Implementation of Experimental Design Techniques to Optimize Immunoglobulins Detection with Ultrasensitive Sandwich Immunoassays

The ultrasensitive measurement of protein markers plays a pivotal role in the early diagnosis of infectious and progressive diseases. Recently, digital methods such as those enabled by the Simoa Planar Array technology (SP-X System) have made significant progress in reaching ultrasensitive detection with clinically relevant protein biomarkers. The elicited Simoa technology is based on printing high-density capturing antibodies layers on the bottom of the wells of a microtiter plate, followed by a standard sandwich-type immunometric chemiluminescent detection. Such assay, reaching limit-of-detections (LODs) in the low femtomolar range, can be conveniently customized. An optimized Simoa SP-X assay for detecting and quantifying immunoglobulin M (IgM, non-specific indicator of inflammation) is developed herein and optimized. A full factorial experimental design is undertaken to optimize the assay, leading to a reduced experimental effort and increased quality of the information obtained concerning the traditional one-variable-at-a-time approach. The optimization process leads to an IgM LOD of 4 fm that compares well with those achieved with commercially available Simoa Planar Array kits. Remarkably, depositing both the capturing and detecting layer from a solution (0.1 μg mL−1) one order of magnitude less concentrated than in standard kits is needed, and the assay’s cost is sizably reduced.