WO2004113921A9 - Reagent for assaying anti-treponema pallidum antibody and method of assaying anti-treponema pallidum antibody - Google Patents
Reagent for assaying anti-treponema pallidum antibody and method of assaying anti-treponema pallidum antibodyInfo
- Publication number
- WO2004113921A9 WO2004113921A9 PCT/JP2004/008902 JP2004008902W WO2004113921A9 WO 2004113921 A9 WO2004113921 A9 WO 2004113921A9 JP 2004008902 W JP2004008902 W JP 2004008902W WO 2004113921 A9 WO2004113921 A9 WO 2004113921A9
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- Prior art keywords
- treponema pallidum
- antigen
- antibody
- kda
- molecular weight
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- 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
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- 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-syphilis antibodies in 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 N-S-transferase has been disclosed! / In addition, other methods have been reported for the measurement of anti-Treponemal antibody against syphilis using an antigen having a specific molecular weight prepared by a specific method.
- Patent Document 1 Japanese Patent Application Laid-Open No. 2-500403
- Patent Document 2 JP-A-7-287017
- Non-Patent Document 1 Journal of Immunology, vol. 129, pp. 1287—1291, 1982 Disclosure of Invention
- 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 measuring an anti-Treponemal anti-Syphilis antibody utilizing an antigen-antibody reaction, wherein the 15K antigen comprising a protein in which j3-galactosidase is fused to a surface antigen having a molecular weight of 15 kDa of syphilis and Z or
- This is an anti-treponema pallidum antibody measurement reagent containing a 17K antigen consisting of a protein in which ⁇ -extragalactosidase is fused to a surface antigen with a molecular weight of 17 kDa.
- the present inventors have conducted intensive studies and found that among the surface antigens of Treponema pallidum, a 15K antigen and / or a 17kDa surface antigen consisting of a protein in which ⁇ -galactosidase is fused to a surface antigen having a molecular weight of 15 kDa.
- a 15K antigen and / or a 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 ⁇ -galactosidase as an antigen, it was found that the anti-Treponemal anti-Syphilis antibody could be measured with significantly higher sensitivity than the conventional method, but with extremely high accuracy.
- the present invention has been completed.
- the anti-Treponemal anti-Syphilis antibody measuring reagent of the present invention has a molecular weight of 15 kDa of Treponema pallidum.
- Corrected form (Rule 91) 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 is as follows: 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.
- Both the 15K antigen consisting of a protein in which ⁇ -galactosidase is fused to a surface antigen having a molecular weight of 15 kDa and the 17K antigen consisting of a protein in which a galactosidase is fused to a surface antigen having a molecular weight of 17 kDa are known.
- these are used for immunoassay of anti-Treponemal syphilis antibodies their sensitivity is dramatically improved as compared with the case where a surface antigen not fused to ⁇ -galatatosidase is used, and In addition, a decrease in detection accuracy due to a non-specific reaction can be suppressed.
- 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 consisting of 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 obtained by fusing ⁇ -galactosidase to a surface antigen having a molecular weight of 15 kDa on a well of a microtiter plate or the like and a molecular weight of 15 kDa and / or the molecular weight of 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 17K antigen comprising a protein obtained by fusing ⁇ -galactosidase to a surface antigen having a molecular weight of 15
- 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 to react with the antigen.
- an anti-human immunoglobulin antibody labeled with an enzyme such as peroxidase and a chemiluminescent substrate are used.
- 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 particle carrier such as animal erythrocytes such as chickens, goats, sheep, poma, and porch;
- 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 measurement 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
- Table 1 Furthermore, based on the data shown in Table 1, the reaction amount is shown on the vertical axis, and the reaction amount is shown in FIG. 1 for syphilis-positive serum and in FIG. [0032] Table 1 shows that when the anti-treponema pallidum antibody assay reagents prepared in Examples 1 and 2 using a fusion protein fused with galactosidase were used, Comparative Example 1 using an antigen not fused with ⁇ -galactosidase was used. It was found that sera from syphilis patients could be detected with higher sensitivity and that non-specific reactions in the syphilis-negative serum group were lower than those using the prepared anti-treponema pallidum antibody assay reagent.
