Retrocyte Display® technology: generation and screening of a high diversity cellular antibody library.

Over the last nearly three decades in vitro display technologies have played an important role in the discovery and optimization of antibodies and other proteins for therapeutic applications. Here we describe the use of retroviral expression technology for the display of full-length IgG on B lineage cells in vitro with a hallmark of a tight and stable genotype to phenotype coupling. We describe the creation of a high-diversity (>1.0E09 different heavy- and light-chain combinations) cell displayed fully human antibody library from healthy donor-derived heavy- and light-chain gene libraries, and demonstrate the recovery of high affinity target-specific antibodies from this library by staining of cells with a labeled target antigen and their magnetic- and flow cytometry-based cell sorting. The present technology represents a further evolution in the discovery of full-length, fully human antibodies using mammalian display, and is termed Retrocyte Display® (Retroviral B lymphocyte Display).

Insights into proteolytic processing of the major peanut allergen Ara h 2 by endogenous peanut proteases.

BACKGROUND: The major peanut allergens are Ara h 1, Ara h 2 and Ara h 6. Proteolytic processing has been shown to be required for the maturation process of Ara h 6. The aim of this study was to examine whether Ara h 2 undergoes proteolytic processing and, if so, whether proteolytic processing influences its ability to bind human immunoglobulin E (IgE). RESULTS: Ara h 2 isolated from peanut extract under conditions of protease inhibition revealed a single additional peak for its two known isoforms (Ara h 2.01 and Ara h 2.02), corresponding to a C-terminally truncated form lacking a dipeptide (RY). Ara h 2 isolated in the absence of protease inhibition, however, yielded two additional peaks, identified as C-terminally truncated forms lacking either a dipeptide (RY) or a single tyrosine residue. The IgE-binding capacity of the Ara h 2 truncated forms was not altered. CONCLUSION: Ara h 2 undergoes proteolytic processing by peanut proteases that involves C-terminal removal of a dipeptide. Hence Ara h 2 isolated from peanut extract is a complex mixture of two isoforms expressed by different genes, Ara h 2.01 and Ara h 2.02, as well as truncated forms generated by the proteolytic processing of these isoforms.

Quantification of Art v 1 and Act c 1 being major allergens of mugwort pollen and kiwi fruit extracts in mass-units by ion-exchange HPLC-UV method.

A simple ion-exchange HPLC-UV method was developed for determination of major allergens from mugwort pollen and kiwi fruit extracts in mass-units. The separation of Art v 1 and Act c 1 from other components in the extracts was achieved in one step. The extinction coefficients used in the study were theoretically determined and compared to the extinction coefficients determined by gravimetry. We also reported a close correlation of the major allergen contents with the overall allergenic potency of the extracts determined by inhibition ELISA. This method could be a useful tool for standardization of allergenic extracts for clinical use.