Lumiphore’s technology can be adapted for use in a wide variety of applications. The only modifications required are minor changes in the linker in order to optimize the labeling of a wide range of substrates.

Historically, diagnostic assays have relied on heterogeneous formats where at least one component is immobilized and analytical components are added in several stages with numerous washing steps integrated into the protocols. Often, these assays incorporate enzymatic tags such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) that are able to detect molecules at reasonably low concentrations because of the amplification of the signal conferred by the enzyme’s action on a chemiluminescent or colorimetric substrate. Unfortunately, these enzymes are mostly limited to the study of single molecular interactions and are not easily adaptable to multiplexing applications where one might be evaluating the presence of several molecules or interactions simultaneously. Also, heterogeneous assay formats are inherently slow due to limiting rates of diffusion between solution and solid phases.

Luminescent lanthanide complexes are ideally suited for homogeneous assay formats which are preferred for High Throughput Screening (HTS) applications. The small size, multiplexing potential, gated detection and favorable energy transfer properties of lanthanide complexes offer a high level of flexibility in assay design. In addition, the option to accumulate signal through multiple reads amplifies the signal relative to the background, further enhancing the limits of detection.

Fluorescence resonance energy transfer (FRET) between lanthanide donors and organic acceptors has been well studied. The degree of energy transfer between a donor and acceptor can be monitored by exciting the donor and observing emission of the acceptor. The extent of energy transfer (brightness of acceptor) correlates with the relative distance between the donor and acceptor.

A simple FRET experiment schematic is displayed in the figure below. The binding of two different antibodies to a molecule possessing complementary antigens in close proximity can be evaluated by monitoring only the acceptor fluorescence. The transfer only works when the donor and acceptor are in close proximity, so the acceptor emission serves as a measure of the interaction under investigation. If the haptens are monovalent, only the binding event involving the lanthanide is detectable unless the organic acceptor is separately excited. Even then, the lifetime of the acceptor’s fluorescence is short preventing the use of time gated detection and reducing sensitivity.

Appropriately positioning donors and acceptors on two potentially interacting molecules yields information about binding and offers numerous advantages over single label fluorescence assays. No wash steps are required and the long-lived fluorescence of the lanthanide is transferred from the donor to the acceptor eliminating background fluorescence.

Lumiphore’s luminescent reagents offer greatly increased sensitivity and specificity in the biological assay market.