- 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 shows the results of an anti-treponema syphilis antibody assay performed on syphilis-positive sera 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
It is intended to provide a reagent for assaying anti-Treponema pallidum antibody by which anti-Treponema pallidum antibody can be highly sensitively and specifically assayed and an assay method using the same. Namely, a reagent to be employed in assaying anti-Treponema pallidum antibody with the use of an antigen-antibody reaction that is a reagent for assaying anti-Treponema pallidum antibody containing a 15K antigen comprising a protein having β-galactosidase fused with a surface antigen (molecular weight: 15 kDa) of Treponema pallidum and/or a 17K antigen comprising a protein having β-galactosidase fused with a surface antigen (molecular weight: 17 kDa) of Treponema pallidum.
Description
明 細 書 Specification
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 Anti-Treponemal anti-syphilis antibody assay reagent and method for measuring anti-T. Pallidum antibody
技術分野 Technical field
[0001] 本発明は、高い感度 ·特異性で抗梅毒トレポネーマ抗体を測定することができる抗梅 毒トレポネーマ抗体測定試薬及びこれを用いた測定方法に関する。 TECHNICAL FIELD [0001] 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.
背景技術 Background art
[0002] 梅毒は、梅毒トレポネーマ菌（トレポネーマ'パリダム、 Treponema pallidum)の感 染により引き起こされる疾患である。梅毒に罹患しているか否かは血液中に抗梅毒ト レポネーマ抗体が存在するか否力 ^免疫分析により検查することにより診断されてい る。梅毒トレポネーマの表面には多数の表面抗原が存在しており、この表面抗原と検 体中の抗梅毒トレポネーマ抗体との抗原抗体反応を利用する方法により免疫分析が 行われている。力かる梅毒トレポネーマ菌の細胞表面に存在する表面抗原のうち主 なものとしては、分子量 47kDa、 42kDa、 37kDa、 17kDa及び 15kDaのもの力知ら れている (非特許文献 1)。 [0002] Syphilis is a disease caused by infection of Treponema pallidum (Treponema pallidum). Syphilis is diagnosed by detecting the presence of anti-syphilis anti-syphilis antibodies in 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. 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).
[0003] 現在、この免疫分析に用いられている梅毒トレポネーマの表面抗原としては、梅毒ト レポネーマ菌を家兔睾丸で培養し、界面活性剤等で可溶化'抽出し、更に種々の方 法を用いて不要物の除去 ·必要成分の精製を行ったものが用いられている。しかし、 このような家兔を用いた製造方法は、動物を宿主とすることから収量が限られているう え、動物により梅毒トレポネーマ菌の増殖状態がばらつくことから、大量の安定した収 量を確保するのが困難であるという問題があった。一方、現時点では、梅毒トレポネ 一マ菌を直接人工培養することには成功してレ、なレ、。 [0003] 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.
[0004] これに対して、梅毒トレポネーマの表面抗原をコードする遺伝子をクローニングし、遺 伝子工学的に表面抗原を大量生産して免疫分析に用いる方法が提案されている。 例えば、特許文献 1には、梅毒トレポネーマの分子量 47kDaの抗原を遺伝子工学的 に調製し、これを用いて抗梅毒トレポネーマ抗体を免疫測定する方法が記載されて いる。また、特許文献 2には、 15kDa及び 17kDaの表面抗原の N末端にダルタチォ
ンー S—トランスフェラーゼが融合したタンパク質を用いる方法が開示されて！/、る。更 に、この他にも特定の方法で調製した特定の分子量の抗原を用いた抗梅毒トレポネ 一マ抗体の測定方法等が報告されてレ、る。 [0004] On the other hand, a method has been proposed in which a gene encoding a surface antigen of Treponema pallidum is cloned, and the surface antigen is mass-produced by genetic engineering and used for immunoassay. For example, 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 N-S-transferase has been disclosed! / In addition, other methods have been reported for the measurement of anti-Treponemal antibody against syphilis using an antigen having a specific molecular weight prepared by a specific method.
し力もながら、このような方法で調製された梅毒トレポネーマの表面抗原を用いても、 抗梅毒トレポネーマ抗体の測定を行うことは可能であるものの、感度及び精度の点で は必ずしも満足できるものではな力つた。 Although it is possible to measure anti-Treponemal syphilis antibodies using surface antigens of Treponema pallidum prepared by such a method, it is not always satisfactory in terms of sensitivity and accuracy. Helped.
