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PLASTIC RECONSTRUCTIVE AND REGENERATIVE SURGERY

Abstract

Background - RhD-immunoglobulin (RhIg) prevents anti-D alloimmunisation in D-negative pregnant women when the fetus is D-positive, reducing the incidence of haemolytic disease of the fetus and newborn. Manufacturing RhIg is reliant on the limited supply of plasma donations with anti-D antibodies. Monoclonal antibody (mAb) development platforms such as phage display, require blood samples to be collected from anti-D donors, which may be a complicated process. The blood filter chamber (BFC) discarded after an anti-D donor’s donation might provide a source of Ig-encoding RNA. This study aims to evaluate whether used BFCs are a suitable source of Ig-encoding RNA for phage display.
Material and methods - Haemonetics PCS2 BFCs were obtained from 10 anti-D donors for total RNA extraction, cDNA synthesis and amplification of VH and VL IgG sequences for assembly of single-chain variable fragments (scFvs). A scFv-phage display library was constructed and 3 rounds of biopanning were performed using D-positive and D-negative red blood cells (RBCs). Positive phage clones were isolated, Sanger sequenced and, where possible, reformatted into full-length human IgGs to define specificity. The BFC aggregates from 2 anti-D donors underwent a Wright-Giemsa stain and hematological cell count.
Results - Of 10 BFCs, a sufficient yield of total RNA for library construction was obtained from BFCs containing cellular aggregates (n=5). Aggregate analysis showed lymphocytes were the cellular source of Ig-encoding RNA. From the 5 samples with aggregates, scFvs were assembled from amplified IgG variable regions. The library constructed from 1 of these samples resulted in the isolation of clones binding to D-positive RBCs with IGHV3 gene usage. Of the 4 reformatted IgG, 3 were anti-D and 1 had undefined specificity.
Discussion - BFC aggregates are a new and convenient source of Ig-encoding RNA which can be used to construct Ig gene libraries for mAb isolation and discovery via antibody phage display.

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Authors

Eunike C. McGowan - Research and Development, Australian Red Cross Lifeblood, Brisbane, Australia; Australian Research Council Training Centre for Biopharmaceutical Innovation, The University of Queensland, Brisbane, Australia; Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Australia

Robert L. Flower - Research and Development, Australian Red Cross Lifeblood, Brisbane, Australia; Australian Research Council Training Centre for Biopharmaceutical Innovation, The University of Queensland, Brisbane, Australia

Martina L. Jones - Australian Research Council Training Centre for Biopharmaceutical Innovation, The University of Queensland, Brisbane, Australia; Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Australia

David O. Irving - Research and Development, Australian Red Cross Lifeblood, Brisbane, Australia; University of Technology Sydney, Sydney, Australia

Ross T. Barnard - Australian Research Council Training Centre for Biopharmaceutical Innovation, The University of Queensland, Brisbane, Australia;School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia

Catherine A. Hyland - Research and Development, Australian Red Cross Lifeblood, Brisbane, Australia; Australian Research Council Training Centre for Biopharmaceutical Innovation, The University of Queensland, Brisbane, Australia

Stephen M. Mahler - Australian Research Council Training Centre for Biopharmaceutical Innovation, The University of Queensland, Brisbane, Australia; Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Australia

Xuan T. Bui - Research and Development, Australian Red Cross Lifeblood, Brisbane, Australia; Australian Research Council Training Centre for Biopharmaceutical Innovation, The University of Queensland, Brisbane, Australia; Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Australia

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