A new homogeneous chemiluminescent immunoassay method featuring the use of specific binding members separately labeled with an acridan-based chemiluminescent compound and a peroxidase is reported. Formation of an immunocomplex brings the chemiluminescent compound and the peroxidase into close proximity. Without any separation steps, a chemiluminescent signal is generated upon addition of a trigger solution, and the intensity is directly correlated to the quantity of the analyte.
A new class of peroxidase substrates has been developed which
produces chemiluminescence upon
enzymatic oxidation. A wide variety of
N-alkylacridancarboxylic acid derivatives including
esters,
thioesters, and sulfonamides are efficiently oxidized by a peroxidase
and a peroxide to enzymatically
produce the corresponding chemiluminescent acridinium compound. In
conjunction with a
peroxidase enhancer, continuous light emission with high light
intensities and an extended duration
are produced. Alternately, an appropriately designed acridan
substrate produces a stable acridinium
ester intermediate which can be accumulated and the chemiluminescence
elicited as a burst of
light by raising the pH. The effects of leaving groups and
substitution on the acridan ring on the
mechanism of light production are discussed. Peroxidase-catalyzed
oxidation in the presence of a
peroxide permits the detection of enzyme with subattomolar sensitivity
and a broad linear dynamic
range.
We have designed and constructed an inexpensive imaging system based on charge-coupled device (CCD) technology and utilized it to demonstrate the sensitivity and rapid detection possible with Lumigen chemiluminescent reagents. We also report the development of two new chemiluminescent reagents, Lumi-Phos Plus and Lumigen PS. Lumi-Phos Plus is an enhanced formulation for the rapid detection of alkaline phosphatase on membranes and in solution. It provides excellent images in blotting applications with exposures of under a minute. Lumigen PS represents a new generation of peroxidase detection reagents. The wide dynamic range with excellent linearity, higher signal and lower background than other chemiluminescent reagents make Lumigen PS of unsurpassed value in enzyme-linked immunoassays and nucleic acid probe assays using HRP conjugates.
Simple cations of the alkali and alkaline‐earth metals have been shown to induce the reversible sequential transfer of two electrons at nearly the same potential in complexes (I) and their diamine Schiff base complexes like (III).
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