High Sensitivity Assay Development on the LSA^®

Key Takeaways

• Enhancing the lower limit of detection (LOD) in the LSA extends the range of assay options available.
• Described here is an approach to enhancing LOD for a cytokine using commercially available reagents.
• LOD was improved from mid-range ng/ml down to single digit pg/ml using this approach.
• The concepts described here are readily adaptable to alternative reagents depending on assay needs.

Introduction

While SPR assays have the advantage of being able to detect binding interactions in a label free manner, this does not preclude the use of signal enhancing label strategies. The lower quantitation limit of surface plasmon resonance (SPR) assays is typically considered to be limited by the mass of the observed analyte and the affinity of the capture system. This limit is typically orders of magnitude less than classic immunological assays using labeled detection systems. When signal enhancement strategies are applied, SPR can achieve lower limit of detection (LOD) levels comparable to or more sensitive than conventional labelled techniques such as Enzyme-Linked Immunosorbent Assay (ELISA). In this example, a commercially available ELISA reagent kit for the measurement of human IL-6 was adapted for use on the Carterra® LSA® instrument. The assay performance exceeded the manufacturer’s reported ELISA sensitivity limit and the LSA allows for many of the assay steps to be automated. This assay utilized a horseradish peroxidase streptavidin conjugate (SA-HRP) and a precipitating substrate mix, 4-chloro-1- naphthol and 3,3′-diaminobenzidine tetrahydrochloride (CN/DAB) for detection. Other signal enhancement strategies are also possible, some which would even allow for regeneration to even further increase the automated throughput possible on the platform.

Posted by Daniel Bedinger, PhD