[Monoclonal antibody against G3BP: preparation, characterization and its application in analysis of human tumors].
OBJECTIVE
To better understand the molecular mechanism of tumorigenesis and progression, the monoclonal antibody against G3BP (Ras-GAP SH3 binding protein), which serves as an important downstream effector of Ras signaling, was prepared, characterized and utilized in analysis of various human tumors.


METHODS
By using the prokaryotic expression vector pGEX-5X1, GST-G3BP fusion protein was expressed in E. coli BL21 under induction of IPTG. Purified GST-G3BP fusion protein was used to immunize BALB/c mice. The monoclonal antibody against G3BP was produced through conventional hybridoma method and characterized by ELISA, Western blot and immunohistochemical staining.


RESULTS
A hybridoma cell line secreting anti-G3BP IgG1 subtype antibody was obtained. Western blot and competitive inhibition assay showed that the antibody was G3BP-specific. Immunohistochemical staining demonstrated that G3BP was over-expressed in formalin-fixed and paraffin-embedded tissues of some human tumors, such as lung cancer, colon cancer, gastric cancer and breast cancer. In breast cancer specimens, the degree of G3BP expression correlated positively with the presence of lymph node metastasis and c-erbB2 expression.


CONCLUSIONS
The G3BP-specific monoclonal antibody derived from recombination protein can be used in ELISA, Western blot and immunohistochemical assay. It may provide an important tool in analysis of G3BP in in vitro and in vivo experiments. Besides, G3BP may serve as another prognostic marker for breast cancer.
Ning
Jun-yu
JY
Department of Pathology, Health Science Center, Peking University, Beijing 100083, China.
You
Jiang-feng
JF
Pei
Fei
F
Wang
Jie-liang
JL
Cui
Xiang-lin
XL
Zheng
Jie
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D014408:Biomarkers, Tumor; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D004265:DNA Helicases; D005260:Female; D005822:Genetic Vectors; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D008207:Lymphatic Metastasis; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D000075223:Poly-ADP-Ribose Binding Proteins; D020365:RNA Helicases; D000067776:RNA Recognition Motif Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D002352:Carrier Proteins; D004265:DNA Helicases; D000075223:Poly-ADP-Ribose Binding Proteins; D020365:RNA Helicases; D000067776:RNA Recognition Motif Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; C116070:G3BP1 protein, human