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A non-immune library of human single chain fragment variable (scFv) antibodies displayed on Sacchar- omyces cerevisiae was screened for binding to the Clostridium botulinum neurotoxin serotype A binding domain (BoNT/A (Hc)) with the goal of identifying scFv to novel epitopes. To do this, an antibody-mediated labeling strategy was used in which antigen-binding yeast clones were selected after label- ing with previously characterized monoclonal antibodies (MAbs) specific to the Hc. Twenty unique scFv clones were isolated that bound Hc. Of these, 3 also bound to full-length BoNT/A toxin complex with affinities ranging from 5 to 48 nM. Epitope binning showed that the three unique clones recognized at least two epitopes distinct from one another as well as from the detection MAbs. After production in E. coli, scFv were coupled to magnetic particles and tested for their ability to capture BoNT/A holotoxin using an Endopep-MS assay. In this assay, toxin captured by scFv coated magnetic particles was detected by incubation of the complex with a peptide containing a BoNT/A-specific cleavage sequence. Mass spectrometry was used to detect the ratio of intact peptide to cleavage products as evidence for toxin capture. When tested individually, each of the scFv showed a weak positive Endopep-MS result. However, when the particles were coated with all three scFv simultaneously, they exhib- ited significantly higher Endopep-MS activity, consistent with synergistic binding. These results demonstrate novel approaches toward the isolation and characterization of scFv antibodies specific to unlabeled antigens. They also provide evidence that distinct scFv antibodies can work synergisti- cally to increase the efficiency of antigen capture onto a solid support. Biotechnol. Bioeng. 2011;108: 2456-2467.

BACKGROUND: Bacterial contamination or platelet (PLT) metabolism can change the pH of stored PLT concentrates (PCs). Measurement of pH for quality control is currently done on a limited basis. An easy noninvasive method was developed to obtain sequential pH measurements over time, without risking contamination and/or consuming PCs.

Pseudo-nitzschia australis Frenguelli is a marine pennate diatom associated with the production of domoic acid— a neuroexcitatory amino acid linked to illness and mortality of humans and wildlife. Distinguishing P. australis from its co-occurring congeners is labor intensive and time consuming because of a requirement for scanning electron microscopy. Here, we apply large-subunit ribosomal RNA (LSU rRNA)-targeted oligonucleotides in whole-cell and sandwich hybridization formats to identify and enumerate this species collected from pure cultures and natural populations. Whole-cell hybridization employed fluorescently labeled probes, filter-based sample processing, and epifluorescence microscopy to enumerate labeled cells. In contrast, sandwich hybridization was accomplished by homogenizing cells in a chaotropic solution and performing two hybridization reactions: capture of LSU rRNA using an oligonucieotide coupled to a macroscopic solid support and binding of signal probe to a region of LSU rRNA near that of the capture site. Sandwich hybrids were detected colorimetrically; color intensity was proportional to the abundance of target species in the original sample. The sandwich hybridization assay was semiautomated with a robotic processor. Both whole-cell and sandwich hybridization are useful techniques for identifying P. australis as it occurs in nature. Sandwich hybridization potentially offers the most rapid and simple means to accomplish this task when screening large numbers of environmental samples.