JPH0464061A - Method and kit for detecting diabetes - Google Patents
Method and kit for detecting diabetesInfo
- Publication number
- JPH0464061A JPH0464061A JP17451890A JP17451890A JPH0464061A JP H0464061 A JPH0464061 A JP H0464061A JP 17451890 A JP17451890 A JP 17451890A JP 17451890 A JP17451890 A JP 17451890A JP H0464061 A JPH0464061 A JP H0464061A
- Authority
- JP
- Japan
- Prior art keywords
- sample
- transferrin
- albumin
- antibody
- sensitized
- 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.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 27
- 206010012601 diabetes mellitus Diseases 0.000 title claims description 17
- 102000009027 Albumins Human genes 0.000 claims abstract description 21
- 108010088751 Albumins Proteins 0.000 claims abstract description 21
- 239000002245 particle Substances 0.000 claims abstract description 20
- 239000012581 transferrin Substances 0.000 claims abstract description 20
- 102000004338 Transferrin Human genes 0.000 claims abstract description 19
- 108090000901 Transferrin Proteins 0.000 claims abstract description 19
- 210000002700 urine Anatomy 0.000 claims abstract description 17
- 238000002156 mixing Methods 0.000 claims abstract description 3
- 230000002776 aggregation Effects 0.000 claims description 9
- 238000004220 aggregation Methods 0.000 claims description 8
- 230000004520 agglutination Effects 0.000 abstract description 6
- 210000004369 blood Anatomy 0.000 abstract description 6
- 239000008280 blood Substances 0.000 abstract description 6
- 241001465754 Metazoa Species 0.000 abstract description 5
- 238000006243 chemical reaction Methods 0.000 abstract description 5
- 239000000427 antigen Substances 0.000 abstract description 4
- 102000036639 antigens Human genes 0.000 abstract description 4
- 108091007433 antigens Proteins 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 239000004816 latex Substances 0.000 description 7
- 229920000126 latex Polymers 0.000 description 7
- 230000002485 urinary effect Effects 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 4
- 238000007796 conventional method Methods 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
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000001235 sensitizing effect Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 241000287828 Gallus gallus Species 0.000 description 2
- 102000017011 Glycated Hemoglobin A Human genes 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 239000002250 absorbent Substances 0.000 description 2
- 230000002745 absorbent Effects 0.000 description 2
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- 235000013330 chicken meat Nutrition 0.000 description 2
- IXZISFNWUWKBOM-ARQDHWQXSA-N fructosamine Chemical compound NC[C@@]1(O)OC[C@@H](O)[C@@H](O)[C@@H]1O IXZISFNWUWKBOM-ARQDHWQXSA-N 0.000 description 2
- 108091005995 glycated hemoglobin Proteins 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- TUSDEZXZIZRFGC-UHFFFAOYSA-N 1-O-galloyl-3,6-(R)-HHDP-beta-D-glucose Natural products OC1C(O2)COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC1C(O)C2OC(=O)C1=CC(O)=C(O)C(O)=C1 TUSDEZXZIZRFGC-UHFFFAOYSA-N 0.000 description 1
- 239000005995 Aluminium silicate Substances 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 229910021555 Chromium Chloride Inorganic materials 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 239000001263 FEMA 3042 Substances 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 101000958041 Homo sapiens Musculin Proteins 0.000 description 1
