JPH0315701B2 - - Google Patents
Info
- Publication number
- JPH0315701B2 JPH0315701B2 JP57088523A JP8852382A JPH0315701B2 JP H0315701 B2 JPH0315701 B2 JP H0315701B2 JP 57088523 A JP57088523 A JP 57088523A JP 8852382 A JP8852382 A JP 8852382A JP H0315701 B2 JPH0315701 B2 JP H0315701B2
- Authority
- JP
- Japan
- Prior art keywords
- human hemoglobin
- denatured
- antibody
- hemoglobin
- immobilized
- 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
- 102000001554 Hemoglobins Human genes 0.000 claims description 40
- 108010054147 Hemoglobins Proteins 0.000 claims description 40
- 210000004369 blood Anatomy 0.000 claims description 14
- 239000008280 blood Substances 0.000 claims description 14
- 238000012360 testing method Methods 0.000 claims description 14
- 238000004925 denaturation Methods 0.000 claims description 9
- 230000036425 denaturation Effects 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 239000002253 acid Substances 0.000 claims description 2
- 239000003513 alkali Substances 0.000 claims description 2
- 239000000243 solution Substances 0.000 description 15
- 208000032843 Hemorrhage Diseases 0.000 description 11
- 210000003608 fece Anatomy 0.000 description 10
- 102000008015 Hemeproteins Human genes 0.000 description 9
- 108010089792 Hemeproteins Proteins 0.000 description 9
- 208000034158 bleeding Diseases 0.000 description 9
- 230000000740 bleeding effect Effects 0.000 description 9
- 229920002451 polyvinyl alcohol Polymers 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 239000004372 Polyvinyl alcohol Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- INGWEZCOABYORO-UHFFFAOYSA-N 2-(furan-2-yl)-7-methyl-1h-1,8-naphthyridin-4-one Chemical compound N=1C2=NC(C)=CC=C2C(O)=CC=1C1=CC=CO1 INGWEZCOABYORO-UHFFFAOYSA-N 0.000 description 4
- 102000003992 Peroxidases Human genes 0.000 description 4
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 108040007629 peroxidase activity proteins Proteins 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 239000000872 buffer Substances 0.000 description 3
- 238000004040 coloring Methods 0.000 description 3
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 210000001035 gastrointestinal tract Anatomy 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 210000002784 stomach Anatomy 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 2
- 102000003670 Carboxypeptidase B Human genes 0.000 description 2
- 108090000087 Carboxypeptidase B Proteins 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 210000001198 duodenum Anatomy 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- NUIURNJTPRWVAP-UHFFFAOYSA-N 3,3'-Dimethylbenzidine Chemical compound C1=C(N)C(C)=CC(C=2C=C(C)C(N)=CC=2)=C1 NUIURNJTPRWVAP-UHFFFAOYSA-N 0.000 description 1
- BMUDPLZKKRQECS-UHFFFAOYSA-K 3-[18-(2-carboxyethyl)-8,13-bis(ethenyl)-3,7,12,17-tetramethylporphyrin-21,24-diid-2-yl]propanoic acid iron(3+) hydroxide Chemical compound [OH-].[Fe+3].[N-]1C2=C(C)C(CCC(O)=O)=C1C=C([N-]1)C(CCC(O)=O)=C(C)C1=CC(C(C)=C1C=C)=NC1=CC(C(C)=C1C=C)=NC1=C2 BMUDPLZKKRQECS-UHFFFAOYSA-K 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000283977 Oryctolagus Species 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 210000000436 anus Anatomy 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 229940109738 hematin Drugs 0.000 description 1
- 238000000760 immunoelectrophoresis Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
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
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
Description
【発明の詳細な説明】
本発明は、人の糞、尿のような排泄物中の潜血
を選択的に検出し、さらに潜血中の変性度の異な
るヒトヘモグロビンを区別して検出することによ
り、消化管内の出血個所の診断に役立つ潜血反応
検査用の固定化抗体に関する。Detailed Description of the Invention The present invention selectively detects occult blood in human excreta such as human feces and urine, and furthermore distinguishes and detects human hemoglobin with different degrees of denaturation in the occult blood. This invention relates to an immobilized antibody for use in occult blood reaction tests that are useful for diagnosing bleeding sites within blood vessels.
