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TOXOPLASMA 抗原分離와 家兎抗血淸과의 免疫血淸學的 硏究

Title
TOXOPLASMA 抗原分離와 家兎抗血淸과의 免疫血淸學的 硏究
Other Titles
Fractionation of Toxoplasma antigen and immunoserological studies of rabbits inoculated with Tosoplasma gondii
Authors
李玉蘭
Issue Date
1976
Department/Major
대학원 생물학과
Publisher
이화여자대학교 대학원
Degree
Master
Advisors
崔源永
Abstract
Serological studies on toxoplasmosis were conducted with rabbits sera that were immunized with RH-strain or infected with Beverley-strain of Tgoxoplasma gondii. Complement fixation tests, Agar-gel double diffusion tests and Agar-gel immunoelectrophoresis were performed. Toxoplasma crude antigen was prepared from the organism in mouse peritoneal fluids, which were infected with RH-strain of Toxoplasma gondii. The organisms were suspended in saline volume originally exudated and counted in hemocytometer for purity of the organisms over 99%. These suspended organisms were prepared by sonication, and the solution was centrifuged for 30 min. at 10,000 rpm in 4℃. These supernatant fluids were used, as crude antigen. On the other hand, Purified antigens were fractionated on Sephadex G-200 gel filtration. A Sephadex G-200 column, 80 by 4 cm, equilibrated, with Tris-HC1(0.1 M)-NaCl(1.0M) buffer, PH 8.0, was used. The elute fractions were collected in 3 ml per hour and the absorbance at 280 nm was measured with a Beckmari Du-2 spectrophotometer. Each tube is pooled into 6 fractions by protein density graph. For immunization of rabbits, crude antigen was emulsified with an equal amount of incomplete Freund's adjuvant and 1 ml of mixture was injected, subcutaneously into them once a week for 5 successive weeks. Antisera were obtained at an interval of a week after the first immunization, while several rabbits were infected with Beverley-strain of Toxoplasma gondii by inoculating about 200 cysts and antisera were obtained from them serially at a week interval. The results were as follows: 1. The immunized sera or infected sera against the crude antigen showed the first positive reactions in 2 or 3 weeks after the immunization or infection in complement fixation tests. Maximum tilers appeared in 4 or 5 weeks after immunization and in 7 or 9 weeks after infection respectively. Complement fixation tests in fractionated antigens are showed, the positive reactions in the immunized sera against the fractionated antigens II, III, IV, V and VI; moreover, antigen IV fraction showed, the highest titer. On. the other hand, in the infected sera against the fractionated antigens II, III and IV showed the positive reaction: especially, fractionated antigen IV showed the highest titer. 2. In agar-gel double diffusion -tests, immunized sera and infected sera against the crude antigen appeared the precipitin bands 2 weeks after the immunization or infection and the immunized sera showed 2 or 5 precipitin bands after 5-8 weeks and infected sera shoved the 1 or 2 precipitin bands after 6 weeks. Immunized sera against the fractionated, antigens II, III, IV, V and VI showed the precipitin bands, and the sera from the infected sera against the fractionated antigens II, III, and IV showed the precipitin bands. Especially antigen IV was the strongest reaction against the immunized sera and infected sera. 3. In agar-gel immunoelectrophoresis, the immunized sera against the crude antigen showed 8 arcs. But the infected sera against the crude antigen showed 4 or 5 arcs. The immunized sera against the fractionated antigers II, III, IV, V, VI showed arcs, and against the fractionated antigen IV showed 6arcs and in the antigens II, III, V, VI showed 1 or 2 arcs only. On the other hand, the infected sera against the fractionated antigens IV showed 4 arcs, II and III showed 1 arc, which was the most weak of all. Qn the other hand, Uhe a.iafect&d seg"a against ttie fyac- tionatect arrtigeiis IV ghia'sed 4 aycg, II U..rs.iI III ishov!e3 USos 1 arc, whj-oh was the most weak of all ;본 논문은 Toxoplasma症의 혈청학적 연구로서 Toxoplasma gondii RH株抗原(粗抗原)을써서 토끼에 면역시키고 한편 Beverley株蟲體로 다른 토끼群에 감염시켜서 補體結合反應, 寒天二重擴散反應, 寒天免疫電氣泳動反應으로 항원 - 항혈청 반응을 관찰한것이다. 항혈청에 대응할 항원으로蟲體가 含有된 생리식염수를 Sonicator로 분쇄하여 10,000rpm으로 원심분리한 후 그 상청액을 粗抗原(蛋白농도5.0mg/㎖) Sephadex G-200 Gel에 Tris-HCL(0.1M)-Nacl(1.0M) 완충액으로 유출시켜 단백농도에 의해 6개의 分劃으로 분리한 것 을 사용하였다. 면역혈청은 粗抗原 0.5㎖와 incomplete Freund's adjuvant 0.5㎖를 합한 1㎖를 1회 주사량으로 하여 1주 간격으로 토끼에 5회 피하 주사하여 첫번주사후 1주 후부터 1주 간격으로 채혈하여 얻었다. 감염혈청은 Beverley株씨스트 약 200個가 함유되어 있는 마우스 뇌를 토끼 복강에 주사하여 얻은 혈청이다. 실험결과를 요약하면 다음과 같다. 1. 補體結合反應은 면역군과 감염군이 粗항원과의 반응에서 양군 모두 면역과 감염 후 2~3주에 陽性反應을 나타내기 시작하였고 면역군은 면역후 4~5주에 최고의 力價에 달했으며 감염군은 7~9주에 최고치를 나타내었다. 한편 분리항원과의 반응은 면역혈청과는 Ⅱ Ⅲ Ⅳ Ⅴ Ⅵ 分晝에서 陽性反應이었으며 감염혈청과는 分劃 Ⅱ Ⅲ Ⅳ分劃이 양성반응이었다. 양군 모두 Ⅳ分劃이 가장 높은 力價를 나타내었다. 2. 寒天二重擴散反應에서 보면 粗抗原과 항혈청반응은 면역군과 감염군 모두 면역과 감염 후 2주 혈청에서 침강대를 만들었고 면역군은 5~8주에 침강대 2~5개로 가장 강한 염색반응을 나타내었다. 감염군은 6주 혈청에서 강한 반응을 보였다. 한편 분리항원과는 Ⅱ Ⅲ Ⅳ Ⅴ Ⅵ 分劃이 면역군에 대해 침강반응을 나타내었고 감염군에 대해서는 Ⅱ Ⅲ Ⅳ分劃에서 침강반응을 나타내었다. 3. 免疫電氣泳動反應에서 粗抗原과 免疫群은 8개의 침강대를 만들었으며 감염군에 대해서는 4~5개의 침강대를 나타내었다. 분리항원과 면역군은 Ⅳ分劃과 6개의 침강대가 만들어졌고 Ⅱ Ⅲ Ⅴ Ⅵ分劃에서는 1~2개의 침강대를 만들었다. 감염군과 Ⅳ分劃은 약한 4개의 침강대, Ⅱ Ⅲ分劃에서는 각각 약한 1개의 침강대를 만들었다. 이상의 결과로 보아 가장 강한 감수성을 나타낸 Ⅳ分劃중에는 특이항원성물질(特異抗原性物質) 이 존재한다고 생각된다.
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