The optimum number of chemiluminescent tags on the species to be detected is best determined empirically. Even though more labels should theoretically provide more photons, quenching effects can occur if labels are too closely spaced. The surface area and the number of derivatizable groups, typically amino or sulfhydryl groups, provide further limitations. In practice 10-20 labels, at most, can be incorporated onto a large molecule. Moreover, the more labels on the surface of a binding molecule, the more likely that the label will interfere with its binding properties.
Signal modulation can, in principle, be any of several types.
For example, the complement to the chemiluinescent labeled partner can be labeled with a quencher or a wavelength-shifting agent or a substance which affects the time course of emission. In practice, such chemiluminescent assays are very difficult to design. It is necessary that two critical components for the reaction be separately linked to the respective binding partners and that they interact to modulate the production of chemiluminescence only when the binding event occurs and not when the two labeled components are both physically present in the assay mixture in the unbound state. The discrimination between the two signal states must be at least 3-4 orders of magnitude. It is far simpler to design heterogeneous assays in which labeled binding pair complexes are separated from unbound labeled reactants.