**5.2. Surface chemistry**

The microarray surface chemistry plays a critical role in determining protein microarray quality. Slide surfaces vary: aldehyde and epoxy-derivatized glass surfaces are used for random attachment through amines, whereas nitrocellulose, hydrogel or metal surfaces for attachment of affinity-purified proteins. An ideal surface chemistry should resist nonspecific adsorption, whilst preserving the folded structure of the arrayed proteins [1].

Common challenges associated with slide surface chemistry include high background and incorrect protein orientation or conformation of proteins, whereby all functional binding sites are not readily available for interaction. Proteins have various hydrophobic domains and charged patches, so tend to adsorb non-specifically to most solid surfaces resulting in the disruption of protein 3-D structure and eventually complete loss of activity. This indirectly gives rise to the second issue - the loss of protein conformation upon immobilisation. In particular, when the functional domains interact excessively with a solid surface, the orientation of the proteins may be altered or completely lost, resulting in the subsequent disruption of the functional domains and loss of discontinuous epitopes [66]. Partial or complete denaturation of proteins on the arrayed surface is also deleterious for downstream autoantibody binding since it is well known that antibodies tend to bind non-specifically to exposed hydrophobic epitopes, giving rise to false positive signals in autoantibody profiling assays.

rapid culture conditions, and are highly scalable. In addition, many parameters can be altered to optimise expression levels of protein. However, inefficient disulfide bond formation, insolubility, aggregation and poor folding of proteins have been reported using this method, as

Expression of proteins in yeast is a common alternative to prokaryotic expression systems as it is a well-defined and economical eukaryotic expression system. Commonly used yeast strains include *Saccharomyces cerevisiae* and *Pichia pastoris*, although other yeast strains have also been reported. Proteins expressed using both strains fold efficiently and numerous posttranslational modifications can occur; *P. pastoris* typically gives better protein yields than *S. cerevisiae* [65]. However, a major disadvantage of the yeast expression systems is that they do not mimic protein glycosylation patterns from mammalian cells, with proteins tending to be hyperglycosylated due to the presence of large mannose glycans. Furthermore, lysis conditions for yeast are typically harsh and induce many endogenous proteases, meaning that the

Baculoviruses belong to a diverse group of large double-stranded DNA viruses that infect many different species of insects as their natural hosts but are highly species-specific and are not known to propagate in any non-invertebrate host. Baculoviral expression systems yield good expression levels, especially for intracellular proteins, and typically produce functionally active, recombinant mammalian proteins that are properly folded and oligomerised and which contain correct disulfide bonds, as well as mammalian-like post-translational modifications, including glycosylation, so are both structurally and functionally similar to their native

Mammalian expression systems are preferred by some researchers as they produce more 'humanised' proteins, with the most biologically-relevant post-translational modifications and native folding. Amongst the most widely used mammalian cells include HeLa, human embryonic kidney-derived (HEK293) epithelial cells, Chinese hamster ovary cells (CHOs) and African green monkey kidney cells (COS). However, mammalian protein expression systems require more demanding culture conditions compared to other systems [65] so are signifi-

The microarray surface chemistry plays a critical role in determining protein microarray quality. Slide surfaces vary: aldehyde and epoxy-derivatized glass surfaces are used for random attachment through amines, whereas nitrocellulose, hydrogel or metal surfaces for attachment of affinity-purified proteins. An ideal surface chemistry should resist nonspecific adsorption,

Common challenges associated with slide surface chemistry include high background and incorrect protein orientation or conformation of proteins, whereby all functional binding sites are not readily available for interaction. Proteins have various hydrophobic domains and charged patches, so tend to adsorb non-specifically to most solid surfaces resulting in the disruption of protein 3-D structure and eventually complete loss of activity. This indirectly

well as very minimal capability in performing post-translational modifications [65].

extracted recombinant proteins are often significantly proteolysed.

cantly more challenging for high throughput expression purposes.

whilst preserving the folded structure of the arrayed proteins [1].

counterparts [65].

174 Autoantibodies and Cytokines

**5.2. Surface chemistry**

Proteins can be immobilised onto a microarray surface via encapsulation, surface adsorption, covalent attachment or affinity binding (**Figure 4**), which are further described below [67].

Encapsulation of a purified protein on a solid surface involves suspending the protein in a random orientation within a 3D gel pad (e.g. acrylamide or agarose) on an array surface; this approach provides a high capacity for immobilisation and thereby enhances the sensitivity of subsequent assays. A drawback of the technique, however, is that the size of the protein or other ligand applied may restrict diffusion into the gel, resulting in stronger signals at the periphery of the gel pad. This challenge may be surmounted when using different crosslinkers that can improve the porosity of the gel pads [68].

