Study on hybridoma clones secreting anti-toxoplasma gondii antibody

의학과/박사[한글] Toxoplasmosis의 진단을 위해 많은 면역학적 진단방법이 연구 개발되어 이용되고 있으나(Jacobs, 1976), 낮은 감수성과 특이성 때문에 실제 임상이용에 있어 많은 문제점들을 내포하고 있다. 최근 Kohler와 Milstein(1975)은 어떤 항원으로 면역시킨 마우스의 비장 임파구와 마우스 myeloma세포를 융합하여 얻은 hybrid세포를 계속 배양함으로써 특이항체를 다량 계속적으로 분비한다고 보고하였다. 이 hybridoma 항체를 이용하여 기생충감염에 대한 보다 좋은 면역학적 진단방법의 개발과 숙주자체를 면역시킬 수 있는 vaccine개발 의 가능성을 열어 놓았다. 본 실험에서는 마우스의 myeloma 세포와 Toxoplasma gondii에 면역된 마우스의 비장임파구를 융합시켜 hybrid cell을 얻고 이를 계속 배양하여 얻은 hybridoma로부터 Toxoplas ma gondii의 항원에 반응하는 hybridoma항체를 간접형광항체법으로써 측정하고자 하였다. Toxoplasma gondii RH주를 ICR마우스 복강내에 계대감염시켜 얻은 복수액을 채취하여 원심침전한 후 그 침사를 10% formaldehyde로 고정하고 생리식염수로 여러번 세척하여 만든 세포부유액을 항원으로 사용하였다. 이 항원을 BALB/c마우스 복강내에 1주에 한번씩, 한번에 T. gondii영양형 1×10**6개씩 되게 4∼5회 주사하여 면역시켰다. 마우스 myeloma세포인 P3/NSI/1/AG4/1(NS-1)을 20% fetal calf serum(이하 FCS로 약함) 함유 RPMI 1640배지(이하 RPMI배지로 약함)를 사용하여 5% CO^^2 항온기에서 배양하였다. 세포융합은 Galfre등(1977), Kohler등(1976) 및 Littlefield(1964)의 융합 방법을 참고하였다. 면역된 마우스 비장조직으로부터 제조된 비장세포 부유액을 원심침전시켜 상충액 을 제거하고 그 침사에 적혈구를 제거하기 위해 0.75% NH^^4 Cl함유 0.Ol6M tris완충액(pH 7.25)을 첨가한 후 실온에서 5분간 방치하였다. 20% FCS함유 RPMI배지를 가하고 원심침전하여 그 침사를 20% FCS함유 RPMI배지로 부유시켜 단핵구세포(mononuclear cell)수가 ml당 5.0x10**5이 되게 한 후 96wells로 된 polystyrene plate의 각 well에 100μl씩 넣고 37℃ 5% CO^^2 항온기에서 1주야 방치하였다. 이것이 "feeder layer"이다. 동일한 방법으로 만든 임파구와 배양된 myeloma cell NS-1의 수가 10:1의 비율로 되게 잘 혼합하였다. 이때 이 세포들은 FCS이 포함 안된 배양액으로 제조하였다. 원심침전하여 가라앉은 세포층을 잘 흔들고 37℃수조에 보관하였다. polyethylene glycol(분자량 4,00 0) 1gm을 autoclave하고 RPMI배지 1ml를 가하여 잘 흔들고 이중 1ml를 세포 부유액에 천천히 1분간에 걸쳐 첨가하고, 1분간 방치하고, 다음 1분간에 걸쳐 흔들면서 RPMI배지 1ml를 첨가하였다. 그 다음 5분간에 RPMI배지 20ml을 첨가하였다. 원심침전하여 가라앉은 세포층에 20% FCS함유 RPMI배지를 가하여 임파구 수가 ml 당 0.5×10**7되게 부유액을 만들었다. 이 부유액 100μl씩을 "feeder layer"의 각 well에 첨가시키고 37℃ 5% CO^^2 항온기에서 배양하였다. 다음날 20% FCS와 hypoxanthine, aminopterin, thymidine이 함유된 HAT RPMI배지에서 배양하였다. 세포융합을 실시하고 10∼14일 후 현미경으로 관찰하여 96 well plate에서 hydrid cell의 존재여부를 확인하고 세포수가 많아진 후 24 well plate로 옮겼다. Hydrid cell 배양액을 채취하여 간접형광항체법을 실시하여 항체의 존재여부를 관찰하였다. 사용된 항원은 10% formaldehyde로 고정된 T. gondii영양형이었고, fluorescein conjugate는 rabbit anti-mouse IgG(SYCCO)였다. 면역시킨 마우스의 retro-orbital plexus에서 채취한 혈액으로부터 분리한 혈청의 형광항체가는 1:1,024였다. 즉 면역방법에는 이상이 없음을 확인하였다. 둥글고 투명한 hybrid cell을 융합후 7일∼14일 후에 관찰할 수 있었다. 이같은 융합은 5번에 걸쳐 시행하였고, T. gondii로 면역된 마우스 비장세포에서 유래된 hybridoma 82개를 얻을 수 있었다. Hybridoma배양액을 각각 채취하여 간접형광항체법에 의하여 형광항체가 검출되는 hybridoma는 82개중 3개였으며 그 항체가는 1:2∼1:16이었다. 항체를 분비하는 hybrid cell 10**6개씩을 BALB/C마우스 복강내에 주입하고 3주 후 채취한 복수에서의 형광항체가는 1:64∼1:128이었다. Study on Hybridoma Clones Secreting Anti-Toxoplasma gonii antibody Kwan-Chul Tark Department of Medical Science, The Graduate School, Yonsei University (Directed by Professor Chin-Thack Soh, M.D.) Toxoplasma gondii isthe etiological agent of the world wide human and animal toxoplasmosis. IN Korea, both congenital toxoplasmosis cases and cases with high anti-toxoplasma antibody titer have been reported(Choi et al., 1980; Chung et al., 1980; Soh et al., 1975). A number of immunological procedures are available for measuring the different antibody responses to this protozoon(Jacobs, 1976). However, practical application of immunodiagnostic methods of toxoplasmosis remains unsatisfactory because of low sensitivity and specificity. Kohler and Milstein(1975) demonstrated that fusion of myeloma cells with spleen cells from immunized animals results in hybrid cell lines which secrets antibodies against the immunizing antigen. This technical improvement introduced a new era in the production and use of antibodies as research and diagnostic tools. Therefore, although the immediate use of monoclonal antibody in parasitic infection would probably be related to immunodiagnosis, such reagents may prove as an important tools for the development of vaccine against parasitic diseases. The present study is aimed to establish hybridoma clones of Toxoplasma gondii, confirm whether the clones produce anti-toxoplasma antibodies which may contribute in prophylaxis or diagnosis of toxoplasmosis. The immunization of BALB/c mice wart accomplished by weekly intraperitoneal injection of 1x10**5 Toxoplasma gondii trophozoites, fixed in 10% formaldehyde over 4∼5 weeks. Myeloma cells, P3/NS1/1/AG4/1(NS-1), were cultured in medium RPMI 1640 with 20% fetal calf serum(FCS) in CO^^2 incubator. Immunized spleen cells were fused by polyethylene glycol with NS-1 myeloma cells. Conditions for fusion were essentially as deacribed by Galfre et dl. (1977) and Kohler et at.