JP3550614B2 - Measurement method of glycated hemoglobin - Google Patents
Measurement method of glycated hemoglobin Download PDFInfo
- Publication number
- JP3550614B2 JP3550614B2 JP10619896A JP10619896A JP3550614B2 JP 3550614 B2 JP3550614 B2 JP 3550614B2 JP 10619896 A JP10619896 A JP 10619896A JP 10619896 A JP10619896 A JP 10619896A JP 3550614 B2 JP3550614 B2 JP 3550614B2
- Authority
- JP
- Japan
- Prior art keywords
- hemoglobin
- monoclonal antibody
- glycated hemoglobin
- human
- glycated
- 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 - Fee Related
Links
Images
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Description
【０００１】
【発明の属する技術分野】
本発明は、糖化ヘモグロビンの測定方法に関する。
【０００２】
【従来の技術】
糖化ヘモグロビン（ＨｂＡ１Ｃ）は、糖尿病患者において、疾病の進行度に応じ血中濃度が増加することが知られており、糖化ヘモグロビンの測定は血糖値の測定と併用して糖尿病の診断及び治療に広く利用されている。従来、糖化ヘモグロビンの測定は、検体中の糖化ヘモグロビンを直接固相に結合させ、酵素標識抗糖化ヘモグロビン抗体を反応させるエンザイムイムノアッセイ（ＥＩＡ）により行われていた〔Ｊ．Ｉｍｍｕｎｏｌ．Ｍｅｔｈｏｄｓ，９９，９５（１９８７）〕。しかしながら、この方法では、それぞれの検体中の糖化ヘモグロビンを固相に直接結合させるという前処理が必要であり、大量の検体を迅速かつ正確に測定するには不適当な方法であった。
【０００３】
【発明が解決しようとする課題】
本発明者は、簡便、迅速、かつ正確にヒト糖化ヘモグロビンの測定を行うことができる方法を開発するべく鋭意研究した結果、２種の特定のモノクローナル抗体と不溶性担体粒子との結合体の組み合わせを用いると、検体中のヘモグロビンの妨害を受けずに、糖化ヘモグロビンの特異的な測定を行うことができることを見出した。
本発明はこうした知見に基づくものである。
【０００４】
【課題を解決するための手段】
本発明は、単独では検体中の糖化ヘモグロビン及びヘモグロビンと凝集しない特性をもつ抗ヘモグロビンモノクローナル抗体結合粒子と、抗糖化ヘモグロビンモノクローナル抗体結合粒子の混合粒子と、糖化ヘモグロビンを含む検体とを接触させ、得られる凝集反応によって糖化ヘモグロビンを測定する方法に関する。
【０００５】
【発明の実施の形態】
本発明方法において使用する抗ヘモグロビンモノクローナル抗体結合粒子は、それ単独で、ヘモグロビン及び糖化ヘモグロビンを含有する検体と接触させても、ヘモグロビン及び糖化ヘモグロビンのいずれに対しても凝集反応を示さない抗体結合粒子である。また、本発明方法で使用する抗糖化ヘモグロビンモノクローナル抗体結合粒子も、それ単独で、ヘモグロビン及び糖化ヘモグロビンを含有する検体と接触させても、ヘモグロビン及び糖化ヘモグロビンのいずれに対しても、同様に凝集反応を示さない抗体結合粒子である。しかしながら、前記の抗ヘモグロビンモノクローナル抗体結合粒子と、抗糖化ヘモグロビンモノクローナル抗体結合粒子との組合せを、ヘモグロビン及び糖化ヘモグロビンを含有する検体と接触させると、ヘモグロビンに対しては凝集反応を示さないが、糖化ヘモグロビンに対しては特異的に凝集反応を示す。
【０００６】