[0005] 特許文献 1 :特表平 2— 500403号公報 [0005] Patent Document 1: Japanese Patent Application Laid-Open No. 2-500403
特許文献 2 :特開平 7— 287017号公報 Patent Document 2: JP-A-7-287017
非特許文献 1 Journal of Immunology, vol. 129, pp. 1287— 1291， 1982 発明の開示 Non-Patent Document 1 Journal of Immunology, vol. 129, pp. 1287—1291, 1982 Disclosure of Invention
発明が解決しょうとする課題 Problems to be solved by the invention
[0006] 本発明は、上記現状に鑑み、高い感度 ·特異性で抗梅毒トレポネーマ抗体を測定す ることができる抗梅毒トレポネーマ抗体測定試薬及びこれを用いた測定方法を提供 することを目的とするものである。 [0006] In view of the above circumstances, 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.
課題を解決するための手段 Means for solving the problem
[0007] 本発明は、抗原抗体反応を利用した抗梅毒トレポネーマ抗体の測定に用いる試薬で あって、梅毒トレポネーマの分子量 15kDaの表面抗原に j3—ガラクトシダーゼが融 合したタンパク質からなる 15K抗原及び Z又は梅毒トレポネーマの分子量 17kDaの- 表面抗原に β—ガラ外シダーゼが融合したタンパク質からなる 17K抗原を含有する 抗梅毒トレポネーマ抗体測定試薬である。 [0007] The present invention relates to a reagent used for measuring an anti-Treponemal anti-Syphilis antibody utilizing an antigen-antibody reaction, wherein the 15K antigen comprising a protein in which j3-galactosidase is fused to a surface antigen having a molecular weight of 15 kDa of syphilis and Z or This is an anti-treponema pallidum antibody measurement reagent containing a 17K antigen consisting of a protein in which β-extragalactosidase is fused to a surface antigen with a molecular weight of 17 kDa.
以下に本発明を詳述する。 Hereinafter, the present invention will be described in detail.
[0008] 本発明者らは、鋭意研究の結果、梅毒トレポネーマの表面抗原のうち、分子量 15kD aの表面抗原に β一ガラクトシダ一ゼが融合したタンパク質からなる 15K抗原及び/ 又は分子量 17kDaの表面抗原に β—ガラクトシダーゼが融合したタンパク質からな る 17K抗原を抗原として用レ、ることにより、従来の方法よりも有意に高い感度で、しか も極めて高い精度で抗梅毒トレポネーマ抗体を測定できることを見いだし、本発明を 完成するに至った。 The present inventors have conducted intensive studies and found that among the surface antigens of Treponema pallidum, a 15K antigen and / or a 17kDa surface antigen consisting of a protein in which β-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 β-galactosidase as an antigen, it was found that the anti-Treponemal anti-Syphilis antibody could be measured with significantly higher sensitivity than the conventional method, but with extremely high accuracy. The present invention has been completed.
[0009] 本発明の抗梅毒トレポネーマ抗体測定試薬は、梅毒トレポネーマの分子量 15kDa [0009] The anti-Treponemal anti-Syphilis antibody measuring reagent of the present invention has a molecular weight of 15 kDa of Treponema pallidum.
訂正された用紙 （規則 91)
の表面抗原に β一ガラクトシダーゼが融合したタンパク質からなる 15K抗原及び/又 は梅毒トレポネーマの分子量 17kDaの表面抗原に β一ガラクトシダーゼが融合した タンパク質からなる 17Κ抗原を含有するものである。 Corrected form (Rule 91) It contains a 15K antigen consisting of a protein in which β-galactosidase is fused to the surface antigen and / or a 17Κ antigen consisting of a protein in which β-galactosidase is fused to the surface antigen of molecular weight 17 kDa of Treponema pallidum.
これらの抗原のうち、いずれか一方が含まれていてもよいし、両方が含まれていても よい。特に両方が含まれる場合には、検出感度が更に高まるので好ましい。 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.
[0010] 上記梅毒トレポネーマの分子量 15kDaの表面抗原及び分子量 17kDaの表面抗原 については、既にコードする遺伝子がクローユングされており、また、これらのアミノ酸 配列も決定されている（INFECTION AND IMMUNITY, vol. 57, NO. 12, p p. 3708-3714, 1989 ; Molecular Microbiology, vo. 4， NO. 8， ppl 371— 1 379, 1990 ; INFECTION AND IMMUNITY, vol. 61， NO. 4， pp. 1202- 1210, 1993)。従って、これらを常法により遺伝子工学的に生産して用いることがで きる。 [0010] Regarding the surface antigen with a molecular weight of 15 kDa and the surface antigen with a molecular weight of 17 kDa of the above-mentioned Treponema pallidum, the genes encoding them have already been cloned, and their amino acid sequences have also been determined (INFECTION AND IMMUNITY, vol. 57). , NO. 12, pp. 3708-3714, 1989; Molecular Microbiology, vo. 4, NO. 8, ppl 371—1379, 1990; INFECTION AND IMMUNITY, vol. 61, NO. 4, pp. 1202-1210 , 1993). Therefore, these can be produced and used in a conventional manner by genetic engineering.