Immobilisation of purified proteins via noncovalent adsorption is a straightforward, reversible method that involves protein attachment onto a solid support through weak, non-specific interactions, including van der Waals hydrophobic interactions and electrostatic interactions. Commonly used surfaces here include nitrocellulose-coated and amine-terminated glass slides. Although this approach can provide high protein loading onto the surface, the orientation of the immobilised protein cannot be controlled, resulting in variable reaction efficiency, accuracy and reproducibility of the resultant arrays [69]. Furthermore, the underlying surfaces tend to be relatively denaturing towards the arrayed proteins [1].

Covalent attachment takes place by chemically cross-linking proteins to the surface through the nucleophilic residues lysine or cysteine. These residues are cross-linked to surface-bound ligands that are terminated with aldehyde, epoxy, or N-hydroxysuccinamide moieties. Irreversible immobilisation of a wide range of proteins to the carrier surfaces are feasible using covalent attachment, but the non-specific modification of surface residues on the arrayed protein carries the risk of altering the activity and folded structure of those proteins [70].

Affinity capture is a particularly advantageous way to immobilise proteins, since it circumvents many of the limitations of other approaches described above. Typical affinity capture methods include use of biotinylated, hexa-His-tagged, glutathione S transferase tagged or Halo tagged recombinant proteins [1], with orientation of the immobilised protein being controlled via the tag, thereby aiding in preserving the structure and function of the arrayed proteins.

Numerous human protein microarray platforms are available today for autoantibody research, including Immunome arrays (Sengenics, Singapore), Nucleic Acid Programmable Arrays (BioDesign Institute, Arizona), Human Protein Atlas Protein Fragment Arrays (SciLifeLab, Sweden), HuProt arrays (CDI Laboratories, USA) and ProtoArrays (ThermoFischer, USA). These various human protein microarray platforms have differing protein content and make

**5.3. Sensitivity and reproducibility**

Quantification of autoantibody biomarkers using a protein microarray starts with the production of recombinant proteins, printing of the proteins onto a solid support, probing them with serum or plasma samples and finally capturing interactions using fluorescent-labelled secondary antibodies. Protein microarrays have thus often been referred to as miniaturised version of ELISA. Miniaturisation allows a high overall sensitivity as analyte measurement is conducted while retaining the highest concentration per unit volume attainable for the given sample, with decreased reaction times due to short diffusion distances [71]. Furthermore, fluorescent-based signal detection in protein microarrays offers lower limits of detection (as low as 1 pg/mL; [72]) and greater dynamic range (up to 5 orders of magnitude; [73]) than colourimetric readouts in typical ELISAs. In addition to their greater sensitivity compared to ELISA, protein arrays are also superior in terms of multiplexing, as thousands of proteins can

Autoantibody-Based Diagnostic Biomarkers: Technological Approaches to Discovery and Validation

http://dx.doi.org/10.5772/intechopen.75200

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Given the capacity for multiplexing, as well as the high-throughput, low sample consumption, remarkable sensitivity and reproducibility of protein arrays, this platform is rapidly proving now to be very well suited to the challenges of autoantibody biomarker discovery. However, when choosing the optimal platform for discovery research, important factors such as the protein expression system used and the surface chemistry of the platform should be considered carefully to ensure that only biologically-meaningful autoantibody biomarkers

Microarrays fabricated with proteins derived from tissues or cell-line, or recombinant proteins have been used in many studies to identify potential autoantibody biomarkers for can-

**Figure 5.** Depiction of the slide surface chemistry of the immunome protein array. Individually-purified BCCP-tagged proteins are immobilised onto customised hydrogel-coated surfaces such that they retain folded structure and function

be printed onto glass slides in replicates and analysed simultaneously.

that have the potential to be translated into clinical use will be discovered.

**5.4. Protein microarray-based autoantibody discovery**

in an aqueous environment and behave as if they are in free solution.

cer, a few examples of which follow.

**Figure 4.** Various methods of protein immobilisation onto a solid support; encapsulation, surface adsorption, covalent cross-linking and affinity attachment.

different use of the various protein expression systems, surface chemistries and immobilisation strategies described above, all of which gives rise to differences in technical performance, as has been reviewed recently [1].

By way of example, proteins on the Immunome array are expressed in a baculoviral system as in-frame fusions to a biotin carboxyl carrier protein (BCCP) folding marker, that itself becomes biotinylated *in vivo* or *in vitro* only when the fusion protein is correctly folded. Immunome's surface chemistry is based on a hydrogel polymer that dramatically reduces non-specific background binding to the array surface whilst providing an aqueous-like environment for the arrayed proteins. The hydrogel matrix is derivatised with a low density of streptavidin molecules that are held away from the underlying array substrate, providing a selective surface for binding of biotinylated proteins (**Figure 5**). This helps to ensure that each protein immobilised on the array retains its native conformation, correctly folding and functionality on the array surface.
