2. Total cellular RNA was isolated from oligoclonal cell populations positive for anti-CD4 Ab production (RNeasy mini kit, Qiagen). cDNAs were synthesized and amplified by PCR with specific primers for human Ig μ-, γ-, λ-, and κ-chains. Only the μ- and κ-chains were amplified from HO538 selleck and HO702 cultures and cloned into the pFab1-His2 vector, generating bacterial Fab-expression

libraries 30. The pFab libraries were screened for the production of CD4-reactive Fab by ELISA. The Fab fragments were purified using an anti-Fab Ab affinity column. The eluted Fab was dialyzed against PBS and concentrated by centrifugation (VIVASPIN concentrator, Vivascience AG). The purity of the Fab Ab was greater than 95% as determined by SDS-PAGE analysis (data not shown). Surface plasmon resonance analyses were performed using BIACORE 3000 (GE Healthcare). The hrCD4 was immobilized onto CM5 sensor chips using standard amine-coupling chemistry. The purified Fab was diluted in a running buffer (10 mM HEPES, 0.15 M NaCL, 3 mM EDTA, surfactant P 20, pH 7.4) to 0.3–20 μg/mL and injected at a rate of 20–30 μL/min. The Fab was allowed to associate and dissociate for 120–270 s. B-LCL and 293 T cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 (Sigma) supplemented with 10% fetal bovine serum

Acalabrutinib supplier (Japan Bioserum), penicillin, and streptomycin (Invitrogen). The primary mononuclear cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, penicillin, streptomycin, 5 μg/mL plasmocin (InvivoGen), 10 mM HEPES, 5 μg/mL anti-CD3 mAb (OKT3, Janssen Pharmaceutical), 70 U/mL recombinant

human IL-2 (Shionogi Pharmaceutical), GlutaMax-I (Invitrogen), insulin–transferrin–selenium-A (Invitrogen), and 10 mM HEPES (Invitrogen). Cells were incubated at 37°C in a humidified 5% CO2 atmosphere. Procedures for monitoring HIV-1 replication 31 and membrane floatation assays 32 were described ADP ribosylation factor previously. Standard auto-Ab was tested by the clinical laboratory testing service SRL (Tokyo, Japan). The authors thank Hideo Tsukamoto for BIACORE analysis. This work was supported by the Japan Health Science Foundation, the Japanese Ministry of Health, Labor and Welfare (H18-AIDS-W-003 to JK), and the Japanese Ministry of Education, Culture, Sports, Science and Technology (18689014 and 18659136 to JK). Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. “
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