Porcine IgE in the context of experimental food allergy: purification and isotype-specific antibodies.
Measurement of allergen-specific immunoglobulin E (IgE) is a common practice in the investigation of allergy. It has not been possible to measure porcine IgE due to unavailability of anti-porcine IgE. This study was undertaken to purify and characterize porcine IgE from sera of allergic pigs, identify heterologous anti-IgE reactive with pig IgE and to use purified heavy (H) chain of porcine IgE to generate rabbit anti-IgE. A four-step process for the purification of porcine IgE is reported using ammonium sulphate precipitation, Protein G affinity chromatography, DEAE cellulose anion-exchange chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to obtain IgE H chain. The resultant IgE was evaluated for purity using SDS-PAGE and immunoreactivity was detected by Prausnitz-Küstner (PK) tests and passive cutaneous anaphylaxis with the allergen, crude peanut extract, used to induce experimental allergy. Cross-reactivity with anti-mouse and anti-human IgE antibodies were confirmed in western blot and enzyme-linked immunosorbent assays (ELISA). The H chain of IgE was excised from SDS-PAGE gels and used to develop rabbit anti-porcine IgE antisera. Antiserum obtained from rabbits immunized with porcine IgE, as well as heterologous murine and human-specific anti-IgE, induced reverse cutaneous anaphylaxis in pig skin and detected allergen-specific IgE in ELISA but did not react with IgG H chain in western blots. These results confirm allergy-associated bioactivity of porcine IgE and describe both homologous and heterologous anti-pig IgE suitable for use in allergen-specific and other assays. This will enhance utility of pig allergy models and provide an additional measure of type-2 immune response in pigs.
Rupa
Prithy
P
Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada.
Hamilton
Korinne
K
Cirinna
Melissa
M
Wilkie
Bruce N
BN
D000818:Animals; D000833:Animals, Suckling; D000918:Antibody Specificity; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004195:Disease Models, Animal; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology* / Q000302:isolation & purification; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D007132:Immunoglobulin Isotypes / Q000276:immunology*; D010323:Passive Cutaneous Anaphylaxis / Q000276:immunology; D021183:Peanut Hypersensitivity / Q000276:immunology*; D011897:Random Allocation; D013047:Specific Pathogen-Free Organisms; D013552:Swine
D007143:Immunoglobulin Heavy Chains; D007132:Immunoglobulin Isotypes; D007073:Immunoglobulin E