すなわち、本発明方法において使用する前記の組合せ、すなわち、前記の抗ヘモグロビンモノクローナル抗体結合粒子と抗糖化ヘモグロビンモノクローナル抗体結合粒子との組合せは、糖化ヘモグロビン１分子に対して２個のエピトープをもつので凝集するのに対し、ヘモグロビン１分子に対して１個のエピトープしかもたないので凝集しない。従って、本発明による前記の組合せを用いると、凝集反応により、検体中の糖化ヘモグロビンを特異的に非常に迅速に測定することができる。
本発明において、検体とは、糖化ヘモグロビンを含有するおそれのある生物学的試料であれば特に限定されないが、特には、血液、血清、血漿又は尿である。なお、本明細書において、生物学的試料は特にヒトの試料であり、従って、ヘモグロビンは、特にヒトヘモグロビンであり、糖化ヘモグロビンは、特にヒト糖化ヘモグロビンである。また、抗ヘモグロビンモノクローナル抗体は、特に抗ヒトヘモグロビンモノクローナル抗体であり、抗糖化ヘモグロビンモノクローナル抗体は、特に抗ヒト糖化ヘモグロビンモノクローナル抗体である。
【０００７】
本発明方法で用いることのできる粒子は、凝集反応のモノクローナル抗体用担体として、通常使用されている水不溶性担体粒子であれば特に限定はされないが、ポリスチレン粒子が最も好ましい。ポリスチレン粒子の平均粒径（直径）も特に限定されないが、好ましくは０．１〜１μｍである。ポリスチレン粒子の平均粒径が０．１μｍ未満になると、遠心分離が困難であり、１μｍを越えると均一に分散させにくい。
【０００８】
抗ヘモグロビンモノクローナル抗体それ自体、及び抗糖化ヘモグロビンモノクローナル抗体それ自体は、従来より周知であり、それぞれ周知の方法で調製することができる。しかし、本発明方法で使用することのできる抗ヘモグロビンモノクローナル抗体は、それを粒子と結合させ、その結合粒子を単独でヘモグロビン及び糖化ヘモグロビンと反応させた場合に、いずれとも凝集反応をしないという性質をもつことが必要である。更に、この抗体のエピトープが本発明方法で使用するもう一方の抗糖化ヘモグロビンモノクローナル抗体のエピトープと相互に立体障害を起こさない位置にあることが必要である。
【０００９】
従って、本発明方法で使用することのできるモノクローナル抗体は、前記の抗ヘモグロビンモノクローナル抗体及び抗糖化ヘモグロビンモノクローナル抗体の中から、目的に沿って選択する。すなわち、各抗体を粒子と結合させ、抗ヘモグロビンモノクローナル抗体結合粒子及び抗糖化ヘモグロビンモノクローナル抗体結合粒子を調製し、それぞれ単独でヘモグロビン及び糖化ヘモグロビンと反応させ、いずれとも凝集反応をしないモノクローナル抗体を選択する。次に、選ばれたモノクローナル抗体結合粒子の中から、ヘモグロビンを含む検体と接触させても凝集しないが、糖化ヘモグロビンを含む検体と接触させると凝集を起こす、抗ヘモグロビンモノクローナル抗体結合粒子と抗糖化ヘモグロビンモノクローナル抗体結合粒子との組合せを選択する。こうして本発明方法で用いる結合粒子の組み合わせを得ることができる。
【００１０】
本発明で用いるモノクローナル抗体と不溶性担体との結合は、周知の方法で実施することができる。例えば、ラテックスと抗体含有緩衝液とを、撹拌下に混合し、遠心分離して得られる沈渣を適当な緩衝液に懸濁する。得られた感作ラテックスはこの懸濁液で保存することができる。
【００１１】
本発明方法で用いる抗ヘモグロビンモノクローナル抗体結合粒子と抗糖化ヘモグロビンモノクローナル抗体結合粒子との組合せにおいて、両者の使用比率は、特に限定はされないが、抗ヘモグロビンモノクローナル抗体結合粒子：抗糖化ヘモグロビンモノクローナル抗体結合粒子の比が、好ましくは１：１である。
また、検体に対して使用する抗ヘモグロビンモノクローナル抗体結合粒子及び抗糖化ヘモグロビンモノクローナル抗体結合粒子の添加量は、特に限定されないが、好ましくは１：１（ｖ：ｖ）である。
【００１２】
本発明方法は、スライドグラス上での肉眼による凝集検査だけでなく、自動分析機による分光学的自動分析にも適用することができる。
【００１３】
【実施例】
以下、実施例によって本発明を具体的に説明するが、これらは本発明の範囲を限定するものではない。
実施例１：ヒトヘモグロビンに対するモノクローナル抗体の作製
ヒトヘモグロビン（２ｍｇ／ｍｌ）をフロイント完全アジュバンド（体積比１：１）に充分に分散させた。この分散液１００μｌをＢａｌｂ／ｃマウスに一回免疫し、４週間後から２週間毎に、生理食塩水で１ｍｇ／ｍｌの濃度にしたヒトヘモグロビン液１００μｌにより４回免疫した。
