Protein microarrays: The future of allergy diagnosis? Optimization of coating and printing of allergen arrays used with fluorescent humanised basophil reporter cell line NFAT-DsRed RBL

Abstract

Up-to-date, in vitro allergy diagnosis techniques rely on the detection of allergen-specific Immunoglobulin E (sIgE). Protein microarrays enable the simultaneous screening of hundreds of sIgEs using only a small amount of serum. However, they do not always report clinically relevant sIgE. Combining protein arrays with humanised rat basophilic leukaemia (RBL) reporter cells that provide a biological readout, we aim to develop a high-throughput amenable, clinically relevant allergy diagnosis platform. Here, we demonstrate the effectiveness of different extracellular matrix (ECM) proteins to facilitate RBL cell attachment on various polymer-coated slide surfaces. We also determine printed protein stability using time-of flight secondary ion mass spectrometry (ToF-SIMS). Lastly, we demonstrate the ability of this system to detect an allergic response. Aldehyde-, epoxy- and amine-coated glass slides were coated with 10 μg/mL vitronectin, fibronectin or laminin for 48 hours at 37 ͦ C and with 10 μg/mL collagen for 48 hours at 4 ͦ C. After incubation, NFAT-DsRed RBL cells were cultured on each surface for 72 hours. The number of adherent cells was assessed every 24 hours using 1 μg/mL Hoechst 33342 solution. Next, the following proteins diluted in PBS to a final concentration of 1mg/mL, goat anti-human IgE, timothy grass pollen extract, human serum albumin, k-casein from bovine milk or the above mentioned proteins diluted with 10% fibronectin, were printed in triplicate onto the different polymer-coated slides using a XYZ3200 dispensing workstation (Biodot). Printed proteins were characterized by ToF-SIMS before and after washing the surfaces with deionized water. Finally, cells were sensitized overnight with human IgE. Cell activation was assessed using fluorescence microscopy after 24h incubation onto the protein printed surfaces. Treatment of the aldehyde- and epoxy-coated slides with different ECM proteins had no significant effect on cell adhesion compared to control (cells only). The amine coated surfaces showed higher printed protein stability compared to aldehyde and epoxy ones. Lastly, cell activation was higher on the epoxy slides after printing 10% fibronectin containing protein solution compared to control (protein only). The epoxy-coated slides would consist the optimum platform to assess cell activation and potentially detect an allergic response.