, then move from column B to column 0, and move one after another until one column is complete. As a result, the test sample g is serially diluted 2 times and tested, so that the dilution W in one row is less than the multiple number 1024 times.
■ 別に被験液の代わりに、陽性コントロールおよび隅
柱コントロールについても、同様にして希釈系列をつく
る。■ Separately, instead of the test solution, prepare a dilution series for the positive control and corner column control in the same way.
■ ｄｒｏｐｐｅｒ を用い、すべての穴に抗ヒトＨ
ＭＡＰ抗体感作ヒツジ赤血球のり／酸緩衝１代懸７蜀孜
を２５μｔずり加える。■ Use a dropper to fill all holes with anti-human H.
Add 25 μt of MAP antibody-sensitized sheep red blood cell glue/acid buffer 1st generation 7 Shu Kei.
（６） プレートば、ｍｉｃｒｏｍｉｘｅｒで約１０
秒間振盪後、室温で２時開イノギュベートする。(6) Plate, about 10 minutes using a micromixer
After shaking for seconds, incubate at room temperature for 2 hours.
■ コノトロールの観察
陽性コノドロー・しについては、ＪａＩ材６４倍布釈以
上でべ（集を認める。陰性コノトロールは、Ａ〜Ｊの−
［べてにおいて穴の底に赤血球の沈降物を認める（測定
が良好な場合、陰性像は小さく明瞭である）。■ For conotrol observations, positive conotrols are observed with a magnification of more than 64 times the JaI material. Negative conotrols are -
[In all cases, a sediment of red blood cells is observed at the bottom of the hole (if the measurement is good, the negative image is small and clear).
■ 試験結果
被靜欣につき、陰性コノトロールと比較して凝集像を凪
める最高希釈倍数をもってヒ）ＢＭＡＰ量とした１゜
参考例１
人胎盤（４００個）の生理食塩液 懸濁液の遠心沈渣（
遠心沈渣■）を０．１Ｍ炭酸塩ｂｕｆｆｅｒ（＋）８９
．５）ｌ容でホモジナイズし、その上清に０．４Ｍ１ｉ
ｉ酸ｂａｆｆｅｒ （ｐ）１４．８ ）２Ｇｋ加えて
酸沈＃させる。沈渣を、再ひ０．１Ｍ炭酸塩ｂｕｆｆｅ
ｒ（ｐ）１９．５）２谷を加えてホモジナイズし、得ら
れ禽上信につき、３０％飽和硫′ケ分ｉｌ！１１を行う
。硫安沈澱物を０．０５Ｍ リン酸ｂｕｆｆｅｒ （
ｐ１′１７．０）に対して透析することにより、混在す
る硫安を除去したのち、ＤＥＡＥ−セルロース、カラム
クロマトグラフィーにかける。予め、０．０５Ｍ リ
ン酸ｂｕｆ ｆ ｅｒ（ｐｉ−１７，０１で平衡化して
おいた樹Ｊｍに、す／グルｔｔＸ充填したのら、Ｏ，ｌ
Ｍ リン酸１）ｕｆｆｅｒ （ｐＨ７，０）で十分洗
浄して、夾雑蛋白を除いたあと、０、 ｌ ５ ｈｘ
リン酸ｂｕｆ （ｅｒ （ｐ Ｈ７，０１で目的物を
溶量させる。のち、仇ＮＨ５（１成ヒト血清）抗体セフ
ァロースカラムによるアフイニデイ りロマトグラフイ
ーを行って、丈に混在する夾雑蛋白をカラムに吸看させ
、目的と１−るＢＭＡＰを溶出オ青鋭する。得られたＨ
ＭＡＰの精製品Ｉｉ４．５ｒでありた。■ Subject to test results, the highest dilution factor that reduces the agglutination image compared to negative Conotrol was used as the amount of BMAP.Reference Example 1 Centrifugation of a saline suspension of human placenta (400 pieces) sediment(
Centrifugal sediment ■) in 0.1M carbonate buffer (+) 89
．． 5) Homogenize in 1 volume and add 0.4M1i to the supernatant.
Add acid buffer (p) 14.8) 2Gk and precipitate with acid. The sediment was rehydrated in 0.1M carbonate buffe.
r (p) 19.5) and homogenize, and the resulting mixture contains 30% saturated sulfuric acid! Do step 11. Ammonium sulfate precipitate was added to 0.05M phosphoric acid buffer (
After removing contaminating ammonium sulfate by dialysis against p1'17.0), the mixture is subjected to DEAE-cellulose column chromatography. After filling the tree Jm which had been equilibrated with 0.05M phosphoric acid buf fer (pi-17,01) with S/Glue ttX, O,L
After thoroughly washing with M phosphoric acid 1) buffer (pH 7,0) to remove contaminant proteins, 0, l 5 hx
Dissolve the target product with phosphate buf (pH 7.01). After that, perform affine day chromatography using an anti-NH5 (adult human serum) antibody Sepharose column to absorb contaminant proteins mixed in the column into the column. The target BMAP is eluted.The obtained H
It was a purified product of MAP Ii4.5r.
参考例２
えて、ＨＭＡＰの抽出を行う。Ｂ Ｍ Ａ Ｐｆ′ｉ０
．８★Ｓ Ｄ Ｓと１．０膏ＳＤＳとの間でｉｆ＠性画
分に現わ７′Ｌる。その上清に、２０％飽相饋安沈嫂を
行う。Reference Example 2 Then, HMAP is extracted. B M A Pf'i0
．． It appears in the if@sexual fraction between 8★SDS and 1.0SDS. The supernatant was subjected to 20% enrichment and precipitation.