(1976). The presence of hybrid cells on the 96 well plates was established microscopically 10-14 days after fusion. Hybridoma clones hove grown up to be sufficiently confluent, and it was subcultured to 2 ml in a 24 well plate. Hybrid cell supernates were pooled for antibody assay by indirect immunofluorescent method using Toxoplasma gondii trophozoite antigen and rabbit anti-mouse immunoglobulin G(SYCCO) as fluorescein conjugrate. Hybrid cells were also injected intraperitoneally into mice in order to obtain ascitic fluid. The fluorescent antibody tiler of serum collected from the mice immunized with Toxoplasma gondii was 1:1.024. It was confirmed that immunization schedule of mice used for cell fusion was fair. Clustering of round cells was observed on the 96 well plate 10∼14 days after the fusion technique. Antibody was demonstrated in 3 of 82 hybridoma clones. Antibody titers were 1:2∼1:16 in hybridoma medium and 1:64∼1:128 in ascitic fluids of mice inoculated with hybrid cells. [영문] Toxoplasma gondii isthe etiological agent of the world wide human and animal toxoplasmosis. IN Korea, both congenital toxoplasmosis cases and cases with high anti-toxoplasma antibody titer have been reported(Choi et al., 1980; Chung et al., 1980; Soh et al., 1975). A number of immunological procedures are available for measuring the different antibody responses to this protozoon(Jacobs, 1976). However, practical application of immunodiagnostic methods of toxoplasmosis remains unsatisfactory because of low sensitivity and specificity. Kohler and Milstein(1975) demonstrated that fusion of myeloma cells with spleen cells from immunized animals results in hybrid cell lines which secrets antibodies against the immunizing antigen. This technical improvement introduced a new era in the production and use of antibodies as research and diagnostic tools. Therefore, although the immediate use of monoclonal antibody in parasitic infection would probably be related to immunodiagnosis, such reagents may prove as an important tools for the development of vaccine against parasitic diseases. The present study is aimed to establish hybridoma clones of Toxoplasma gondii, confirm whether the clones produce anti-toxoplasma antibodies which may contribute in prophylaxis or diagnosis of toxoplasmosis. The immunization of BALB/c mice wart accomplished by weekly intraperitoneal injection of 1x10**5 Toxoplasma gondii trophozoites, fixed in 10% formaldehyde over 4∼5 weeks. Myeloma cells, P3/NS1/1/AG4/1(NS-1), were cultured in medium RPMI 1640 with 20% fetal calf serum(FCS) in CO^^2 incubator. Immunized spleen cells were fused by polyethylene glycol with NS-1 myeloma cells. Conditions for fusion were essentially as deacribed by Galfre et dl. (1977) and Kohler et at.(1976). The presence of hybrid cells on the 96 well plates was established microscopically 10-14 days after fusion. Hybridoma clones hove grown up to be sufficiently confluent, and it was subcultured to 2 ml in a 24 well plate. Hybrid cell supernates were pooled for antibody assay by indirect immunofluorescent method using Toxoplasma gondii trophozoite antigen and rabbit anti-mouse immunoglobulin G(SYCCO) as fluorescein conjugrate. Hybrid cells were also injected intraperitoneally into mice in order to obtain ascitic fluid. The fluorescent antibody tiler of serum collected from the mice immunized with Toxoplasma gondii was 1:1.024. It was confirmed that immunization schedule of mice used for cell fusion was fair. Clustering of round cells was observed on the 96 well plate 10∼14 days after the fusion technique. Antibody was demonstrated in 3 of 82 hybridoma clones. Antibody titers were 1:2∼1:16 in hybridoma medium and 1:64∼1:128 in ascitic fluids of mice inoculated with hybrid cells.restrictio