Production, characterization and applications for Toxoplasma gondii-specific polyclonal chicken egg yolk immunoglobulins.

Toxoplasma gondii may cause abortions, ocular and neurological disorders in warm-blood hosts. Immunized mammals are a wide source of hyperimmune sera used in different approaches, including diagnosis and the study of host-parasite interactions. Unfortunately, mammalian antibodies present limitations for its production, such as the necessity for animal bleeding, low yield, interference with rheumatoid factor, complement activation and affinity to Fc mammalian receptors. IgY antibodies avoid those limitations; therefore they could be an alternative to be applied in T. gondii model.In this study we immunized hens with soluble tachyzoite antigens of T. gondii (STAg) and purified egg yolk antibodies (IgY) by an inexpensive and simple method, with high yield and purity degree. IgY anti-STAg antibodies presented high avidity and were able to recognize a broad range of parasite antigens, although some marked differences were observed in reactivity profile between antibodies produced in immunized hens and mice. Interestingly, IgY antibodies against Neospora caninum and Eimeria spp. did not react to STAg. We also show that IgY antibodies were suitable to detect T. gondii forms in paraffin-embedded sections and culture cell monolayers.Due to its cost-effectiveness, high production yield and varied range of possible applications, polyclonal IgY antibodies are useful tools for studies involving T. gondii

Immunocytochemistry for <i>T. gondii</i> using polyclonal IgY anti-STAg.

<p>HeLa cells infected with tachyzoites were fixed, permeabilized and incubated with IgY anti-STAg and rabbit IgG anti-IgY conjugated to fluorescein isothiocyanate (FITC). In fluorescence photomicrographs, or merged images with phase contrast. tachyzoites were detected into cell cytoplasm around the nucleus (blue, DAPI) with strong staining (arrows).</p

Purification of egg yolk antibodies.

<p>(<b>A</b>) Separation of the water soluble fraction (S1) from a lipid-rich precipitate (P1), after the incubation of crude egg yolk with acid water (pH 5.0–5.2); S1 precipitation by salting-out (19% Na₂SO₄) produced an enriched IgY pellet (P2) and a supernatant with contaminants (S2). (<b>B</b>): Purity degree of IgY samples determined by SDS-PAGE (8%), demonstrating that salting-out protocol produced high purity IgY antibodies. (<b>C</b>) Size-exclusion chromatography of the P2 fraction, with peak of IgY between 13th and 19th fractions, where (<b>D</b>) the 14th fraction presented the highest degree of purity. (<b>E</b>): IgY enrichment conferred by Slot-blot assay, IgY was detected until the concentration of 0.01 µg of protein (box). Bovine sera albumin (BSA) was used as negative control.</p

Immunohistochemistry for <i>T. gondii</i> detection using polyclonal IgY anti-STAg antibodies.

<p>Paraffin-embedded brain sections of mice chronically infected with ME-49 strain were incubated with IgY anti-STAg and rabbit IgG anti-IgY conjugated to fluorescein isothiocyanate (FITC). (<b>A</b> and <b>B</b>), segmented parasitophorus vacuoles were detected into host cell cytoplasm around the DAPI-stained nucleus (blue). (<b>C</b> and <b>D</b>), IgY anti-STAg antibodies strongly recognized antigens on outer walls of tissue cysts and free tachyzoites (<b>C</b>, arrow).</p

Recognition profile of STAg proteins using polyclonal antibodies raised in chicken and mice.

<p>(<b>A</b>) Differential reactivity to <i>Toxoplasma gondii</i> soluble antigens (STAg) of egg IgY antibodies and mouse serum IgG from animals immunized by intramuscular (i.m.) and subcutaneous (s.c.) routes initially was assessed by 1D immunoblotts (WB1D). Antigens with approximately (∼) 30 kDa (blue box) were evenly recognized by all antibodies tested, which was not observed in antigens with ∼40 kDa (green box) and ∼50 kDa (red box), which were mainly recognized by chicken and mouse antibodies, respectively. The same recognition pattern against STAg proteins was noted in two-dimensional immunoblotts (WB2D), probed with chicken IgY (<b>B</b>) or mouse IgG (<b>C</b>). (<b>D</b>) IgY antibodies obtained from eggs of chickens immunized with <i>T. gondii</i>, as well as IgY against <i>Neospora caninum</i> and <i>Eimeria</i> spp., were assayed against STAg-immobilized nitrocellulose membranes in order to check possible antibody cross-reactivity.</p

Production kinetics and avidity of IgY anti-STAg antibodies.

<p>Indirect ELISA showing (<b>A</b>) kinetics of chicken IgY and (<b>B</b>) IgY avidity maturation with 6M urea-treated (•) or untreated (○) immunocomplex. Black arrows indicate the three immunization doses. Samples with ELISA Index (EI) equal or superior to 1.2 were considered positive. (<b>C</b>) 1D-immunoblot presenting recognition kinetics (from 14 to 49 days p.i.) of STAg proteins by IgY and its respective avidity maturation, evaluated by 6M urea treatment of immunocomplexes.</p