- 101000766306 Homo sapiens Serotransferrin Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- LRBQNJMCXXYXIU-PPKXGCFTSA-N Penta-digallate-beta-D-glucose Natural products OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-PPKXGCFTSA-N 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 241000607720 Serratia Species 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- XUGUHTGSMPZQIW-UHFFFAOYSA-N [[4-(4-diazonioiminocyclohexa-2,5-dien-1-ylidene)cyclohexa-2,5-dien-1-ylidene]hydrazinylidene]azanide Chemical compound C1=CC(N=[N+]=[N-])=CC=C1C1=CC=C(N=[N+]=[N-])C=C1 XUGUHTGSMPZQIW-UHFFFAOYSA-N 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 238000005054 agglomeration Methods 0.000 description 1
- 235000012211 aluminium silicate Nutrition 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- QSWDMMVNRMROPK-UHFFFAOYSA-K chromium(3+) trichloride Chemical compound [Cl-].[Cl-].[Cl-].[Cr+3] QSWDMMVNRMROPK-UHFFFAOYSA-K 0.000 description 1
- 239000007822 coupling agent Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 102000046949 human MSC Human genes 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 210000003200 peritoneal cavity Anatomy 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- XNSAINXGIQZQOO-SRVKXCTJSA-N protirelin Chemical compound NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H]1NC(=O)CC1)CC1=CN=CN1 XNSAINXGIQZQOO-SRVKXCTJSA-N 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000004053 quinones Chemical class 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 239000000779 smoke Substances 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- LRBQNJMCXXYXIU-NRMVVENXSA-N tannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-NRMVVENXSA-N 0.000 description 1
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Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は免疫学的方法を用いた糖尿病の検出方法及び検
出用キットに関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method and kit for detecting diabetes using an immunological method.
従来、糖尿病の検出方法には種々の方法がとられてきた
。例えば尿中及び血中のグルコース量、血中グリコヘモ
グロビン量、血中フルクトサミン量を測定する方法があ
った。更に最近、尿中に出現するアルブミン量又はトラ
ンスフェリン量の増大が糖尿病マーカーの１つとして用
いられることが報告されている〔モゲンセンＣ，Ｂ、
（Ｍｏｇｅｎｓｅｎ、 Ｃ，Ｂ、 ） 、キドニー イ
ンターナショナル（Ｋｉｄｎｅｙ Ｉｎｔ、）、第３１
巻、第６７３頁（１９８７） ベルナルトＡ、 Ｍ、
（Ｂｅｒｎａｒｄ。Conventionally, various methods have been used to detect diabetes. For example, there is a method of measuring the amount of glucose in urine and blood, the amount of glycated hemoglobin in blood, and the amount of fructosamine in blood. Furthermore, it has recently been reported that an increase in the amount of albumin or transferrin that appears in the urine can be used as a diabetes marker [Mogensen C, B,
(Mogensen, C, B, ), Kidney Int., No. 31
Vol. 673 (1987) Bernard A, M.
(Bernard.
Ａ、 Ｍ、 ） ら、クリニカル ケミストリー（Ｃ
Ｉｉｎ。A, M, ) et al., Clinical Chemistry (C
Iin.
Ｃｈｅｍ、）、第３４巻、第１９２０頁（１９８８）
’）。Chem, ), vol. 34, p. 1920 (1988)
').
しかし、上記血中のグルコース、グリコヘモグロビン、
フルクトサミンの測定には採血という操作が必要であり
、大量試料が処理される集団検診には、その簡便性、大
量処理能力、コストの点で問題があった。また、上記尿
中のアルブミン又はトランスフェリンの測定も、インス
リン非依存性とインスリン依存性糖尿病とで各陽性率に
差があり、更に両者を測定しようとすれば２種類の試薬
、キットを準備する必要があった。However, the glucose in the blood, glycated hemoglobin,
Measuring fructosamine requires the operation of blood sampling, and there have been problems in terms of simplicity, mass processing capacity, and cost for mass screenings that process large volumes of samples. In addition, when measuring albumin or transferrin in the urine, there is a difference in the positive rate between insulin-independent and insulin-dependent diabetes, and two types of reagents and kits must be prepared to measure both. was there.
更に尿中のグルコース量を含め以上の方法は各々糖尿病
の一病変を反映はしているものの単独で糖尿病の一次マ
ススクリーニング方法として利用できるものではなかっ
た。Furthermore, although each of the above methods including the amount of glucose in the urine reflects one lesion of diabetes, they cannot be used alone as a primary mass screening method for diabetes.
