1 was amplified by PCR using full-length FGFR1 (Accession No. “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_000604″,”term_id”:”13186232″,”term_text”:”NM_000604″NM_000604) those as a template with primers No. 5��-3 [(SEQ ID NO. 3): 5��-ACGGGATCCAGGACCCTGGCTGGAGAGACA-3��] and No. 3��-3 [(SEQ ID NO. 4): 5��-AAGCTCGAGCCGCCGGAACCGCGGCCGGA-3��]. The amplified polynucleotide was inserted into pcDNA3.1 (Invitrogen, Carlsbad, CA, USA) to construct an expression vector that was administered as the immunizing antigen at a dose of 50 ��g/mouse in a 50-��L volume at 1- or 2-week intervals. The antigen for the initial immunization was admixed with complete Freund’s adjuvant, while the antigens for the second and subsequent administrations were admixed with incomplete Freund’s adjuvant.
Spleen monocytic cells from the immunized mouse and a fusion partner, X63-Ag8-653, were fused using polyethylene glycol-mediated cell fusion, which was followed by selection of a hybridoma using the method of Kinebuchi et al. [28]. Cells that had reacted with the immobilized FGFR1 were cultured in serum-free GIT medium (Wako Pure Chemical Industries, Ltd., Osaka, Japan) to produce mAbs until 80% of the cells had died. The cells were then removed from this medium by centrifugation (1,000 rpm, 15 min), and ammonium sulfate was added to 50% saturation and left overnight at 4��C. The resultant precipitates were recovered by centrifugation (1,000 rpm, 30 min) and dissolved in two-fold diluted binding buffer (Protein AMAPS II kit), after which the IgG was adsorbed onto a protein A column (GE Healthcare Life Science).
After eluting the mAbs from the column, the eluate was dialyzed against PBS overnight to purify the antibodies, which yielded a number of mAbs recognizing FGFR1. One of those mAbs was designated A2C9-1 and was confirmed to recognize FGFR1 by Western blotting and FACS using samples of FGFR1 expressed in NIH3T3 cells. Affinity measurement The affinity of anti-FGFR1 mAbs for FGFR1 was determined based on surface plasmon resonance (SPR) using a Biacore 3000 device (Biacore AB, Uppsala, Sweden). The extracellular domain of FGFR1 was covalently coupled to a CM-5 sensor chip at low density (215 response units of FGFR1). Binding kinetics were then assessed using twofold serial dilutions of antibody at concentrations ranging from 500 to 0.08 nM in running buffer (PBS, pH 7.4, 0.
005% (v/v), polysorbate 20 �C filtered and degassed) at 25��C and a flow rate of 25 ml/min. The regeneration Carfilzomib procedure consisted of three injections of 10 ml of 2.5 M guanidinium hydrochloride, after which the sensor chip was flushed for 5 min with running buffer. Statistics and data processing were performed using BIA evaluation software 4.0.1 and GraphPad Prism 4.02 (GraphPad Software Inc., San Diego, CA, USA). All SPR experiments were carried out at Biaffin GmbH & Co. KG (Kassel, Germany).