Journal: International Journal for Parasitology

Article Title: Identification and evaluation of vaccine candidate antigens from the poultry red mite ( Dermanyssus gallinae )

doi: 10.1016/j.ijpara.2015.07.004

Figure Lengend Snippet: Immunoreactive profiles of soluble mite extract of Dermanyssus gallinae separated by ion exchange chromatography. Ion exchange chromatography fractions were pooled into five groups (IEX groups 1–5), with IEX group 1 consisting of the column flow-through (FT) and IEX groups 2–5 of selectively pooled eluted fractions. Two microgram of IEX groups 1–5 (lanes 1–5) and 2 μg of the soluble mite extract (SME, lane 6) were separated on a 12% Bis–Tris Novex gel (GE Healthcare), transferred to nitrocellulose and probed with IgY generated against the soluble mite extract (A) or IgY from a hen that had not been injected with soluble mite extract (B). Bound IgY was detected with rabbit anti-IgY-peroxidase (Sigma) and visualised with SIGMA FAST ™ 3,3′-diaminobenzidine substrate (Sigma).

Article Snippet: After washing three times in PBST, the immobilised proteins were probed with yolk-derived IgY (yolk-IgY, purified using the Pierce™ Chicken IgY Purification Kit, Thermo Fisher Scientific) in PBS (100 μg/ml) from hens that had been immunised with SME.

Techniques: Ion Exchange Chromatography, Generated, Injection

Journal: International Journal for Parasitology

Article Title: Identification and evaluation of vaccine candidate antigens from the poultry red mite ( Dermanyssus gallinae )

doi: 10.1016/j.ijpara.2015.07.004

Figure Lengend Snippet: Identification of Dermanyssus gallinae proteins present in IgY-immunoreactive spots on a two-dimensional gel immunoblot of the soluble mite extract fractionated by ion exchange chromatography (IEX group 1). Those contigs selected for evaluation as vaccine candidates are given in bold.

Article Snippet: After washing three times in PBST, the immobilised proteins were probed with yolk-derived IgY (yolk-IgY, purified using the Pierce™ Chicken IgY Purification Kit, Thermo Fisher Scientific) in PBS (100 μg/ml) from hens that had been immunised with SME.

Techniques: Two-Dimensional Gel Electrophoresis, Western Blot, Ion Exchange Chromatography

Journal: International Journal for Parasitology

Article Title: Identification and evaluation of vaccine candidate antigens from the poultry red mite ( Dermanyssus gallinae )

doi: 10.1016/j.ijpara.2015.07.004

Figure Lengend Snippet: Identification of Dermanyssus gallinae proteins present in IgY-immunoreactive spots on a two-dimensional gel immunoblot of the soluble mite extract fractionated by ion exchange chromatography (IEX group 4). Those contigs selected for evaluation as vaccine candidates are given in bold.

Article Snippet: After washing three times in PBST, the immobilised proteins were probed with yolk-derived IgY (yolk-IgY, purified using the Pierce™ Chicken IgY Purification Kit, Thermo Fisher Scientific) in PBS (100 μg/ml) from hens that had been immunised with SME.

Techniques: Two-Dimensional Gel Electrophoresis, Western Blot, Ion Exchange Chromatography

Journal: International Journal for Parasitology

Article Title: Identification and evaluation of vaccine candidate antigens from the poultry red mite ( Dermanyssus gallinae )

doi: 10.1016/j.ijpara.2015.07.004

Figure Lengend Snippet: Two-dimensional gel and immunoblot analyses of the pooled ion exchange chromatograph fractions (IEX groups 1 and 4) derived from Dermanyssus gallinae soluble mite extracts. (A–C) IEX group 1; (D–F) IEX group 4 proteins were separated on replicate gels with pI ranges of 7–11 (IEX group 1) and 3–10 (IEX group 4). Two replicate gels were electroblotted and screened with yolk-IgY from hens vaccinated three times with their respective IEX group 1 or 4 proteins (A and D, respectively) or with yolk-IgY from group 7 hens that had been immunised with adjuvant only (B and E). Bound IgY was detected with rabbit anti-IgY-peroxidase IgG and visualised with chemiluminescent substrate. A third gel replicate of IEX groups 1 and 4 (C and F) was stained with SimplyBlue™ SafeStain (Thermo Fisher Scientific) and the corresponding locations of the immuno-reactive spots and were identified and excised from the gel for liquid chromatography–electrospray ionisation–tandem mass spectrometry (LC–ESI–MS/MS) (circled and numbered).

Article Snippet: After washing three times in PBST, the immobilised proteins were probed with yolk-derived IgY (yolk-IgY, purified using the Pierce™ Chicken IgY Purification Kit, Thermo Fisher Scientific) in PBS (100 μg/ml) from hens that had been immunised with SME.

Techniques: Two-Dimensional Gel Electrophoresis, Western Blot, Derivative Assay, Staining, Liquid Chromatography, Mass Spectrometry, Tandem Mass Spectroscopy

Journal: International Journal for Parasitology

Article Title: Identification and evaluation of vaccine candidate antigens from the poultry red mite ( Dermanyssus gallinae )

doi: 10.1016/j.ijpara.2015.07.004

Figure Lengend Snippet: 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).

Article Snippet: After washing three times in PBST, the immobilised proteins were probed with yolk-derived IgY (yolk-IgY, purified using the Pierce™ Chicken IgY Purification Kit, Thermo Fisher Scientific) in PBS (100 μg/ml) from hens that had been immunised with SME.

Techniques: Generated, Ion Exchange Chromatography, Affinity Purification, Western Blot, Staining, Liquid Chromatography, Mass Spectrometry, Tandem Mass Spectroscopy