The latex allergen hev B 5 is an antigen with repetitive murine B-cell epitopes

International Archives of Allergy and Immunology
Donald H BeezholdR E O'Hehir

Abstract

Hev b 5 is a potent latex allergen. In this study, we characterize the linear B-cell epitopes for three monoclonal antibodies (mAbs) to Hev b 5. The mAbs included 2 IgG1 (6A10, 3G3) and 1 IgG2b (6F6) isotypes. We used SPOTscan analysis with overlapping octapeptides to identify the binding regions for the antibodies and then methionine substitution analysis to further define the critical amino acids (aa) in each epitope. Site-directed mutagenesis was used to selectively eliminate the IgG binding for each epitope and single and multiple mutations were expressed as recombinant GST fusion proteins. Antibody recognition of the mutant proteins was determined by inhibition ELISA. All three mAbs recognized the same aa sequence by SPOTs analysis with slight variations, and this epitope was repeated 3 times in the Hev b 5 sequence; APETEK (63-68), PAEGEK (120-125), and PAEEEK (126-131). Sequential methionine substitution by SPOTsalogue identified K68, E122, and K131 as critical aa in each epitope to change by site-directed mutagenesis. Inhibition ELISA with the mutant proteins indicated that epitope 126-131 was the dominant epitope, but mutation of epitope 120-125 was also required to eliminate mAb reactivity to Hev b 5. The antibodies d...Continue Reading

References

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Citations

May 24, 2008·Clinical and Experimental Allergy : Journal of the British Society for Allergy and Clinical Immunology·J M Rolland, R E O'Hehir
Dec 15, 2010·Clinical and Experimental Allergy : Journal of the British Society for Allergy and Clinical Immunology·C MadritschR Valenta
Mar 26, 2013·Molecular Nutrition & Food Research·Keity Souza SantosFábio Fernandes Morato Castro
Oct 12, 2011·International Archives of Allergy and Immunology·Han-Chih YuanHo-Jen Peng
Nov 3, 2005·Current Opinion in Allergy and Clinical Immunology·Jennifer M RollandRobyn E O'Hehir
Nov 8, 2005·The Journal of Immunology : Official Journal of the American Association of Immunologists·Isabella SchöllErika Jensen-Jarolim

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