The selection of specific phage antibodies from phage antibody libraries and their expression in different systems.

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Feasibility of a novel multispot nanoarray for antibiotic screening in honey

<p>Practical solutions for multiple antibiotic determination in food are required by the food industry and regulators for cost-effective screening purposes. This study describes the feasibility in development and preliminary performance of a novel multispot nanoarray for antibiotic screening in honey. Using a multiplex approach, the metabolites of the four main nitrofuran antibiotics, including morpholinomethyl-2-oxazolidone (AMOZ), 3-amino-2-oxazolidinone (AOZ), semicarbazide (SEM), 1-aminohydantoin (AHD) and chloramphenicol (CAP), were simultaneously detected. Antibodies specific to the five antibiotics were nano-spotted onto microtitre plate wells and a direct competitive assay format was employed. The assay characteristics and performance were evaluated for feasibility as a screening tool for antibiotic determination in honey to replace traditional ELISAs. Optimisation of the spotting and assay parameters was undertaken with both individual and multiplex calibration curves generated in PBS and a honey matrix. The limits of detection as determined by the 20% inhibitory concentrations (IC₂₀) were determined as 0.19, 0.83, 0.09, 15.2 and 35.9 ng ml^–1 in PBS, 0.34, 0.87, 0.17, 42.1 and 90.7 ng ml^–1 in honey (fortified at the start of the extraction), and 0.23, 0.98, 0.24, 24.8 and 58.9 ng ml^–1 in honey (fortified at the end of the extraction) for AMOZ, AOZ, CAP, SEM and AHD respectively. This work has demonstrated the potential of multiplex analysis for antibiotics with results available for 40 samples within a 90-min period for antibiotics sharing a common sample preparation. Although both the SEM and AHD assay do not show the required sensitivity with the antibodies available for use to meet regulatory limits, with further improvements in these particular antibodies this multiplex format has the potential to show a reduction in cost with reduced labour time in combination with the high-throughput screening of samples. This is the first 96-well spotted microtitre plate nanoarray for the semi-quantitative and simultaneous analysis of antibiotics.</p

Single-chain variable fragments selected on the 57-76 p21Ras neutralising epitope from phage antibody libraries recognise the parental protein

Phage antibodies have been widely prospected as an alternative to the use of monoclonal antibodies prepared by traditional means. Many monoclonal antibodies prepared against peptides are able to recognise the native proteins from which they were derived. Here we show that the same is also true for phage antibodies. We have selected a number of single-chain variable fragments (scFv) from a large phage scFv library against a peptide from the switch region II of p21Ras. This peptide is known to reside in a mobile area of the native protein and is the epitope of a well characterised monoclonal antibody. Selected scFvs were able to recognise native p21Ras in both ELISA and Western blots, indicating that peptides are also likely to be very useful in selecting from phage antibody libraries