糞中の潜血は消化管（胃、十二指腸、小腸、大
腸、直腸等）の潰瘍、癌腫等による出血と密接な
関係があるので、潜血反応検査はこれら疾病の有
力な診断指針となる。しかしながら、従来の検査
法は糞等に含まれるヘム蛋白を区別することなく
ヘマチンを分解し、そのペルオキシダーゼ活性を
測定することによつて行われているが、次のよう
な欠点がある。即ち、糞中には出血に由来するヘ
ム蛋白と食物に由来するヘム蛋白が存在するが、
この検査法では両者の識別が不可能であり、検査
前には動物性食品の摂取を禁じているものの、植
物性食品中にもチトクロール類を含むヘム蛋白が
存在するため、その影響をさけるためにペルオキ
シダーゼ活性の測定感度を下げて行われているの
が現状であり、仮に検査の結果陰性であつても信
頼性にとぼしい。また、潜血中の消化等により変
性したヘモグロビンを区別することなく測定して
いるため、出血個所が胃なのか、腸なのか、肛門
附近なのか判断できない。そこで、出血に由来す
るヘム蛋白のみを選択的に検査する方法及び消化
管内の出血個所の診断にも応用できる方法が強く
望まれている。 Since occult blood in feces is closely related to bleeding due to ulcers, carcinomas, etc. in the gastrointestinal tract (stomach, duodenum, small intestine, large intestine, rectum, etc.), the occult blood reaction test is an effective diagnostic guide for these diseases. However, conventional testing methods are performed by decomposing hematin and measuring its peroxidase activity without distinguishing between heme proteins contained in feces, etc., but they have the following drawbacks. In other words, there are heme proteins derived from hemorrhage and heme proteins derived from food in feces.
This testing method makes it impossible to distinguish between the two, and although the consumption of animal foods is prohibited before the test, heme proteins containing cytochlors are present in plant foods as well, so this is necessary to avoid the effects of heme proteins containing cytochlors. Currently, peroxidase activity is measured at a lower sensitivity, and even if the test results are negative, the reliability is low. Furthermore, since the measurement is performed without distinguishing between hemoglobin that has been denatured due to digestion in occult blood, it is not possible to determine whether the bleeding site is in the stomach, intestines, or near the anus. Therefore, there is a strong desire for a method that selectively examines only heme proteins derived from hemorrhage and a method that can also be applied to diagnosis of bleeding sites within the gastrointestinal tract.
本発明者は、出血に由来するヘム蛋白（ヘモグ
ロビン）は糞として排泄されるまでに出血個所に
より変性度を異にし、例えば肛門近くの直腸での
出血のヘモグロビン分子の蛋白構造は糞中でもそ
れ程破壊されておらず、正常ヘモグロビンに近い
球状構造（α−ヘリツクス含量約70％）である
が、胃又は十二指腸での出血のヘモグロビン分子
の蛋白構造はその高次構造が著しく破壊されて繊
維状構造（α−ヘリツクス含量０％）に変化して
いることに着目し、かつこれら変性度の異なるヘ
モグロビンのそれぞれに対する特異抗体の抗原抗
体反応を利用することによつて、糞中の変性度の
異なるヘモグロビンを区別して、さらに食物由来
のヘム蛋白による影響を排除して排泄物中の潜血
を検査することに成功した。 The present inventor has discovered that the heme protein (hemoglobin) derived from bleeding undergoes different degrees of denaturation depending on the location of the bleeding before being excreted in feces. However, the protein structure of the hemoglobin molecule resulting from bleeding in the stomach or duodenum has its higher order structure significantly destroyed and has a fibrous structure (α-helical content approximately 70%). By focusing on the fact that the α-helix content has changed to 0%) and by using the antigen-antibody reaction of specific antibodies against each of these hemoglobins with different degrees of denaturation, we can detect hemoglobins with different degrees of denaturation in feces. We succeeded in distinguishing between these two types of blood and testing for occult blood in excrement by eliminating the influence of food-derived heme proteins.
