Passive cutaneous anaphylaxis IgE assay

In vivo passive cutaneous anaphylaxis assays were performed using male Sprague-Dawley rats 10 weeks of age. Individual BALB/c serum samples were prepared as serial 1:2 dilutions with a final sample volume of 55 μl. Sprague-Dawley rats were anesthetized via i.p. injection of 4 ml 5.71% chloral hydrate (Sigma-Aldrich) in PBS. Once anesthetized, rats had their back closely shaved to remove all hair, and 50 μl of each sample was injected s.c. down the back. Twenty-four hours later, rats were anesthetized with chloral hydrate and injected i.v. with 2 ml of a 1:1 antigen/dye solution containing 4 mg/ml OVA in PBS and 1% Evans blue dye (Sigma-Aldrich). Blue s.c. injection sites after 15–30 minutes indicate serum samples positive for OVA-specific IgE. The highest positive serum dilution for each sample was recorded, and animals were euthanized with 600 μl Lethabarb (Virbac) intravenously.