Selection of single chain variable fragment (scFv) antibodies from a hyperimmunized phage display library for the detection of the antibiotic monensin.
Concerns over the occurrence of the veterinary antibiotic monensin (MW 671Da) in animal food products and water have given rise to the need for a sensitive and rapid detection method. In this study, four monensin-specific single chain variable fragments (scFvs) were isolated from a hyperimmunized phage-displayed library originating from splenocytes of a mouse immunized with monensin conjugated to bovine serum albumin (BSA). The coding sequences of the scFvs were engineered in the order 5'-V(L)-linker-V(H)-3', where the linker encodes for Gly(10)Ser(7)Arg. Three rounds of selection were performed against monensin conjugated to chicken ovalbumin (OVA) and keyhole limpet hemocyanin (KLH), alternately. In the third round of selection, two different strategies, which differed in the number of washes and the concentration of the coating conjugates, were used to select for specific binders to monensin. A total of 376 clones from round two and three were screened for their specific binding to monensin conjugates and positive clones were sequenced. It was found that 80% of clones from round three contained a stop codon. After removing the stop codon by site-directed mutagenesis, ten binders with different amino acid sequences were subcloned into the vector pMED2 for soluble expression in Escherichia coli HB2151. Four of these scFvs bound to free monensin as determined using competitive fluorescence polarization assays (C-FPs). IC(50) values ranged from 0.031 and 231 microM. A cross-reactivity assay against salinomycin, lasalocid A, kanamycin and ampicillin revealed that the two best binders were highly specific to monensin.
Makvandi-Nejad
Shokouh
S
School of Environmental Sciences, University of Guelph, Guelph, ON N1G2W1, Canada.
Sheedy
Claudia
C
Veldhuis
Linda
L
Richard
Gabrielle
G
Hall
J Christopher
JC
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D005506:Food Contamination; D007117:Immunization, Secondary; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D008985:Monensin / Q000031:analogs & derivatives* / Q000097:blood / Q000138:chemical synthesis / Q000276:immunology; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D011817:Rabbits; D012710:Serum Albumin, Bovine / Q000008:administration & dosage* / Q000138:chemical synthesis; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D019151:Peptide Library; D057127:Single-Chain Antibodies; D012710:Serum Albumin, Bovine; D008985:Monensin