[0011] 上記梅毒トレポネーマの分子量 15kDaの表面抗原及び分子量 17kDaの表面抗原 に β一ガラクトシダーゼを融合させる方法としては特に限定されず常法を用いることが できる。 The method for fusing β-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.
β一ガラクトシダーゼはラタトースの分解及び合成を行う酵素であり、大腸菌では古く 力 ラタトースによって誘導される誘導酵素の典型として研究されている。 β-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.
β一ガラクトシダーゼは分子量約 116kDaであり 4量体を形成する。その構造遺伝子 は lacZと呼ばれ、その遺伝子配列は既知であり（EMBO J 1963； 2 (4)： 593-59 7)、各種のプラスミドに含まれる形で市販されている。市販されているもののうち代表 的なものとしては、例えば、 pUC18、 pUC19 (いずれも東洋紡社製）等が挙げられる が、外来タンパク質と /3 _ガラクトシダーゼとの融合タンパク質が発現し得るものであ れば特に限定されない。また、梅毒トレポネーマの分子量 15kDaの表面抗原及び分 子量 17kDaの表面抗原に j3 -ガラクトシダーゼを融合させる際には、 C末端或いは N末端のいずれからでも良ぐ特には限定されない。 β-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. In addition, when fusing j3-galactosidase to a surface antigen having a molecular weight of 15 kDa and a surface antigen having a molecular weight of 17 kDa of Treponema pallidum, no particular limitation is imposed from either the C-terminal or the N-terminal.
[0012] 上記梅毒トレポネーマの分子量 15kDaの表面抗原及び分子量 17kDaの表面抗原 に β—ガラタトシダーゼを融合させる方法しては、梅毒トレポネーマの分子量 15kDa の表面抗原又は分子量 17kDaの表面抗原の構造遺伝子を lacZ遺伝子に導入すれ
ばよぐその方法は当業者にとっては既知の遺伝子組み換え技術で可能であり、特 に限定されない。 [0012] The method for fusing β-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 is as follows: 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.
[0013] 梅毒トレポネーマの分子量 15kDaの表面抗原又は分子量 17kDaの表面抗原と - ガラクトシダーゼとが融合した融合タンパク質を精製する方法としては特に限定され ず、例えば、 p—ァミノべンジルー 1_チォー j3 _D—ガラクトピラノシドが結合したァガロ ースカラム（例えば、シグマ社製）、又は、抗 j3—ガラクトシダーゼ抗体（例えば、プロメ ガ社製)等を用いて精製する方法等が挙げられる。 [0013] 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. For example, 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).
なお、上記梅毒トレポネーマの分子量 15kDaの表面抗原及び分子量 17kDaの表面 抗原に 一ガラクトシダーゼを融合したタンパク質は、一般に販売されているので、こ れらの市販品を用いることもできる。 In addition, 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.
[0014] これらの梅毒トレポネーマの分子量 15kDaの表面抗原に /3—ガラタトシダーゼが融 合したタンパク質からなる 15K抗原又は梅毒トレポネーマの分子量 17kDaの表面抗 原に β ガラクトシダーゼが融合したタンパク質からなる 17K抗原は、ラテックス粒子 等の適当な担体に固定化してもよい。力かる担体に固定化することにより本発明の抗 梅毒トレポネーマ抗体測定試薬を免疫凝集法等に供することができる。 [0014] 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 β-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.
上記固定化の方法としては特に限定されず、例えば、物理的吸着法、担体表面に設 けた官能基と化学結合する方法等が挙げられる。 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.
