The hallmark of chronic diseases is the production of highly specific autoantibodies (1,2). These autoantibodies are increasingly measured to guide clinical decision making. The goal of these measurements is to define intracellular signal pathway activation profiles. These profiles can be integrated with clinical information to generate “biosignatures” for individual patients, the goal of which is personalized or precision medicine.
Comprehensive autoantibody profiling would revolutionize clinical practice at several levels. Enzyme-linked immunosorbent assays (ELISA) are extensively used in clinical diagnostics for detection of individual analytes in biological samples and are accepted as the “gold standard” against which other assays are measured. Conventional ELISA methods are limited however, as they only detect one target within a single reaction well. Confirming the presence of multiple biomarkers within a sample by conventional ELISA requires a multitude of independent tests, increasing time, cost and the possibility of error. A Microarray is essentially a miniaturized form of ELISA , and offers multiplexing capacity, significantly reducing procedure time, labor and sample quantity requirements, making microarrays the desired format for high-throughput data collection for personalized patient profiling.
Grace Bio-Labs ONCYTE porous nitrocellulose (PNC) offers significant advantages for protein binding over other surface chemistries and is recognized as the surface of choice for protein array applications (3,4,5). Grace’s ArrayCAM platform; a combination of ONCYTE PNC , and NIR fluorescent imaging of extremely bright and highly stable quantum nanocrystals , provides sensitivity and specificity equivalent to ELISA in an automation friendly format for comprehensive, high-throughput data collection.
We used PNC to demonstrate multiplex detection of two powerful diagnostic markers ( IL-6 and IL-1alpha) for inflammation and auto-immune diseases such as Lupus and Osteoarthritis and possibly metastatic cancer (6). Antibodies were spotted on PNC and challenged with a mixture of antigens. We selected antibody pairs for detection that did not cross-react. Detection was without signal amplification ( Table 1 ). Sensitivity and dynamic range of these non-amplified assays was equivalent to TSA-amplified conventional ELISA’s (Table 1, Fig.2). Enzymatic signal amplification may be used to enhance the sensitivity of detection on both assay platforms and is considered the standard method for conventional ELISA. Signal amplification has several disadvantages which include added cost, increased number of assay steps, greater assay time and the potential reduction in linear dynamic range. Table 1 compares the use of signal amplification on LOD and dynamic range.
Expansion of multiplex assays to detect multiple antigens in single microarray spots (level 3 multiplexing), cannot employ signal amplification. Thus, the use of high energy fluorescent tags like Q dots offer significant advantages for multiplex microarrays.
Table 1. Limit of detection and assay range for IL-6 using microarrays compared to conventional ELISA.
Figure 1. Microarray Assays for IL-6 and IL-1α
Figure 2. Multiplexed Microarray Assay for IL-6 and IL-1α
Figure 1. Microarray assay results using ONCYTE® NOVA PNC film slides for detection of either IL-6 or IL-1α from sample buffer spiked with recombinant antigens. Antibody pairs for the sandwich assays were purchased from R&D Systems (IL-6: Cat# MAB406/BAF406; IL-1α: Cat# MAB400/BAF400). Detection was performed after secondary labeling using streptavidin-conjugated QDot800 and imaging with the ArrayCAM microarray imaging system (Grace Bio-Labs). Sensitivity and assay range for both antigens are consistent to those reported for Quantikine ELISA Kits. Reported specifications for sensitivity and assay range are: 1.8 pg/ml and 7.8 – 500 pg/ml (IL-6, R&D Systems Cat# M6000B) and 2.5 pg/ml and 4.69 – 300 pg/ml (IL-1α, R&D Systems Cat# MLA00).
Figure 2. Multiplexed microarray assay results using ONCYTE® NOVA PNC film slides for simultaneous detection of IL-6 and IL-1α from sample buffer spiked with recombinant antigens. Please see Fig. 1 legend for antibody and assay details.
1. Werner, S., et. al (2014) Systematic review: Serum autoantibodies in the early detection of gastric cancer. Int. J. Cancer http://onlinelibrary.wiley.com/doi/10.1002/ijc.28807/abstract
2. Robinson, WH., et. al (2003) Protein arrays for autoantibody profiling and fine-specificity mapping. Proteomics 3,2007-2084 http://utzlab.stanford.edu/publications/robinson_proteomics_03.pdf
3. Liotta, LA et al (2003) Protein Microarrays: Meeting the analytical challenges for clinical applications., Cancer Cell: 2003 http://home.ccr.cancer.gov/ncifdaproteomics/pdf/cancer_cell_protein_arrays.pdf
4. Wagner, JP et al (2013) Receptor Tyrosine Kinases Fall into Distinct Classes Based on Their Inferred Signaling Networks, Science Signaling 6: 204 ra58 https://wolfyadlinlab.gs.washington.edu/drupal/sites/default/files/Wagner%2C%20Wolf-Yadlin%20et%20al.%20Sci%20Sign%202013.pdf
5. Gujral, TS et al (2013) Profiling Phospho-signaling Networks in Breast Cancer Using Reverse Phase Protein Arrays. Oncogene: July 18; 32(29): 3470-3476 http://pubmedcentralcanada.ca/pmcc/articles/PMC3670968/pdf/nihms443637.pdf
6. (2014) First Molecular Target for Triple-Negative Breast Cancer Identified. http://www.genengnews.com/gen-news-highlights/first-molecular-target-for-triple-negative-breast-cancer-identified