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Release date：2019-06-26

JACI

[IF:13.258]

Fast generation of IgE, IgG cloning sharing the same variable region to 1 and IgG4 by PIPE the major birch pollen allergen Bet v 1
DOI: org/10.1016/j.jaci.2018.12.571
Abstract:
RATIONALE: Birch pollen allergy with the single major allergen Bet v 1 is a paradigm to study the molecular interplay of IgE and IgG1 or IgG4 in sensitization and allergen immunotherapy. However, there is a lack in the availability of such antibodies. We here aimed to fuel our antibody PIPEline by using Polymerase Incomplete Primer Extension (PIPE) cloning (Ilieva et al., 2017), the latest method for fast creation of different classes of antibodies sharing the same variable region.
METHODS: Variable region sequences from the literature (Levin et al.,2014) were used to create vectors coding for IgE, IgG1 and IgG4 via PIPE cloning. These self-assembled plasmids were then expressed in the Expi293F system at a cell culture volume of 30 ml. Recombinant antibodies were purified via affinity chromatography and integrity tested in SDS-PAGE. Concentration was determined via BCA protein assay.Specificity to Bet v 1 was determined by dot blot and ISAC112 microarray.
RESULTS: Expression of all three antibody classes in Expi293F cells resulted in high yields from 0.9 mg (IgE) to 6 and 4.4 mg (IgG1 and IgG4,respectively). PIPE cloning delivered correctly assembled IgE, IgG1 and IgG4 antibodies specifically recognizing Bet v 1.
CONCLUSIONS: PIPE cloning proves to be a highly efficient method for the production of recombinant antibodies against Bet v 1. As these antibodies belong to different classes but share the same variable region they will be useful tools for researching mechanisms underlying type 1 allergy as well as allergen immunotherapy.
First Author:
Jill A. Poole
Correspondence:
Nebraska Medical Center, Omaha, NE 68198.
All Authors:
Jill A. Poole, Charles S. Barnes, Jeffrey G. Demain, Jonathan A. Bernstein, Mahesh A. Padukudru, William J. Sheehan, Guillermo Guidos Fogelbach, James Wedner, Rosa Codina, Estelle Levetin, John R. Cohn, Steve Kagen, Jay M. Portnoy, Andre E.

2019-05-08 Article