[Preparation of anti-human retinoblastoma monoclonal antibody by solid tumor cell immunization and study on characteristics of its antigen].
OBJECTIVE
To prepare anti-human retinoblastoma (Rb) monoclonal antibody (McAb) by immunization with solid tumor cells and preliminarily study the characteristics of its antigen.


METHODS
Three Balb/C mice were immunized by intraperitoneal injection of Rb solid tumor cells acquired from enucleation. Spleen lymphocytes were separated from them and fused with myeloma cell line SP2/0. The anti-human Rb McAb was selected by Rb solid tumor cell, SO-Rb(50), SO-Rb(70), etc as antigens. The characteristics of its antigen were preliminarily studied by immunofluorescence, immunohistochemistry and Western blot.


RESULTS
After repeatedly cloning them with micro-manipulation equipment, we successfully established 3 hybridoma cell lines secreting anti-human Rb McAb. Of all, 3C6 hybridoma cells could steadily secrete anti-human Rb McAb after they were frozen, resuscitated and passaged repeatedly for 2 years. Its subgroup was IgG(1). Both immunofluorescence and immunohistochemistry examinations demonstrated that the corresponding antigen of McAb 3C6 was specifically and highly expressed in Rb tumor cell membrane and cytoplasm. The other tumor tissues and human normal eye tissues were negative. Western blot analysis preliminarily demonstrated that McAb 3C6 could bind 25 000 protein band of Rb antigen.


CONCLUSIONS
The anti-human Rb McAb 3C6 established by immunization of solid tumor cells is specifically and highly expressed in Rb tumor cells and has no cross-response with other tumor tissues and normal eyeballs. The antigen molecular weight bounded to this antibody is 25 000 or so, which further shows that it is a possibility for a new unidentified gene to be concerned with the tumor formation of Rb.
Zhong
Xiufeng
X
Zhongshan Ophthalmic Center, Sun Yat-sen University, Eye Laboratory of Ministry of People's Health, Guangzhou 510060, China.
Li
Yongping
Y
Ning
Bo
B
Yan
Jianhua
J
Li
Yongqiang
Y
Luo
Guangwei
G
Lin
Jianxian
J
Ying
Fangwei
F
Feng
Guanguang
G
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification*; D000918:Antibody Specificity; D006367:HeLa Cells; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009368:Neoplasm Transplantation; D012175:Retinoblastoma / Q000276:immunology* / Q000473:pathology; D016160:Retinoblastoma Protein / Q000276:immunology*; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured / Q000276:immunology
D000911:Antibodies, Monoclonal; D016160:Retinoblastoma Protein