Method for measuring cystatin c in human body fluid

A technology of cysteine ​​protease and determination method, applied in the field of determination of cysteine ​​protease inhibitor C in human body fluid, can solve the problem of inability to perform specific determination of cysteine ​​protease inhibitor C, polyclonal antibody Low specificity and other issues, to achieve the effect of low cost and high specificity

Active Publication Date: 2011-10-26
SEKISUI MEDICAL CO LTD
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Problems solved by technology

[0011] In the particle-enhanced immunoassay method of human cystatin C, the general-purpose polyclonal antibo

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  • Method for measuring cystatin c in human body fluid
  • Method for measuring cystatin c in human body fluid
  • Method for measuring cystatin c in human body fluid

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[0035] Hereinafter, a part of the present invention will be described in detail with reference to production examples, evaluation examples, and working examples, but the present invention is not limited to these.

[0036] 〔Example of making〕

[0037] Production of High Affinity Anti-human Cystatin C Monoclonal Antibody

[0038] 100 μg of purified human cystatin C (SCIPAC) was used for one immunization. For the first immunization, 200 μL of an emulsion prepared by mixing equal amounts of human cystatin C and Freund's complete adjuvant was used, and injected into the peritoneal cavity of BALB / c mice. In the booster immunization, 200 μL of the emulsion prepared in the same way using Freund's incomplete adjuvant was used, and in order to obtain high-affinity antibodies, long-term immunization was performed by repeating the booster immunization 6 times at 2-week intervals. The antibody titer in the blood obtained by blood collection from the fundus vein of the mouse was measured ...

example 1

[0042] Selection of anti-human cystatin C monoclonal antibodies with different recognition sites

[0043] Using the reactivity in the sandwich ELISA method as an index, combinations of different recognition sites among the produced anti-human cystatin C monoclonal antibodies were searched. Specifically, 50 μL / well of PBS containing 1 μg / mL of each anti-human cystatin C monoclonal antibody was added, left to stand at room temperature for 2 hours, solid-phased on the plate, and then mixed with 0.05 Wash 3 times with 300 μL of PBS containing % Tween20, add PBS containing 0.05% Tween20 and 1% BSA at 300 μL / well, and let stand at room temperature for 1 hour. Then, after washing three times with PBS containing 0.05% Tween20, 50 μL / well of PBS containing 10 ng / mL human cystatin C was added, and the reaction was carried out at room temperature for 1 hour. After washing with PBS of % Tween20 for 3 times, add 50 μL / well of PBS containing 1 μg / mL of each biotinylated anti-human cystatin...

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Abstract

Disclosed is a particle-enhanced immunoassay method for cystatin C in a human body fluid, which has higher specificity, is more inexpensive, and can be automated more readily compared with conventional assay methods in which polyclonal antibodies having low specificity or monoclonal antibodies having high specificity but having poor aggregability have been used in large quantities. The particle-enhanced immunoassay method utilizes a combination of an antibody-sensitized particle comprising an insoluble carrier particle and an anti-human cystatin C monoclonal antibody having higher affinity and bound to the particle and an antibody-sensitized particle comprising the insoluble carrier particle and an anti-human cystatin C monoclonal antibody having a different recognition site from that of the aforementioned monoclonal antibody and having a relatively lower affinity, wherein the quantity of each of the human cystatin C monoclonal antibodies bound to the insoluble carrier particle is less than 5 wt% relative to the weight of the antibody-sensitized particle.

Description

technical field [0001] The invention relates to a particle-enhanced immunoassay method for cysteine ​​protease inhibitor C in human body fluid and a particle-enhanced immunoassay reagent. Background technique [0002] Cystatin C is a basic low-molecular-weight protein with a molecular weight of 13kDa (isoelectric point pH9.3), which is continuously produced in the nucleated cells of the whole body, and will be secreted in a certain amount without being affected by environmental changes. Extracellular. Therefore, the blood concentration is maintained at a certain level regardless of the influence of inflammation or the like caused by other diseases or the influence of age, sex, exercise, diet or the like. In addition, it is known that cystatin C does not form a complex with other plasma proteins, is filtered in the glomerulus and is reabsorbed in the proximal convoluted tubule, therefore, if the glomerular filtration rate (GFR) decreases, the The blood concentration rises, ...

Claims

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Application Information

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IPC IPC(8): G01N33/53G01N33/543G01N33/577
CPCG01N33/6893G01N2333/8139G01N33/543G01N33/577G01N33/68
Inventor 中山真也高桥弘至中村靖清水知
Owner SEKISUI MEDICAL CO LTD
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