WO2004113921A1 - Reactif pour analyse de l'anticorps anti- treponema pallidium et methode d'analyse de cet anticorps - Google Patents

Reactif pour analyse de l'anticorps anti- treponema pallidium et methode d'analyse de cet anticorps Download PDF

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Publication number
WO2004113921A1
WO2004113921A1 PCT/JP2004/008902 JP2004008902W WO2004113921A1 WO 2004113921 A1 WO2004113921 A1 WO 2004113921A1 JP 2004008902 W JP2004008902 W JP 2004008902W WO 2004113921 A1 WO2004113921 A1 WO 2004113921A1
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WO
WIPO (PCT)
Prior art keywords
treponema pallidum
antigen
antibody
kda
molecular weight
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Application number
PCT/JP2004/008902
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English (en)
Japanese (ja)
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WO2004113921A9 (fr
Inventor
Mie Matsumoto
Yoshitaka Izumoto
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Sekisui Chemical Co., Ltd.
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority claimed from JP2003180087A external-priority patent/JP3936678B2/ja
Application filed by Sekisui Chemical Co., Ltd. filed Critical Sekisui Chemical Co., Ltd.
Publication of WO2004113921A1 publication Critical patent/WO2004113921A1/fr
Publication of WO2004113921A9 publication Critical patent/WO2004113921A9/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/571—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses for venereal disease, e.g. syphilis, gonorrhoea
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the present invention relates to an anti-treponema pallidum antibody measuring reagent capable of measuring an anti-treponema pallidum antibody with high sensitivity and specificity, and a measuring method using the same.
  • Syphilis is a disease caused by infection of Treponema pallidum (Treponema pallidum). Syphilis is diagnosed by detecting the presence of anti-syphilis anti-Treponemal antibodies in the blood by immunoassay. Many surface antigens are present on the surface of Treponema pallidum, and immunoassays are performed by a method utilizing an antigen-antibody reaction between the surface antigen and an anti-Treponemal syphilis antibody in a specimen.
  • Non-Patent Document 1 Among the major surface antigens present on the cell surface of Treponema pallidum, those with a molecular weight of 47 kDa, 42 kDa, 37 kDa, 17 kDa and 15 kDa are known (Non-Patent Document 1).
  • Treponema pallidum As the surface antigen of Treponema pallidum which is currently used in this immunoassay, Treponema pallidum is cultivated in a house testicle, solubilized with a surfactant or the like, and extracted by various methods. Unnecessary substances are removed by use. Purified necessary components are used. However, the production method using such a rabbit is limited in yield due to the use of animals as a host, and the growth state of Treponema pallidum varies among animals. There was a problem that it was difficult to secure them. At the moment, on the other hand, we have succeeded in directly cultivating Treponema syphilis directly.
  • Patent Document 1 describes a method for preparing an antigen having a molecular weight of 47 kDa of Treponema pallidum by genetic engineering and immunoassaying an anti-Treponemal antibody using the antigen.
  • Patent Document 2 discloses that daltathio is added to the N-terminus of 15 kDa and 17 kDa surface antigens. A method using a protein fused with s-transferase is disclosed. Further
  • Patent Document 1 Japanese Patent Publication No. 2-50040
  • Patent Document 2 JP-A-7-63365
  • Non-Patent Document 1 Journal of Immunology, vol. 129, pp. 1287-1291, 1992
  • an object of the present invention is to provide an anti-treponema pallidum antibody measuring reagent capable of measuring an anti-treponema pallidum antibody with high sensitivity and specificity, and a measuring method using the same. Things.
  • the present invention relates to a reagent used for measurement of an anti-treponema pallidum antibody using an antigen-antibody reaction, which is a 15K antigen comprising a protein in which ⁇ -galactosidase is fused to a surface antigen of treponema pallidum having a molecular weight of 15 kDa.
  • the present invention is an anti-treponema pallidum antibody measuring reagent containing a 17K antigen comprising a protein in which ⁇ -galactosidase is fused to a surface antigen having a molecular weight of 17 kDa of Treponema pallidum.
  • the present inventors have conducted intensive studies and found that among the surface antigens of Treponema pallidum, 15K antigen and / or 17kDa surface antigen consisting of a protein in which ⁇ -galactosidase is fused to a surface antigen having a molecular weight of 15 kDa
  • 15K antigen and / or 17kDa surface antigen consisting of a protein in which ⁇ -galactosidase is fused to a surface antigen having a molecular weight of 15 kDa By using the 17K antigen consisting of a protein fused with 3-galactosidase as an antigen, the present inventors have found that an anti-Treponemal antibody can be measured with significantly higher sensitivity and with extremely high accuracy compared to the conventional method, and thus complete the present invention. Reached.