本発明は、上記の知見に基づきなされた排泄物
中の潜血反応の検査に用いるための、ヒトヘモグ
ロビンを酸、アルカリ又は酵素により変性し、未
変性又は変性度の異なるヒトヘモグロビンのそれ
ぞれに対する特異抗体を固定化した固定化抗体で
ある。 The present invention has been made based on the above findings, and has been developed by denaturing human hemoglobin with an acid, alkali, or an enzyme, and producing specific antibodies against each of undenatured and human hemoglobin with different degrees of denaturation, for use in testing for occult blood reactions in excrement. This is an immobilized antibody that has been immobilized.
未変性ヒトヘモグロビン（nHb）は例えば次の
ようにして製造される。４％クエン酸ナトリウム
液1/10容を含む人採血液を3000rpm×５分遠心
し、その沈澱の血球分画を生理食塩水で洗浄後、
約10倍容の水を加えて溶血する。これを
10000rpm×20分遠心してその上澄をとり、未変
性ヒトヘモグロビンを得る。その濃度は414nｍ
での吸収を測定することによりわかる。 Native human hemoglobin (nHb) is produced, for example, as follows. Human blood collection containing 1/10 volume of 4% sodium citrate solution was centrifuged at 3000 rpm for 5 minutes, and the precipitated blood cell fraction was washed with physiological saline.
Add about 10 times the volume of water to lyse. this
Centrifuge at 10,000 rpm for 20 minutes and collect the supernatant to obtain native human hemoglobin. Its concentration is 414nm
This can be determined by measuring the absorption at
変性ヒトヘモグロビンは例えば次のようにして
製造される。カルボキシペプチダーゼＢ変性ヒト
ヘモグロビン（cpHb）は、0.2M炭酸水素ナトリ
ウム液1.5ml中に20mgの未変性ヒトヘモグロビン
を含む液と0.1M食塩水0.5ml中に豚の膵臓から得
たカルボキシペプチダーゼB150μ／mgを含む液と
を混合し、37℃で２時間保温して変性後、凍結乾
燥して得る。また、アルカリ変性ヒトヘモグロビ
ン（adHb）は、水１ml中に未変性ヒトヘモグロ
ビン30mgを含む液を3N水酸化ナトリウム液でPH
13とし、37℃で２時間保温変性後、PBS（リン酸
緩衝液の生理食塩）液９mlを加えてPHを7.5にも
どして得る。また、酸変性ヒトヘモグロビン
（aHb）は、水１mlに未変性ヒトヘモグロビン30
mgを含む液を3N塩酸液でPH１とし、37℃で２時
間保温変性後、PBS液９mlを加えてPHを6.0にも
どして得る。 Denatured human hemoglobin is produced, for example, as follows. Carboxypeptidase B denatured human hemoglobin (cpHb) is a solution containing 20 mg of undenatured human hemoglobin in 1.5 ml of 0.2 M sodium bicarbonate solution and 150 μ/mg of carboxypeptidase B obtained from porcine pancreas in 0.5 ml of 0.1 M saline solution. The mixture is denatured by mixing with a solution containing the above ingredients, kept at 37°C for 2 hours to denature, and then freeze-dried. Alkaline-denatured human hemoglobin (adHb) can be obtained by adding a solution containing 30 mg of undenatured human hemoglobin in 1 ml of water to PH with 3N sodium hydroxide solution.
13, and after incubating and denaturing at 37°C for 2 hours, add 9 ml of PBS (phosphate buffered saline) solution to return the pH to 7.5. In addition, acid-denatured human hemoglobin (aHb) is prepared by adding 30% of undenatured human hemoglobin to 1 ml of water.
Adjust the pH of the solution containing mg to 1 with 3N hydrochloric acid solution, denature it by keeping it at 37°C for 2 hours, and then add 9 ml of PBS solution to return the pH to 6.0.
