Identification of a common Ara h 3 epitope recognized by both the capture and the detection monoclonal antibodies in an ELISA detection kit

Figures

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  Fig 1. P1 and P2 recognize a single antigen in peanut extract. A. SDS-PAGE analysis of peanut protein extract stained with Coomassie brilliant blue R-250 in non-reducing and in reducing conditions. B-C. Immunoblot of peanut protein extract with P1 (B) or P2 (C) mAb in non-reducing (left) and in reducing (right) conditions.

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  Fig 2. Identification of Ara h 3 as the target antigen of P1 and P2. A. Induction of recombinant Ara h 3 protein expression. Lane A, un-induced (vector with DNA insert); Lane B, 20-hr induction; Lane C, supernatant after ultrasonic homogenization; Lane D, precipitation after ultrasonic homogenization. B. Affinity purification of rAra h 3. The recombinant Ara h 3 protein was eluted at 50 mM imidazole concentration visualized through Coomassie blue staining. Lane A, post-centrifugation supernatant; Lane B, flow-through; Lane C, binding buffer eluate; Lane D, 50 mM imidazole eluate; Lane E, 100 mM imidazole eluate; Lane F, 500 mM imidazole eluate. C. Immuno-blotting of recombinant Ara h 3 and crude peanut extract with P1 mAb. D. Competition of P1 binding to native Ara h 3 in peanut extract by recombinant Ara h 3 protein. N: non-reducing; R: reducing. PVDF blots of total peanut extract were probed with mouse mAb P1 in the absence or the presence of the indicated inhibitors.

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  Fig 3. The P1/P2 epitope on the Ara h 3 allergen. The Ara h 3 full length sequence was covered by 167 synthetic peptides of 13-amino acids per peptide, offset from each other by 3 amino acids. These peptides were arranged in an array format and probed with the specific mAbs P1 (A) and P2 (B). The positions of the peptides within the Ara h 3 sequence are shown on the left. C. Peptides at sites 102–103 show recognition with the two mAbs in the membrane. D. The amino acid sequence of the Ara h 3. The shaded areas represent the large subunit of Ara h 3. The positions underlined correspond to the specific peptide targeted as shown in (A) and (B).

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  Fig 4. Alanine scanning of the P1/P2 epitope. A. Membranes containing wild-type (WT) E306-G317 peptide and peptides synthesized with single alanine substitutions at each position were probed with the P1 and P2 mAbs. B. Inhibition of HRP- conjugated P1 by unlabeled P1 and P2 in an inhibition ELISA. The competing antibodies are used in 20x excess relative to the labeled P1 antibody. Experiments were performed in triplicates and standard error bars are shown for all measurements.

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  Fig 5. Structures of hexamer, monomer, and trimer of Ara h 3 proteins. A. The hexameric structure of Ara h3 (PDB: 3C3V). B. The P1/P2 epitope is located on an undefined and presumably flexible loop near the C-terminal end of the large subunit of the Ara h 3. C. Mapping of a 3-D structure of Ara h 3 and the P1/P2 epitopes (also named “epitope 4” in Rabjohn et al.) in a trimer (half molecule) model. Each monomeric subunit is colored differently.