この免疫マウスを屠殺した後、脾臓を摘出し、この脾臓より脾細胞をマウス一匹あたり０．５×１０８ 個得た。この脾細胞１．５×１０８ 個とマウスミエローマ細胞（ＳＰ ２／ｏ）３．０×１０７ 個とを、ＰＥＧ４０００（４５％）存在下で、融合させ培養した。
増殖した細胞の上清を採取し、ＥＬＩＳＡ法により抗ヒトヘモグロビン抗体の有無を調べた。該抗体が陽性の細胞を限界希釈法によりクローニングし、抗ヒトヘモグロビン抗体を産生している細胞８種を確立した。
これにより得られた細胞８種を抗ヒトヘモグロビンマウスモノクローナル抗体産生細胞（ハイブリドーマ）として、それぞれ大量に培養し、マウス腹腔中に注射した。２週間後に腹水を採取し、抗ヒトヘモグロビンモノクローナル抗体８種（Ｈ−１、Ｈ−２、Ｈ−３、Ｈ−４、Ｈ−５、Ｈ−６、Ｈ−７、Ｈ−８）を得た。得られたモノクローナル抗体の免疫グロブリンサブクラスは、以下の表１に示すとおりであった。
【００１４】
【表１】
実施例２：抗ヒトヘモグロビンモノクローナル抗体結合ラテックスの調製とそのラテックス反応性
ポリスチレンラテックス〔セラダイン社製（米国）；１０％，直径０．４８９μｍ〕０．２ｍｌを、実施例１で作製した抗ヒトヘモグロビンモノクローナル抗体それぞれを含む５０ｍＭトリス塩酸緩衝液（ｐＨ８．０）１．８ｍｌ（それぞれの抗体濃度０．９ｍｇ／ｍｌ）に混合し、マグネチックスターラーで攪拌した。
混合液を遠心分離（２０，０００ｇ×１０分間）し、０．０５％ＮａＮ３ を含む蒸留水で４回洗浄し、１ｍｇ／ｍｌのＢＳＡ（ウシ血清アルブミン）を含む０．１Ｍトリス塩酸緩衝液（ｐＨ８．０）に懸濁させ（１ｗ／ｖ％）、保存した。各感作ラテックスと種々の濃度のヒトヘモグロビンを混合し、凝集の有無を確認した。結果を表２に示す。以下の各表において「Ｌ−」は、モノクローナル抗体Ｈ−１〜Ｈ−８とラテックスとの結合体であることを意味し、「＋」は凝集有り、「−」は凝集無しを示す。
【００１６】
【表２】
【００１７】
実施例３：糖化ヘモグロビンに対するモノクローナル抗体の作製
糖化ヘモグロビンをフロイント完全アジュバンドに充分に分散させ、この分散液１００μｌ（糖化ヘモグロビン濃度１ｍｇ／ｍｌ）でマウスに３週間毎に３回免疫した。
この免疫マウスを屠殺した後、脾臓を摘出し、この脾臓から一匹あたり１．５×１０８ 個の脾細胞を得た。この脾細胞１．５×１０８ 個とマウスミエローマ細胞（ＳＰ ２／ｏ）３．０×１０７ 個とを、ＰＥＧ４０００（４５％）存在下で、融合させ培養した。
増殖した細胞の上清を採取し、ＥＬＩＳＡ法により抗ヒト糖化ヘモグロビン抗体の有無を調べた。該抗体が陽性の細胞を限界希釈法によりクローニングし、抗ヒト糖化ヘモグロビン抗体を産生している細胞１種を確立した。
これにより得られた細胞１種を抗ヒト糖化ヘモグロビンマウスモノクローナル抗体産生細胞（ハイブリドーマ）として大量に培養し、マウス腹腔中に注射した。２週間後に腹水を採取し、抗糖化ヒトヘモグロビン抗体１種（ｇＨ−１）を得た。得られたモノクローナル抗体ｇＨ−１の免疫グロブリンサブクラスは、ＩｇＧ１ κであった。
【００１８】
実施例４：抗ヒト糖化ヘモグロビンモノクローナル抗体結合ラテックスの調製とそのラテックスの反応性
ポリスチレンラテックス〔セラダイン社製（米国）；１０％，直径０．４８９μｍ〕０．２ｍｌと、実施例３で作製した抗ヒト糖化ヘモグロビンモノクローナル抗体（ｇＨ−１）を含む５０ｍＭトリス塩酸緩衝液（ｐＨ８．０）１．８ｍｌ（抗体濃度０．９ｍｇ／ｍｌ）とを混合し、マグネチックスターラーで撹拌した。
混合液を遠心分離（２０，０００ｇ×１０分間）し、０．０５％ＮａＮ３ を含む蒸留水で４回洗浄し、１ｍｇ／ｍｌのＢＳＡを含む０．１Ｍトリス塩酸緩衝液（ｐＨ８．０）に懸濁させ（１ｗ／ｖ％）、保存した。この感作ラテックスと種々の濃度のヒト糖化ヘモグロビン、又はヒトヘモグロビンとを混合し、凝集の有無を確認した。結果を表３及び表４に示す。
【００１９】
【表３】
【表４】
【００２１】
実施例５：抗ヒトヘモグロビンモノクローナル抗体結合ラテックスと抗ヒト糖化ヘモグロビンモノクローナル抗体結合ラテックスの混合ラテックスの反応性