本発明において、変性度の異なるヒトヘモグロ
ビンには未変性及び上記の変性ヒトヘモグロビン
の外、種々の変性条件（例えばトリプシン、キモ
トリプシによる消化等）により製造されたものを
包服し、これら変性ヒトヘモグロビンと出血個所
により異なる検体中の変性ヒトヘモグロビンとの
相関は適宜実験により求められる。 In the present invention, human hemoglobin with different degrees of denaturation includes undenatured human hemoglobin and the above-mentioned denatured human hemoglobin, as well as those produced under various denaturing conditions (for example, digestion with trypsin, chymotrypsy, etc.), and these denatured human hemoglobins The correlation between this and denatured human hemoglobin in the specimen, which differs depending on the bleeding site, can be determined by appropriate experiments.
上記未変性又は変性ヒトヘモグロビンに対する
特異抗体は例えば次のようにして製造される。上
記の方法により得た未変性又は変性ヒトヘモグロ
ビン２mgを含む１ml液と完全フロイント・アジユ
バンド１mlを含む液を、日本白色家兎（2.5〜3.5
Kg）の背中の皮内約10ヶ所及び後足の筋肉約10ヶ
所に分けて注射する。１週間間隔で同薬量をさら
に３回注射し、最終注射２週間後に耳の静脈より
採血する。採血液から血清を分離し、この血清を
33％飽和硫安で塩析し、２時間室温に静置して
IgG分画を沈澱として採取する。この沈澱を
3000rpm×５分遠心後、生理食塩水に溶解し、
0.1M硼酸緩衝液（PH7.8）で透析し、精製抗体を
得る。抗体の精製には硫安分画を繰り返すか又は
DEAE−セフアデツクスに通すことにより、さら
に交叉性の向上及び高力価のものが得られる。得
られた抗体はウフタロニーテスト又は免疫電気泳
動によりそれぞれのヒトヘモグロビンに対する特
異性を確認することができる。 The above-mentioned specific antibody against native or denatured human hemoglobin is produced, for example, as follows. A Japanese white rabbit (2.5-3.5
Divide the injection into approximately 10 locations intradermally on the back of a person (Kg) and approximately 10 locations in the muscles of the hind legs. The same dose was injected three more times at one-week intervals, and blood was collected from the ear vein two weeks after the final injection. Separate serum from collected blood and use this serum as
Salted out with 33% saturated ammonium sulfate and left at room temperature for 2 hours.
Collect the IgG fraction as a precipitate. This precipitate
After centrifuging at 3000 rpm for 5 minutes, dissolve in physiological saline,
Dialyze with 0.1M borate buffer (PH7.8) to obtain purified antibody. For antibody purification, repeat ammonium sulfate fractionation or
By passing through DEAE-Sephadex, cross-reactivity is further improved and a high titer can be obtained. The specificity of the obtained antibodies to human hemoglobin can be confirmed by Uftalony test or immunoelectrophoresis.
上記のようにして得られた特異抗体を常法によ
り固定化する。例えば、ポリビニルアルコール
（）のような支持体を臭化シアンで処理し活性
化ポリビニルアルコール（）を得、これに上記
抗体（）を加えて抗体分子中のアミノ基と結合
させ固定化抗体（）を調整する。 The specific antibody obtained as described above is immobilized by a conventional method. For example, a support such as polyvinyl alcohol () is treated with cyanogen bromide to obtain activated polyvinyl alcohol (), and the above-mentioned antibody () is added to this and bound to the amino group in the antibody molecule to immobilize the antibody (). Adjust.
支持体としては、ポリビニルアルコール（例え
ば日本合成化学製Bovlon）の外、セルロース等
のプレート、シート又はセンイが用いられる。 As the support, in addition to polyvinyl alcohol (for example, Bovlon manufactured by Nippon Gosei Kagaku), plates, sheets, or fibers such as cellulose can be used.
