Monoclonal antibodies against selected peanut allergens: Production and use as affinity agents

Abstract

An extract of dry-roasted commercial peanut mix (CPE) was examined for allergenic activity in peanut-sensitive individuals, using skin tests and radioallergosorbent (RAST) assays. Proteins in the extract were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The proteins were electroeluted in three fractions in the ranges 15-25, 26-58 and 65 kDa. The 15-25 kDa molecular weight fraction produced the most reactive skin tests in peanut-sensitive subjects and was chosen for monoclonal antibody production. Six hybridoma cell lines secreting peanutspecific antibodies of the IgM isotype (kappa light chain) were produced. Immunopurified CPE proteins were then subjected to SDS-PAGE, resulting in five major bands with approximate molecular weights of 14, 25, 38, 40 and 44 kDa. Immunoblotting of these separated proteins revealed: (1) three bands with approximate molecular weights of 38, 44 and 65 kDa, which bound IgE from peanut-sensitive patients; and (2) that the monoclonal antibodies recognized epitopes in bands at approximate molecular weights of 12, 14, 23 and 25 kDa. RAST inhibition assays showed that the affinity-purified proteins were able to inhibit the binding of serum IgE from peanut-allergic individuals to solid-phase CPE.

Identification and partial characterization of multiple major allergens in peanut proteins

“…Hefle et al . [ 22] described IgE‐binding to six proteins with a MW range from 15 to 25 kDa which in addition showed skin test reactivity in 12 PA patients. This substantiates that this MW range contains at least three major allergens (Ara h2 included) which are also bioreactive [ 22].…”

“…[ 22] described IgE‐binding to six proteins with a MW range from 15 to 25 kDa which in addition showed skin test reactivity in 12 PA patients. This substantiates that this MW range contains at least three major allergens (Ara h2 included) which are also bioreactive [ 22]. In order to study these previously described allergens, Ara h1 and Ara h2 were purified using ionexchange chromatography developed with a linear salt gradient, resulting in a higher purity ( Fig.…”

“…Several food allergens inducing IgE‐mediated disorders in humans, have been identified as in cod fish [ 8], shrimp [ 9–11] hen's egg [ 12, 13], cow's milk [ 14, 15], soybean [ 16] and peanut [ 17–22]. Peanut is an oil‐legume containing about 44–56% of oil and 22–30% of protein [ 23].…”

Identification of peanut and hazelnut allergens by native two-dimensional gel electrophoresis

“…Cleangels were rehydrated in 0.7% w/v CHAPS, 20% w/v glycerol, 3 [9]. Strips were focused on a Mulitphor II, under silicone oil for a total of 66 000 Vh, with stepwise increments from 300 V for 10 min, followed by 1000, 2000 and 3000 V for 30 min each and then 3500 V overnight.…”