  • the anti-treponema pallidum antibody measuring reagent of the present invention is a syphilis treponemal whose molecular weight is 15 kDa. It contains a 15K antigen consisting of a protein in which ⁇ -galactosidase is fused to the surface antigen and / or a 17 ⁇ antigen consisting of a protein in which ⁇ -galactosidase is fused to the surface antigen of molecular weight 17 kDa of Treponema pallidum.
  • Either one of these antigens may be contained, or both may be contained. In particular, when both are included, it is preferable because the detection sensitivity is further increased.
  • the method for fusing ⁇ -galactosidase to the surface antigen having a molecular weight of 15 kDa and the surface antigen having a molecular weight of 17 kDa of the Treponema pallidum is not particularly limited, and an ordinary method can be used.
  • ⁇ -galactosidase is an enzyme that decomposes and synthesizes ratatose, and has been studied in Escherichia coli as a typical inducible enzyme induced by the force ratatose.
  • ⁇ -galactosidase has a molecular weight of about 116 kDa and forms a tetramer. Its structural gene is called lacZ, and its gene sequence is known (EMBO J 1963; 2 (4): 593-597) and is commercially available in various plasmids. Representative examples of commercially available ones include pUC18 and pUC19 (both manufactured by Toyobo Co., Ltd.), and those that can express a fusion protein of a foreign protein and / 3-galactosidase. It is not particularly limited.
  • the method for fusing ⁇ -galatatosidase to the surface antigen having a molecular weight of 15 kDa and the surface antigen having a molecular weight of 17 kDa of the Treponema pallidum may be performed by converting the structural gene of the surface antigen having a molecular weight of 15 kDa or the surface antigen having a molecular weight of 17 kDa to the lacZ gene. Introduce to Such a method can be performed by a gene recombination technique known to those skilled in the art, and is not particularly limited.
  • a method for purifying a fusion protein obtained by fusing -galactosidase with a surface antigen having a molecular weight of 15 kDa or a surface antigen having a molecular weight of 17 kDa of Treponema pallidum is not particularly limited.
  • p-aminobenziru 1_thio j3 _D examples include a method of purifying using an agarose column to which galactopyranoside is bound (for example, manufactured by Sigma) or an anti-j3-galactosidase antibody (for example, manufactured by Promega).
  • proteins obtained by fusing monogalactosidase to the surface antigen having a molecular weight of 15 kDa and the surface antigen having a molecular weight of 17 kDa of the Treponema pallidum are generally sold, and therefore, these commercially available products can also be used.
  • the 15K antigen composed of a protein obtained by fusing / 3-galatatosidase to a surface antigen having a molecular weight of 15 kDa and the 17K antigen composed of a protein obtained by fusing a ⁇ -galactosidase to a surface antigen having a molecular weight of 17 kDa is obtained from It may be immobilized on a suitable carrier such as latex particles. By immobilizing it on a strong carrier, the reagent for measuring an anti-treponema pallidum antibody of the present invention can be subjected to an immunoagglutination method or the like.
  • the method of immobilization is not particularly limited, and examples thereof include a physical adsorption method and a method of chemically bonding to a functional group provided on the surface of the carrier.
  • the 15K antigen consisting of a protein in which ⁇ -galactosidase is fused to the surface antigen of 15 kDa molecular weight of the above-mentioned Treponema pallidum and the 17K antigen consisting of a protein in which -galatatosidase is fused to the surface antigen of 17 kDa in molecular weight of Treponema pallidum are both known.
  • a method for measuring an anti-treponema pallidum antibody in a sample using an antigen-antibody reaction wherein the antigen is a 15K antigen consisting of a protein in which / 3_galactosidase is fused to a surface antigen having a molecular weight of 15 kDa of trepidome syphilis, and 15K antigen and Z or syphilis.
  • a method for measuring an anti-treponema pallidum antibody using a 17K antigen comprising a protein obtained by fusing / 3_galactosidase to a surface antigen having a molecular weight of 17 kDa of treponemal is also one of the present invention.
  • the method for measuring the anti-treponema pallidum antibody of the present invention is not particularly limited as long as the reagent for measuring the anti-treponema pallidum antibody of the present invention is used, and can be performed by a usual method. That is, if classified according to the reaction format, the sandwich method, agglutination method, etc. can be used.If classified according to the label, the enzyme immunoassay, fluorescence analysis, radioimmunoassay, etc. can be used. . Among them, the latex immunoagglutination method, which is easy to operate, has good sensitivity, and is suitable for processing a large number of samples, is more preferable.
  • Specific examples of the method for measuring an anti-treponema pallidum antibody of the present invention include, for example, a 15K antigen comprising a protein in which ⁇ -galatatosidase is fused to a surface antigen having a molecular weight of 15 kDa and / or the molecular weight of treponema pallidum.
