Identification of a truncated ratp28-related protein expressed in kidney

Generation of a monoclonal human single chain antibody fragment to hepatic stellate cells – a potential mechanism for targeting liver anti-fibrotic therapeutics

“…The molecular weight of the peptide was checked by mass spectrometry (data not shown). Peptides were chemically conjugated to bovine serum albumin (BSA) using 3-maleimidoacetic acid-N-hydroxysuccinimide (MBS) [21]. SDS-PAGE and MALDI-TOF (Applied Biosystems Voyager-DE STR) analyses confirmed that the peptides had been conjugated to BSA-typically on an average w4 peptide molecules per molecule of albumin (data not shown).…”

“…Human single-fold scFv phagemid (pIT2) libraries (Tomlinson I C J, MRC, UK) [22,23] were used to screen for phage-antibodies with specificity for the peptide sequences essentially as previously outlined [21]. A total of three rounds of panning were carried out against each peptide sequence.…”

“…After extensive dialysis at 4 8C against PBS, scAb was stored in aliquots at K20 8C. The purified scAb was quantified by capture ELISA via the human Ck domain using a whole human IgG assay standard as previously outlined [21].…”

“…The scAb was expressed in IPTG-treated cells as outlined [21] and purified via the hexahistidine C-terminal tag tail using Ni 2C charged IMAC Membrane spanning regions (background grey) were predicted (for human sequence) using TMpred software (EMBnet.org). Sequences in bold indicate peptide 1 and 2 sequences used as targets in phage display.…”

“…Purified polyclonal phage enriched for binders to peptides were assayed by ELISA as previously outlined [21].…”