Identification of a common Ara h 3 epitope recognized by both the capture and the detection monoclonal antibodies in an ELISA detection kit (1)
Peanut (Arachis hypogaea L.) is one of the most common inducers of type I IgE-mediated food allergy.
Peanut flour and 1 X extraction buffer were mixed at a ratio of 1:20 and incubated at 62 ̊C for 10 min with shaking at 2 min intervals. The aqueous peanut protein extract was centrifuged at 3000 x g for 10 min with saving of the supernatant.
Peanut protein extract revealed multiple major protein fractions under non-reducing and reducing conditions. the two mAbs. Both P1 and P2 bound to one band about 60 kDa in non-reducing conditions and one band about 40 kDa in reducing conditions.
His-tagged rAra h 3 was expressed in E. coli and purified to homogeneity by affinity purification. Immunoblotting with P1 mAb con- firmed that the rAra h 3 exhibited the same strong binding to P1 as the native Ara h 3 protein in the peanut extract, although the additional tag slightly altered the gel mobility of the recombinant protein. soluble rAra h 3 could inhibit P1 binding to native Ara h 3 present in the peanut extract in a dosage-dependent manner.
Both mAbs recognized the same two adjacent spots (#102 and #103 on the array), which represent a single linear peptide on the large subunit with a sequence of YEYDEEDRRR on167 overlapping solid phase peptides, constituting the entire length of the Ara h 3 large and small subunits.
A mutagenesis study by synthesizing a series of peptides, each with a different single alanine substitution (alanine scanning) to cover each position of the mapped epitope was demonstrated. The two antibodies showed remarkable similarities in the binding patterns in this alanine scanning peptide array at positions 308, 310, 311, 313, 314, and 317. Alanine replacement at positions 309, 312, 315, and 316 showed a somewhat reduced binding whereas alanine substitution at position 306 had no effect on binding.
Ara h 3 is structurally a member of the cupin superfamily of proteins and composed of six identical subunits, each of which is heterodimeric itself. The 11S storage protein Ara h 3 fulfills these requirements as it is a hexamer consisting of six identical subunits.
Molecular modeling reveals that the epitopes are in a flexible loop located on the periphery of the two adjacent trimeric rings that comprise the molecule. Consequently at least three and possibly as many as six copies of this same epitope would be available for binding in the fully assembled hexamer.