Assay and purification of Fv fragments in fermenter cultures: design and evaluation of generic binding reagents.
Fv fragments whose genes have been cloned using common PCR primers carry identical peptide motifs at their termini. We have raised antibodies against the C-terminal motif of the VH chain GQGTTVTVSS and evaluated their utility as reagents for the assay and purification of Fvs in the fermenter culture. Three different Fvs were included in the investigation. We found that the motif was exposed and available for capture when Fv fragments were blotted onto nitrocellulose paper or adsorbed directly onto microtiter plates. In contrast, the motif was either partially or totally obscured when the Fv was complexed with immobilised antigen or when free in solution. This reactivity profile enabled us to develop a general-purpose assay for Fv protein, but not a general-purpose assay for monitoring active Fv. The apparent inaccessibility of the C-terminus of VH conflicts with currently held views on the three-dimensional structure of these molecules.
Berry
M J
MJ
Biosciences Division, Sharnbrook, Bedford, UK.
Wattam
T A
TA
Willets
J
J
Lindner
N
N
de Graaf
T
T
Hunt
T
T
Gani
M
M
Davis
P J
PJ
Porter
P
P
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D015151:Immunoblotting; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000276:immunology; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007128:Immunoglobulin Fragments; D009842:Oligopeptides; D011994:Recombinant Proteins; C055966:immunoglobulin Fv