Novel applications of acridinium compounds and derivatives in homogeneous assays

A homogenous assay for detection or quantitation of an analyte in a sample, comprising the following steps:

• (a) conjugation of the analyte to a chemiluminescent acridinium compound selected from the group consisting of an acridinium ester with electron donating functional groups at the C2 and/or C7 position on the acridinum nucleus or an acridinium sulfonamide with or without electron donating functional groups at the C2 and/or C7 position on the acridinium nucleus;
• (b) adding a predetermined amount of the conjugate to the sample containing the unknown concentration of the analyte;
• (c) adding an antibody specific to the analyte to form a binding complex with either the analyte or its acridinium conjugate;
• (d) incubating the solution of the binding complex;
• (e) triggering the chemiluminescence of the binding complex reaction mixture by adding chemiluminescence triggering reagents in a pH range of about 6 to 10 to cause light emission;
• (f) measuring the amount of light emission with a luminometer;
• and
• (g) calculating the concentration of the analyte by comparing the amount of light emitted from the reaction mixture with a standard dose response curve which relates the amount of light emitted to a known concentration of the analyte.

A homogenous resonance energy transfer sandwich assay for the detection or quantitation of a macromolecular analyte comprising the following steps:

• (a) conjugation of an antibody specific for the macromolecular analyte with a chemiluminescent acridinium compound;
• (b) conjugation of a second antibody specific for the same macromolecular analyte with an acceptor molecule;
• (c) complexation of the conjugates with the macromolecular analyte in a sample to from a reaction mixture;
• (d) triggering the chemiluminescence of the binding complex reaction mixture by adding chemiluminescence triggering reagents in a pH range of about 6 to 10 wherein excited state energy is transferred from the chemiluminescent acridinium compound of the first conjugate to the acceptor molecule of the second conjugate, thereby causing light emission from the acceptor molecule or light attenuation;
• (e) measuring the amount of light with a luminometer;
• and
• (f) calculating the concentration of the macromolecular analyte by comparing the amount of light emitted from the reaction mixture with a standard dose response curve which relates the amount of light emitted to a known concentration of the macromolecular analyte.