Views
Section11:Instrumentation
From Assay Guidance Wiki
Contents |
Instrument Linearity and Performance
The instrument used to read the output of the immunoassay should be tested initially for both linearity and performance. Instrument performance should be regularly calibrated according to manufacturer’s specifications. The majority of plate readers employ UV-Vis Absorbance, fluorescence or chemiluminescence signals as the measured response, since the products of enzyme labels are chromophores, fluorophores or emit luminescent signals. Linearity in response of the specific enzyme product of an ELISA should be checked at the appropriate wavelengths and instrument settings.
Spectrophotometric/Colorimetric Plate Readers
Lamp sources and Photomultiplier tubes (PMT) vary in quality and performance in many plate readers. The linear range of many plate readers is generally between 0-2.5 Absorbance units (AU), but other instruments have a linear range up to 4.0 AU. A malfunctioning lamp source or photomultiplier tube can significantly affect the linear response range.
Fluorescence Plate Readers
These readers employ excitation and emission filter sets in addition to excitation lamp sources and photomultiplier tubes (PMT’s). In addition to the lamps and PMT’s, the filter sets also vary in quality, light throughput and bandwidth. Fluorescence signals are generally in Relative Fluorescence Units (RFUs) and linearity should be verified with appropriate filter sets for the fluorophores employed according to instrument specifications.
Chemiluminescence Readers
These instruments have sensitive photomultipliers to detect light emitted from a chemical reaction. No Lamp sources are necessary. These readers usually have a much larger dynamic range, thus allowing for the increase in sensitivity. Signals or responses are measured in Relative Light Units (RLU) and can be significantly different depending on their instrument design.
