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

<div><p>Allergy to peanuts has become a common and severe problem, especially in westernized countries. In this study, we evaluated the target and epitope specificity of the capture and detection mouse monoclonal antibodies (mAbs) used in a commercial peanut allergen detection platform. We first identified the target of these antibodies as Ara h 3 and then used an overlapping peptide array of Ara h 3 to determine the antibody-binding epitopes. Further amino acids critical for the binding via alanine substitutions at individual amino acid residues within the epitope were mapped. Finally, inhibition ELISA and inhibition immunoblotting using a recombinant Ara h 3 protein were performed to confirm these results. Surprisingly, the capture and detection mAbs showed identical binding characteristics and were presumed to represent two isolates of the same clone, a notion supported by both isoelectric focusing electrophoresis and Liquid chromatography–mass spectrometry experiments. The simultaneous binding of a pair of identical mAbs to an individual allergen such as Ara h3 is attributed to the multivalency of the analyte and has implications for developing diagnostic assays for additional multimeric allergens.</p></div

Structures of hexamer, monomer, and trimer of Ara h 3 proteins.

<p>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.</p

Genetic variation of Sporothrix globosa

Sporothrix globosa is the main causative agent of sporotrichosis, a common mycosis that usually affects the skin, in China. Despite increasing efforts in the molecular identification of this fungal pathogen, its modes of transmission and epidemiology remain poorly understood. The goals of this study were to assess the genetic diversity of S. globosa using amplified fragment length polymorphism (AFLP) analysis and to assess the correlation of AFLP profiles with the geographic origins, growth rates, clinical forms, and antifungal susceptibilities of S. globosa isolates. AFLP analysis of 225 clinical S. globosa isolates from eight provinces or municipalities in China identified eight distinct clustering groups (I–VIII), with groups I, II and IV being the most common. The AFLP genotypes showed distinct distribution patterns among different regions within Jilin Province and between northern and southern China, but there was no obvious association between the AFLP genotypes and the growth rates, clinical forms or antifungal susceptibilities of the S. globosa isolates. These results expand our understanding of the genetic variation of S. globosa and suggest that AFLP analysis is a potentially useful tool for studying the epidemiology of this fungal pathogen.Emerging Microbes & Infections (2017) 6, e88; doi:10.1038/emi.2017.75; published online 11 October 201

Identification of Ara h 3 as the target antigen of P1 and P2.

<p>A. Induction of recombinant Ara h 3 protein expression. Lane <i>A</i>, un-induced (vector with DNA insert); Lane <i>B</i>, 20-hr induction; Lane <i>C</i>, supernatant after ultrasonic homogenization; Lane <i>D</i>, 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 <i>A</i>, post-centrifugation supernatant; Lane <i>B</i>, flow-through; Lane <i>C</i>, binding buffer eluate; Lane <i>D</i>, 50 mM imidazole eluate; Lane E, 100 mM imidazole eluate; Lane <i>F</i>, 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.</p

P1 and P2 recognize a single antigen in peanut extract.

<p>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.</p

Alanine scanning of the P1/P2 epitope.

<p>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.</p