単独ではヒトヘモグロビンと凝集しない抗ヒトヘモグロビンモノクローナル抗体ラテックスと、単独ではヒト糖化ヘモグロビン及びヒトヘモグロビンと凝集しない抗ヒト糖化ヘモグロビンモノクローナル抗体結合ラテックスを混合して、前記の抗原との反応性を調べた。結果を表５及び表６に示す。
【００２２】
【表５】
【表６】
表５及び表６に示すとおり、抗ヒト糖化ヘモグロビンモノクローナル抗体結合ラテックスＬ−ｇＨ−１と抗ヒトヘモグロビンモノクローナル抗体結合ラテックスＬ−Ｈ−２との混合ラテックスだけがヒト糖化ヘモグロビンと凝集反応を示した。この混合ラテックスを用いて、光学的測定機器ＬＰＩＡ−２００〔登録商標；三菱化学（株）〕により、検体中の糖化ヘモグロビンを分光学的反応速度法で測定した結果を図１に示す。なお、図１のＶ値とは、凝集反応速度である。図１から明らかなとおり、本発明方法によれば、自動分析装置を利用する定量も可能である。
【００２５】
【発明の効果】
本発明方法によれば、従来法に比べて遥かに短時間（１０分以内）で、検体中のヘモグロビンの妨害を受けずに、糖化ヘモグロビンの特異的な測定を行うことができる。
【図面の簡単な説明】
【図１】本発明による混合ラテックスを用いて、光学的測定機器により糖化ヘモグロビン濃度を測定した結果を示すグラフである。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for measuring glycated hemoglobin.
[0002]
[Prior art]
Glycated hemoglobin (HbA1C) is known to increase in blood concentration in diabetic patients according to the degree of disease progression. Measurement of glycated hemoglobin is widely used in the diagnosis and treatment of diabetes in combination with the measurement of blood sugar level. It's being used. Conventionally, glycated hemoglobin has been measured by an enzyme immunoassay (EIA) in which glycated hemoglobin in a sample is directly bound to a solid phase and reacted with an enzyme-labeled anti-glycated hemoglobin antibody [J. Immunol. Methods, 99, 95 (1987)]. However, this method requires a pretreatment of directly binding glycated hemoglobin in each sample to a solid phase, and is unsuitable for quickly and accurately measuring a large number of samples.
[0003]
[Problems to be solved by the invention]
The present inventors have conducted intensive studies to develop a method that can easily, quickly and accurately measure human glycated hemoglobin, and as a result, have found that a combination of a conjugate of two specific monoclonal antibodies and insoluble carrier particles can be used. It has been found that, when used, specific measurement of glycated hemoglobin can be performed without interference of hemoglobin in the sample.
