Modification of AlphaLISA Excitation Wavelength Leads to Improved Assay Sensitivity for Photosynthetic Tissue Samples.

Abstract

For the purposes of high-throughput immunoassay screening, PerkinElmer's AlphaLISA technology offers many benefits over traditional enzyme-linked immunosorbant assay (ELISA). However, its 680 nm excitation wavelength coincides with a wavelength of peak photosynthetic pigment absorbance, hindering the technology's utility within the plant biotechnology industry. In assays containing photosynthetic matrices, it is proposed that excitation of chlorophyll leads to the production of singlet oxygen, which initiates a pigment-associated background signal, reducing assay sensitivity. A customized donor bead, modified for excitation outside the range of photosystem absorbance, was tested for its capacity to improve assay sensitivity with extracts containing photosynthetic pigments. In three assays designed against crystalline domain insecticidal protein targets, use of the customized donor bead along with its altered excitation wavelength led to the elimination of pigment-associated signal and improved separation between target-positive and null samples. Reduction in null photosynthetic extract signal led to a 16× sensitivity improvement in a quantitative assay. The customized donor bead was also found to be photostable under ambient laboratory lighting, potentially improving the overall utility of AlphaLISA technology. The customized donor bead enables sensitive, high-throughput immunoassay screening of photosynthetic tissues within the plant biotechnology industry using a convenient, photostable protocol.