High quality purification of German cockroach vitellin/vitellogenin from whole body acetone defatted powder

Strader, Michael Brad; Blank, Alexander; Somers, Joshua; Schal, Coby

doi:10.1016/j.jaci.2021.12.713

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“…Ascertaining that Vg/Vn is immunopathogenic in GCr-allergic individuals requires having adequate quantities of reasonably pure allergen, and our earlier attempts to purify Vg/Vn from oothecae (unpublished) and GCr powder by conventional methods [21,22] were successful but with extremely low yield. The availability of a high-titer rabbit antiserum to GCr Vg/Vn made it possible to obtain much greater yields of pure material by affinity chromatography.…”

Section: Discussionmentioning

confidence: 99%

Vitellin/Vitellogenin Is an Important Allergen in German Cockroach

Hadiwinarta,

Blank,

Somers

et al. 2024

Int Arch Allergy Immunol

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Introduction: German cockroach (GCr) aeroallergens are associated with allergic rhinitis and asthma. Vitellogenin (Vg) and vitellin (Vn) are abundant proteins in GCr blood and eggs (including egg cases), respectively, and are possible high molecular mass allergens. Prior efforts to purify Vg/Vn yielded amounts too small for subsequent studies. In this study, we report the affinity purification of Vg/Vn from whole-body defatted GCr powder and determination of the binding of Vg/Vn to anti-GCr IgE. Method: New Zealand white rabbits were immunized with pure Vg/Vn in Freund’s adjuvant, and IgG was purified from the rabbit sera and conjugated to cyanogen bromide (CNBr)-activated Sepharose. Aqueous extracts from GCr powder were passed over the column. After extensive washing, putative Vg/Vn was eluted in low-pH buffer, neutralized, and analyzed by SDS-PAGE and liquid chromatography high-resolution mass spectrometry (LC-HRMS). IgE binding of Vg/Vn was evaluated by inhibition of IgE binding to GCr-ImmunoCAP(I6) in sera from 10 GCr-allergic individuals. In addition, Vg/Vn was biotinylated and bound to ImmunoCAP-streptavidin, and direct IgE antibody binding to the immobilized Vg/Vn was determined in sera from 26 GCr-allergic individuals. Results: Vg/Vn isolated by affinity chromatography was 91% pure by LC-HRMS; contaminants included Bla g 3 (0.9%), human keratin (6%), and rabbit IgG. Vg/Vn inhibited IgE binding to GCr-ImmunoCAP(I6) in 8 of 10 sera. In direct-binding experiments, 21/26 (80%) sera had anti-Vg/Vn IgE at >0.10 kUA/L, while 11/26 (42%) sera were >0.35 kUA/L. Conclusions: We affinity-purified Vg/Vn and demonstrated that Vg/Vn-specific IgE antibody is a major component of GCr-specific IgE.

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Section: Discussionmentioning

confidence: 99%