With a goal of making assays bigger, better, and faster, multiplexing is gaining momentum as it tackles challenges in the research and diagnostics arenas. The latter market is projected to reach $6 billion by 2013, according to Scienion. Cutting-edge approaches in this rapidly expanding field include new technologies for biomarker development, reducing bias among platforms, improving instrumentation for delivering tiny volumes, and enhancing the rigor of statistical data evaluation.
Some multiplexing assays seek to identify disease-specific biomarkers. This can be a slow and painful process, according to Niro Ramachandran, Ph.D., R&D manager, protein technologies, Life Technologies.
“The only way to speed up biomarker development is to standardize the workflow. It is critical to find out quickly if the lead you are pursuing is valid or not. We have developed a hybrid method for doing this that allows evaluation of more than 50 potential biomarker leads within a three-month period.”
According to Dr. Ramachandran, the company recently developed an antigen-based Luminex assay that complements its ProtoArray® microarray platform. “By coupling these two assays we are enabling everyday scientists to evaluate hundreds to thousands of proteins. Many researchers are familiar with antibody-coated Luminex beads, but a problem with antibody-coated beads is that they can cross react. We prepared beads in the opposite orientation, for example, with the protein targets of antibodies.”
The Luminex xMAP technology itself consists of color-coded beads in 500 distinct sets. Each bead set can be coated with a reagent specific for an assay that subsequently allows capture and detection of analytes within a sample. The ProtoArray is a high-density microarray that contains more than 9,000 unique human proteins spotted onto a 1 inch by 3 inch nitrocellulose-coated glass slide.
“We performed studies that demonstrated how the two technologies in combination (and accessing broad content) could be employed to develop immune response signatures in systemic lupus erythematosus (SLE) and mesothelioma patients.
“For the SLE study, serum samples of 40 SLE patients and 40 matched controls were evaluated on a ProtoArray. This yielded 200 possible markers that were further trimmed to 50 markers. We made proteins to the 50 markers and attached them to Luminex beads. We tested whether these markers were specific for SLE by also testing sera from other diseases such as multiple sclerosis and Crohn disease.
“In the end, we derived 20 markers that performed well and were specific for SLE. This is a great example of how to standardize workflow with high confidence results.”
Although it is possible to utilize several multiplexing platforms to develop and validate biomarkers, a critical factor is how much bias is contained within each platform. “You can lose a lot of time developing biomarkers on several platforms each of which has bias. This compounds the problems and may lead to false positives. We spent a lot of time evaluating this issue and determined that these two platforms nicely complement each other while greatly reducing bias.”
Protein microarrays can also be coated with capture antibodies. LightArray Biotech Research Center uses a 2,470 microarray printing system and can reportedly have up to 16 capture antibodies printed in each well of 96 or 384 plates with strict assay validation to ensure no cross reactivity. The company’s system employs a cool CCD imager and its SignaturePLUS and ProArray analysis software specially designed for chemiluminescent assays. “With this system we are able to provide the quantified protein concentration with a sensitivity averaging 0.1 pg/mL to 1 pg/mL.”