Design of acridinium-9-carboxamides and anti-acridinium antibodies for chemiluminescent signal enhancement

A novel system of signal enhancement is presented in which every labeled an tibody is capable of generating a signal. Three chemiluminescent acridinium -9-carboxamide haptens (1, 2, and 3) which incorporated differences in char ge and location of the linker were designed and synthesized. Antiacridinium polyclonal antibodies for each hapten were screened using surface plasmon resonance instrumentation to determine specificity for each hapten. Anti-ac ridinium 2 antibodies were found to be non-cross-reactive to acridinium 1. This property was exploited to design secondary antibody conjugates which w ould bind to primary antibodies labeled with 2 yet could still be labeled w ith the structurally similar acridinium 1. Consequently, both layers contri buted to the overall chemiluminescent signal. This format is an advance ove r other signal amplification formats which employ nonsignal-generating, lab eled antibodies to construct multilayered systems.