この変性度の異なるヒトヘモグロビンのそれぞ
れに対する特異抗体を固定化した固定化抗体は、
例えばプレート又はシートの場合１cm角に切断し
て公知の方法で潜血反応検査に用いられる。検査
に際しては、少量の糞を水に懸濁させ、この検体
液中に前記変性度の異なるヒトヘモグロビンのそ
れぞれに対する特異抗体を固定化した１組の固定
化抗体を浸漬し、37℃でゆるやかに振とうして一
定時間保つた後、取り出し生理食塩水で洗浄して
試料を得る。それぞれの固定化抗体は検体中の対
応する未変性又は変性ヒトヘモグロビンのみと抗
原抗体反応によつて複合物を形成しており、他の
ヘム蛋白は結合しない。このようにして検体中か
ら分離し固定化抗体に結合した未変性又は変性ヒ
トヘモグロビンのそれぞれはペルオキシダーゼ法
又は放射免疫測定法により測定することにより、
各種変性ヒトヘモグロビンを区別して定量するこ
とができ、それにより消化管中出血個所を推測す
ることができる。 These immobilized antibodies are immobilized with specific antibodies for human hemoglobin with different degrees of denaturation.
For example, in the case of a plate or sheet, it is cut into 1 cm square pieces and used for occult blood reaction testing using a known method. For testing, a small amount of feces is suspended in water, and a set of immobilized antibodies with specific antibodies for each of the human hemoglobins with different degrees of denaturation is immersed in this sample solution, and then slowly incubated at 37°C. After shaking and keeping it for a certain period of time, it is taken out and washed with physiological saline to obtain a sample. Each immobilized antibody forms a complex only with the corresponding native or denatured human hemoglobin in the specimen through an antigen-antibody reaction, and does not bind to other heme proteins. Native or denatured human hemoglobin separated from the specimen and bound to the immobilized antibody in this way is measured by peroxidase method or radioimmunoassay.
Various types of denatured human hemoglobin can be distinguished and quantified, thereby making it possible to estimate the location of bleeding in the gastrointestinal tract.
ペルオキシダーゼ法は、0.5ｍＭオルトトリジ
ン、10Mホルムアミド及び80ｍＭ過酸化水素を含
む３mlの発色溶液中に、固定化抗体に検体中の変
性ヒトヘモグロビンを結合させた前記試料を入れ
２〜４分撹拌し、発色が安定した後、溶液の
620nｍの吸光度を測定する。あるいはまた、前
記試料を上記の発色試薬をしみ込ませた試験紙上
に置き、試験紙の発色を肉眼的に又は測光的に測
定する。測定結果は標準曲線又は標準着色表から
それぞれの未変性又は変性ヒトヘモグロビン量を
求めることができる。 In the peroxidase method, the sample in which denatured human hemoglobin in the specimen is bound to the immobilized antibody is placed in 3 ml of a coloring solution containing 0.5mM orthotolidine, 10M formamide, and 80mM hydrogen peroxide, and stirred for 2 to 4 minutes to develop the color. After stabilizing, the solution
Measure the absorbance at 620 nm. Alternatively, the sample is placed on a test paper impregnated with the above coloring reagent, and the color development of the test paper is measured visually or photometrically. As for the measurement results, the amount of undenatured or denatured human hemoglobin can be determined from a standard curve or a standard coloring table.
また、放射免疫測定法は、糞１ｇの懸濁液にあ
らかじめ 125Iで標識した対応する未変性又は変
性ヒトヘモグロビン（ヘモグロビン10μｇを1mCi
で標識）1μｇを加え、この懸濁液に本発明の１
組の固定化抗体を浸漬し、前述したと同様に処理
して固定化抗体に検体中の未変性又は変性ヒトヘ
モグロビン及ひ標識した未変性又は変性ヒトヘモ
グロビンを競争的に結合させ試料を得る。この糞
から分離した未変性又は変性ヒトヘモグロビンの
125Iの放射能をｒ−カウンターで測定し、標準
曲線からそれぞれの未変性又は変性ヒトヘモグロ
ビン量を求めることができる。 In the radioimmunoassay method, a suspension of 1 g of feces is mixed with the corresponding undenatured or denatured human hemoglobin (10 μg of hemoglobin is added to 1 mCi of 125 I-labeled suspension).
1 μg of the present invention was added to this suspension.
A set of immobilized antibodies is immersed and treated in the same manner as described above to competitively bind native or denatured human hemoglobin in the specimen and labeled native or denatured human hemoglobin to the immobilized antibodies to obtain a sample. Native or denatured human hemoglobin isolated from this feces.
The radioactivity of 125 I is measured with an r-counter, and the amount of native or denatured human hemoglobin can be determined from the standard curve.
