| Title |
|
|
|
Selection of scFv phages specific for chloramphenicol acetyl transferase (CAT), as alternatives for antibodies in CAT detection assays
| |
| Author |
|
|
|
| |
| 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 promoteroperator 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 (Kd) 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. | | |
| Language |
|
|
|
English
| |
| Source (journal) |
|
|
|
Journal of applied toxicology. - Chichester | |
| Publication |
|
|
|
Chichester : 2012
| |
| ISSN |
|
|
|
0260-437X
| |
| Volume/pages |
|
|
|
32:10(2012), p. 783-789
| |
| ISI |
|
|
|
000308082800005
| |
| Full text (Publisher's DOI) |
|
|
| | |
| Full text (publisher's version - intranet only) |
|
|
| | |
|