SBIR-STTR Award

Solid-phase, enzyme-linked immunosorbent assays (ELISAs), such as the ELISA AIDS screening test, depend on the diffusion of macromolecules and organic substrates across a quiescent liquid boundary layer prior to adsorption to the solid phase or reactivity with solid-phase-bound macromolecules. Time-dependent diffusion often results in lengthy incubation times, or elevated concentrations of expensive or rare reagents to reduce incubation time. Stirring by vigorous microplate agitation is only marginally effective in improving diffusion, cannot be done while the enzyme-substrate reaction is read in fast kinetic applications, and presents potential biohazard and instrument malfunction problems. The goal of this research is to determine the efficacy of acoustic microstreaming for improving ELISA performance. The aims are: (1) to use an existing acoustic microstreaming prototype device to determine optimum conditions to enhance binding of ELISA immunoreactants to microplate wells, both at room and elevated temperatures; and (2) to determine the value of acoustically enhanced ELISA performance as measured by increased sensitivity, decreased incubation time, and decreased reagent expenditure.The long-term objective (Phase II) is to develop an acoustic microplate acoustic microstreaming device with elevated temperature incubator.

Anticipated Results:
As a result of Phase I, the Sonata Opus 1 Acoustic Diffusion Accelerator has been developed and is now offered for sale to research and diagnostic laboratories. New Opus-series instruments with elevated temperature incubation, mechanized transports, and capacity for multiple microplates, for use in stand-alone instruments and as components for microplate readers and washers, are planned.National Institute of Allergy And Infectious Diseases

In this Phase II application Dr. Boraker proposes to continue his studies enhancing ELISA performance using AM technology. The rate-limiting factor in solid-phase immunoassays (such as the enzyme-linked immunosorbent assay or ELISA) is diffusion. When a cylindrical polystyrene probe is immersed in the microwell contents and caused to oscillate to and fro in a transverse linear fashion, a stable pattern of 8 high-velocity vortices appears in the well (called "acoustic microstreaming," or); the rapid liquid motion produced by these vortices reduces diffusion layer thickness and thereby enhances transport of macromolecules onto the microwell surface. During the Phase I study, it was observed that the acoustic probe itself, although only l9 percent of the surface area, produced 60-90 percent of the signal of unstirred control wells. The investigators propose, therefore, to optimize acoustic probe ELISA by studying the independent and interdependent variables of probe shape, diameter, frequency and amplitude to maximize diffusion rate. They then propose to build l) a manual AM instrument capable of enhancing ELISA instrument which would process the equivalent of 5 microplates simultaneously, and 2) a prototype automated ELISA instrument which would process the equivalent of 5 microplates by performing all steps from sample incubation to chromophore development. Advantages of this innovation are: reduced reliance on individual well timing, simplified, rapid probe washing; "batch" processing in conjugate; retention of original sample; elimination of the "quenching" step; ease of servicing and cleaning, and reduced cost. Such an automated instrument could have wide appeal to diagnostic companies as world-wide demand for ELISA automation continues to drive the nonistopic immunoassay marketplace.

Thesaurus Terms:
biomedical equipment development, enzyme linked immunosorbent assay, nonclinical biomedical equipment, sound