title: Selection of scFv phages specific for chloramphenicol acetyl transferase (CAT), as alternatives for antibodies in CAT detection assays.
abstract: Reporter gene assays are commonly used in applied toxicology to measure the transcription of genes involved in toxic responses. In these reporter gene assays, transgenic cells are used, which contain a promoter-operator region of a gene of interest fused to a reporter gene. The transcription of the gene of interest can be measured by the detection of the reporter protein. Chloramphenicol acetyl transferase (CAT) is frequently used as a reporter protein in mammalian reporter gene assays. Although CAT can be measured by different detection systems, like enzymatic and immune assays, most of these tests are expensive, time-consuming and labor-intensive. The excellent characteristics of phages, like their high affinity and specificity, their fast, cheap and animal-friendly manufacturing process with low batch-to-batch variations and their stability, make them appropriate as alternatives for antibodies in detection assays. Therefore, in this study single-chain variable fragment (scFv) phages were selected with affinity for CAT. Several scFv phages were selected that showed affinity towards CAT in a screening ELISA. Surface plasmon resonance analyses showed that the tested scFv phages have an affinity for CAT with a dissociation constant (K(d)) around 1 µM. The selected scFv phages in this study could be used as capture elements in a highly sensitive sandwich ELISA to detect CAT concentration as low as 0.1 ng ml⁻¹ or 4 pM. This low detection limit demonstrates the potential of the scFv phages as an alternative for capturing antibodies in a highly sensitive detection test to measure CAT concentrations in reporter gene assays.
authors.affiliation: University Antwerp, Department of Biology, Laboratory for Ecophysiology, Biochemistry and Toxicology, Groenenborgerlaan 171, B-2020, Antwerp, Belgium. bieke.vandorst@ua.ac.be
authors.forename: Bieke
authors.initials: B
authors.lastname: Van Dorst
authors.forename: Jaytry
authors.initials: J
authors.lastname: Mehta
authors.forename: Elsa
authors.initials: E
authors.lastname: Rouah-Martin
authors.forename: Jelke
authors.initials: J
authors.lastname: Backeljau
authors.forename: Wim
authors.initials: W
authors.lastname: De Coen
authors.forename: Dominique
authors.initials: D
authors.lastname: Eeckhout
authors.forename: Geert
authors.initials: G
authors.lastname: De Jaeger
authors.forename: Ronny
authors.initials: R
authors.lastname: Blust
authors.forename: Johan
authors.initials: J
authors.lastname: Robbens
mesh_terms: D000915:Antibody Affinity; D000918:Antibody Specificity; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001435:Bacteriophages / Q000187:drug effects* / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D015723:Gene Library; D017930:Genes, Reporter / Q000187:drug effects; D006801:Humans; D057230:Limit of Detection; D009875:Operator Regions, Genetic / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000187:drug effects; D040081:Protein Array Analysis; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D020349:Surface Plasmon Resonance; D014116:Toxicology / Q000379:methods*; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D015500:Chloramphenicol O-Acetyltransferase