Analysis of the Effector Activity of Ara h 2 and Ara h 6 by Selective Depletion from a Crude Peanut Extract (1)
The effect of removing a specific allergen on the effector activity of an extract can be measured using a number of in vitro model systems such as the humanized RBL cell assay and ex vivo models such as basophil histamine release (BHR), the basophil activation test (BAT), or release of leukotrienes (LT). RBL SX-38 cells are rat basophilic leukemia cells that stably express approximately 70,000 copies per cell of the human high affinity receptor for IgE, FcεRI.
Candidate peptides for production of antibodies to Ara h 6 were chosen for 1) uniqueness and 2) hydrophilicity and antigenicity using a commercial algorithm program (MacVector). The most hydrophilicity, antigenic index surface probability, and uniqueness of the sequence within the peanut proteome as discerned by protein BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Affinity purified antibodies that were raised against the unique Ara h 6 peptide, EQEQYDSYDIRSTRSSDQ (amino acids 37–55) linked to thyroglobulin (THY) were consistently capable of removing Ara h 6 without removing Ara h 2. Antibodies raised to the same peptide conjugated to keyhole limpet hemocyanin (KLH) were not successful at immunodepletion of Ara h 6.
Since the CPE consists of approximately 4% Ara h 2 and 6% Ara h 6 as measured by a competitive ELISA, approximately 4 mg of this anti-Ara h 6 antibody linked to 1 ml of packed beads was, with two passes, able to remove approximately 540 μg of Ara h 6 from our CPE.
Immunoblot analysis with both rabbit anti-peptide antibodies and with IgE from the serum of a pool of peanut allergic subjects confirmed that each protein was specifically depleted by ~99% when assessed either with rabbit anti-peptide antibodies or with human anti-peanut IgE.
immunodepletion of both Ara h 2 and Ara h 6 together significantly reduced the effector activity of peanut extract.
