Fig. 6.

Immunoaffinity enrichment of Dermanyssus gallinae proteins using immobilised antigen-specific IgY. Yolk-IgY generated against ion exchange chromatography group 4 proteins was cross-linked to a HiTrap N-hydroxysuccinimide (NHS)-activated column and IEX group 4 proteins were selectively bound to the column, washed to remove unbound material and then eluted into an affinity enriched fraction with 0.1 M Glycine, 6 M Urea, pH 2.5. (A) The IEX group 4 proteins prior to affinity purification (lanes 1 and 3) and the eluted affinity-enriched material (lanes 2 and 4) were separated on a 12% Bis–Tris Novex gel (GE Healthcare) and transferred to nitrocellulose. Lanes 1 and 2 of the immunoblot were probed with a 1:200 dilution of sera from naive hens that had not been immunised with the soluble mite extract and lanes 3 and 4 were probed with a 1:200 dilution of post-vaccination sera from hens immunised with IEX group 4. Bound IgY was detected with rabbit anti-IgY-peroxidase (Sigma) and visualised using SIGMA FAST™ 3,3′-diaminobenzidine substrate (Sigma). (B) The eluted immunoaffinity enriched IEX group 4 material was concentrated fivefold by lyophilisation and 30 μl was electrophoretically separated as before and stained with SimplyBlue™ SafeStain (lane 5). The gel lane was sectioned into 24 equally-sized horizontal slices and the proteins extracted and subjected to liquid chromatography–electrospray ionisation–tandem mass spectrometry (LC–ESI–MS/MS).