[0015] 上記梅毒トレポネーマの分子量 15kDaの表面抗原に β ガラクトシダーゼが融合し たタンパク質からなる 15K抗原と梅毒トレポネーマの分子量 17kDaの表面抗原に —ガラタトシダーゼが融合したタンパク質からなる 17K抗原とはいずれも公知のもので あつたが、驚くべきことに、これらを用いて抗梅毒トレポネーマ抗体を免疫測定すると 、その感度は β—ガラタトシダーゼと融合していない表面抗原を用いた場合よりも飛躍 的に向上し、かつ、非特異的反応による検出精度の低下を抑制することができる。 抗原抗体反応を利用して検体中の抗梅毒トレポネーマ抗体を測定する方法であって 、抗原として、梅毒トレポネーマの分子量 15kDaの表面抗原に /3 _ガラクトシダーゼ が融合したタンパク質からなる 15K抗原及び Z又は梅毒トレポネーマの分子量 17k Daの表面抗原に /3 _ガラクトシダーゼが融合したタンパク質からなる 17K抗原を用い る抗梅毒トレポネーマ抗体の測定方法もまた、本発明の 1つである。
[0016] 本発明の抗梅毒トレポネーマ抗体の測定方法としては、本発明の抗梅毒トレポネー マ抗体測定試薬を用いるのであれば特に限定されず、通常の方法により行うことがで きる。即ち、反応形式で分類すればサンドイッチ法、凝集法等を利用することができ、 また標識で分類すれば、酵素免疫測定法、蛍光分析測定法、放射免疫分析等を採 用すること力 sできる。なかでも、操作が簡便で感度も良好であり、かつ多数の検体の 処理に適してレ、るラテックス免疫凝集法がより好ましレ、。 [0015] Both the 15K antigen consisting of a protein in which β-galactosidase is fused to a surface antigen having a molecular weight of 15 kDa and the 17K antigen consisting of a protein in which a galactosidase is fused to a surface antigen having a molecular weight of 17 kDa are known. Surprisingly, surprisingly, when these are used for immunoassay of anti-Treponemal syphilis antibodies, their sensitivity is dramatically improved as compared with the case where a surface antigen not fused to β-galatatosidase is used, and In addition, a decrease in detection accuracy due to a non-specific reaction can be suppressed. 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 consisting of 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. [0016] 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.
[0017] 本発明の抗梅毒トレポネーマ抗体の測定方法の具体例としては、例えば、梅毒トレポ ネーマの分子量 15kDaの表面抗原に β—ガラタトシダーゼが融合したタンパク質か らなる 15K抗原及び/又は梅毒トレポネーマの分子量 17kDaの表面抗原に /3—ガ ラタトシダーゼが融合したタンパク質からなる 17K抗原をラテックス粒子に固定化し、 所定の操作を得てラテックス試薬として調製した後、これに検体を加えて反応させ、 所定の時間間隔での濁度変化を視覚的、光学的又は電気化学的に検出する方法 が挙げられる。 [0017] 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 β-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.
[0018] また、別の具体例としては、例えば、マイクロタイタープレート等のゥエルに梅毒トレポ ネーマの分子量 15kDaの表面抗原に β一ガラクトシダーゼが融合したタンパク質か らなる 15K抗原及び/又は梅毒トレポネーマの分子量 17kDaの表面抗原に -ガ ラタトシダーゼが融合したタンパク質からなる 17K抗原を固定化し、非特異的吸着部 位をブロッキング後、洗浄し、これに検体を加えて反応させ、洗浄後、ペルォキシダー ゼ等の酵素で標識した抗ヒト免疫グロブリン抗体を反応させ、洗浄後、標識抗体の基 質を加えて酵素反応を行レ、発色させ、反応停止後の発色の程度を吸光度測定によ り測定する方法が挙げられる。 [0018] Further, as another specific example, for example, a 15K antigen comprising a protein obtained by fusing β-galactosidase to a surface antigen having a molecular weight of 15 kDa on a well of a microtiter plate or the like and a molecular weight of 15 kDa and / or the molecular weight of 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. Can be
[0019] 更に、別の具体例としては、例えば、フェライト磁性粒子等の粒子に梅毒トレポネー マの分子量 15kDaの表面抗原に β一ガラクトシダーゼが融合したタンパク質からなる 15K抗原及び/又は梅毒トレポネーマの分子量 17kDaの表面抗原に /3 _ガラクトシ ダーゼが融合したタンパク質からなる 17K抗原を固定化し、これに検体を加えて反応 させ、ペルォキシダ-ゼ等の酵素で標識した抗ヒト免疫グロブリン抗体と化学発光基 質とを反応させる化学発光酵素免疫測定法 (CLEIA)が挙げられる。 Further, as another specific example, for example, a 15K antigen consisting of a protein in which β-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 to react with the antigen.An anti-human immunoglobulin antibody labeled with an enzyme such as peroxidase and a chemiluminescent substrate are used. And a chemiluminescent enzyme immunoassay (CLEIA).
