An allergen immobilization platform based on material-binding peptides for highly sensitive detection of allergies

Detection of antibodies in allergy diagnostics requires allergens to be immobilized, e.g., on microtiter plates; thereby, immobilization without capturing antibodies remains a challenge. An immobilization platform, based on the material-binding peptide Snakin-1, addresses the outlined challenge for ELISA-based allergy detection on the example of Bet v 1 from birch pollen. Bet v 1 from birch pollen is the main cause of allergic rhinitis in Europe. Snakin-1 was selected among five material-binding peptides due to its polystyrene (PS)-binding properties, productive immobilization in a fusion protein with Bet v 1, and lack of interference with the primary and secondary antibody of the assay. The developed ELISA covers the whole range of antibody detection in diagnostics of the anti-Bet v 1 antibody. The reached sensitivity of the Bet v 1-Snakin-1 ELISA on standard PS plates is comparable to the Bet v 1 signal on high-binding PS plates, and the Bet v 1-Snakin-1 ELISA outperforms the Bet v 1 ELISA on high-binding plates (1.3-fold). SPR measurements of binding Bet v 1 and Bet v 1-Snakin-1 to PS, as well as subsequent antibody detection, confirm the role of Snakin-1 in productive immobilization of the Bet v 1 allergen on PS. The necessity to block the microtiter plate with BSA could be omitted, and a detection limit of at least 0.333 ng/mL (0.14 kU/L) sIgE in serum was achieved. The latter results make the scalable coating of allergens on untreated PS plates from aqueous solutions, in combination with ELISA, a highly attractive platform for allergy diagnostics.