[Preparation and identification of monoclonal antibodies against human programmed cell death 10 (PDCD10)].
OBJECTIVE
To obtain monoclonal antibodies against programmed cell death 10 (PDCD10) for further study of the structure and function of PDCD10 protein.


METHODS
Balb/c mice were immunized with recombinant PDCD10, hybridoma cell lines secreting monoclonal antibodies against PDCD10 were screened by regular cell fusion and subcloning approach. The specificities of these monoclonal antibodies were determined by ELISA, Western blotting and Immunofluorescence assay.


RESULTS
Three hybridoma cell lines (5G1, 4F7 and 3H5) stable in secreting specific monoclonal antibodies were successfully obtained. Subclass of IgG belonged to IgG1 (4F7 and 5G1) and IgG2b (3H5), respectively. The ascite titers of these monoclonal antibodies reached 1:10(7). They could specifically bind to recombinant PDCD10 and endogenous and overexpressed PDCD10 proteins proved by ELISA and Western blotting. They failed to react with E.coli lysates and glutathione S-transferase (GST). In addition, these three monoclonal antibodies could recognize different epitopes of PDCD10 proteins assessed by immune fluorescence competitive binding assay. Both endogenous and overexpressed PDCD10 protein mainly located in the nucleus.


CONCLUSIONS
Monoclonal antibodies against PDCD10 with high titers and specificity have been successfully prepared, which has laid the foundation for further study of PDCD10 protein.
Chen
Yao-yao
YY
Department of Immunology, Peking University School of Basic Medical Sciences, Beijing 100083, China.
Zhao
Yun-gang
YG
Zhang
Ting
T
Xu
Lan-jun
LJ
Ma
Xi
X
Zhao
Hong-shan
HS
Chen
Ying-yu
YY
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000276:immunology*; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D051017:Apoptosis Regulatory Proteins; D008565:Membrane Proteins; C495390:PDCD10 protein, human; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins