[Generation and characterization of monoclonal antibody against human PIK3IP1].
OBJECTIVE
To obtain monoclonal antibody against PIK3IP1 for further study of the structure and biological function of PIK3IP1 protein.


METHODS
BALB/c mice were immunized with recombinant GST-PIK3IP1(62-168), Hybridoma cell lines secreting monoclonal antibodies against PIK3IP1 were screened by regular cell fusion and subcloning approach. The specificities of the monoclonal antibody was determined by ELISA, Western blot and Immunofluorescence assay.


RESULTS
One hybridoma cell line (5C6) stable in secreting specific monoclonal antibody was successfully obtained. The subclass of IgG belonged to IgG1. The ascite titers of this monoclonal antibody reached 1:10(7). It could specifically bind to recombinant GST-PIK3IP1(62-168); protein and overexpressed PIK3IP1 and variant PIK3IP1-v1 proteins proved by Western blot. This antibody failed to react with E.coli lysates and glutathione S transferase (GST). At the same time, endogenous PIK3IP1 was not detected using 5C6 antibody. Immunofluorescence results revealed that overexpressed PIK3IP1 and variant PIK3IP1-v1 protein located in cytoplasm and distributed in fleck manner.


CONCLUSIONS
Monoclonal antibody against PIK3IP1 with high titer and specificity has been successfully generated, which could be utilized as a useful reagent for the analysis of biochemical, structural, and functional properties of PIK3IP1.
Chang
Ying
Y
Department of Immunology, Peking University School of Basic Medical Sciences, Peking University Center for Human Disease Genomics, Beijing 100191, China.
Xu
Dong
D
Chen
Yao-yao
YY
Mo
Xiao-ning
XN
Xu
Lan-jun
LJ
Chen
Ying-yu
YY
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D006825:Hybridomas; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012995:Solubility
D000911:Antibodies, Monoclonal; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C521076:PIK3IP1 protein, human; D011993:Recombinant Fusion Proteins