Use of micro-emulsion technology for the directed evolution of antibodies.

Affinity reagents, such as antibodies, are needed to study protein expression patterns, sub-cellular localization, and post-translational modifications in complex mixtures and tissues. Phage Emulsion, Secretion, and Capture (ESCape) is a novel micro-emulsion technology that utilizes water-in-oil (W/O) emulsions for the identification and isolation of cells secreting phage particles that display desirable antibodies. Using this method, a large library of antibody-displaying phage will bind to beads in individual compartments. Rather than using biopanning on a large mixed population, phage micro-emulsion technology allows us to individually query clonal populations of amplified phage against the antigen. The use of emulsions to generate microdroplets has the promise of accelerating phage selection experiments by permitting fine discrimination of kinetic parameters for binding to targets. In this study, we demonstrate the ability of phage micro-emulsion technology to distinguish two scFvs with a 300-fold difference in binding affinities (100nM and 300pM, respectively). In addition, we describe the application of phage micro-emulsion technology for the selection of scFvs that are resistant to elevated temperatures.

Automated enumeration of Eimeria oocysts in feces for rapid coccidiosis monitoring.

Coccidiosis represents a major driver in the economic performance of poultry operations, as coccidia control is expensive, and infections can result in increased feed conversion ratios, uneven growth rates, increased co-morbidities with pathogens such as Salmonella, and mortality within flocks. Shifts in broiler production to antibiotic-free strategies, increased attention on pre-harvest food safety, and growing incidence of anti-coccidial drug resistance has created a need for increased understanding of interventional efficacy and methods of coccidia control. Conventional methods to quantify coccidia oocysts in fecal samples involve manual microscopy processes that are time and labor intensive and subject to operator error, limiting their use as a diagnostic and monitoring tool in animal parasite control. To address the need for a high-throughput, robust, and reliable method to enumerate coccidia oocysts from poultry fecal samples, a novel diagnostic tool was developed. Utilizing the PIPER instrument and MagDrive technology, the diagnostic eliminates the requirement for extensive training and manual counting which currently limits the application of conventional microscopic methods of oocysts per gram (OPG) measurement. Automated microscopy to identify and count oocysts and report OPG simplifies analysis and removes potential sources of operator error. Morphometric analysis on identified oocysts allows for the oocyst counts to be separated into 3 size categories, which were shown to discriminate the 3 most common Eimeria species in commercial broilers, E. acervulina, E. tenella, and E. maxima. For 75% of the samples tested, the counts obtained by the PIPER and hemocytometer methods were within 2-fold of each other. Additionally, the PIPER method showed less variability than the hemocytometer counting method when OPG levels were below 100,000. By automated identification and counting of oocysts from 12 individual fecal samples in less than one hour, this tool could enable routine, noninvasive diagnostic monitoring of coccidia in poultry operations. This approach can generate large, uniform, and accurate data sets that create new opportunities for understanding the epidemiology and economics of coccidia infections and interventional efficacy.

Rational library design by functional CDR resampling.

Successful antibody discovery relies on diversified libraries, where two aspects are implied, namely the absolute number of unique clones and the percentage of functional clones. Instead of pursuing the absolute quantity thresholded by current display technology, we have sought to maximize the effective diversity by improving functional clone percentage. With the combined effort of bioinformatics, structural biology, molecular immunology and phage display technology, we devised a bioinformatic pipeline to construct and validate libraries via combinatorial assembly of sequences from a database of experimentally validated antibodies. Furthermore, we showed that the libraries constructed as such yielded a significantly increased success rate against different antigen types and generated over 20-fold more unique hits per targets compared with libraries based on traditional degenerate nucleotide methods. Our study indicated that predefined CDR sequences with optimized CDR-framework compatibility could be a productive direction of functional library construction for in vitro antibody development.

AXM mutagenesis: an efficient means for the production of libraries for directed evolution of proteins.

Affinity maturation is an important part of the recombinant antibody development process. There are several well-established approaches for generating libraries of mutated antibody genes for affinity maturation, but these approaches are generally too laborious or expensive to allow high-throughput, parallel processing of multiple antibodies. Here, we describe a scalable approach that enables the generation of libraries with greater than 10(8) clones from a single Escherichia coli transformation. In our method, a mutated DNA fragment is produced using PCR conditions that promote nucleotide misincorporation into newly synthesized DNA. In the PCR reaction, one of the primers contains at least three phosphorothioate linkages at its 5' end, and treatment of the PCR product with a 5' to 3' exonuclease is used to preferentially remove the strand synthesized with the non-modified primer, resulting in a single-stranded DNA fragment. This fragment then serves as a megaprimer to prime DNA synthesis on a uracilated, circular, single-stranded template in a Kunkel-like mutagenesis reaction that biases nucleotide base-changes between the megaprimer and uracilated DNA sequence in favor of the in vitro synthesized megaprimer. This method eliminates the inefficient subcloning steps that are normally required for the construction of affinity maturation libraries from randomly mutagenized antibody genes.

Phage ESCape: an emulsion-based approach for the selection of recombinant phage display antibodies.

Antibody phage display technology is a well established method for selecting specific antibodies against desired targets. Although phage display is the most widely used method of generating synthetic antibodies, it is laborious to perform multiple selections with different antigens simultaneously using conventional manual methods. We have developed a novel approach to the identification and isolation of cells secreting phage encoding desirable antibodies that utilizes compartmentalization and Fluorescence Activated Cell Sorting (FACS). This method, termed Phage Emulsion, Secretion, and Capture (ESCape), allows us to individually query each phage against the antigen. Here, we demonstrate the ability of Phage ESCape to identify novel scFvs against a phosphopeptide epitope of the Her2 kinase from a phage display library containing approximately 10(8) synthetically diversified antibodies. Clones were analyzed by monoclonal phage ELISA against the Her2 phosphopeptide, and positive binders were identified as those showing a signal greater than 3-fold higher than the background signal against an irrelevant antigen. We isolated antibodies recognizing the phosphopeptide in a single round of selection by Phage ESCape, but the strength and specificity of the hits was substantially improved when the library was pre-enriched by a single round of biopanning. By minimizing the selection rounds required for phage display and using a FACS machine as a 'colony picker' equivalent, Phage ESCape has the potential to dramatically increase the throughput of in vitro screening methods.