本発明の目的は、精度が高く、糖尿病の診断効率が高く
、かつ簡便で大量試料が処理できる安価な糖尿病の検出
方法及びキットを提供することにある。An object of the present invention is to provide an inexpensive method and kit for detecting diabetes that has high accuracy, high efficiency in diagnosing diabetes, and is simple and capable of processing a large amount of samples.
本発明を概説すれば、本発明の第１の発明は糖尿病の検
出方法に関し、ヒト尿試料をアルブミン及びトランスフ
ェリンに対する抗体を感作した担体粒子と混合し、担体
粒子の凝集状態を判定することを特徴とする。また、第
２の発明は糖尿病の検出用キットに関し、アルブミン及
びトランスフェリンに対する抗体を感作した担体粒子を
含有することを特徴とする。To summarize the present invention, the first invention relates to a method for detecting diabetes, which includes mixing a human urine sample with carrier particles sensitized with antibodies against albumin and transferrin, and determining the aggregation state of the carrier particles. Features. Furthermore, the second invention relates to a kit for detecting diabetes, which is characterized by containing carrier particles sensitized with antibodies against albumin and transferrin.
本発明者らは、上記現状にかんがみて鋭意研究を重ね、
逆受身凝集反応を利用した尿中のアルブミン及びトラン
スフェリンの検出方法を開発し、この方法が糖尿病の検
出方法及びスクリニング方法として適当であることを見
出して本発明を完成した。In view of the above-mentioned current situation, the present inventors have conducted extensive research,
The present invention was completed by developing a method for detecting albumin and transferrin in urine using a reverse passive agglutination reaction and finding that this method is suitable as a method for detecting and screening diabetes.
本発明で用いられる試料は、ヒト尿から得られるもので
あり、通常は尿そのものを、また場合によっては一定量
のろ紙などの吸収材に染みこませ、ここから抽出する方
法などによって調製し、用いることもできる。The sample used in the present invention is obtained from human urine, and is usually prepared by soaking the urine itself or, in some cases, in a certain amount of absorbent material such as filter paper, and extracting it from there. It can also be used.
抗体を作製するための抗原としてはヒト血液由来のアル
ブミン及びトランスフェリンが用いられる。Albumin and transferrin derived from human blood are used as antigens for producing antibodies.
抗体は、ウサギ、ヤギ、馬、モルモット、ニワトリなど
の温血動物に体重１ ｋｇ当り、０．３〜２ｍｇ程度の
上記抗原を１〜数回背中皮下、フットパッド、大腿筋等
にアジュバントと共に注射して、当該動物の体内に形成
させる。抗体は十分に精製して用いるのが良く、この精
製方法としてはアフィニティークロマトグラフィーが特
に有効である。The antibody is injected with an adjuvant into warm-blooded animals such as rabbits, goats, horses, guinea pigs, and chickens at a dose of 0.3 to 2 mg per 1 kg of body weight once or several times subcutaneously on the back, foot pad, thigh muscle, etc. and form within the body of the animal. Antibodies are preferably used after being sufficiently purified, and affinity chromatography is particularly effective as a method for this purification.
一方、この抗体はモノクローナル抗体とじて公知の方法
で取得することもできる。この場合には、例えばマウス
に上記抗原をアジュバントと共に数回腹腔等に注射し、
膵臓細胞を取り出してポリエチレングリコール等を用い
てマウスミエローマ細胞と融合させる。そして、融合細
胞の中から当該抗体を産生ずるものをクローニングし、
増殖させ、マウス腹腔内で培養することによりモノクロ
ーナル抗体を大量に製造することができる。On the other hand, this antibody can also be obtained by a known method as a monoclonal antibody. In this case, for example, the above antigen is injected into the abdominal cavity of a mouse several times together with an adjuvant,
Pancreatic cells are taken out and fused with mouse myeloma cells using polyethylene glycol or the like. Then, clone those that produce the antibody from among the fused cells,
Monoclonal antibodies can be produced in large quantities by multiplying and culturing in the peritoneal cavity of mice.
