3.3. Evaluation of Autoimmune Responses
In autoimmune diseases, antibodies, known as “auto-antibodies,” are often generated by the humoral immune system against self-proteins in response to many pathological processes. This kind of antibody follows a specific pathway to recognition by the immune system, including antigen over-expression, mutation, and/or altered PTM released from damaged tissues [18,19]. The presence of these autoantibodies is related to the development of certain diseases such as diabetes. Thus, they can be useful as diagnostic/prognostic biomarkers [20]. As diagnostics they have several key advantages: (i) they can be detected even before the appearance of clinical symptoms; (ii) even if the antigen that induced them is absent or present at very low amounts, the antibodies can be readily detected; (iii) they can be measured from easily obtained sources such as blood other body fluids; (iv) they are very stable in standardized collection vessels; and (v) they are straightforward to measure using many available chemistries.
In 2007 Anderson and colleagues employed NAPPA arrays for serological screening in breast cancer. After NAPPA design, they tested p53 together with other three negative control antigens (S100A7, p21 and ML-IAP) with positive and negative p53 sera confirming the expression for all the proteins printed and checking the detection of antibodies against p53. Moreover, they determined differences in p53-expression levels between healthy donors and breast cancer patients, and also within disease stages. In addition, they confirmed that many regions of the protein expressed were accessible on the arrays. To extend the study to autoantibody biomarker detection, they built a high density NAPPA array printing 1117 cancer related genes of which 539 were implicated on breast cancer and tested them against melanoma, ovarian and breast cancer sera [21]. Later, they increased the number of novel autoantibodies to be tested in breast cancer (around 4988 candidate antigens). Finally they identify 28 autoantibodies that could distinguish between benign breast disease and invasive cancer in a blinded study [22].
Recently, LaBaer et al. developed several NAPPA studies for juvenile idiopathic arthritis (JIA) [23] and type 1 diabetes (T1D). In both cases, serological autoantibodies (AAbs) from the disease were screened using a two-stage method. Firstly, more than 6000 unique proteins were displayed in NAPPA arrays which were incubated with 50 sera from T1D patients and 20 from controls allowed the elimination of uninformative antigens. In the second stage, 750 remaining genes were printed in duplicate and 26 proteins were identified as novel AAbs (TBCA, CDK4, CDK6, TBRG4, among others) with p < 0.005 [24]. For juvenile idiopathic arthritis, they assessed the levels of antibodies present in the systemic circulation and synovial joint of a small cohort of juvenile arthritis patients as a pilot study. Their results showed a strong correlation between the circulating antibody levels and those of the inflamed joint.
In 2009, Wong et al. adapted NAPPA technology to the Luminex suspension bead array platform to monitor the humoral immunity. To accomplish this, they expressed the proteins and captured them with the Luminex beads through anti-tag antibodies. After mixing the antigen-loaded beads, serum was added and human IgG was detected with standard secondary detection reagents. Protein arrays are a useful method for testing a moderate number of clinical samples against thousands of candidate proteins. The advantage of the Luminex approach is that it allows testing hundreds of clinical samples against a moderate number of candidate antigens, i.e., mesoscale. They concluded that detection of antibodies was highly reproducible and the specificity and limits of detection of the platform were comparable to standard ELISAs [25].
Recently, Henjes and Lourido have performed an analysis of auto-antibody profiles in osteoarthritis (OA) using comprehensive protein arrays concepts. In this work, NAPPA arrays and antigen arrays have been used to characterize differential autoantibody profiles in a set of 62 samples from OA, rheumatoid arthritis (RA), and healthy controls. An untargeted screen was performed on 3840 protein fragments spotted on planar antigen arrays, and 373 antigens were selected for validation on bead-based arrays. In the NAPPA approach, a targeted screening was performed on 80 preselected proteins. The autoantibody targeting CHST14 was validated by conventional ELISA assays in the same set of patients. Altogether, nine and seven disease-related autoantibody target candidates were identified, respectively, and this work demonstrated a combination of these two array concepts for biomarker discovery and their usefulness for characterizing disease-specific autoantibody profiles [26].