The present invention is based on such findings.
[0004]
[Means for Solving the Problems]
The present invention provides a glycated hemoglobin in a sample alone and an anti-hemoglobin monoclonal antibody-bound particle having the property of not aggregating with hemoglobin, a mixed particle of anti-glycated hemoglobin monoclonal antibody-bound particles, and a sample containing glycated hemoglobin, The present invention relates to a method for measuring glycated hemoglobin by an agglutination reaction obtained.
[0005]
BEST MODE FOR CARRYING OUT THE INVENTION
The anti-hemoglobin monoclonal antibody-bound particles used in the method of the present invention, alone, when contacted with a sample containing hemoglobin and glycated hemoglobin, do not exhibit an agglutination reaction with respect to any of hemoglobin and glycated hemoglobin. It is. Further, the anti-glycated hemoglobin monoclonal antibody-bound particles used in the method of the present invention, alone, even when brought into contact with a sample containing hemoglobin and glycated hemoglobin, similarly to any of hemoglobin and glycated hemoglobin, agglutination reaction No antibody binding particles. However, when the combination of the above-described anti-hemoglobin monoclonal antibody-bound particles and the anti-glycated hemoglobin monoclonal antibody-bound particles is brought into contact with a sample containing hemoglobin and glycated hemoglobin, no agglutination reaction with hemoglobin is exhibited, but glycation is not observed. shows the specific agglutination reaction for hemoglobin.
[0006]
That is, the combination used in the method of the present invention, that is, the combination of the anti-hemoglobin monoclonal antibody-bound particles and the anti-glycated hemoglobin monoclonal antibody-bound particles has two epitopes per glycated hemoglobin molecule, and thus has an aggregation. On the other hand, since there is only one epitope per one hemoglobin molecule, no aggregation occurs. Therefore, using the above-described combination according to the present invention, glycated hemoglobin in a sample can be specifically and very quickly measured by an agglutination reaction.
In the present invention, the specimen is not particularly limited as long as it is a biological sample which may contain glycated hemoglobin, but is particularly blood, serum, plasma or urine. In the present specification, the biological sample is particularly a human sample, and therefore, hemoglobin is particularly human hemoglobin, and glycated hemoglobin is particularly human glycated hemoglobin. The anti-hemoglobin monoclonal antibody is particularly an anti-human hemoglobin monoclonal antibody, and the anti-glycated hemoglobin monoclonal antibody is particularly an anti-human glycated hemoglobin monoclonal antibody.
[0007]
The particles that can be used in the method of the present invention are not particularly limited as long as they are water-insoluble carrier particles that are generally used as a carrier for a monoclonal antibody in an agglutination reaction, but polystyrene particles are most preferred. The average particle size (diameter) of the polystyrene particles is not particularly limited, but is preferably 0.1 to 1 μm. If the average particle size of the polystyrene particles is less than 0.1 μm, centrifugation is difficult, and if it exceeds 1 μm, it is difficult to uniformly disperse the particles.
[0008]
The anti-hemoglobin monoclonal antibody itself and the anti-glycated hemoglobin monoclonal antibody itself have been known in the art, and can be prepared by well-known methods. However, the anti-hemoglobin monoclonal antibody that can be used in the method of the present invention has a property that when it is bound to particles and the bound particles are reacted alone with hemoglobin and glycated hemoglobin, they do not undergo agglutination. It is necessary to have. Furthermore, it is necessary that the epitope of this antibody be located at a position that does not cause mutual steric hindrance to the epitope of the other anti-glycated hemoglobin monoclonal antibody used in the method of the present invention.