担体粒子は間接凝集反応用のものを用いれば良く例えば
ヒツジ、ニワ） ＩＪ等の動物の赤血球、セラチア菌な
どの微生物菌体、ゼラチン粒子、ポリスチレンラテック
ス、カオリン、炭素末などを使用することができる。Carrier particles for indirect agglutination reactions may be used, such as red blood cells of animals such as sheep and chicken (IJ), microbial cells such as Serratia bacteria, gelatin particles, polystyrene latex, kaolin, carbon powder, etc. .
アルブミン及びトランスフェリンに対する抗体を担体に
感作する方法は一般の抗体を感作する公知の方法によれ
ば良く、例えばタンニン酸、グルタルアルデヒド、ビス
ジアゾベンジジン、カルボジイミド類、キノン類、塩化
クロム類等のいわゆるカップリング剤を使用する方法、
あるいは物理吸着させる方法などによって行うことがで
きる。更に、抗アルブミン抗体及び抗トランスフェリン
抗体を適当な比で同一担体上に感作することもできるし
、また、別々に各々の抗体を感作した担体を適当な比で
混合して使用することもできる。The method for sensitizing a carrier with antibodies against albumin and transferrin may be any known method for sensitizing general antibodies, such as tannic acid, glutaraldehyde, bisdiazobenzidine, carbodiimides, quinones, chromium chloride, etc. A method using a so-called coupling agent,
Alternatively, it can be carried out by a method of physical adsorption. Furthermore, anti-albumin antibodies and anti-transferrin antibodies can be sensitized on the same carrier in appropriate ratios, or carriers sensitized with each antibody separately can be mixed in appropriate ratios. can.
こうして得られた感作粒子は通常は懸濁液で、又は常法
により凍結乾煙標品とし、必要に応じ、例えば希釈用液
、未感作担体、非特異反応吸収剤、標準液などと組合せ
、糖尿病検出用キットとして用いる。The sensitized particles obtained in this way are usually in the form of a suspension or freeze-dried smoke preparation by a conventional method, and are mixed with a diluting solution, a non-sensitized carrier, a non-specific reaction absorbent, a standard solution, etc. as necessary. The combination is used as a diabetes detection kit.
このようなキットを用いて尿試料中アルブミン及びトラ
ンスフェリンを測定する方法は逆受身反応、ラテックス
凝集方法に通常用いられる方法によれば良く、例えばマ
イクロプレートのウェルに被検試料を入れてその希釈系
列をつくり、各ウェルに前記の感作粒子を加えて混合し
、通常１〜２時間静置し、この感作粒子の凝集像を観察
し、判定すれば良い。この方法のほか、例えば被検試料
と感作粒子をスライド板上で混ぜ合せ、１〜２分後の凝
集状態を判定することもできる。A method for measuring albumin and transferrin in a urine sample using such a kit may be a method normally used for reverse passive reaction or latex agglutination. For example, a test sample is placed in a well of a microplate and its dilution series The above-mentioned sensitized particles are added to each well, mixed, and usually allowed to stand for 1 to 2 hours, and the aggregation image of the sensitized particles is observed to make a judgment. In addition to this method, it is also possible, for example, to mix the test sample and sensitized particles on a slide plate and determine the state of aggregation after 1 to 2 minutes.
これらの場合、感作粒子が凝集するときを陽性、凝集を
認めないときを陰性とする。また、この凝集状態は光電
管を使用して自動的に判定させることもできる。その場
合においては、感作粒子の凝集速度が、例えば健常人尿
試料での平均凝集速度より大きいときを陽性とすれば良
い。In these cases, the test is positive when the sensitized particles aggregate, and the test is negative when no aggregation is observed. Moreover, this aggregation state can also be automatically determined using a phototube. In that case, a positive test may be taken when the aggregation rate of the sensitized particles is higher than the average aggregation rate of, for example, a healthy human urine sample.
