228–230 Intradermal skin testing. (228) Sequential intradermal injections of allergen using a 1 ml syringeand a 28 gauge needle are made into a site on the lateral thorax. The site should be carefully clipped, nochemical skin preparation used and areas of lesional skin should be avoided. (229) Approximately 0.05 ml of each allergen is injected. The wheal response is compared to a positive control of histamine and a negative control of sterile saline. The test is read at 15–20 minutes for immediate hypersensitivity responses; delayed reactions (8–48 hours) may be recorded as well (230).

ELISA for detection of allergen-specific IgE.(231) In the ELISA for allergen-specific IgE, diluted serum from the patient is added to a series of plastic microtitre wells that have been coated with individual allergens in a regional panel. After a period of incubation, unbound serum antibody is removed by washing and an enzyme-conjugated (e.g. alkaline phosphatase) antiserum specific for canine or feline IgE added. Following this second incubation the wells are again washed, before addition of enzyme substrate.Colour change after a defined interval is read spectrophotometrically using an ELISA plate reader. Positive reactions are generally defined relative to positive and negative control wells and a numerical grading system is sometimes employed. (232) A completed ELISA demonstrating the colour change that occurs using alkaline phosphatase as a substrate. The wells in column 1 have been left empty (plate blank) and the negative control wells are in row A (numbers 2–7).