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

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. AR patients.

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.