[0020] この他にも、上記ラテックス粒子、プラスチックプレート、フェライト磁性粒子以外の不
溶化担体、例えば、特公昭 63-29223号公報に開示されているゼラチン等からなる 人工担体；ニヮトリ、ャギ、ヒッジ、ゥマ、ゥシ等の動物赤血球等の粒子担体に固定化 し、この担体の懸濁液に検体をカ卩えて粒子が凝集するか否力を測定する凝集免疫 分析法も挙げられる。 [0020] In addition, other than the latex particles, the plastic plate, and the ferrite magnetic particles, A solubilized carrier, for example, an artificial carrier composed of gelatin or the like disclosed in JP-B-63-29223; immobilized on a particle carrier such as animal erythrocytes such as chickens, goats, sheep, poma, and porch; There is also an 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.
[0021] 本発明の抗梅毒トレポネーマ抗体の測定方法に供される検体としては特に限定され ず、梅毒の診断を行うべきヒトゃ動物の血清等の体液及びその希釈物を挙げることが できる。 [0021] 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.
[0022] 本発明の抗梅毒トレポネーマ抗体の測定方法は、本発明の抗梅毒トレポネーマ抗体 測定試薬を用いることにより、極めて高い感度 ·精度で抗梅毒トレポネーマ抗体を測 定することができ、より正確な梅毒の診断を行うことができる。 [0022] 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.
発明の効果 The invention's effect
[0023] 本発明によれば、高い感度 ·特異性で抗梅毒トレポネーマ抗体を測定することができ る抗梅毒トレポネーマ抗体測定試薬及びこれを用いた測定方法を提供することがで きる。 According to the present invention, 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.
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
[0024] 以下に実施例を掲げて本発明を更に詳しく説明するが、本発明はこれら実施例のみ に限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to only these Examples.
[0025] (実施例 1) (Example 1)
ラテックス液 (積水化学工業社製：平均粒径 0. 3 μ ΐη、固形分 10%) 0. 05mLを試 験管内で攪拌しながら、梅毒トレポネーマの分子量 15kDaの表面抗原に ーガラタト シダ—ゼを融合したタンパク質溶液（Bios Pacific社製、 100 μ g/mL) lmLを添加 し、ラテックス粒子の表面に融合タンパク質を固定化した。 Latex solution (manufactured by Sekisui Chemical Co., Ltd .: average particle size: 0.3 μΐη, 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 µg / mL) was added to immobilize the fusion protein on the surface of the latex particles.
[0026] 得られた融合タンパク質固定化ラテックス粒子の抗原未吸着部分を 1%ゥシ血清ァ ノレブミン（Bovine Serum Albumin:以下、 BSAともいう）を含む PBSバッファーに てブロッキングし、未吸着抗原を遠心洗浄にて除去し、充分攪拌した後、 1%BSAを 含む PBSバッファ一にて 0. 1 %に希釈して、抗梅毒トレポネーマ抗体測定試薬とし た。 [0026] 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.
[0027] (実施例 2)
梅毒トレポネーマの分子量 15kDaの表面抗原に β一ガラクトシダーゼを融合したタン パク質溶液の代わりに、梅毒トレポネーマの分子量 17kDaの表面抗原に -ガラタト シダーゼを融合したタンパク質溶液（Bios Pacific社製、 100 μ g/mL)を用いた以 外は実施例 1と同様にして、抗梅毒トレポネーマ抗体測定試薬を調製した。 (Example 2) Instead of a protein solution obtained by fusing β-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 μg / mL) was used in the same manner as in Example 1 to prepare a reagent for measuring an anti-Treponemal antibody.
[0028] (比較例 1) (Comparative Example 1)
梅毒トレポネーマの分子量 15kDaの表面抗原に β—ガラタトシダ―ゼを融合したタン パク質溶液の代わりに、梅毒トレポネーマの分子量 15kDaの表面抗原溶液（GST1 5抗原、 Biokit社製、 100 x gZmL)を用いた以外は実施例 1と同様にして、抗梅毒 トレポネーマ抗体測定試薬を調製した。 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 β-galatatosidase to a surface antigen of 15 kDa molecular weight of Treponema pallidum. Except for the above, an anti-syphilis treponemal antibody measurement reagent was prepared in the same manner as in Example 1.
