Identifying the stability of a new wheat gliadin extract by protein analysis, skin tests and cell degranulation assay

Abstract

Background: The commercial wheat extract for skin prick test (SPT) provides less sensitivity to predict wheat allergy, compared to in-house gliadin extracts. SPT is a preferred method to study extract stability as it is the aim of developing extract. The role of cell degranulation assay, a functional assay with the same mechanism as SPT, is not widely used to determine extract stability. Objective: To study the stability of in-house gliadin extracts stored at different periods, by using protein analysis, SPT and degranulation assay of humanized rat basophilic-leukemia (RBL-SX38) cells. Methods: Patients with a history of wheat allergy and positive SPT to wheat, were recruited. The gliadin extracts stored for 1, 6, 9, and 12 months at 2–8°C were used in SDS-PAGE, SPT and cell degranulation assay. The cell degranulation was determined by β-hexosaminidase release. Results: Forty children were recruited. The gliadin extract stored for 9 and 12 months provided lighter protein bands than 1 and 6 months. However, the wheal diameters from SPT using extracts stored at different periods, were not significantly different (p = 0.09). There were also no significant differences of the β-hexosaminidase released using 0.1 and 1 μg/mL of gliadin extracts stored at different periods (p > 0.05). The 10 μg/mL of gliadin extracts stored at longer periods, significantly stimulated higher β-hexosaminidase release (p = 0.01). The extracts were sterile at all storage times. Conclusions: To determine the stability of in-house gliadin extracts, SPT or cell degranulation assay provided additional information to SDS-PAGE. The extracts were stable for up to 12 months.