A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope

Abstract

Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv) antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1), carp (Cyp c 1) and rainbow trout (Onc m 1) parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients’ sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. 1H/15N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes.

Solution structure of the major fish allergen parvalbumin Sco j 1 derived from the Pacific mackerel

“…An NMR structural and dynamics study of Gad m 1 revealed that its structure and the accessibility of putative IgE epitopes are similar to those of other parvalbumin types 14 . In a recent study, an interaction between Gad m 1 and the scFv fragment of the antibody was established using a 1 H- 15 N HSQC chemical shift perturbation method 15 .…”

Mitochondrial haplogroup J associated with higher risk of obesity in the Qatari population

“…Both the above-mentioned studies by Nardelli et al and Ebner et al reported that T haplogroup was associated with a risk of obesity in Caucasians from Southern Italy and Austria, respectively, whereas our study showed that J haplogroup was associated with obesity in Arabs from Qatar. It is interesting to note that J and T are sister haplogroups and share polymorphisms 14 , such as MT:4216 T > C, which was significantly correlated with risk of obesity in our study (Table 4 ). However, the frequency of haplogroup T was not significantly different between our study groups with and without obesity (Fig.…”

“…A novel mitochondrial tRNA Cys mutation at MT:5802A > G observed in 3 matrilineal relatives has been attributed as the probable cause for obesity in the family with matrilineally inherited obesity 13 . Furthermore, the mitochondrial haplogroup T has been suggested to increase the risk of obesity in 2 independent association studies on Austrian 14 and Southern Italian 15 populations. Maternal haplogroups X and H have been proposed to reduce the risk of obesity in Caucasians of northern European origin in the United States (US) 16 as well as in Arabs living in Kuwait 17 , respectively.…”

Generation of scFv-Monoclonal Antibody Avian Influenza Diagnostic tests

“…Comparable approach to the present study had been used by previous studies in an attempt to isolate scFv recognizing parvalbumin allergen from various species of fish (Bublin et al 2015). For that purpose, the group carried out three sequential panning on cod, carp and rainbow-trout parvalbumins, respectively.…”