[0009]
Therefore, the monoclonal antibody that can be used in the method of the present invention is selected from the above-mentioned anti-hemoglobin monoclonal antibody and anti-glycated hemoglobin monoclonal antibody according to the purpose. That is, each antibody is bound to the particles, anti-hemoglobin monoclonal antibody-bound particles and anti-glycated hemoglobin monoclonal antibody-bound particles are prepared, and reacted alone with hemoglobin and glycated hemoglobin, respectively, and a monoclonal antibody that does not agglutinate with any is selected. . Next, among the selected monoclonal antibody-bound particles, anti-hemoglobin monoclonal antibody-bound particles and anti-glycated hemoglobin do not aggregate when contacted with a sample containing hemoglobin, but cause aggregation when contacted with a sample containing glycated hemoglobin. Select the combination with the monoclonal antibody binding particles. Thus, a combination of the binding particles used in the method of the present invention can be obtained.
[0010]
The binding between the monoclonal antibody used in the present invention and the insoluble carrier can be carried out by a known method. For example, the latex and the antibody-containing buffer are mixed with stirring, and the precipitate obtained by centrifugation is suspended in an appropriate buffer. The resulting sensitized latex can be stored in this suspension.
[0011]
In the combination of the anti-hemoglobin monoclonal antibody-bound particles and the anti-glycated hemoglobin monoclonal antibody-bound particles used in the method of the present invention, the use ratio of both is not particularly limited, but the anti-hemoglobin monoclonal antibody-bound particles: the anti-glycated hemoglobin monoclonal antibody-bound particles Is preferably 1: 1.
The amount of the anti-hemoglobin monoclonal antibody-bound particles and anti-glycated hemoglobin monoclonal antibody-bound particles used for the specimen is not particularly limited, but is preferably 1: 1 (v: v).
[0012]
The method of the present invention can be applied not only to the agglutination test with the naked eye on a slide glass, but also to automatic spectroscopic analysis by an automatic analyzer.
[0013]
【Example】
Hereinafter, the present invention will be described specifically with reference to Examples, but these do not limit the scope of the present invention.
Example 1: Preparation of monoclonal antibody against human hemoglobin Human hemoglobin (2 mg / ml) was sufficiently dispersed in Freund's complete adjuvant (1: 1 by volume). 100 μl of this dispersion was immunized once into Balb / c mice, and every four weeks after four weeks, four times with 100 μl of human hemoglobin solution adjusted to a concentration of 1 mg / ml with physiological saline.
After sacrifice of the immunized mouse, the spleen was removed, and 0.5 × 10 8 spleen cells were obtained from the spleen per mouse. 1.5 × 10 8 spleen cells and 3.0 × 10 7 mouse myeloma cells (SP 2 / o) were fused and cultured in the presence of PEG 4000 (45%).
The supernatant of the proliferated cells was collected, and the presence or absence of an anti-human hemoglobin antibody was examined by ELISA. Cells positive for the antibody were cloned by the limiting dilution method to establish eight cells producing anti-human hemoglobin antibodies.
Eight types of the cells thus obtained were cultured in large amounts as anti-human hemoglobin mouse monoclonal antibody-producing cells (hybridomas), and injected into the peritoneal cavity of mice. Two weeks later, ascites was collected to obtain eight anti-human hemoglobin monoclonal antibodies (H-1, H-2, H-3, H-4, H-5, H-6, H-7, H-8). Was. The immunoglobulin subclasses of the obtained monoclonal antibodies were as shown in Table 1 below.
[0014]
[Table 1]
Example 2: Preparation of anti-human hemoglobin monoclonal antibody-bound latex and its latex reactivity 0.2 ml of polystyrene latex (Ceradyne (USA); 10%, 0.489 μm in diameter) was prepared in Example 1. Each of the obtained anti-human hemoglobin monoclonal antibodies was mixed with 1.8 ml of 50 mM Tris-HCl buffer (pH 8.0) (concentration of each antibody 0.9 mg / ml) and stirred with a magnetic stirrer.
The mixture was centrifuged (20,000 g × 10 minutes), washed four times with distilled water containing 0.05% NaN 3, and 0.1 M Tris-HCl buffer containing 1 mg / ml BSA (bovine serum albumin). (PH 8.0) (1 w / v%) and stored. Each sensitized latex was mixed with various concentrations of human hemoglobin, and the presence or absence of aggregation was confirmed. Table 2 shows the results. In each of the following tables, "L-" means a conjugate of the monoclonal antibodies H-1 to H-8 and latex, "+" indicates aggregation, and "-" indicates no aggregation.
