Development of a protein microarray-based diagnostic chip mimicking the skin prick test for allergy diagnosis

Abstract

Protein microarrays have been successfully used for detection of allergen-specific IgE in patient sera. Here, we demonstrate proof-of-concept of a solid-phase technique coupling the high-throughput potential of protein microarrays with the biologically relevant readout provided by IgE reporter cells, creating a novel allergic sensitization detection system. Three proteins (κ-casein, timothy grass pollen extract, polyclonal anti-human IgE) were printed onto three different polymer-coated surfaces (aldehyde-, epoxy- and NHS ester-coated). ToF–SIMs analysis was performed to assess printed protein stability and retention during washing steps. NFAT-DsRed rat basophil leukemia cell attachment and retention during washing steps was assessed after treatment with various extracellular matrix proteins. NFAT-DsRed IgE reporter cells were sensitized with serum of an allergic donor, incubated on the printed slides, and cell activation determined using a microarray laser scanner. NFAT DsRed IgE reporter cell binding was significantly increased on all polymer surfaces after incubation with fibronectin and vitronectin, but not collagen or laminin. All surfaces supported printed protein stability during washing procedure, with epoxy- and NHS ester-coated surfaces showing best protein retention. Cell activation was significantly higher in NHS ester-coated slides after timothy grass pollen extract stimulation appearing a suitable substrate for further development of an automated allergy diagnosis system.

The Humanised NPY-mRFP RBL Reporter Cell Line Is a Fast and Inexpensive Tool for Detection of Allergen-Specific IgE in Human Sera

“…As fluorescence is released during degranulation, this reporter system cannot be used in array format, as it would disconnect the signal from its location on the array. For use on arrays, we developed another humanised reporter system called RBL NFAT-DsRed FCER1G, where fluorescence is cytosolic and can be measured in array format [ 10 ]. The second potential issue is inherent to the signal transduction pathway.…”

“…These reporters are very sensitive, allowing detection of as little as 15 pg of IgE or 1 fg/mL of egg white protein using appropriate sera of hen egg white allergic donors [ 7 ]. Since then, fluorescent variants of the RS-ATL8 reporter have been created [ 8 ] and improved [ 9 ], which enable their use in solid-phase-based (e.g., allergen array) format [ 10 ]. The advantages and disadvantages of RBL-based, humanised IgE reporters have been discussed in detail before [ 3 , 11 ].…”

Pushing the detection limits: strategies towards highly sensitive optical-based protein detection

“…This method can offer advantages such as low reagent and antibody consumption, high sensitivity, and multiplexity [ 19 ]. Through miniaturization of the assay spots and thus concentrating the analyte molecules into micro-scale dimensions for signal production, antibody microarrays have been able to provide a LOD in the ng/mL or even middle pg/mL range [ 21 ]. The technology and applications of the “standard” protein or antibody microarrays have been previously reviewed [ 22 – 24 ].…”

Are humanized IgE reporter systems potential game changers in serological diagnosis of human parasitic infection?

“…While rat basophilic leukemia cells stably transfected with the human IgE receptor have been used for many years (Falcone et al 2015), the last decade has seen the rather cumbersome and insensitive beta-hexosaminidase determination replaced by fluorescent or chemiluminescent reporter assays. These powerful IgE reporter cell lines can be used in a variety of formats, ranging from 384-well plates (Ali et al 2017) to protein arrays (Kalli et al 2020). The prototype, and to date still best performing IgE reporter system, was (Falcone et al 2018).…”