  • a 17K antigen consisting of a 17-kDa surface antigen fused with / 3-galatatosidase is immobilized on latex particles, prepared as a latex reagent by a specified procedure, and a sample is added to it for reaction for a specified time.
  • a method of visually, optically, or electrochemically detecting a change in turbidity at intervals may be used.
  • a 15K antigen comprising a protein in which ⁇ -galactosidase is fused to a surface antigen having a molecular weight of 15 kDa to a well such as a microtiter plate or the like, and a molecular weight of the Treponema pallidum
  • a 17kDa surface antigen is immobilized with a 17K antigen consisting of a protein fused with -galatasidase, blocking nonspecific adsorption sites, washing, adding a sample to react, washing, and enzyme such as peroxidase. Reacting with an anti-human immunoglobulin antibody labeled with, washing, adding the substrate of the labeled antibody, performing an enzymatic reaction, causing color development, and measuring the degree of color development after termination of the reaction by absorbance measurement.
  • a 15K antigen consisting of a protein in which ⁇ -galactosidase is fused to a surface antigen having a molecular weight of 15 kDa of syphilis treponema on particles such as ferrite magnetic particles, and / or a molecular weight of 17 kDa of syphilis treponema
  • a 17K antigen consisting of a protein fused with 3 / 3-galactosidase is immobilized on the surface antigen of, and a sample is added thereto and allowed to react.
  • Anti-human immunoglobulin antibody labeled with an enzyme such as peroxidase and a chemiluminescent substrate And a chemiluminescent enzyme immunoassay (CLEIA).
  • a solubilized carrier for example, an artificial carrier composed of gelatin or the like disclosed in JP-B-63-29223; immobilized on a particulate carrier such as animal erythrocytes such as chickens, goats, sheep, sesame, and pest;
  • agglutination immunoassay method in which a sample is collected in a suspension of a carrier and the force of agglutination of the particles is measured.
  • the sample to be used in the method for measuring an anti-treponema pallidum antibody of the present invention is not particularly limited, and examples thereof include a body fluid such as serum of a human animal to be diagnosed with syphilis and a dilution thereof.
  • the method for measuring an anti-treponema pallidum antibody of the present invention can measure an anti-treponema pallidum antibody with extremely high sensitivity and accuracy by using the reagent for measuring an anti-treponema pallidum antibody of the present invention. Diagnosis of syphilis can be made.
  • an anti-treponema pallidum antibody measuring reagent capable of measuring an anti-treponema pallidum antibody with high sensitivity and specificity, and a measuring method using the same can be provided.
  • Latex solution (manufactured by Sekisui Chemical Co., Ltd .: average particle size: 0.3 ⁇ , solid content: 10%) While stirring 0.05 mL in a test tube, fructose-tidase is fused to the surface antigen of Treponema pallidum having a molecular weight of 15 kDa. LmL of the resulting protein solution (Bios Pacific, 100 ⁇ g / mL) was added to immobilize the fusion protein on the surface of the latex particles.
  • the antigen-unadsorbed portion of the obtained fusion protein-immobilized latex particles is blocked with a PBS buffer containing 1% bovine serum albumin (BSA), and the unadsorbed antigen is centrifuged. After removal by washing and sufficient stirring, the mixture was diluted to 0.1% with a PBS buffer containing 1% BSA to obtain an anti-Treponemal antibody measurement reagent.
  • BSA bovine serum albumin
  • Example 2 Instead of a protein solution obtained by fusing ⁇ -galactosidase to a surface antigen having a molecular weight of 15 kDa with Treponema pallidum, a protein solution obtained by fusing -galatatosidase to a surface antigen having a molecular weight of 17 kDa with Treponema pallidum (Bios Pacific, 100 ⁇ g / mL) was used in the same manner as in Example 1 to prepare a reagent for measuring an anti-Treponemal antibody.
  • a 15 kDa surface antigen solution (GST15 antigen, Biokit, 100 x gZmL) of T. syphilis was used instead of a protein solution obtained by fusing ⁇ -galatatosidase to a surface antigen of 15 kDa molecular weight of Treponema pallidum. Except for the above, an anti-syphilis treponemal antibody measuring reagent was prepared in the same manner as in Example 1.
  • a surface antigen solution of 17 kDa molecular weight of Treponema pallidum (GST17 antigen, Biokit, 100 ⁇ g / mL) was used instead. Except for using, a reagent for measuring anti-Syphilis treponemal antibody was prepared in the same manner as in Example 2.
  • a fully automatic analyzer (Hitachi, Model 7170) was used to determine syphilis as a syphilis positive serum from clinical symptoms. Serum was diagnosed as syphilis-negative serum, and 20 cases of non-syphilis normal human serum were tested as samples.
  • a sample dilution buffer PBS buffer containing 1% BSA
  • an anti-treponema pallidum antibody measuring reagent capable of measuring an anti-treponema pallidum antibody with high sensitivity and specificity, and a measuring method using the same.
  • FIG. 1 is a diagram showing the results of an anti-treponema pallidum antibody measurement test performed on syphilis-positive serum in Examples.
  • FIG. 2 is a graph showing the results of an anti-treponema antibody assay for syphilis performed in Examples.