[0016]
[Table 2]
[0017]
Example 3: Preparation of monoclonal antibody against glycated hemoglobin Glycated hemoglobin was sufficiently dispersed in Freund's complete adjuvant, and mice were immunized three times every three weeks with 100 µl of this dispersion (glycated hemoglobin concentration 1 mg / ml). did.
After sacrifice of the immunized mouse, the spleen was removed, and 1.5 × 10 8 spleen cells were obtained from the spleen per animal. 1.5 × 10 8 spleen cells and 3.0 × 10 7 mouse myeloma cells (SP 2 / o) were fused and cultured in the presence of PEG 4000 (45%).
The supernatant of the proliferated cells was collected, and the presence or absence of an anti-human glycated hemoglobin antibody was examined by ELISA. Cells positive for the antibody were cloned by the limiting dilution method to establish one cell producing an anti-human glycated hemoglobin antibody.
One of the cells thus obtained was cultured in large quantities as anti-human glycated hemoglobin mouse monoclonal antibody-producing cells (hybridomas) and injected into the peritoneal cavity of mice. Two weeks later, ascites was collected to obtain one kind of anti-glycated human hemoglobin antibody (gH-1). The immunoglobulin subclass of the obtained monoclonal antibody gH-1 was IgG 1 κ.
[0018]
Example 4: Preparation of anti-human glycated hemoglobin monoclonal antibody-bound latex and reactivity of the latex 0.2 ml of polystyrene latex (Ceradyne (USA); 10%, 0.489 μm in diameter) and Example 3 Was mixed with 1.8 ml of 50 mM Tris-HCl buffer (pH 8.0) (antibody concentration: 0.9 mg / ml) containing the anti-human glycated hemoglobin monoclonal antibody (gH-1) prepared in the above, and stirred with a magnetic stirrer.
The mixture is centrifuged (20,000 g × 10 minutes), washed four times with distilled water containing 0.05% NaN 3, and 0.1 M Tris-HCl buffer (pH 8.0) containing 1 mg / ml BSA. (1 w / v%) and stored. This sensitized latex was mixed with various concentrations of human glycated hemoglobin or human hemoglobin, and the presence or absence of aggregation was confirmed. The results are shown in Tables 3 and 4.
[0019]
[Table 3]
[Table 4]
[0021]
Example 5: Reactivity of mixed latex of anti-human hemoglobin monoclonal antibody-bound latex and anti-human glycated hemoglobin monoclonal antibody-bound latex Anti-human hemoglobin monoclonal antibody latex that does not aggregate with human hemoglobin alone, and human glycated hemoglobin alone And a mixture of anti-human glycated hemoglobin monoclonal antibody-bound latex that does not aggregate with human hemoglobin was tested for reactivity with the antigen. The results are shown in Tables 5 and 6.
[0022]
[Table 5]
[Table 6]
As shown in Tables 5 and 6, only the mixed latex of anti-human glycated hemoglobin monoclonal antibody-bound latex L-gH-1 and anti-human hemoglobin monoclonal antibody-bound latex L-H-2 showed an agglutination reaction with human glycated hemoglobin. . Using this mixed latex, the results of measurement of glycated hemoglobin in a sample by a spectroscopic reaction rate method using an optical measuring device LPIA-200 (registered trademark; Mitsubishi Chemical Corporation) are shown in FIG. In addition, the V value in FIG. 1 is an agglutination reaction speed. As is clear from FIG. 1, according to the method of the present invention, quantification using an automatic analyzer is also possible.
[0025]
【The invention's effect】
According to the method of the present invention, a specific measurement of glycated hemoglobin can be performed in a much shorter time (within 10 minutes) than in the conventional method without interference of hemoglobin in the sample.
[Brief description of the drawings]
FIG. 1 is a graph showing the results of measuring the glycated hemoglobin concentration using an optical measurement device using the mixed latex according to the present invention.
