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4 表表 2 3 図1 量の1/100加 Sephadex えて,4 G-25お 0.14M NaCl 4時間conjugateしよびDEAE 0.01M PB使子を取り除いて,さたこれを cellulose 用)処らにAceton column (ph 7.4, 理を行ない非特異因 mouse liver powderで吸収操作を行なつて精製した (表3) 4) 上記精製標識抗体についてF/P ratioの測定,梅毒患者血清および非梅毒血清を用いて非特異反応の検討を行なつた成績図1,写真1は1/3飽和硫安3回 cellulose column処 M 用)血清(C)お PB使 ulose bumin, acetate膜 7.5, 0.015M よび全血清(A)に α1-, α2-, β-, γ-globulinと cellulose処処理血清(B), β-,γ-globulinを写真ついてCell血清はAl- 分かれたが1/3飽和硫認めたまたDEAE 理のものでは γ2(γss)-globulinのれなかつた DEAE NaCl 電気泳動を行なつた像で,全安処理のものでは 186 理(pH みしかみらつぎにOuchterlony法では図2,写抗原側に1/3飽和硫安3回処理血清(B)お cellulose処理血清(C)を,抗清(Anti は3本 A=AA)を真2に示す如くよびDEAE 体側に抗ヒト血清家兎血置いて沈降反応を行なつた B側にの沈降線がみられたがC側には明らかに1本の沈降線しかみとめられない

cellulose column処 M 用)血清(C)お PB使 ulose bumin, acetate膜 7.5, 0.015M よび全血清(A)に α1-, α2-, β-, γ-globulinと cellulose処処理血清(B), β-,γ-globulinを写真1 0.

5 日本細菌学雑誌20 (4), 1965 図4 図2 注:AA=抗ヒト血清家兎血清 B=1/3飽和硫安3回 C=DEAE 処理血清 cellulose処理血清写真4 また免疫電気泳動法では図3,写ヒト血清を抗体側(溝)に Bは γ2-globulin γ1-globulin以みられたがCは真3に示すように抗おきAは12 13本 γ2-globulin の沈降線を, 外に2 3本 1本の沈降線がのみしか証明できなかつたさらに精製抗7S 抗7S びNHS 写真2 γ-globulin血 γ-globulin血 (A)を清を,穴泳動清の検討として,溝に7S γ-globulin させた図4,写に明らかに対称的に γ2-globulinのに (C)お真4によ示すよう位置にそれぞれ1本の沈降線をみたこのことから純粋な抗7S γ-globulin 抗体を作製できたとみてよい精製FITC標識抗体について,ま定したところでずF/P ratioを測あつたこの数値は螢光抗体法に充分使用しうるものであるつぎに実際に梅毒血清および非梅毒血清のものを標準血清とし,標 Aを識抗体を4倍から128倍行ない検討した表4にまで希釈してFT 示すように64倍まで3+ 表4 図3 で,128倍で2+と 2単位すなわち32倍したまた4倍なつた 64倍のものを1単位として希釈にして実験に使用することに希釈のものでも非特異染色は認められなかつたがBackgroundのNonspecific stainingはかなり強かつた写真3 実験II 各期梅毒患者血清中の7S型梅毒抗体の推 187

称的に γ2-globulinのに (C)お真4によ示すよう位置にそれぞれ1本の沈降線をみたこのことから純粋な抗7S γ-globulin 抗体を作製できたとみてよい精製FITC標識抗体について,ま定したところ1.

12 Serodiagnosis of Syphilis by Fluorescent Treponemal Antibody Test (FTA Test) 2. On the Transition of Antibodies of 7S ƒá Type in the Syphilitic Sera in Each Stage and the Specificity of FTA Test Tatsuo SASAHIRA Department of Microbiology, Kyoto Prefectural University of Medicine (Director: Prof. S. Suzuki, M.D.) Even if serodiagnosis of syphilis with FTA test, using fluorescent antibody to human ƒá-globulin has the advantages of being convenient, specific and highly sensitive, still it remains the problem of so-called biological false positive reaction in a debate. In order to investigate in detail the specificity of this test we tried first to study the distribution of antibodies, especially antibodies of 7S ƒá type in syphilitic sera in each stage. Two kinds of the antiserum were used as anti-human ƒá-globulin. In order to get purified 7S ƒá-globulin 8 00mg of the same globulin was separated by chromatography on DEAE-cellulose as follows: 200ml of normal human serum was salted out with 33% saturated (NH4)2SO4 three times, whereafter it was transfered to a column (4.0 ~25.0cm) of DEAE-cellulose equilibrated with 0.01M phosphate buffer, ph 7.5, containing 0.015M NaCl and eluted with the same buffer. Elution with this buffer gave a pure ƒá2-globulin peak as shown by cellulose acetate electrophoresis, Ouchterlony agar diffusion method and immunoelectrophoresis using LKB's immunophor. Then, anti-7s ƒá-globulin was prepared in rabbits following a course of immunization which included intramuscular injections of alum precipitated antigen (purified 7S ƒá-globulin preparation) and intravenous injections of 1% aqueous solution of the antigen and then ƒá-globulin fraction of thus raised antiserum was conjugated with fluorescein isothiocyanate (FITC). Its F/P ratio was estimated as 1.32 ~10-3. Immunoelectrophoresis with normal human serum showed a strong 7S ƒá line only. We used FTA tests with this FITC-labeled anti-7s ƒá-globulin and with FITC-labeled anti-ƒá-globulin, in which the latter ƒá-globulin was prepared by routine ammonium sulfate precipitation and thus it contained both globulins of 7S ƒá and 19S ƒá types. Comparative studies were undertaken to investigate the transition of antibodies of 7S ƒá and 19S ƒá types in the syphilitic sera by both methods. From our results it was found that 7S ƒá class antibodies appeared always in the sera of primary stage (2 cases), secondary stage (5 cases), tertiary stage (6 cases) and congenital syphilis (4 cases), but in contrast to this 19S ƒá class antibody was found only in the sera of primary syphilis. These findings seem likely to show the evidence that in the very early stages of syphilis macroglobulin (ƒá1m-globulin) antibodies appeared, but later predominantly 7S ƒá-globulin (ƒá2-globulin) antibodies. One hundred thirty seven sera which were sent to our laboratory for FTA test, were tested also by RPCF, W.R. and VDRL tests, comparing with the results of this test. It was found that discrepancies of 20%, were encountered between FTA test and several other tests for syphilis (RPCF, W.R. and VDRL tests). The present results show that FTA test is found to be most specific compared with the other three tests for syphilis, and also show the most promising results also in the cases of biological false positive reaction.

(Director: Prof. S. Suzuki, M.D.) Even if serodiagnosis of syphilis with FTA test, using fluorescent antibody to human ƒá-globulin has the advantages of being convenient, specific and highly

semen quality or those without WBC in semen. In the patients with azoospermia and normal FSH levels (normogonadotropic azzospermia), the antibody (IgG

semen quality or those without WBC in semen. In the patients with azoospermia and normal FSH levels (normogonadotropic azzospermia), the antibody (IgG CLINICAL STUDIES OF UROGENITAL INFECTIONS WITH CHLAMYDIA TRACHOMA TIS Report 2. The Epidemiology of Chlamydial Infections in Okayama District in Japan and Detection of Antibodies to Chlamydiae in the Sera