[Show abstract][Hide abstract] ABSTRACT: Vascular endothelial growth factor receptor 3 (VEGFR-3) is a receptor for the vascular endothelial growth factor C and D (VEGF-C and D) and plays a critical role in the development of embryonic vascular system and regulation of tumor lymphangiogenesis. In this report, we generated a novel panel of 17 monoclonal antibodies (mAbs) against human VEGFR-3 and determined their ability to inhibit the proliferation of human erythroleukemia (HEL) cells and angiogenesis of chick embryo chorioallantoic membrane (CAM). Among these mAbs, BDD073 was demonstrated to inhibit the interaction of soluble VEGFR-3 with VEGF-D and the proliferation of HEL cells. Furthermore, in chick embryo CAM angiogenesis experiments, the angiogenesis induced by recombinant glutathione-S-transferase-VEGF-D was decreased in the presence of antibody BDD073. These data suggest that this novel neutralizing antibody against human VEGFR-3 could be a tool for the investigations into the biology of VEGFR-3, and potentially a reagent for blocking VEGF-D-induced angiogenesis and lymphogenesis.
[Show abstract][Hide abstract] ABSTRACT: Analysis of the mitochondrial proteome would provide valuable insight into the function of this important organelle, which plays key roles in energy metabolism, apoptosis, free radical production, thermogenesis, and calcium signaling. It could also increase our understanding about the mechanisms that promote mitochondrial disease. To identify proteins that are antigenically dominant in human liver mitochondria, we generated >240 hybridoma cell lines from native mitochondrial proteins after cell fusion, screening, and cloning. Antibodies that recognized mitochondrial proteins were identified by screening human liver cDNA expression libraries. In this study, we identified 6 major antigens that were recognized by at least 2 different monoclonal antibodies (mAbs). The proteins that were antigenically dominant were: acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl-Coenzyme A thiolase), aldehyde dehydrogenase 1 family member A1, carbamoyl phosphate synthetase 1, dihydrolipoamide S-acetyltransferase (E2 component of pyruvate dehydrogenase complex), enoyl coenzyme A hydratase 1, and hydroxysteroid (11-beta) dehydrogenase 1. We also determined the subcellular localizations of these enzymes within the mitochondria using immunohistocytochemistry. We believe that these well-characterized antibodies will provide a valuable resource for the Human Liver Proteome Project (HLPP), and will make studies aimed at investigating liver mitochondrial function far easier to perform in future. Our results provide strong evidence that, (i) depletion of dominant proteins from liver mitochondrial samples is possible and, (ii) the approaches adopted in this study can be used to explore or validate protein-protein interactions in this important organelle.
[Show abstract][Hide abstract] ABSTRACT: Monoclonal antibodies (mAbs) have the potential to be a very powerful tool in proteomics research to determine protein expression, quantification, localization and modification, as well as protein-protein interactions, especially when combined with microarray technology. Thus, a large amount of well-characterized and highly qualified antibodies are needed in proteomics. Purified antigen, which is not always available, has proven to be one of the rate-limiting steps in mAb large-scale generation. Here we describe our strategies to establish a murine hybridoma cell bank for human liver mitochondria using unknown native proteins as the immunogens. The antibody-recognized mitochondrial proteins were identified by MS following immunoprecipitation (IP), and by screening of human liver cDNA expression library. We found that the established antibodies reacted specifically with a number of important enzymes in mitochondria. The subcellular localization of these antigens in mitochondria was further confirmed by immunohistocytochemistry. A panel of antibodies was also tested for their ability to capture and deplete the targeting proteins and complexes from the total mitochondrial proteins. We believe these well-characterized antibodies would be useful in various applications for Human Liver Proteome Project (HLPP) when the scale of this hybridoma cell bank is enlarged significantly in the near future.
[Show abstract][Hide abstract] ABSTRACT: Construction of a monoclonal antibody (mAb) bank containing a vast variety of antibodies against human tissue proteins is important for proteomic research. A novel strategy of subtractive immunization using fractionated native proteins was developed for high throughput generation of mAb against human plasma proteins. By this novel approach, the bottleneck of antigen preparation can be overcome by combining repeated immunization of animals with subtracted fractions of plasma or tissue proteins and identification of target antigen by immunoprecipitation/mass spectrum strategies. Plasma freshly collected from healthy adults was pooled and three fractions were prepared by size exclusion chromatography. Mice were immunized with the fractionated plasma proteins, and 205 strains of hybridomas secreting mAb were obtained after two-round subtractive immunizations and cell fusions. In the first round, 110 strains of hybridomas were established, in which 77 strains secreting mAb were identified against 10 human plasma high-abundant proteins. In the second round, plasma fraction I was absorbed with mAb against IgM, IgG, ceruloplasmin and haptoglobin. The absorbed fraction I was used as immunogen for the second round immunization and cell fusion. Ninety-five strains of hybridomas secreting mAb were obtained. Although the target antigens of mAb from 82 strains of hybridomas were identified as IgM, IgA, alpha2-macroglobulin and fibrinogen, about 85% antibodies obtained from this round were identified as new antibodies when compared with mAb obtained in the first round immunization with plasma fraction I. The results suggest that subtractive immunization with fractionated plasma proteins followed by identification of antigens with immunoprecipitation/mass spectrum may be an effective approach for rapid preparation of mAb against high-and medium-abundant plasma or tissue proteins.