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.