以下、本発明を実施例により更に具体的に説明するが、
本発明はこれら実施例により限定されるものではない。Hereinafter, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to these Examples.
実施例１
市販のウサギ抗ヒトアルブミン抗体液〔ダコ（ＯＡＫＯ
）社製）をリン酸緩衝生理食塩水（ＰＢＳ）で予め平衡
化したヒト・アルブミン固定化セファロース４Ｂカラム
（ファルマシア社製）に流し、次いで吸着画分を溶出し
、ＰＢＳに透析し、感作原の抗体を調製した。次に市販
のウサギ抗ヒトトランスフェリン抗体液（ダコ社製）を
同様にヒト・トランスフェリン固定化セファロース４Ｂ
カラムを用いて感作原の抗体を調製した。Example 1 Commercially available rabbit anti-human albumin antibody solution [OAKO
) was applied to a human albumin-immobilized Sepharose 4B column (Pharmacia) equilibrated in advance with phosphate buffered saline (PBS), and the adsorbed fraction was then eluted and dialyzed against PBS for sensitization. The original antibody was prepared. Next, a commercially available rabbit anti-human transferrin antibody solution (manufactured by Dako) was added to human transferrin-immobilized Sepharose 4B in the same manner.
The sensitizing antibody was prepared using a column.
これら抗ヒトアルブミン抗体と抗ヒトトランスフェリン
抗体を重量比で１＝３となるように混合し、この抗体混
合液をラテックス粒子（漬水化学社製）に常法により感
作した。このようにして作製したラテックス試薬の感度
はヒトアルブミンで３０μｇ７ｍｌ、ヒトトランスフェ
リンで１μｇ／ｍｌであった。These anti-human albumin antibodies and anti-human transferrin antibodies were mixed at a weight ratio of 1=3, and latex particles (manufactured by Tsukisui Kagaku Co., Ltd.) were sensitized with this antibody mixture by a conventional method. The sensitivity of the latex reagent thus prepared was 30 μg/ml for human albumin and 1 μg/ml for human transferrin.
実施例２
実施例１で調製した感作ラテツクスを用いて尿試料中ア
ルブミン及びトランスフェリン測定キットを作製した。Example 2 Using the sensitized latex prepared in Example 1, a kit for measuring albumin and transferrin in urine samples was prepared.
キットの内容（１回分）を以下に示す。The contents of the kit (one dose) are shown below.
１）抗体感作ラテックス ２０μ１２）試料
希釈液（ＰＢＳ） ２００μ１３） 陽
性コントロール
実施例３
実施例２で調製したキットを用いて健常人及び糖尿病患
者の尿中アルブミン及びトランスフェリン量を測定した
。なお同一試料について従来方法である尿中グルコース
、尿中アルブミン、尿中トランスフェリンを各々、プレ
テスト９Ａ（和光紬薬工業社製）、マイクロアルブミン
−ＨＡ テストヮコ−（和光紬薬工業社製） グバー
に、 （Ｇｕｂａｒ、 Ｋ）らの免疫比濁方法〔クリニ
カル ケミストリー、第３２巻、第１１４３頁（１９８
６） ］により測定し、両方法の関係を検討した。1) Antibody sensitized latex 20μ12) Sample diluent (PBS) 200μ13) Positive control Example 3 Using the kit prepared in Example 2, the amounts of albumin and transferrin in the urine of healthy subjects and diabetic patients were measured. The same sample was tested using conventional methods such as urinary glucose, urinary albumin, and urinary transferrin using Pretest 9A (manufactured by Wako Tsumugi Pharmaceutical Industries, Ltd.) and Microalbumin-HA Testwaco (manufactured by Wako Tsumugi Pharmaceutical Industries, Ltd.). (Gubar, K) et al. [Clinical Chemistry, Vol. 32, p. 1143 (198
6)] and examined the relationship between both methods.
