Standardization of RP-HPLC methods for the detection of the major peanut allergens Ara h 1, Ara h 2 and Ara h 3

Authors: SINGH, HARMIT - California Polytechnic State University; CANTORIA, MARY JO - California State University; MALAVE, POONAM - California State University; SAPUTA, DENNY - California State University; Maleki, Soheila

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/8/2015
Publication Date: 1/1/2016
Citation: Singh, H., Cantoria, M., Malave, P., Saputa, D., Maleki, S.J. 2016. Standardization of RP-HPLC methods for the detection of the major peanut allergens Ara h 1, Ara h 2 and Ara h 3 . Journal of Agricultural and Food Chemistry. 194:383-390.

Interpretive Summary: Crude peanut extract (CPE) was analyzed for three major allergens (Ara h 1, h 2, and h 3) using different chromatographic methods. The presence and level of the allergens were measured with different methods including the use of dissolving solutions. The purification of the individual allergens with chromatography were compared for retention time, resolution or separation, and levels. CPE samples were spiked with addition of pure allergens to enhance their presence and to make it easier to identify and separate the corresponding to allergens. The separated fractions of corresponding allergens were collected and freeze-dried in order to perform protein analysis and immunological assays. The best method identified to separate the allergens from one another was the one with a shorter retention time, better resolution, and greater level of an allergen as compared with the other methods. The major disadvantage of using an alternate chromatographic purification methodod tested here was the need for two sets of conditions to identify the 3 allergens. The method developed in this study allows for purification or separation of all three allergens in one run therefore saving time and resources.

Technical Abstract: Crude peanut extract (CPE) was analyzed for three major allergens (Ara h 1, h 2, and h 3) using a C12 and a C18 column at two wavelengths (280 and 220 nm) and under different solvent conditions. HPLC profiles were compared for retention time, resolution, and peak heights. CPE samples were spiked with pure allergens to identify the peaks corresponding to allergens. The HPLC fractions of corresponding allergens were collected and freeze-dried in order to perform SDS–PAGE and immunoblotting tests. The best method identified was the one with a shorter retention time, better resolution, and greater peak height as compared with the other methods. In general, the peak heights were greater at 220 nm than at 280 nm. The major disadvantage of the C12 column was the need for two sets of conditions to identify the allergens as compared to the C18 column where all three allergens could be identified in one run.