RESUMO
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 ß-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 ß-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 ß-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.
RESUMO
PURPOSE: Tolerance to shrimp has been reported in some patients with a history of shrimp allergy. The predictors of the natural resolution of shrimp allergy have not been widely explored. This study aimed to investigate the role of specific IgE (sIgE) and specific IgG4 (sIgG4) to shrimp extracts and the cross-reactive shrimp allergens tropomyosin (TM), arginine kinase (AK) and myosin light-chain (MLC), as markers of persistent or resolved shrimp allergy (PSA or RSA). METHODS: Seventeen patients with a 10-year history of allergy to Penaeus monodon (Pm) and/or Macrobachium rosenbergii (Mr) were recruited. Oral shrimp challenges identified 10 patients with PSA and 7 patients with RSA. Sera from these patients were evaluated for sIgE and sIgG4 to Mr and Pm extracts as well as to TM, AK and MLC. RESULTS: The levels of sIgE to Mr and Pm extracts were lower in the RSA than in the PSA groups (P = 0.05 and P = 0.008, respectively), but sIgG4 or sIgG4:sIgE ratio did not show statistical significance. The sIgE to AK and MLC, but not TM, were lower in the RSA group than in the PSA group (P = 0.009 and P = 0.0008, respectively). There was no difference in sIgG4 to TM, AK and MLC between both groups. The ratio of sIgG4:sIgE to MLC, but not TM or AK, was higher in the RSA than in the PSA group (P = 0.02). A higher diversity of sIgE to shrimp components was found in the PSA group than in the RSA group (P = 0.006). CONCLUSIONS: Specific bioassays can be used to identify patients with RSA. Oral shrimp challenges in these patients may provide a higher rate of passing the challenges and finally reintroducing shrimp in their diet.