キットの操作方法は以下のようにして行った。The kit was operated as follows.
１）試料１０μｌをスライド板上のサークル内にとる。1) Take 10 μl of the sample in a circle on the slide plate.
２）抗体感作ラテックス試薬を軽く振り均一にしてから
、スライド板のサークル内に１０μｍ滴下する。2) Gently shake the antibody-sensitized latex reagent to make it uniform, then drop it 10 μm into the circle on the slide plate.
３）かくはん棒でよくかくはん混合後、振とう装置にの
せ、振とう開始２分後に凝集の有無を判定する。3) After stirring thoroughly with a stirring rod, place on a shaking device and determine the presence or absence of agglomeration 2 minutes after the start of shaking.
４）同時に陽性コントロールを用いて同様に操作を行い
、陽性となることを確認する。4) At the same time, perform the same operation using a positive control and confirm that it is positive.
その結果、第１表に示すように、本発明による測定方法
は、従来の尿中グルコース測定方法、尿中アルブミン測
定方法、尿中トランスフェリン測定方法に比べ糖尿病患
者の高い陽性率を示した。As a result, as shown in Table 1, the measuring method according to the present invention showed a higher positive rate in diabetic patients than the conventional methods for measuring urinary glucose, urinary albumin, and urinary transferrin.
本発明の方法及びキットを用いることにより、尿試料中
のアルブミン及びトランスフェリンを高感度で正確かつ
簡便に検出することができる。By using the method and kit of the present invention, albumin and transferrin in a urine sample can be detected accurately, easily and with high sensitivity.
その結果、短時間に大量の試料の測定が可能となり集団
検診等における糖尿病の一次スクリーニングの労力、コ
ストが軽減される。As a result, it becomes possible to measure a large amount of samples in a short period of time, reducing the labor and cost of primary screening for diabetes in mass medical examinations and the like.
Claims (1)
対する抗体を感作した担体粒子と混合し、担体粒子の凝
集状態を判定することを特徴とする糖尿病の検出方法。 ２、糖尿病の検出用キットであって、アルブミン及びト
ランスフェリンに対する抗体を感作した担体粒子を含有
することを特徴とする糖尿病の検出用キット。[Claims] 1. A method for detecting diabetes, which comprises mixing a human urine sample with carrier particles sensitized with antibodies against albumin and transferrin, and determining the aggregation state of the carrier particles. 2. A kit for detecting diabetes, which comprises carrier particles sensitized with antibodies against albumin and transferrin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP17451890A JPH0464061A (en) | 1990-07-03 | 1990-07-03 | Method and kit for detecting diabetes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP17451890A JPH0464061A (en) | 1990-07-03 | 1990-07-03 | Method and kit for detecting diabetes |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0464061A true JPH0464061A (en) | 1992-02-28 |
Family
ID=15979922
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP17451890A Pending JPH0464061A (en) | 1990-07-03 | 1990-07-03 | Method and kit for detecting diabetes |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0464061A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102128924A (en) * | 2010-01-12 | 2011-07-20 | 上海景源医疗器械有限公司 | Agent kit for measuring urinary transferrin by latex-enhanced immunoturbidimetry and preparation method thereof |
| CN105301257A (en) * | 2015-05-02 | 2016-02-03 | 王贤俊 | Detection method for microalbuminuria (mAlb) |
-
1990
- 1990-07-03 JP JP17451890A patent/JPH0464061A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102128924A (en) * | 2010-01-12 | 2011-07-20 | 上海景源医疗器械有限公司 | Agent kit for measuring urinary transferrin by latex-enhanced immunoturbidimetry and preparation method thereof |
| CN105301257A (en) * | 2015-05-02 | 2016-02-03 | 王贤俊 | Detection method for microalbuminuria (mAlb) |
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