[0029] (比較例 2) (Comparative Example 2)
梅毒トレポネーマの分子量 17kDaの表面抗原に β—ガラタトシダ―ゼを融合したタン パク質溶液の代わりに、梅毒トレポネーマの分子量 17kDaの表面抗原溶液（GST1 7抗原、 Biokit社製、 100 μ g/mL)を用いた以外は実施例 2と同様にして、抗梅毒 トレポネーマ抗体測定試薬を調製した。 Instead of a protein solution obtained by fusing β-galatatosidase to a surface antigen of 17 kDa molecular weight of Treponema pallidum, a surface antigen solution of 17 kDa molecular weight of Treponema pallidum (GST17 antigen, Biokit, 100 μg / 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.
[0030] 実施例 1、 2及び比較例 1、 2で調製した抗梅毒トレポネーマ抗体測定試薬を用いて 全自動分析装置（日立社製、 7170形）により、梅毒陽性血清として臨床症状から梅 毒と診断された患者血清 20例を、梅毒陰性血清として各種血清検査等から非梅毒 である正常ヒト血清 20例を検体として試験を行った。 [0030] Using the anti-treponema pallidum antibody assay reagents prepared in Examples 1 and 2 and Comparative Examples 1 and 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.
即ち、検体希釈用緩衝液（1 %BSAを含む PBSバッファー） 120 レ抗梅毒トレポ ネーマ抗体測定試薬 60 μ L及び検体 15 μ Lを加え、 20— 34分間の溶液の濁度の 変化を 700nmの吸光度の変化量として測定し、これを反応量とした。 That is, a sample dilution buffer (PBS buffer containing 1% BSA) 120 Add 60 μL of anti-Syphilis anti-Treponemal antibody measurement reagent and 15 μL of sample, and change the turbidity of the solution for 20-34 minutes to 700 nm It was measured as the amount of change in absorbance, and this was used as the reaction amount.
結果を表 1に示した。 The results are shown in Table 1.
[0031] [表 1]
更に、表 1のデータより縦軸を反応量として梅毒陽性血清の場合を図 1に、梅毒陰性 血清の場合を図 2に示した。
[0032] 表 1より、 ガラクトシダーゼが融合した融合タンパク質を用いた実施例 1、 2で作製 した抗梅毒トレポネーマ抗体測定試薬を用いた場合には、 β ガラクトシダーゼを融 合しない抗原を用いた比較例 1、 2作製した抗梅毒トレポネーマ抗体測定試薬を用い た場合に比べ、高感度で梅毒患者血清を検出することができ、かつ、梅毒陰性血清 群における非特異反応も低いことがわかった。 [0031] [Table 1] Furthermore, based on the data shown in Table 1, the reaction amount is shown on the vertical axis, and the reaction amount is shown in FIG. 1 for syphilis-positive serum and in FIG. [0032] Table 1 shows that when the anti-treponema pallidum antibody assay reagents prepared in Examples 1 and 2 using a fusion protein fused with galactosidase were used, Comparative Example 1 using an antigen not fused with β-galactosidase was used. It was found that sera from syphilis patients could be detected with higher sensitivity and that non-specific reactions in the syphilis-negative serum group were lower than those using the prepared anti-treponema pallidum antibody assay reagent.
産業上の利用可能性 Industrial applicability
[0033] 本発明によれば、高い感度 ·特異性で抗梅毒トレポネーマ抗体を測定することができ る抗梅毒トレポネーマ抗体測定試薬及びこれを用いた測定方法を提供することがで きる。 According to the present invention, it is possible 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.
図面の簡単な説明 Brief Description of Drawings
[0034] [図 1]実施例において梅毒陽性血清について行った抗梅毒トレポネーマ抗体測定試 験の結果を示す図である。 FIG. 1 shows the results of an anti-treponema syphilis antibody assay performed on syphilis-positive sera in Examples.
[図 2]実施例にぉレ、て梅毒陰性血清にっレ、て行った抗梅毒トレポネーマ抗体測定試 験の結果を示す図である。
FIG. 2 is a graph showing the results of an anti-treponema antibody assay for syphilis performed in Examples.