実施例
ポリビニルアルコールのシート２ｇを臭化シア
ン２ｇを含む水溶液80ml中に浸し、激しく振とう
しながら3N水酸化ナトリウム液を滴下しPHを11
に調節後、ポリビニルアルコールを取出し、冷水
１で遊離臭化シアンを洗い流して活性ポリビニ
ルアルコールを得た。この活性ポリビニルアルコ
ールを未変性又は変性ヒトヘモグロビンに対する
特異抗体120mgを0.1Mホウ酸緩衝液（PH7.8）に
溶解した液に浸漬し、室温で12時間反応させて抗
体を固定化した。固定化抗体を取出し、上記緩衝
液で洗浄し、未反応の活性基を安定化させるため
に1Mエタノールアミン50ml中で室温で12時間振
とうした。得られた固定化抗体はPBS液で洗浄
し、４℃で保存する。Example: 2 g of polyvinyl alcohol sheet was immersed in 80 ml of an aqueous solution containing 2 g of cyanogen bromide, and while shaking vigorously, 3N sodium hydroxide solution was added dropwise to bring the pH to 11.
After adjusting the temperature, the polyvinyl alcohol was taken out and free cyanogen bromide was washed away with cold water 1 to obtain active polyvinyl alcohol. This activated polyvinyl alcohol was immersed in a solution in which 120 mg of a specific antibody against undenatured or denatured human hemoglobin was dissolved in 0.1 M borate buffer (PH7.8), and reacted at room temperature for 12 hours to immobilize the antibody. The immobilized antibody was removed, washed with the above buffer, and shaken for 12 hours at room temperature in 50 ml of 1M ethanolamine to stabilize unreacted active groups. The obtained immobilized antibody is washed with PBS solution and stored at 4°C.
Claims (1)
より変性し、未変性又は変性度の異なるヒトヘモ
グロビンのそれぞれに対する特異抗体を固定化し
た潜血反応検査用の固定化抗体。1. An immobilized antibody for an occult blood reaction test, in which human hemoglobin is denatured with acid, alkali, or enzyme, and specific antibodies for undenatured or human hemoglobin with different degrees of denaturation are immobilized thereon.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8852382A JPS58205854A (en) | 1982-05-25 | 1982-05-25 | Immobilized antibody for occult blood test |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8852382A JPS58205854A (en) | 1982-05-25 | 1982-05-25 | Immobilized antibody for occult blood test |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS58205854A JPS58205854A (en) | 1983-11-30 |
| JPH0315701B2 true JPH0315701B2 (en) | 1991-03-01 |
Family
ID=13945189
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP8852382A Granted JPS58205854A (en) | 1982-05-25 | 1982-05-25 | Immobilized antibody for occult blood test |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS58205854A (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60173471A (en) * | 1984-02-20 | 1985-09-06 | Konishiroku Photo Ind Co Ltd | Detection of antigen |
| CA1339952C (en) * | 1984-10-29 | 1998-07-14 | William J. Knowles | Immunoassays for denatured protein analytes, particularly hb alc, and monoclonal antibodies thereto |
| CA1292092C (en) * | 1985-08-21 | 1991-11-12 | Biotope, Inc. | Methods and devices for separating and detecting components in specific binding assays |
| JPH0240558A (en) * | 1988-07-29 | 1990-02-09 | Kyoto Ikagaku Kenkyusho:Kk | Detecting method of occult blood in feces |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS54145210A (en) * | 1978-05-01 | 1979-11-13 | Univ Rockefeller | Hemogulobin aic radioimmunoassay |
| JPS56106154A (en) * | 1980-01-17 | 1981-08-24 | Suovaniemi Finnpipette | Detection method of hemoglobin in night soil |
-
1982
- 1982-05-25 JP JP8852382A patent/JPS58205854A/en active Granted
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS54145210A (en) * | 1978-05-01 | 1979-11-13 | Univ Rockefeller | Hemogulobin aic radioimmunoassay |
| JPS56106154A (en) * | 1980-01-17 | 1981-08-24 | Suovaniemi Finnpipette | Detection method of hemoglobin in night soil |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS58205854A (en) | 1983-11-30 |
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