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Protein assay method specific to tracp-5b (tartrate resistant acid phosphatase 5b)

一种tracp-5b、NITEBP-01866的技术，应用在抗酶免疫球蛋白、抗动物/人类的免疫球蛋白、特定肽等方向，达到辅助诊断的效果

Active Publication Date: 2017-08-01
NITTO BOSEIKI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a result, although an antibody with certain selectivity was successfully produced, there is room for improvement in its selectivity, and further improvement is necessary for use in clinical trials

Method used

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  • Protein assay method specific to tracp-5b (tartrate resistant acid phosphatase 5b)
  • Protein assay method specific to tracp-5b (tartrate resistant acid phosphatase 5b)
  • Protein assay method specific to tracp-5b (tartrate resistant acid phosphatase 5b)

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Experimental program
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Embodiment approach

[0089] The monoclonal antibody of the present invention can be obtained by using recombinant human TRACP-5b as an immunogen. In the examples described later in this specification, the hosts for purification from genetically recombinant silkworms are not limited as long as they are cultured cells, Escherichia coli , etc., as long as they can express human TRACP-5b.

[0090] The monoclonal antibody of the present invention is produced, for example, by a hybridoma obtained by immunizing an animal with purified human TRACP-5b as an immunogen, and fusing anti-human TRACP-5b antibody-producing cells produced by the animal with myeloma cells.

[0091] The above-mentioned hybridomas can be obtained by the following method. That is, by mixing the human TRACP-5b obtained as described above using Freund's complete and incomplete adjuvants, aluminum hydroxide adjuvants, pertussis adjuvants, etc., to prepare an adjuvant solution for sensitization, fraction Animals such as mice and rats ar...

Embodiment 1

[0123] (1) Selection and preparation of antigens for monoclonal antibody production

[0124] As an antigen for preparing anti-human TRACP monoclonal antibody, recombinant TRACP-5b produced in the silk gland of genetically recombinant silkworm was prepared. The preparation method of the recombinant TRACP-5b was prepared by the method described in Japanese Patent No. 5177431.

[0125] The silk gland was extracted from the genetically recombinant silkworm containing human recombinant TRACP-5b to obtain 300 g of silk gland. The silk glands were suspended in 5600 mL of buffer solution (50 mM Tris-HCl, pH 7.5), and homogenized with a rotor stator type homogenizer. Thereafter, centrifuge at 10,000 rpm for 20 minutes and apply the supernatant to a CM-Sepharose column (GE Healthcare), the adsorbed protein was eluted with a linear concentration gradient (0-1.0 M NaCl) of the above-mentioned Tris buffer containing NaCl. The tartrate-resistant acid phosphatase activity was measured us...

Embodiment 2

[0179] Epitope analysis of TrK-126 and TrK-127

[0180] (1) Epitope analysis of TrK-126 and TrK-127 by Replitope

[0181] Based on the above results, epitope analysis of TrK-126 and TrK-127 was carried out by the luminescence method.

[0182] Specifically, the above-mentioned peptide slides were blocked at room temperature for 60 minutes using SuperBlock (PIERCE). Thereafter, as the primary antibody, TrK-126 or TrK-127 was reacted at a concentration of 1 μg / mL at room temperature for 60 minutes. After washing with PBS-T 3 times, the secondary antibody attached to Amersham ECL Prime (GE Healthcare) was added and reacted at room temperature for 60 minutes. After washing 3 times with PBS-T again, add the detection reagent attached to the kit. After a certain period of incubation, the degree of luminescence of the plaques was confirmed by ImageQuant LAS4000 (GE Healthcare).

[0183] As a result of the analysis, neither TrK-126 nor TrK-127 showed reactivity with each sequence p...

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Abstract

The purpose of the present invention is to provide a monoclonal antibody that is useful in specifically assaying tartrate resistant acid phosphatase 5b (TRACP-5b). A hybridoma producing a monoclonal antibody against TRACP-5b, said monoclonal antibody showing higher reactivity with TRACP-5b than with tartrate resistant acid phosphatase 5a (TRACP-5a) and, therefore, being specific to TRACP-5b, is obtained by cell fusion using, as an antigen, human recombinant TRACP-5b purified from silkworm silk gland. By using this monoclonal antibody, TRACP-5b in a specimen can be highly sensitively and specifically detected.

Description

[0001] 【Technical field】 [0002] The present invention relates to production of a monoclonal antibody specific for tartrate-resistant acid phosphatase 5b (TRACP-5b: alias, osteoclast-derived tartrate-resistant acid phosphatase), hybridomas of the monoclonal antibody, and use of the monoclonal antibody The detection method of TRACP-5b and the kit used therein. [0003] The monoclonal antibody of the present invention is extremely effective as a bone resorption marker in the field of medical treatment or clinical examination of bone diseases. [0004] 【Background technique】 [0005] Tartrate-resistant acid phosphatase (TRACP: Tartrate Resistant acid Phosphatase EC3.1.3.2) in serum is mostly acid phosphatase derived from osteoclasts, and its measurement is useful as an index for evaluating the function of osteoclasts, and as a bone resorption Markers are of interest (Non-Patent Document 1). On the one hand, acid phosphatase in serum is electrophoresed on polyacrylamide gel, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/00C07K16/40C12N5/10C12N15/02G01N33/53G01N33/543G01N33/545G01N33/553G01N33/577C12N9/16C12P21/08
CPCC12N9/16C12N15/00C12N15/02G01N33/53G01N33/543G01N33/545G01N33/553G01N33/577C12N5/12C12N5/163A61K38/00C07K16/40C12Y301/03002G01N2333/916C07K16/18A61K2039/505G01N33/5008C07K16/30C07K2317/14C07K2317/33G01N33/573G01N2800/10
Inventor 菊地涉笹川久美子野田健太
Owner NITTO BOSEIKI CO LTD
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