Allergenicity attributes of different peanut market types (1)
The validation of the effectiveness of cleaning protocols on shared equipment or in shared processing facilities can be monitored using immunoassays to detect peanut residue on equipment surfaces and quantifying peanut residue in food samples. Such immunoassays have different sensitivities for specific peanut allergens. The four main market types (Runner, Virginia, Spanish, and Valencia) had comparable contents of the major allergens, Ara h 1 and Ara h 2.
The intact peanut kernels contained protein levels ranging from 20.7 to 29.7% (relative to total weight) with a mean protein content for all samples of 25.2 ± 2.7%. Extractable protein for peanut as determined by the Bradford method was somewhat lower that the theoretically expected value based on the nitrogen content of peanut kernels.
Purified reference peanut allergens shown in the first four lanes were used to assign the bands. Ara h 1, peanut vicilin (7S globulin), migrates at both non-reducing and reducing conditions at approximately 63 kDa. Ara h 2 and Ara h 6, peanut conglutin (2S albumin), migrate at approximately 15/17 and 13 kDa, respectively, under non- reducing conditions and at 17/19 and 15 kDa under reducing con- ditions due to unfolding of the tight protein core. Upon reduction of Ara h3, the two chains are dissociated resulting in 14, 42/ 45 kDa (N-terminal, acidic chain) and 25 kDa (C-terminal, basic chain) bands. Under non-reducing conditions, Ara h1 and Ara h3 comigrate,while Ara h2 and Ara h6 are well separated from Ara h 3 (basic 25 kDa chain).
All of the peanut extracts contained Ara h 1, Ara h 2, Ara h 3 and Ara h 6. Qualitatively, the intensities of these allergens are similar be- tween different peanut extracts, although only semi-quantitative statements can be made based on the SDS-PAGE data.
The protein profiles of the extracts obtained with SDS-containing buffer (Fig. 2) show similar protein bands to those of the aqueous extract.
These allergens appear as separate peaks in the chromatograms of the samples, and exhibit the same elution characteristics of the reference proteins Ara h 1, Ara h 2, Ara h 3 and Ara h 6. Ara h 1 ranges from 11.7 to 23.7%, with a mean (±SD) of 17.1 (±3.4) %. Ara h 2 ranges from 3.5 to 8.0% with a mean (±SD) of 6.2 (±1.3) %. Ara h 3 is clearly the most abundant protein in all samples, with an estimated range from 57.7 to 83.5% with a mean (±SD) of 70.6 (±8.6) %. Ara h 6 content ranges from 2.5 to 9.7% with a mean (±SD) of 5.8 (±1.8) %.
A clinical study applying skin-prick tests demonstrated that peanut allergens Ara h 2 and Ara h 6 are 100- to 1000-fold more potent than Ara h 1 and Ara h 3.
The ranges of reactivity of the samples are from 2.8 to 4.5 mg/mL (mean ± SD 1⁄4 4.0 ± 0.5) for the Neogen kit, and 0.44 to 0.92 (mean ± SD 1⁄4 0.6 ± 0.1) mg/ml for the Morinaga kit. The IC50 values which ranged from 0.20 to 0.41 mg/mL. Attempts to make allergen extracts or allergens hypo-allergenic, for example by chemical modification or biotechnological modification, aim to reduce the IgE-binding at least 100-fold, preferably more.
