Specific antibody directed to active hepatocyte growth factor activator and method for using the same

Inactive Publication Date: 2006-04-06
NAKA DAIJI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0013] Further, the inventors found for the first time that, when the method and kit for specifically measuring active HGFA of the present invention are used, there could be observed a markedly increased amount of active HGFA in blood of patients with organ derangement including glomerular nephritis and cancer patients compared with that of healthy subjects. Further, they also found for the first time that thrombosis such as angina pectoris, myocardial infarction and cerebral infarction could be accurately predicted by using the increase in the amount of active HGFA in blood as an index.
[0014] Meanwhile, when the amount of active HGFA existing in a biological component including human blood is measured, there is required a method of stably collecting active HGFA existing therein with good reproducibility. The inventors of the present invention examined addition of various protease inhibitors and so forth in this method and found that active HGFA could be extremely stably measured with good reproducibility by adding argatroban, which is a selective thrombin inhibitor. Further, as a result of many detailed investigations, they also found that argatroban was effective even when human blood such as whole blood, serum or plasma was collected and that an extremely favorable result could be obtained by using citrated plasma among blood components.

Problems solved by technology

However, uniformly strong reactivity was observed in all human blood including that of normal subjects by the ELISA measurement system using these monoclonal antibodies, and no reactivity specific to human diseases, that is, no marked changes in the HGFA blood level could be detected.
Thus, it was found that the ELISA measurement system using existing monoclonal antibodies could not reveal the relationship between HGFA and human diseases at all.

Method used

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  • Specific antibody directed to active hepatocyte growth factor activator and method for using the same
  • Specific antibody directed to active hepatocyte growth factor activator and method for using the same
  • Specific antibody directed to active hepatocyte growth factor activator and method for using the same

Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of Inactive HGFA and Active HGFA

[0108] Inactive HGFA was prepared by using HGFA cDNA coding for the HGFA precursor described in Japanese Patent Laid-open Publication No. 6-153946 according to the method described in Japanese Patent Laid-open Publication No. 6-153966 to produce recombinant inactive HGFA. That is, the HGFA cDNA coding for the full length of the inactive HGFA precursor, 655 amino acids, was inserted into pcDNA3.1 (Invitrogen), which is an animal cell expression vector, downstream from a CMV promoter in a conventional manner.

[0109] The obtained expression vector was introduced into a CHO cell, which is an animal cell strain derived from a Chinese hamster ovary cell, by using a Transfectam (BioSepra) according to the attached instruction. Then, CHO cells expressing HGFA cDNA were selected by utilizing a property of a neomycin resistant gene existing on the introduced expression vector. That is, cells that could grow in an ERDF medium (Kyokuto Seiyaku) conta...

example 3

Preparation of Active HGFA Specific Monoclonal Antibody

[0115] A solution containing 100 μg of the 36 kDa HGFA or 100 μg of the 98 kDa HGFA, which were prepared in Example 1, was subcutaneously and intraperitoneally administered to a Balb / c mouse with the same volume of complete Freund's adjuvant 6 times with 2-week intervals. After production of antibodies in the serum of the mouse was confirmed, a solution containing 100 μg of HGFA was administered into the caudal vein. Three days later, spleen was removed and spleen cells were fused with myeloma cells P3U1 by using polyethylene glycol 1500 according to “Monoclonal Antibody Experimental Manual” (Kodansha Scientific, 1987), introduced into wells of 96-well plate, added with HAT medium and cultured for 14 days.

[0116] Subsequently, hybridomas producing monoclonal antibodies specific to respective active HGFA in the medium were selected. That is, culture supernatant of a hybridoma subjected to selection was added to an ELISA plate fo...

example 5

Measurement of Dissociation Constant of Active HGFA Specific Monoclonal Antibody

[0123] Among active HGFA specific monoclonal antibodies prepared and purified in the Example 3, the dissociation constant was measured for the monoclonal antibody derived from the hybridoma clone AHGA-A. An active HGFA immobilized plate on which active HGFA was immobilized and blocked with BSA was added with the antibodies at different antibody concentrations and allowed to sufficiently react until equilibrium was reached (2 hours or longer).

[0124] Subsequently, the plate was sufficiently washed with a PBS(-) solution containing 0.05% Tween 20 (hereafter, abbreviated as “PBST solution”), and then 100 μl of PBS(-) containing 1 μg / ml of HRP (horseradish peroxidase) conjugated goat anti-mouse IgG / Fc polyclonal antibody (ICN) and 1% BSA was added to each well and further allowed to react at room temperature for 1 hour. The plate was sufficiently washed with a PBST solution, and then a citrate-phosphate buf...

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Abstract

An antibody which recognizes an active hepatocyte growth factor activator (HGFA) and does not substantially recognize inactive HGFA is provided. Also disclosed is a monoclonal antibody thereof, and a hybridoma cell line for producing the monoclonal antibody. There is further provided a method for measuring active HGFA using the antibody, and a method for detecting a disease, by detecting or measuring active HGFA using the antibody.

Description

[0001] This application is a divisional of application Ser. No. 10 / 000,096, filed Dec. 4, 2001, currently pending.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention relates to an antibody or a monoclonal antibody used for specifically measuring active hepatocyte growth factor activator (HGFA), a method for using the same and a measurement kit. The present invention also relates to a method for detecting a disease of patient in a pathological condition, in particular, organ inflammation, glomerular nephritis, cancer, myocardial infarction, angina pectoris or thrombosis, by using the active HGFA as an index and further relates to a method for collecting a biological component and blood, which is suitable for performing the method. [0004] 2. Description of the Related Art [0005] Protease (protein decomposition enzyme) is a protein having a function of hydrolyzing a peptide bond in a protein or a peptide and it is deeply involved in organic functio...

Claims

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Application Information

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IPC IPC(8): G01N33/53C12N5/06C07K16/22C07K16/40
CPCA61K2039/505C07K16/40Y10S435/81
Inventor NAKA, DAIJINAGAIKE, KAZUHIKO
Owner NAKA DAIJI
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