Claims (4)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10619896A JP3550614B2 (en) | 1996-04-03 | 1996-04-03 | Measurement method of glycated hemoglobin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10619896A JP3550614B2 (en) | 1996-04-03 | 1996-04-03 | Measurement method of glycated hemoglobin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH09274038A JPH09274038A (en) | 1997-10-21 |
| JP3550614B2 true JP3550614B2 (en) | 2004-08-04 |
Family
ID=14427483
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP10619896A Expired - Fee Related JP3550614B2 (en) | 1996-04-03 | 1996-04-03 | Measurement method of glycated hemoglobin |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3550614B2 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH11258238A (en) * | 1998-03-10 | 1999-09-24 | Tokuyama Corp | Reagent kit for measuring immunoagglutination |
| JP3854716B2 (en) * | 1998-04-20 | 2006-12-06 | 株式会社日立製作所 | Liquid chromatograph |
| JP4796893B2 (en) * | 2006-05-23 | 2011-10-19 | 積水化学工業株式会社 | Method for measuring hemoglobin A1c |
| CN110702894A (en) * | 2019-10-10 | 2020-01-17 | 南京欧凯生物科技有限公司 | Detection method for directly measuring content of glycosylated hemoglobin |
| CN111157747A (en) * | 2020-01-17 | 2020-05-15 | 上海高踪医疗器械科技有限公司 | Glycosylated hemoglobin detection kit and production process thereof |
-
1996
- 1996-04-03 JP JP10619896A patent/JP3550614B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH09274038A (en) | 1997-10-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5807300B2 (en) | Method and reagent for measuring C-reactive protein in sample | |
| EP0573972B1 (en) | Method for measuring glycated hemoglobin | |
| JP5199067B2 (en) | Immunoagglutination reagent kit and antigen measurement method | |
| EP0166623A2 (en) | Antibody lectin sandwich assay | |
| EP2322562A1 (en) | Monoclonal antibody, and immunoassay using same | |
| EP0645630A2 (en) | Method of determining the presence of a pivka and a reagent therefor | |
| JPS61500315A (en) | How to detect immune complexes in serum | |
| JPWO2020067396A1 (en) | Method for measuring glycated hemoglobin (%) | |
| JP3550614B2 (en) | Measurement method of glycated hemoglobin | |
| JPWO2012161226A1 (en) | Method for suppressing non-specific reaction in PIVKA-II measuring reagent | |
| JP2018173334A (en) | Method for immunological measurement and reagent kit used for the same | |
| JPWO2020075691A1 (en) | Leucine-rich α2 glycoprotein immunoassay method and measurement reagent | |
| CN111239404A (en) | Detection kit capable of simultaneously detecting retinol binding protein in urine sample and serum sample | |
| CN114835808A (en) | Blocking agent capable of directionally eliminating false positive and preparation method thereof | |
| CN111239403B (en) | Beta 2 microglobulin latex enhanced immunoturbidimetry kit and application | |
| JP2596321B2 (en) | Measurement method of glycated hemoglobin | |
| JPH0666796A (en) | Anti-glycated hemoglobin monoclonal antibody and method for measuring glycated hemoglobin | |
| JP2684425B2 (en) | Latex reagent | |
| JPH0448265A (en) | Immunoassay | |
| JP7543575B2 (en) | Free hemoglobin measuring reagent, free hemoglobin measuring method, and anti-hemoglobin antibody | |
| JPH02141665A (en) | Detection of hemoglobin in dejection | |
| JP3225248B2 (en) | Detection method | |
| JP2759365B2 (en) | Immunological assay method and reagents | |
| RU2262703C1 (en) | Method for preparing control sample used in immunoenzyme analysis | |
| JPH02259569A (en) | Method for measuring rheumatism factor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20031222 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20040106 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20040305 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20040406 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20040407 |
|
| R150 | Certificate of patent or registration of utility model |
Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| S531 | Written request for registration of change of domicile |
Free format text: JAPANESE INTERMEDIATE CODE: R313531 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20080514 Year of fee payment: 4 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20090514 Year of fee payment: 5 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20100514 Year of fee payment: 6 |
|
| S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313111 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20100514 Year of fee payment: 6 |
|
| R371 | Transfer withdrawn |
Free format text: JAPANESE INTERMEDIATE CODE: R371 |
|
| S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313111 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20100514 Year of fee payment: 6 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20110514 Year of fee payment: 7 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20120514 Year of fee payment: 8 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20130514 Year of fee payment: 9 |
|
| LAPS | Cancellation because of no payment of annual fees |