Peanut protein extraction conditions strongly influence yield of allergens Ara h 1 and 2 and sensitivity of immunoassays

- Defatting peanut meal with n-hexane gave significantly more Ara h 1 and 2 than with diethyl ether.
- Buffer type and pH affected extraction yields and detection rates of Ara h 1 and 2 in ELISAs.
- Extraction yields of crude peanut protein correlated poorly with yields of Ara h 1 and Ara h 2.
- Western blots of allergens extracted with PBS were more sensitive than those extracted with TBS or Tris.
- Common extraction buffers could retain allergens in peanut meal; e.g. TBS (pH 8.5) retained Ara h 3.

The clinical importance of peanut (Arachis hypogaea) allergies demands standardized allergen extraction protocols. We determined the effectiveness of common extraction conditions (20 buffers, defatting reagents, extraction time/temperatures, processing, extraction repeats) on crude protein and Ara h 1 and 2 yields. Despite similar 1D-gel profiles, defatting with n-hexane resulted in significantly higher yields of crude protein, Ara h 1, and Ara h 2 than with diethyl ether. The yields were affected by the composition and pH of the extraction buffers and other conditions, but crude protein yield did not always correlate with Ara h 1 and 2 yields. Denaturants, reducing agents, acidic buffers, and thermal processing of peanuts perturbed allergen quantification in ELISAs, probably via exposure of additional epitopes. Allergen detection in 2D-Western blots with PBS resulted in greater sensitivity than with TBS or Tris. We recommend that allergen extraction conditions be selected based on the research question being investigated.