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Jun 1, 2009 (Vol. 29, No. 11)

Novel Antibody Microarray Technology

Mass Spec Alternative Detects Biomarker Signatures in Tissues, Blood, and Cell Culture

  • Click Image To Enlarge +
    SQI Diagnostics' QuantiSpot Array can reportedly deliver up to 12 quantitative results in each of 76 patient wells.

    The current generation of antibody microarray technologies is providing highly sensitive new tools for screening proteins in complex biological samples. Offering an alternative to mass spectrometry (MS) and 2-D gel electrophoresis or chromatography, antibody-based affinity platforms have been developed as high-throughput approaches to the detection of disease-related biomarker signatures in tissues, blood, and cell culture. Last month in Stockholm, researchers revealed their latest microarray-related developments at Select Biosciences’ “Advances in Microarray Technology” meeting.

    Providing proof of the commercial viability of truly multiplexed antibody array technologies, SQI Diagnostics’ QuantiSpot™  Rheumatoid Arthritis Assay has been approved in Canada, is CE marked in Europe, and is currently progressing through FDA 510(k) certification. The assay has been developed as a fully automated, highly sensitive, and specific microarray-based fluorescence test for the simultaneous detection and quantification of up to four common rheumatoid arthritis (RA) antibody markers.

    SQI reports that the technology represents a major advance in RA diagnosis and monitoring, compared with standard, single-analyte immunoassay technologies and even the more recent advances in multiplexing. “Although multiplex technologies have been developed that can differentiate between rheumatoid factor (RF) and cyclic citrullinated peptide (CCP) antibodies, the systems we are aware of are only capable of demonstrating the presence of antibodies (over a threshold concentration), rather than their concentrations, and don’t provide truly quantitative data,” explained Andrew Morris, SQI’s CFO.

    “Clinicians today are interested not only in the presence of anti-RF and anti-CCP  but in the concentrations of these subclasses of antibodies, which are increasingly being used to help predict disease progression and make decisions about disease management,” he added.

    To address the need for simultaneous quantitation of multiple analytes in multiple samples, SQI’s immunoassay technology has been developed to provide semi-quantitative measurement of anti-CCP IgG, coupled with quantitative measurement of RF-IgA, RF-IgG, and RF-IgM, for up to 76 patients, on an industry-standard 96-well microarray assay plate.

    Carried out on the company’s SQiDworks™ platform, the printed CCP peptide and RF antigen microspots capture autoimmune antibodies present in RA patients, and each well includes capture, normalization, and control subarrays. The three internal assay calibrations comprise calibrated concentrations of purified human IgG, IgM, and IgA, to provide an internal dynamic reference standard.

    When variations occur, algorithms automatically normalize the capture signal to ensure a comparable signal response across equivalent test spots in different wells and on different plates. Normalized values are then translated to a standardized result.

    The technology is fully automated, including sample dilution, incubation, washing, reporter tagging incubation, and final plate conditioning prior to scanning and analysis. SQI maintains the overall process is 80% more efficient than current methods when taking into consideration the four multiplexed analytes. Capable of running up to three plates in a batch analyzing 12 analytes, with a run-time of 270 minutes, the process equates to a throughput of 10.1 patient results per minute.

    High signal-to-noise ratios are essential to enable diagnostic-grade precision and accuracy, Morris pointed out. This is in part enabled by high-precision spot printing that binds capture spots to glass substrates, to ensure reproducible arrays of test spots are generated for every well and every plate. This is carried out in the manufacturing process for the kits, which are built at SQI and shipped ready to go.

    “Our platform not only represents a fully automated analytical technology for quantitative measurement of antibodies in multiple patients, it importantly includes some 31 confidence tests in each patient well,” he stressed. “Typical ELISA plate technologies may calibrate the device measuring the luminosity signal just once a day. Our technology calibrates for every well, every patient, and every antibody. We essentially overwhelm the test for statistical significance.”

    SQI Diagnostics was established about 10 years ago, on the back of technology developed by SQI’s founder and CSO, Peter Lea, Ph.D., for capturing the signal from multiple analytes simultaneously. “The technology is all about generating the best signal-to-noise ratios and getting rid of as much of the background noise as possible. We focused initially on autoimmune diseases, as it was felt the multiplexing technology would be lower risk to develop,” Morris said.

    “Development of the RA assay involved optimizing the technology in terms of plate coating, spot detection and analysis, and automation. Now that this has been achieved, we expect development of further tests should be relatively rapid. Our development pipeline includes multiple antibody tests for celiac (6-plex), autoimmune thyroid (3-plex), antiphospholipid syndrome (9-plex), and Crohn’s disease (6-plex).”


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