Methods: The parvalbumin-specific scFvs were selected from the human synthetic scFv phage library ETH-2 by alternating the parvalbumin from cod (Gad m 1), carp (Cyp c 1) and trout (Onc m 1) during three rounds of sequential biopanning. Based on their reactivity to parvalbumins by ELISA, two clones were expressed in Escherichia coli. The ability of the two scFv antibodies to inhibit the binding of parvalbumin-specific IgE from fish allergic patients’ sera was showed by ELISA competition experiments and the rat basophilic leukemia mediator release assay.
Results: Based on ability to bind different parvalbumins and sequence analysis, phage clones scFv-gco9 and scFv-goo8 were selected for production of soluble scFv antibodies. We obtained 1 mg of scFv-gco9 and 1.3 mg of scFv-goo8 per litre of bacterial culture. The scFv-gco9 was able to detect all three parvalbumins at a concentration of 10 ng/ml. The scFv-goo8 bound to cod parvalbumin, but not to carp and trout parvalbumin. The detection limit for 1 µg/ml of the scFv-gco9 was 0.01 µg/ml of the Gad m 1 and 0.2 µg/ml of Onc m 1 or Cyp c 1. We found that scFv-gco9 dose-dependently blocked the binding of IgE to immobilized Gad m 1, Cyp c 1 and Onc m 1. At a concentration of 5 µg/ml of scFv-gco9 binding of IgE to the three parvalbumins was inhibited by approximately 40%, and at a concentration of 20 µg/ml the IgE binding was inhibited to ~ 70%. In the case of the scFv-goo8, inhibition of IgE binding to Gad m 1 was about 15%. The inhibition of degranulation of basophils was 55% in the presence of 2 µg/ml scFv-gco9.
Conclusions: This work, supported by grant SFB-F01802, revealed that the scFv antibodies can be used for the standardization of protein extracts used for allergy diagnosis and for IgE epitope mapping. Epitope characterization enables the engineering of parvalbumin molecules with reduced IgE binding for allergen-specific immunotherapy.