**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 be printed onto glass slides in replicates and analysed simultaneously.

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

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

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,

**Figure 4.** Various methods of protein immobilisation onto a solid support; encapsulation, surface adsorption, covalent

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

as has been reviewed recently [1].

cross-linking and affinity attachment.

176 Autoantibodies and Cytokines

functionality on the array surface.

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 cancer, a few examples of which follow.

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

*Native protein microarrays*: Nam et al. demonstrated the feasibility of manufacturing a native protein microarray using chromatographic techniques and microarray printing technology. Here, a crude cell lysate was resolved in 2 dimensions using liquid-based isoelectric focusing followed by reverse-phase liquid chromatography, resulting in 1760 fractions which were then printed on a nitrocellulose surface and used to screen sera from cancer patients vs. healthy controls to identify fractions containing cancer-specific reactive autoantigens. Fractions corresponding to reactive spots were analysed using mass spectrometry to identify cancer-specific autoantigens, which revealed that 9/15 colon cancer patients, but neither of the healthy controls, produced autoantibodies against ubiquitin *C*-terminal hydrolase isozyme (UCH-L3). Autoantibody production against UCH-L3 was confirmed by Western blot in 19 of 43 (44%) additional colon cancer patients [74].

and 5 down-regulated in the case group. Identified autoantigens included Nuclear factor I subtype A (NFIA), Platelet-derived growth factor B (PDGFB) and Eukaryotic translation initia-

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

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

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Other protein microarray platforms, including nucleic acid programmable protein arrays (NAPPA), HuProt arrays, Protoarrays and Human Protein Atlas Protein Fragment Arrays, have also found utility in autoantibody biomarker discovery applications across a wide variety of disease areas, including a broad spectrum of cancers and autoimmune diseases, as well as several neurological disorders and inflammatory disorders, as recently reviewed else-

Biomarkers can be used for variety of purposes including disease prediction, diagnosis and treatment monitoring. However, while there are thousands of papers reporting discovery of potential biomarkers, very few of these have been validated and approved by the Food and Drug Administration (FDA) for clinical use (**Table 2**), despite preliminary reports of good sensitivity and specificity. This highlights the reality that biomarker validation is a challenging process with multiple criteria that need to be fulfilled before the markers can be approved use in clinical settings. There are also multiple stages where attrition can occur in the validation process, including poor study design, variations in sample collection, and the simple failure of the biomarkers in blinded validations, as dis-

A key requirement for all biomarker validation is that the biomarker demonstrates a correlation with specific pathophysiological processes or serves as a surrogate endpoint in a clinical trial. Diagnostic precision and accuracy are key technical parameters, since inaccurate or variable results, as well as false positive and false negative results, could lead to misdiagnosis that

Typical biomarker discovery programs are initially set up as case–control studies, with clearly defined and well-separated clinical groups. However, in real world settings, the diagnostic challenge is often not to distinguish diseased from healthy, but to differentiate amongst people with similar clinical symptoms but different underlying disorders. As a first step towards validation therefore, once candidate biomarkers have been identified from an initial discovery study, a scientifically sound and statistically-powered validation cohort needs to be designed to test the diagnostic power of the biomarkers in the context of 'diseased patients' and 'other disease' controls. Power calculations are used to determine the sample size required to identify reproducible, precise and accurate biomarkers that qualify for clinical utilisation and this cohort is then typically sub-divided into a training cohort and a larger blinded validation cohort. Typically, the clinical sensitivity and specificity of a larger set of candidate biomarkers from the discovery research is first assessed in the training cohort and the best performing markers that survive are taken forward for further evaluation in the blinded validation cohort.

tion factor 4A3 (elFA3) [78].

**6. Biomarker validation**

cussed further below:

could bring about unwanted sequelae.

where [1].

*Antibody microarrays*: in order to identify prostate cancer-associated autoantibodies, wellcharacterised monoclonal antibodies were arrayed onto nanoparticle slides to capture native antigens from prostate cancer cells, which were subsequently incubated with fluorescentlylabelled IgG from patients with prostate cancer and benign prostate hyperplasia (BPH). The study revealed that prostate cancer patients had higher autoantibody levels against TLN1, TARDBP, LEDGF, CALD1, and PARK7 when compared to patients with BPH. The study concluded that PSA alone produced sensitivity- and specificity-values of 12.2 and 80%, respectively, whereas the collective panel produced sensitivity- and specificity-values of 95 and 80%, respectively [75].

*Functional protein microarrays*: a cancer antigen microarray, comprising 123 full length, folded, recombinant tumour-associated antigens expressed in insect cells was used to identify autoantibodies that differentiate prostate cancer patients from benign prostatic hyperplasia (BPH) and other disease controls. The study identified 41 potential diagnostic/therapeutic antigen biomarkers for prostate cancer and found that autoantibody titres against GAGE1, ROPN1, SPANX1 and PRKCZ were high in prostate cancer patients, whereas autoantibody titres against MAGEB1 and PRKCZ were higher in BPH controls. Of the 41 potential antigens identified, FGFR2, COL6A1 and CALM1 were identified in urine from the same patients by shotgun proteomics [76].

Functional protein microarrays have also been used to identify autoantibodies against autoantigens in a number of other infectious or autoimmune-related diseases, including malaria and Parkinson's disease (PD). In malaria, *Plasmodium knowlesi* infection results in an autoimmune-like response in some individuals that has been hypothesised to play a protective role against malarial infection. Using the Sengenics Immunome protein array comprising 1636 correctly folded human antigens, 24 antigens with high reactivity to serum autoantibodies were identified, which may serve as potential biomarkers for asymptomatic malaria, mild malaria, or predictive biomarkers for severe malaria [77].

PD is a chronic and progressive neurodegenerative disorder, and a positive correlation is associated with *Helicobacter pylori* (*H. pylori*) and PD motor severity. The Sengenics Immunome protein array was used to screen *H. pylori*-seropositive PD patients and *H. pylori*-seronegative PD patients in a study that identified 13 significant autoantibodies, of which 8 were up-regulated and 5 down-regulated in the case group. Identified autoantigens included Nuclear factor I subtype A (NFIA), Platelet-derived growth factor B (PDGFB) and Eukaryotic translation initiation factor 4A3 (elFA3) [78].

Other protein microarray platforms, including nucleic acid programmable protein arrays (NAPPA), HuProt arrays, Protoarrays and Human Protein Atlas Protein Fragment Arrays, have also found utility in autoantibody biomarker discovery applications across a wide variety of disease areas, including a broad spectrum of cancers and autoimmune diseases, as well as several neurological disorders and inflammatory disorders, as recently reviewed elsewhere [1].
