ELISA Test Transfer
Serum NSE levels can be measured with commercial ELISA kits. Though quite sensitive, ELISA is time consuming and best suited for a large number of samples. Although a good research tool, ELISA lacks the reproducibility required of a clinical test and because of the needs for a calibration curve for each sample, it is considered too expensive for clinical care.
For this research study, Axela scientists applied the principles of the ELISA method to develop a dotLab NSE assay and improve the throughput characteristics and robustness of the assay. Based on the NSE ELISA, the Axela dotLab System demonstrated its use as a rapid alternative to ELISA analysis.
Standard ELISA techniques use a pair of monoclonal anti-NSE antibodies that recognize different nonoverlapping epitopes on the antigen. One of the antibodies is designated as a capture antibody and labeled with biotin residues for immobilization onto avidin-coated ELISA plates. NSE from samples binds to the capture antibody on the plate. HRP-tagged detector antibodies are used in the next step. The signal is visualized by appropriate enzymatic reaction and detected by spectrophotometer.
In the dotLab System, affinity reagents (e.g., avidin or amine reactive substrates) are prepatterned on the surface of the inexpensive, disposable dotLab Sensor. For the dotLab NSE assay, biotinylated monoclonal anti-NSE capture antibody (CAb) binds strongly to the avidin surface of the dotLab Sensors that create the diffraction grating. An HRP-conjugated detector monoclonal anti-NSE antibody (DAb) is preincubated with NSE to form a detection complex. The resulting complex binds to the capture antibody on the surface of the sensor through the antigen, as the CAb recognizes a nonoverlapping epitope of the antigen. (Figure 1).
The dotLab System automatically delivers samples and reagents from a standard 96-well plate or bulk containers. Complex matrices including blood and plasma can normally be used directly without labeling or purification. The patterned capture molecules on the dotLab Sensor interact with sample in a 10 µL flow channel.
Diffractive optics technology (dot®) quantitatively measures the change in diffraction as sample molecules bind to the sensor surface in real-time (Figure 2). As sample molecules bind to the sensor surface, diffraction efficiency improves in proportion to size and concentration of the target. A prism molded into the base of the disposable sensor allows operation in total internal reflection mode, effectively eliminating interference and the need for a separate reference channel. Binding is determined quantitatively by monitoring the change in intensity of the diffractive signal.