Generation of a Highly Reactive Chicken-Derived Single-Chain Variable Fragment against Fusarium verticillioides by Phage Display

Fusarium verticillioides is the primary causal agent of Fusarium ear and kernel rot in maize, producing fumonisin mycotoxins that are toxic to humans and domestic animals. Rapid detection and monitoring of fumonisin-producing fungi are pivotally important for the prevention of mycotoxins from entering into food/feed products. Chicken-derived single-chain variable fragments (scFvs) against cell wall-bound proteins from F. verticillioides were isolated from an immunocompetent phage display library. Comparative phage enzyme-linked immunosorbant assays (ELISAs) and sequencing analyses identified four different scFv antibodies with high sensitivity. Soluble antibody ELISAs identified two highly sensitive scFv antibodies, FvCA3 and FvCA4, with the latter being slightly more sensitive. Three-dimensional modeling revealed that the FvCA4 may hold a better overall structure with CDRH3, CDRL1 and CDRL3 centered in the core region of antibody surface compared with that of other scFvs. Immunofluorescence labeling revealed that the binding of FvCA4 antibody was localized to the cell walls of conidiospores and hyphae of F. verticillioides, confirming the specificity of this antibody for a surface target. This scFv antibody was able to detect the fungal mycelium as low as 10(-2) μg/mL and contaminating mycelium at a quantity of 10(-2) mg/g maize. This is the first report that scFv antibodies derived from phage display have a wide application for rapid and accurate detection and monitoring of fumonisin-producing pathogens in agricultural samples.     

Labeling of Anti-MUC-1 Binding Single Chain Fv Fragments to Surface Modified Upconversion Nanoparticles for an Initial in Vivo Molecular Imaging Proof of Principle Approach

In vivo optical Imaging is an inexpensive and highly sensitive modality to investigate and follow up diseases like breast cancer. However, fluorescence labels and specific tracers are still works in progress to bring this promising modality into the clinical day-to-day use. In this study an anti-MUC-1 binding single-chain antibody fragment was screened, produced and afterwards labeled with newly designed and surface modified NaYF(4):Yb,Er upconversion nanoparticles as fluorescence reporter constructs. The MUC-1 binding of the conjugate was examined in vitro and in vivo using modified state-of-the-art small animal Imaging equipment. Binding of the newly generated upconversion nanoparticle based probe to MUC-1 positive cells was clearly shown via laser scanning microscopy and in an initial proof of principal small animal optical imaging approach.     

A White Plaque,Associated with Genomic Deletion,Derived from M13KE-Based Peptide Library Is Enriched in a Target-Unrelated Manner during Phage Display Biopanning Due to Propagation Advantage

The nonspecific enrichment of target-unrelated peptides during biopanning remains a major drawback for phage display technology. The commercial Ph.D.^TM-7 phage display library is used extensively for peptide discovery. This library is based on the M13KE vector, which carries the lacZα sequence, leading to the formation of blue plaques on IPTG-X-gal agar plates. In the current study, we report the isolation of a fast-propagating white clone (displaying WSLGYTG peptide) identified through screening against a recombinant protein. Sanger sequencing demonstrated that white plaques are not contamination from environmental M13-like phages, but derive from the library itself. Whole genome sequencing revealed that the white color of the plaques results from a large 827-nucleotide genomic deletion. The phenotypic characterization of propagation capacity through plaque count- and NGS-based competitive propagation assay supported the higher propagation rate of Ph-WSLGYTG clone compared with the library. According to our data, white plaques are likely to arise endogenously in Ph.D. libraries due to mutations in the M13KE genome and should not always be viewed as exogenous contamination. Our findings also led to the conclusion that the deletion observed here might be an ancestral mutation already present in the naïve library, which causes target-unrelated nonspecific enrichment of white clone during biopanning due to propagation advantage.