As a consequence o f environmental protection and legal restrictions, increasing efforts are made t o avoid radioactivity. One alternative is the labelling of ligands with chemiluminescent acridinium esters such as 2,6,-dimethyl-4-(N-succinimidyloxy-carbony1)phenyl 10-methylacridinium-9-carboxylate methosulphate (DMAE-NHS). When exposed t o hydrogen peroxide in a basic solution, the DMAE-moiety decays with emission o f a short-lasting chemiluminescent flash.
With the goal o f replacing the radioactive label in protein ligands with a DMAE label, and o f increasing the efficiency by using microtitre plate technology for DMAE detection, w e compared the receptor binding properties o f iodinated interleukin-la ('251-IL-1 a ) , interleukin-10 ('251-IL-1fJ) and interferon--y ('251-IFN-y) w i t h the corresponding DMAE-labelled ligands. The luminescence signal was assessed in a single-tube luminometer and in the prototype o f a chemiluminescent microtitre plate reader. Derivatization o f the three proteins with DMAE-N-hydroxy-succinimide resulted in photon yields o f up t o 100,000 counts per femtomole. As shown by Scatchard analysis, no significant loss o f receptor binding affinity was observed, which might have been expected as a consequence o f the chemical modification o f the proteins. The use of DMAE labelling o f proteins has the following advantages as compared t o iodination: (i) the coupling reaction and binding assay can be performed in a normal laboratory, (ii) since there is no radiolysis, the DMAE-labelled proteins remain stable, (iii) the detection sensitivity may be improved as a consequence o f higher specific activity o f the DMAE label. Thus, the method could be used t o replace the standard lZ5l label in receptor screening assays as well as other applications.