The quantification of proteins is an integral part of biopharmaceutical R&D. From the crude quantitation of hybridoma expression profiles of monoclonal antibodies, to quantitative determination of biologic drugs and protein biomarkers in clinical trials, ligand binding assays have become a research standard. Low equipment cost requirements, sensitivity, and the fact that samples do not usually require substantial cleanup are the principle advantages.
Ligand binding assays, however, can be time consuming, suffer from matrix effects, and be challenging to validate to current ICH/FDA guidelines. A reduction in analysis time, sample volume, and time to transfer and validate assays would provide a significant advantage to the progression of biopharmaceutical portfolios within the industry. We recently evaluated the Gyrolab® from Gyros, a semi-automated, low-volume assay system, for its ability to address these issues.
Instead of performing the assay in microtiter plates, Gyrolab system uses injection-molded microfluidic channels on the surface of a plastic disc, identical in dimensions to the standard compact disc (CD). Liquids are added to microstructures by a robotic microfluidic handling system. By a combination of capillary action, hydrophobic barriers, and centrifugal force (produced by spinning the disc), liquids are delivered to desired parts of each microstructure (Figure 1).
Each microstructure contains a Streptavidin-conjugated bead resin column, 15 nanoliters in volume. Step-wise addition of biotinylated capture reagent, samples, and then fluorophore-labeled detection reagent forms the classic sandwich assay. The fluorescent signal on each column is then read upon excitation by the red laser. Alexa Fluor® 647 (Invitrogen/Life Technologies) is commonly employed as the detection label, although other dyes with similar spectral properties can also be used, e.g., DyLight™ 649 (Thermo Fisher Scientific).
The use of such fluorophores facilitates the possibility of generating a sensitive assay using small sample volumes. There are three different discs that one can use to define different sample volumes applied to the affinity-capture columns in the micro-structures. Each disc contains sample definition chambers above the affinity column of either 20 nL, 200 nL, or 1,000 nL in volume.
Therefore, for sample analysis where the analyte of interest is present in high concentrations, i.e., 10 µg/mL or greater, the 20 nL CD should be used, to effectively shift the working range of the assay. For analysis of biomarkers, whose likely concentration in a sample is in the order of 50 pg/mL, the 1,000 nL CD is more effective, as it ensures a larger amount of sample is exposed to the affinity column.
Calibration standards, QC samples or unknown sample replicates are analyzed in individual microstructures. Eight microstructures are contained within each segment. They are connected to common reagent channels for the application of capture and detection reagents within the same segment. As a result, it is possible to run different assays on different segments of a disc (i.e., reagent screening applications).
As each microstructure is processed in parallel with all others on the disc, the time required to run an assay on 112 data points (capacity of both 20 nL and 200 nL CDs, 96 data points on the 1,000 nL CD) is just under one hour. This provides a time saving of at least two hours over conventional immunoassays. In addition, as the assay is automated, no further operator input is required once the disc and samples are loaded into the machine.