But it was higher than that for hAQP4\M1 cells, also supporting the bivalent binding hypothesis

But it was higher than that for hAQP4\M1 cells, also supporting the bivalent binding hypothesis. item BPH-173-103-s002.pdf (365K) GUID:?4B28D9EE-97F3-44F3-8626-2910D9BC9188 Supporting info item BPH-173-103-s003.pdf (99K) GUID:?7FC40F74-564A-46A2-833A-543F59BDA315 Supporting info item BPH-173-103-s004.pdf (1.8M) GUID:?4C73625E-98BD-4ABC-A03B-F8108772EBD5 Supporting info item BPH-173-103-s005.pdf (207K) GUID:?41211A32-4721-4C29-94C2-BB44C6D26BFB Supporting info item BPH-173-103-s006.pdf (2.1M) GUID:?329C58F2-48AB-4D25-8553-48F07CBC1177 Supporting info item BPH-173-103-s007.mp4 Rabbit polyclonal to ALOXE3 (2.9M) GUID:?86BDCB74-0202-488E-8AA8-A96C6A21355C Supporting info item BPH-173-103-s008.mp4 (2.9M) GUID:?20BF0A3B-3B87-4933-A03B-32B731950993 Supporting info item BPH-173-103-s009.mp4 (2.9M) GUID:?E3166079-C57A-4818-8540-F5A016A7616F Supporting info item BPH-173-103-s010.docx (63K) GUID:?2EE695B5-ED2F-4604-91C5-188CF2050360 Abstract Background and Purpose Most of the cases of neuromyelitis optica (NMO) are characterized by the presence of an autoantibody, NMO\IgG, which recognizes the extracellular domains of the water channel, aquaporin\4. Binding of NMO\IgG to aquaporin\4 expressed in end\feet of astrocytes leads to Trelagliptin complement\dependent disruption of astrocytes followed by demyelination. One therapeutic option for NMO is to prevent the binding of NMO\IgG to aquaporin\4, using high\avidity, non\pathogenicCchimeric, monoclonal antibodies to this water channel. We describe here the development of such antibodies. Experimental Approach cDNAs encoding variable regions of heavy and light chains of monoclonal antibodies against the extracellular domains of human aquaporin\4 were cloned from hybridoma total RNA and fused to those encoding constant regions of human IgG1 and Ig respectively. Then mammalian expression vectors were constructed to establish stable cell lines secreting mature chimeric antibodies. Key Results Original monoclonal antibodies showed high avidity binding to human aquaporin\4, as determined by ELISA. Live Trelagliptin imaging using Alexa\Fluor\555\labelled antibodies revealed that the antibody “type”:”entrez-nucleotide”,”attrs”:”text”:”D15107″,”term_id”:”286299″,”term_text”:”D15107″D15107 more rapidly bound to cells expressing human aquaporin\4 than others and strongly enhanced endocytosis of this water channel, while “type”:”entrez-nucleotide”,”attrs”:”text”:”D12092″,”term_id”:”2148886″,”term_text”:”D12092″D12092 also bound rapidly to human aquaporin\4 but enhanced endocytosis to a lesser degree. Chimeric “type”:”entrez-nucleotide”,”attrs”:”text”:”D15107″,”term_id”:”286299″,”term_text”:”D15107″D15107 prevented complement\dependent cytotoxicity induced by NMO\IgG from patient sera (Miyazaki genomic DNA using the basic local alignment search tool (National Center for Biotechnology Information) to identify V gene segments highly homologous with each cDNA (Supporting Information Figure Trelagliptin S2). Then sense primers 5’\TCTAACCATGGGATGGAGCTGGATCTTTC\3′ for the heavy chain of C9401; 5’\GAGCCCCCATCAGAGCATGGCTGTC\3′ for the heavy chains of “type”:”entrez-nucleotide”,”attrs”:”text”:”D12092″,”term_id”:”2148886″,”term_text”:”D12092″D12092 and “type”:”entrez-nucleotide”,”attrs”:”text”:”D15107″,”term_id”:”286299″,”term_text”:”D15107″D15107 and 5’\TCTCAGAGATGGAGACAGACACACTCCTG\3′ for the light chains of C9401, “type”:”entrez-nucleotide”,”attrs”:”text”:”D12092″,”term_id”:”2148886″,”term_text”:”D12092″D12092 and “type”:”entrez-nucleotide”,”attrs”:”text”:”D15107″,”term_id”:”286299″,”term_text”:”D15107″D15107 were designed according to the sequence of exon 1 of each corresponding V gene segment of C57BL/6 genome to