Friday, March 21, 2014

Using Raman Emission to Improve ELISA Reactions

The enzyme-linked immunosorbent assay (ELISA) is one of the most commonly used assay technologies in the biomedical field today. The ELISA format typically uses a microplate to quantitate an analyte in a liquid sample through its interaction with a specific antibody. The technology combines the specificity of antibodies with a standardized assay process and format that is very amenable to automation. Because of its popularity, efforts are continually being made to improve assay performance. Toward that end, Sword Diagnostics has developed a substrate for ELISA reactions that produces a Raman emission that can be used as a direct replacement substrate for commonly used peroxidase conjugates.

Energy Level Diagram of Raman and Fluorescence Signals.
Figure 1.  Energy Level Diagram of Raman and Fluorescence Signals.
Raman emission is a light scattering event where photons interact with the vibrational modes of a molecule’s electrons to gain or lose energy from the interaction and scatter at shifted frequencies (Figure 1). Unlike fluorescence there is no electronic transition and spontaneous emission of a photon. The Raman shift is dependent on the structure of the interacted target bonds and the environment of the bonds that the incident light interacted with. Resonance Raman is a special scattering event where the excitation wavelength is carefully tuned to be very close an electronic transition. Such overlap can result in scattering intensities which are increased significantly. The main difference between Raman scattering and fluorescence is the excited state lifetime. Fluorescence excited states are longer-lived than the 'virtual' states associated with Raman scattering.

Comparison of Raman and Absorbance signal IL-6 ELISA Dose Response Curves.
Figure 2.  Comparison of Raman and Absorbance signal IL-6 ELISA Dose Response Curves. 
The data obtained using an absorbance-based TMB substrate and a Raman-based Sword peroxidase substrate both generate sigmoidal shaped calibration curves that can effectively be described using either a 4- or 5-parameter logistic fit (Figure2). However, the dose response of the Raman based reagents is significantly shifted leftward to lower concentrations. This indicates that the Raman substrate is capable of detecting approximately five-fold lower sample concentrations as compared to the absorbance based substrate with no other changes to the assay.

Raman and resonance Raman scattering measurements are usually performed using an expensive dedicated tunable laser based instrument with detection primarily taking place in the IR portion of the spectrum. Sword peroxidase chemistry employs reagents that have emissions in the near IR spectrum that an appropriately configured Synergy H4 Hybrid Multi-Mode Microplate Reader from BioTek Instruments is capable of detecting with high sensitivity. This combination of chemistry and hardware provides an increase in ELISA sensitivity without the additional costs of a dedicated instrument.

By: BioTek Instruments, Paul Held, PhD., Laboratory Manager

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