Claims
[1] 抗原抗体反応を利用した抗梅毒トレポネーマ抗体の測定に用いる試薬であって、梅 毒トレポネーマの分子量 15kDaの表面抗原に β—ガラタトシダーゼが融合したタンパ ク質からなる 15K抗原及び Ζ又は梅毒トレポネーマの分子量 17kDaの表面抗原に /3—ガラクトシダーゼが融合したタンパク質からなる 17K抗原を含有することを特徴と する抗梅毒トレポネーマ抗体測定試薬。 [1] A reagent used for the measurement of an anti-treponema pallidum antibody utilizing an antigen-antibody reaction, comprising a 15K antigen consisting of a protein in which β-galatatosidase is fused to a surface antigen of 15 kDa molecular weight of treponema pallidum and ト レ or treponema pallidum 1. A reagent for measuring an anti-treponema pallidum antibody, comprising a 17K antigen comprising a protein obtained by fusing a / 3-galactosidase to a surface antigen having a molecular weight of 17kDa.
[2] 抗原抗体反応を利用した抗梅毒トレポネーマ抗体の測定に用いる試薬であって、担 体と、前記担体に固定化された梅毒トレポネーマの分子量 15kDaの表面抗原に 一 ガラクトシダーゼが融合したタンパク質からなる 15K抗原及び/又は梅毒トレポネー マの分子量 17kDaの表面抗原に β一ガラクトシダーゼが融合したタンパク質からなる 17K抗原とからなることを特徴とする抗梅毒トレポネーマ抗体測定試薬。 [2] A reagent used for measuring an anti-treponema pallidum antibody using an antigen-antibody reaction, comprising a carrier and a protein obtained by fusing one galactosidase to a surface antigen having a molecular weight of 15 kDa of treponema pallidum immobilized on the carrier. A reagent for measuring an anti-treponema pallidum antibody, comprising: a 15K antigen and / or a 17K antigen consisting of a protein in which β-galactosidase is fused to a surface antigen having a molecular weight of 17 kDa of treponema pallidum.
[3] 抗原抗体反応を利用して検体中の抗梅毒トレポネーマ抗体を測定する方法であって 、抗原として、梅毒トレポネーマの分子量 15kDaの表面抗原に 一ガラクトシダーゼ が融合したタンパク質からなる 15K抗原及び/又は梅毒トレポネーマの分子量 17k Daの表面抗原に —ガラクトシダーゼが融合したタンパク質からなる 17K抗原を用い ることを特徴とする抗梅毒トレポネーマ抗体の測定方法。 [3] A method for measuring an anti-treponema pallidum antibody in a sample using an antigen-antibody reaction, wherein the antigen is a 15K antigen and / or a protein obtained by fusing one galactosidase to a surface antigen having a molecular weight of 15 kDa of treponema syphilis. A method for measuring an anti-treponema pallidum antibody, which comprises using a 17K antigen comprising a protein fused with galactosidase to a surface antigen having a molecular weight of 17 kDa of Treponema pallidum.
[4] 免疫凝集法により抗梅毒トレポネーマ抗体を測定する方法であって、担体に固定化 された梅毒トレポネーマの分子量 15kDaの表面抗原に β—ガラタトシダーゼが融合 したタンパク質からなる 15K抗原及び/又は梅毒トレポネーマの分子量 17kDaの表 面抗原に 一ガラクトシダーゼが融合したタンパク質からなる 17K抗原を用いることを 特徴とする請求項 3記載の抗梅毒トレポネーマ抗体の測定方法。
[4] A method for measuring an anti-treponema pallidum antibody by an immunoagglutination method, wherein the 15K antigen and / or treponema pallidum are composed of a protein in which β-galatatosidase is fused to a surface antigen having a molecular weight of 15 kDa and immobilized on a carrier. 4. The method for measuring an anti-treponema anti-syphilis antibody according to claim 3, wherein a 17K antigen consisting of a protein obtained by fusing one galactosidase to a surface antigen having a molecular weight of 17 kDa is used.
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| JP2003-180087 | 2003-06-24 | ||
| JP2003180087A JP3936678B2 (en) | 2002-07-19 | 2003-06-24 | Anti-syphilis treponema antibody measurement reagent and anti-syphilis treponema antibody measurement method |
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| JP3831759B2 (en) * | 1995-12-20 | 2006-10-11 | 富士レビオ株式会社 | Method and apparatus for immunoassay of anti-syphilis treponema antibody |
| JP4318092B2 (en) * | 2000-12-28 | 2009-08-19 | 株式会社シノテスト | Measuring reagent for measuring substance and method for stabilizing measuring reagent for measuring substance |
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