RF-SJ1 was shown to have 100-fold higher affinity for IgG than the clonally related RF close to germline. In this B cell selection process base-pair substitutions as well as deletions of triplets in CDR regions, leaving the transcripts in frame, were involved. Together, these data provide Rivastigmine further evidence for an Ag-driven immune response in the terminal differentiation into RF-producing PCs in patients with RA, including growth of clonally related B cells, selection and isotype switching, all hallmarks of a GC reaction. secreting PCs . Furthermore, EBV immortalization targets less than 1% of the B cell portion and EBV cloning and somatic heterohybridization are inefficient in humans [16,17]. So, it is debatable whether the RFs of EBV-transformed B cells can be considered representatives of the RFs produced XL1-Blue cells . After each round of selection, phages were rescued from single ampicilin-resistant colonies of infected XL1-Blue cells. Binding to HuIgG Fc was verified by ELISA, using bacterial supernatants made up of monoclonal phage antibodies (moPhabs) or moPhabs which were purified and concentrated by polyethylene glycol/NaCl precipitation and Rivastigmine resuspended in PBS made up of 1% (w/v) BSA. Enzyme-linked immunosorbent assay (ELISA) Binding of moPhabs to HuIgG Fc was assessed by ELISA Rivastigmine using plates (Titertek, Flow Laboratories, Zwanenburg, the Netherlands) coated Mouse monoclonal to EphA3 overnight at room heat with HuIgG Fc fragments (10 g/ml in a carbonate buffer, pH 96). A monoreactive moPhab directed against group B Streptococcae (kind gift of Dr J. de Kruif, Department of Immunology, University or college Hospital Utrecht, Utrecht, the Netherlands) or a representative moPhab which does not bind to HuIgG Fc was used as a negative control. MoPhabs binding to antigen were detected using horseradish peroxidase (HRP)-conjugated sheep anti-M13 antibody and 2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) substrate (Detection Module Recombinant Phage Antibody System, Pharmacia Biotech AB) according to the manufacturer’s recommendations with the modification that PBS made up of 005% (v/v) Tween 20 and 1% (v/v) newborn bovine serum (Circulation Laboratories, Irvine, Scotland) was utilized for blocking. The colour reaction was read at 415 nm in an ELISA reader (EL 312e Bio-kinetics Reader, Bio-Tek Devices, Inc., Winooski, VT, USA). DNA fingerprinting of clones The diversity of moPhabs with HuIgG Fc-binding activity was assessed by MvaI DNA fingerprinting of clones. The scFv place was amplified Rivastigmine by PCR using primers M13 reverse (5-AAC AGC TAT GAC CAT G-3) and PHENSEQ (5-CTA TGC GGC CCC ATT CA-3) . Reactions, preceded with an incubation at 95C for 12 min, were carried out for 30 cycles (60 s at 94C, 60 s at 55C and 120 s at 72C) on Rivastigmine a thermal cycler. Subsequently, the samples were incubated at 72C for 7 min. All PCR reactions were performed in a volume of 25 l made up of 50 mm KCl, 10 mm Tris-HCl pH 83, 2 mm MgCl2, 250 m of each dNTP, 10 pmol of each primer and 05 U Ampli Taq Platinum DNA polymerase (Perkin-Elmer). Amplified DNA was digested with the frequent-cutting enzyme MvaI (MBI Fermentas, Amherst, NY, USA) and analysed on a 3% agarose gel. Soluble scFv production Soluble single chain (sc) Fv fragments were produced in E. coli amber nonsuppressor strain SF110, altered to contain the F episome of E. coli XL1-Blue, as explained [26,27], likely resulting in the production of a mixture of scFv monomers and dimers . Integrity and quantity of monoclonal scFv fragments was assessed by Western blotting using antimyc mAb 9E10 (CRL-1729, ATCC, Rockville, MD) which recognizes the C-terminal peptide tag and rabbit antimouse Ig-HRP antibodies (DAKO, Glostrup, Denmark). Blots were assayed using the enhanced chemiluminescence (ECL) detection system (Amersham, Little Chalfont, UK). Binding of soluble monoclonal scFv fragments to HuIgG Fc was determined by ELISA. ScFv fragments bound to antigen were detected using the mouse antimyc mAb 9E10, a rabbit antimouse Ig-HRP antibody (DAKO) and ABTS. The colour reaction was read at 415 nm in an ELISA reader. Nucleic acid sequence analysis Nucleotide sequence analysis.