第１法と同様にし−Ｃ，ＩＪノ酸ｂｕｆｆｅｒで透析し
たノチ、抗ＢＭＡＰ抗体−セファロースによるアフイ二
云イ クロマトグラフィーを行って（〆離削として、３
．５Ｍ千オシアン醒ナトリウム（ｐＨ８，０）全１４１
いる）、Ｂ Ｍ Ａ Ｐを得る。得られた精製り仏Ｐｔ
ｄ、２．７？であっだ。In the same manner as in the first method, anti-BMAP antibody was dialyzed with C, IJ acid buffer, and chromatography using anti-BMAP Sepharose was performed (as a final step,
．． 5M 1,000 ocyanate sodium (pH 8,0) total 141
), obtain B M A P. Obtained purified Buddha Pt
d, 2.7? Oh yeah.
参考例３ 参考例２の１％５ＩＪＳ抽出、上的５００−を。Reference example 3 1% 5IJS extraction of Reference Example 2, above 500-.
抗ＢＭＡＰ抗皿ｆＲ（ウサギ）２５ｍｌに加えて、不溶
性の抗原−抗体・緩合物を侍る１、３．５Ｍ千オ／ア／
飯ナトリウＡ（ｐ）１８．０）でこのり合物ケＩＩＮ、
離したのう、セファデックスＧ−１５０ケル濾過により
抗原としてのＢＭＡＰと抗体と全分収ラーる。In addition to 25 ml of anti-BMAP anti-dish fR (rabbit), add 1,3.5 M 1,000 ml of insoluble antigen-antibody mixture.
Rice Natoriu A (p) 18.0) and Konori Aimono Ke IIN,
After separation, BMAP as an antigen and the antibody were completely separated by Sephadex G-150 gel filtration.
ａ＋’ｒ襞ＢＭＡＰ１ま１，５ｓｙ、精袈抗ＢＭＡＰ抗
俸は４８〜得られた。The a+'r fold BMAP1 was 1.5sy, and the seminal BMAP resistance was 48~.
ここで得られた精製ＢＭＡＰは参考＠ｌ及び２で侍られ
た精製品と同様Ｐ）ｉＡ試薬の感作用抗原として、また
、ここで得られた精装抗ＢＭＡＰ抗体は、Ｒｉ’ｆ（Ａ
試薬の感作用抗体として利用し得るものである。。The purified BMAP obtained here was used as the sensitizing antigen for the P)iA reagent, similar to the purified products served in Reference @1 and 2.
It can be used as a sensitizing antibody for reagents. .
特ｄ［−出願人 株式会社ミドリ寸字
手続袖正書（自発）
昭和５８年９月１３ｒａ
昭４１１”８ 年 箱 ｄ′Ｉ 随筆０１ｊ ８
０ ：３７号；３ 禎ｉｌｌをするり
、１１１’ｌノｌとの関１系 １、旨′Ｉ出ル１１
人ｆ１）すｌ
ｉに ’ｉ″（ｌ１４’） 株式穴（、、、ミｐす
＋７゜７？ｌｌｉ正の対象Special d[-Applicant Midori Co., Ltd. Sleeve Procedures Sleeve Letter (self-motivated) September 13ra, 1980 1980 411'' Box d'I Essay 01j 8
0: No. 37; 3 The relationship with Ill, 111'l No. 1 series 1, Uma'I output 11
person f1) sl i 'i''(l14') stock hole (,,, mips+7゜7?lli positive object
Claims (1)
物赤血球に感作して得た感作赤血球を含むことを特徴と
する一ｉ１！！受身抗体赤血琢凝集反応用ヒト胎盤由来
基底幌関連蛋白質測定用拭楽。1i1!, characterized in that it contains sensitized red blood cells obtained by sensitizing animal red blood cells with a specific anti-human placenta-derived basal ray associated protein HMAP antibody; ! Wipe for passive antibody red blood agglutination reaction and measurement of basal hood-related protein derived from human placenta.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP58088037A JPS59212768A (en) | 1983-05-18 | 1983-05-18 | Reagent for measuring protein associated with basement membrane bmap originating in human placenta |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP58088037A JPS59212768A (en) | 1983-05-18 | 1983-05-18 | Reagent for measuring protein associated with basement membrane bmap originating in human placenta |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS59212768A true JPS59212768A (en) | 1984-12-01 |
Family
ID=13931621
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP58088037A Pending JPS59212768A (en) | 1983-05-18 | 1983-05-18 | Reagent for measuring protein associated with basement membrane bmap originating in human placenta |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS59212768A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0664453A1 (en) * | 1988-12-12 | 1995-07-26 | Bard Diagnostic Sciences, Inc. | Detection of basement membrane components as diagnostic of cancer and other diseases |
| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
-
1983
- 1983-05-18 JP JP58088037A patent/JPS59212768A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0664453A1 (en) * | 1988-12-12 | 1995-07-26 | Bard Diagnostic Sciences, Inc. | Detection of basement membrane components as diagnostic of cancer and other diseases |
| US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11609043B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
| US11609042B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| US11740019B2 (en) | 2019-03-14 | 2023-08-29 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11747082B2 (en) | 2019-03-14 | 2023-09-05 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| US11815311B2 (en) | 2019-03-14 | 2023-11-14 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
| US11994343B2 (en) | 2019-03-14 | 2024-05-28 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
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