Multiplexing of miniaturized planar antibody arrays for serum protein profiling – a biomarker discovery in SLE nephritis

Abstract

In the quest to decipher disease-associated biomarkers, miniaturized and multiplexed antibody arrays may play a central role in generating protein expression profiles, or protein maps, of crude serum samples. In this conceptual study, we explored a novel, 4-times larger pen design, enabling us to, in a unique manner, simultaneously print 48 different reagents (antibodies) as individual 78.5 μm(2) (10 μm in diameter) sized spots at a density of 38,000 spots cm(-2) using dip-pen nanolithography technology. The antibody array set-up was interfaced with a high-resolution fluorescent-based scanner for sensitive sensing. The performance and applicability of this novel 48-plex recombinant antibody array platform design was demonstrated in a first clinical application targeting SLE nephritis, a severe chronic autoimmune connective tissue disorder, as the model disease. To this end, crude, directly biotinylated serum samples were targeted. The results showed that the miniaturized and multiplexed array platform displayed adequate performance, and that SLE-associated serum biomarker panels reflecting the disease process could be deciphered, outlining the use of miniaturized antibody arrays for disease proteomics and biomarker discovery.

Generation of miniaturized planar ecombinant antibody arrays using a microcantilever-based printer

“…choice of solid support, spotting conditions) for recombinant antibody array production in future efforts, it is very likely that this key parameter can be even further improved [5]. In comparison, we observed mean/ median CV-values of 12% (range 5-27%) and 4% (range 0-11%) for the 1st and second generation of miniaturized recombinant antibody arrays produced based on DPN technology (NLP3000) [33,34]. Noteworthy, the CV-value has been found to be <30% for conceptual antibody nanoarrays [14-23, 25, 30], while conventional antibody microarrays (established platforms) regularly display CVs of <10% (recombinant antibody microarrays) [49] and <20% (polyclonal and monoclonal antibody microarrays [50,51].…”

“…Noteworthy, the CV-value has been found to be <30% for conceptual antibody nanoarrays [14-23, 25, 30], while conventional antibody microarrays (established platforms) regularly display CVs of <10% (recombinant antibody microarrays) [49] and <20% (polyclonal and monoclonal antibody microarrays [50,51]. The array-to-array and slide-to-slide reproducibility were found to be higher, which could be expected in evaluating non-normalized data [5,33,34]. Being a pilot study, we identified the efforts to apply such normalization strategies [5,33,34] to be more suited to follow-up studies, where the aim would be to evolve the platform from a proof-of-concept set-up to an optimized (established) methodology aiming to run (clinical) applications.…”

“…Being a pilot study, we identified the efforts to apply such normalization strategies [5,33,34] to be more suited to follow-up studies, where the aim would be to evolve the platform from a proof-of-concept set-up to an optimized (established) methodology aiming to run (clinical) applications. In this context, it might still be of interest to note that CV values in the range of 5% was observed for the second generation of miniaturized antibody arrays (DPN technology) [33,34], while CV values in the same range [5][6][7]13] or better (1.6%) (Wingren et al unpublished data) regularly have been observed for established conventional antibody microarray set-ups. In comparison, the inter-and intra-assay CV values for well-optimized conventional ELISAs are frequently ⩽15%.…”

“…Using a DPN-based desktop printer (NLP3000 TM ), we have produced the first 12-and 48-plex planar recombinant antibody arrays, based on 78.5 um 2 (Ø 10 μm)-sized spots at a density of 38 000 spots cm −2 , interfaced with a fluorescent-based readout [33,34]. To the best of our knowledge, only one other complete microprinter system is currently at hand, the Nano eNabler TM , relying on a microcantilever-based surface patterning tool for printing [25,32].…”

“…To the best of our knowledge, only one other complete microprinter system is currently at hand, the Nano eNabler TM , relying on a microcantilever-based surface patterning tool for printing [25,32]. Noteworthy, a novel microspotting tool, Bioplume TM -consisting of an array of micromachined silicon cantilevers with integrated microfluidics channels-was recently introduced [34][35][36][37]. This spotting tool thus represents a novel opportunity for producing multiplex, miniaturized antibody arrays spots sized 5-20 μm in diameter.…”