Abstract: A biological substance is determined with the use of a marker comprising a lanthanide or other metal ion coupled to the biological substance by means of chelate-forming compound. The chelate complex is dissociated by reduction of the ambient pH to a sufficiently low level to liberate the metal ion and the metal ion is then chelated with a separate chelating chromophore with an increase in the pH to a value closer to the optimum for determination of the metal-chelated chromophore by fluorescence spectroscopy, luminometry, colourometry or the like.