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Luminophores are frequently utilized probe labels for detecting biological analytes. Multiple fluorescent luminophores, or fluorophores, can be readily distinguished from one another based on different energy excitation and emission wavelengths and lifetimes. However, suitable methods and reagents for distinguishing multiples of the much more sensitive chemically initiated luminophores have been limited. Herein we describe a new class of hybrid luminophore probes that emit light of distinct wavelength ranges and intensities upon energy transfer (ET) from an in-common, acridinium ester chemiluminophore to a covalently conjugated fluorophore. This format supports rapid, rational design of spectrally resolvable, chemically initiated probes. Time-resolved spectrographic and luminescence characterizations indicate that ET is not dependent on overlap in the emission spectrum of the luminophore and the absorption spectra of acceptors, suggesting a non-Förster resonance ET mechanism. Analysis of a combination of the chemiluminophore and new chemiluminophore-acceptor conjugate probes demonstrates the benefits of their use in sensitive, multiplex quantification of nucleic acid sequences indicative of environmentally relevant microbes without prior enzymatic amplification.

A novel method is described for simultaneous detection and quantification of attomoles or a few femtomoles of two (or potentially more) nucleic acid targets, without need for amplification. The technique depends on spectral-temporal resolution of chemiluminescence emitted from independent hybridization-induced chemiluminescent signal probes. The probes are internally quenched except in the presence of their specific targets, thereby allowing detection limits up to 10,000 times lower than with fluorescent probes. This is sufficient to obviate the need for amplification in many cases. The utility of the technique has been demonstrated by use of resolvable N-linked acridinium and 2,7-dimethoxyacridinium ester labeled probes in a homogeneous assay for sensitive and simultaneous independent quantification of pan-bacterial and pan-fungal target sequences in seawater.

Chemiluminescent acridinium esters (AEs) permit the development of high sensitivity ligand binding assays due to a combination of high intensity light emission and very low backgrounds. Here these advantages are exploited for use in homogeneous nucleic acid hybridisation assays using quenched chemiluminescence. AE chemiluminescence is conventionally initiated at highly alkaline pH. Novel "active" AEs were designed that permit initiation under conditions compatible with maintenance of nucleic acid hybrids (i.e. pH less than 9). Methyl red was found to be a dark quencher species capable of functioning at this pH. Practical application of the chemiluminescence quenching assay system has been demonstrated using two model nucleic acid hybridisation assays based on intra- and intermolecular emitter/quencher pairs.

Chemiluminescence immunoassays have now achieved a recognized place in the diagnostic laboratory. The advantages of this non-isotopic technology derive from the use of acridinium esters which can be used to label antigens and antibodies to high specific activities, as well as from optimized immunochemistry. The availability of simple, reliable instrumentation for chemiluminescence measurement together with a range of assay kits offers a logical alternative to traditional radioimmunoassay.

A chemiluminescent acridinium ester has been synthesized that reacts spontaneously with proteins to yield stable, immunoreactive derivatives of high specific activity. The compound has been used to prepare chemiluminescent monoclonal antibodies to human alpha 1-fetoprotein having average incorporation ratios as great as 2.8 mol of label per mole of antibody, which corresponds to a detection limit of approximately 8 X 10(-19) mol. These antibodies have been used in the preliminary development of a two-site immunochemiluminometric assay for human alpha 1-fetoprotein, which requires only a 30-min incubation and a quantification time of 5 s per sample.

A sensitive chemiluminescence based immunoassay is described for measuring antibody to staphylococcal peptidoglycan in blood and dialysates from patients undergoing continuous ambulatory peritoneal dialysis (CAPD). Peptidoglycan was isolated from a strain of S. epidermidis obtained from the dialysate of a CAPD patient with peritonitis and after sonication used to coat polystyrene beads. The coated beads were incubated with standard or sample and bound IgG was detected by the addition of affinity-purified goat anti-human IgG labelled with acridinium ester. After a wash stage 0.1 M nitric acid containing 0.1% hydrogen peroxide was added to the beads. Subsequently the chemiluminescence produced following the addition of 0.3 M sodium hydroxide was measured over a 2 s time interval with an automatic luminescence analyser. Using this technique the optimum dilution of serum for detecting antibodies to peptidoglycan was found to be 1/800 and for overnight effluent from CAPD patients the dilution was 1/8. Initial values of serum and dialysate antibody levels from 34 subjects are presented. This method has the advantage that it will detect concentrations of anti-peptidoglycan which are less than 1% of those in sera, the reagents remain stable for long periods and large numbers of samples can be processed on the same day.

A 2-site immunochemiluminometric assay is described for the measurement of complement component C9 concentrations in biological fluids as an aid in the diagnosis and management of immune-based diseases. The assay utilises 2 monoclonal antibodies one of which is labelled with a chemiluminescent acridinium ester and one of which is covalently coupled to reprecipitated aminoaryl cellulose. The incubation time is 1 h with simultaneous reagent addition. The working range of the assay is 10-2500 micrograms/1 at CVs less than or equal to 10% and the results are in excellent agreement with those of an immunoradiometric assay. The assay exhibits superior performance to other immunochemical and immunoassay techniques and has the advantage of using stable, non-radioactive reagents.