obtain full\length cDNAs encoding each variable region of heavy and light chains (Supporting Information Figure S2). As a result, “type”:”entrez-nucleotide”,”attrs”:”text”:”D15129″,”term_id”:”286321″,”term_text”:”D15129″D15129 was found to be identical with “type”:”entrez-nucleotide”,”attrs”:”text”:”D15107″,”term_id”:”286299″,”term_text”:”D15107″D15107, so we used only “type”:”entrez-nucleotide”,”attrs”:”text”:”D15107″,”term_id”:”286299″,”term_text”:”D15107″D15107 for subsequent experiments. To connect each variable region of the heavy chain to the constant region of human IgG1, an NheI site was introduced at the junction of the chimeric heavy chain by PCR using antisense primers 5’\CCGCGGAGCTAGCTGCAGAGACAGTGACCAGAGTC\3′ for C9401 and “type”:”entrez-nucleotide”,”attrs”:”text”:”D15107″,”term_id”:”286299″,”term_text”:”D15107″D15107 and 5’\CCGCGGAGCTAGCTGYAGAGACAGTGACCAGAGTC\3′ for “type”:”entrez-nucleotide”,”attrs”:”text”:”D12092″,”term_id”:”2148886″,”term_text”:”D12092″D12092. To connect each variable region of the light chain to the constant region of human Ig, an XhoI site was introduced by PCR at the junction of the chimeric light chain using antisense primers 5’\CTCGAGCTTGGTGCCTCCACCGAACGTC\3′ for C9401, 5’\CTCGAGCTTGGTCCCAGCACCGAACGTG\3′ for “type”:”entrez-nucleotide”,”attrs”:”text”:”D12092″,”term_id”:”2148886″,”term_text”:”D12092″D12092 and 5’\CTCGAGCTTTGTCCCCGAGCCGAACGTG\3′ for “type”:”entrez-nucleotide”,”attrs”:”text”:”D15107″,”term_id”:”286299″,”term_text”:”D15107″D15107, and sense primers for Ig constant region 5’\CTCGAGATCAAACGGACTGTGGCTGCACCATCTGTC\3′ to connect C9401 and “type”:”entrez-nucleotide”,”attrs”:”text”:”D15107″,”term_id”:”286299″,”term_text”:”D15107″D15107 and 5’\CTCGAGCTGAAACGGACTGTGGCTGCACCATCTGTC\3′ to connect “type”:”entrez-nucleotide”,”attrs”:”text”:”D12092″,”term_id”:”2148886″,”term_text”:”D12092″D12092. Obtained cDNAs for each chimeric heavy chain and those for each chimeric light chain were inserted into a pEBMulti\Puro and a pEBMulti\Hyg vector (Wako Pure Chemical Industries), respectively, to establish stable CHO cell lines. The cDNA encoding the C9401 chimeric light chain was also inserted into a pIRES2\EGFP vector as described previously. Purification of chimeric antibodies To obtain chimeric antibodies secreted into the culture supernatant, CHO cells stably Trelagliptin Trelagliptin expressing each antibody were cultured in CD\CHO medium (Life Technologies Corporation) for a week. Then culture supernatants were collected and concentrated with Amicon Ultra 15?mL 10?k (Merck Millipore, Billerica, MA, USA). Concentrated antibody solution was applied to PD\10 desalting columns (GE Healthcare, Little Chalfont, UK) to change buffer to binding buffer (20?mM sodium phosphate, pH7.0, GE Healthcare) to purify with HiTrap rProtein A FF column (GE Healthcare). Bound antibodies were eluted with elution buffer (100?mM glycine\HCl, pH2.7, GE Healthcare) followed by neutralization with neutralizing buffer (1?M TrisCHCl, pH9.0, GE Healthcare). Purified antibodies were subjected again to PD\10 desalting columns to change the buffer to PBS. ELISA Baculovirus and CHO cells expressing AQP4 were immobilized and were seeded, respectively, onto 96\well plates. For baculovirus expressing AQP4, virus particles were suspended in PBS (0.05?mg protein??mL?1) followed by addition to 96\well plates (2.5?g protein per well). The plates were incubated at 4C overnight then washed twice with PBS containing 0.05% Tween 20. Washed plates were blocked with 40% Block Ace (DS Pharma Biomedical Co., Ltd., Osaka, Japan) in PBS at room temperature for 1?h then incubated with various concentrations of mAb in 40% Block Ace at 4C overnight. CHO cells stably expressing AQP4 were seeded at a density of 1 1??105 cells per well. For binding.