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METHOD FOR PRODUCING REAGENT FOR ANTIBODY DETECTION AND USE THEREOF

外国特許コード F130007660
整理番号 S2012-0582-N0
掲載日 2013年10月17日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2013JP059692
国際公開番号 WO 2013147233
国際出願日 平成25年3月29日(2013.3.29)
国際公開日 平成25年10月3日(2013.10.3)
優先権データ
  • 特願2012-082735 (2012.3.30) JP
発明の名称 (英語) METHOD FOR PRODUCING REAGENT FOR ANTIBODY DETECTION AND USE THEREOF
発明の概要(英語) The present invention provides the following: a method for efficiently producing a reagent for detecting an antibody that specifically binds with an insoluble antigen protein present in a liquid sample; a reagent for antibody detection produced by the production method; and a use of the antibody. In a step for solubilizing an antigen protein, it is possible to efficiently solubilize and recover the antigen protein by using a cationizing agent; therefore, when compared to conventional methods, it is possible to efficiently produce a reagent for detecting an antibody that has bound to multiple antigen protein molecules in a carrier.
従来技術、競合技術の概要(英語) BACKGROUND ART
Included in the liquid sample the method of detection of the antibody, for example, radioimmunoassay or ELISA(Enzyme-linked immunosorbent assay; enzyme-linked immunosorbent assay) and the like are present. ELISA is, a specific antigen on the microplate was immobilized, the sample containing the antibodies were serially diluted and then, the antigen on the microplate, followed by a reaction of the antibody, the antibody bound to the enzyme-labeled secondary antibody is detected by the method of the invention. In this method, the inspection of the individual antibody to the antigen, the assay plate was used to assess the individual is required.
As a method to solve the above problem, a carrier that holds the immobilized reporter microbeads is used, a wide variety of antigens can be simultaneously analyzed by the multiplex technique is attracting attention.
Even in the case where any of the technique, a variety of antigens may be prepared as a water-soluble essential. In the prior art, can be prepared as a water-soluble protein or a chemically synthesized Native structure of an example of using a polypeptide fragment in most cases.
For example, antibodies contained in the blood of cancer patients for the detection described in Patent Document 1 or 2 respectively. Epitope of the antigen (antigenic peptide to the number of amino acids) immobilized on the beads, beads in contact with the blood component of the subject, the antigen contained in the blood of a subject antibody specific for an epitope is a method for the detection. However, the epitope to which differ depending on the type of HLA portion, as described in Patent Document 1 or 2 is to use the method, the type of HLA examining the subject, to match the subject's HLA type peptides that have been identified with various conditions such as when necessary to clear.
Of antibodies to a particular antigen in the preparation of detection reagents, 1 kinds of the surface of the beads 1 of the kind of antigen derived from a protein that includes an epitope, with high sensitivity and stably detected for the preparation of the reagent, water-soluble antigen having the flexible structure is preferably obtained. However, a number of denatured protein, a protein or an insoluble membrane proteins such as the physical properties of a protein is unstable and easily coagulated. For this reason, conventionally, a partial peptide may exhibit stable physical properties has often been used.
In the case of using the partial peptide, to cover all of the epitope for the synthesis of a wide variety of overlapping peptides is required, many types of beads as well as the need to prepare, to provide seamless peptides that it is difficult in practice. In addition, the overall length of the antigen of the structure may be bio-Native can be acquired, generally the hydrophobic portion is embedded in a higher-order structure is formed and therefore, not exposed to the antibody that reacts with the epitope may not.
Furthermore, the solubilized protein was used to create the reagent for detection of antibodies, with the passage of time, are included in the protein thiol groups form disulfide bonds and thus, affect the detection of the antibody is a possibility.
For example, to Patent Document 3, a human C-type hepatitis virus (HCV) using a long-chain polypeptide, anti-HCV antibody contained in the blood serum of a subject for the detection have been described. Patent Document 3 is, in a protein that occurs with the elapse of time or a disulfide linkage between the protein, it is possible to reduce the detection sensitivity of the antibodies have been described. Solve the problem, before detection or a detection reagent in a disulfide bond between the protein using a protein in or is opened, the detection sensitivity of the antibodies have been described can also be enhanced. That is, a solubilized protein, precipitate with the lapse of time and a possibility that, in order to eliminate the need to take some means.
As the compound to solubilize the protein, TAPS-Sulfonate (trimethylammonio propyl methane thiosulfonate, hereinafter, referred to as TAPS) has been known. TAPS is, bound to a thiol group of a protein, the protein can be reversibly cations (for example, Non-Patent Document 1, reference 2).
Cationized protein is improved solubility in water. In addition, the binding of the protein and TAPS reversible reaction and thus, when incorporated into cells, the protein can be shifted from a TAPS, refolding occurs, exerts the original function of the protein are known. However, in the step of manufacturing a reagent for detecting antibody, TAPS soluablizing using the attempt to use heretofore has not been made. In addition, a sugar chain-modified antibodies bind to the protein are known, such as TAPS artificially synthesized compound bound to the antibody can be bound to the protein is not reported whether or not.
  • 出願人(英語)
  • ※2012年7月以前掲載分については米国以外のすべての指定国
  • NATIONAL UNIVERSITY CORPORATION OKAYAMA UNIVERSITY
  • MEDINET CO., LTD.
  • 発明者(英語)
  • FUTAMI Junichiro
  • KAKIMI Kazuhiro
  • MAEKAWA Ryuji
  • SHIRAKI Masato
国際特許分類(IPC)
指定国 National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LA LC LK LR LS LT LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

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