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Abstract

L'invention concerne un réactif permettant d'analyser l'anti-corps anti-Treponema pallidum spécifiquement et avec une grande sensibilité ainsi qu'une méthode d'analyse correspondante. Plus précisément, l'invention concerne un réactif utilisé pour analyser l'anti-corps anti-Treponema pallidum au moyen d'une réaction antigène-anticorps, c'est-à-dire un réactif permettant d'analyser un anti-corps anti-Treponema pallidum qui contient un antigène 15K comprenant une protéine dont la β-galactosidase est fusionnée avec un antigène de surface (poids moléculaire: 15kDa) de Treponema pallidum et/ou un antigène 17k comprenant une protéine dont la β-galactosidase est fusionnée avec un antigène de surface (poids moléculaire: 17 kDa) de Treponema pallidum.
PCT/JP2004/008902 2003-06-24 2004-06-24 Reactif pour analyse de l'anticorps anti- treponema pallidium et methode d'analyse de cet anticorps WO2004113921A1 (fr)

Applications Claiming Priority (2)

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JP2003-180087 2003-06-24
JP2003180087A JP3936678B2 (ja) 2002-07-19 2003-06-24 抗梅毒トレポネーマ抗体測定試薬及び抗梅毒トレポネーマ抗体の測定方法

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104698185A (zh) * 2015-02-10 2015-06-10 深圳市新产业生物医学工程股份有限公司 检测梅毒螺旋体抗体的试剂盒及其检测方法和应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07287017A (ja) * 1994-02-28 1995-10-31 Fujirebio Inc 抗梅毒トレポネマ抗体の測定方法
JPH09229938A (ja) * 1995-12-20 1997-09-05 Fujirebio Inc 抗梅毒トレポネーマ抗体の免疫分析方法及び免疫分析装置
JP2002257833A (ja) * 2000-12-28 2002-09-11 Shino Test Corp 測定対象物質の測定試薬及び測定対象物質の測定試薬の安定化方法
JP2003337134A (ja) * 2002-05-20 2003-11-28 Shino Test Corp 非特異反応抑制ペプチド、並びにこれを用いた非特異反応抑制方法及び抗体測定方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07287017A (ja) * 1994-02-28 1995-10-31 Fujirebio Inc 抗梅毒トレポネマ抗体の測定方法
JPH09229938A (ja) * 1995-12-20 1997-09-05 Fujirebio Inc 抗梅毒トレポネーマ抗体の免疫分析方法及び免疫分析装置
JP2002257833A (ja) * 2000-12-28 2002-09-11 Shino Test Corp 測定対象物質の測定試薬及び測定対象物質の測定試薬の安定化方法
JP2003337134A (ja) * 2002-05-20 2003-11-28 Shino Test Corp 非特異反応抑制ペプチド、並びにこれを用いた非特異反応抑制方法及び抗体測定方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104698185A (zh) * 2015-02-10 2015-06-10 深圳市新产业生物医学工程股份有限公司 检测梅毒螺旋体抗体的试剂盒及其检测方法和应用

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