Carboxyl terminated SAMs were deposited onto gold substrates to immobilize the highly stable camel antibody (cAb) which is responsible for prostate specific antigen (PSA) recognition. ToF-SIMS was used in the static mode to follow the surface chemistry at the different stages of surface treatment. First, the thiol (11-mercaptoundecanoic acid) adsorption on the gold substrates was clearly identified by means of the deprotonated molecular fragment (C11H21O2S-) at m/z = 217. Then the activator of carboxylic groups (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, or EDC) was identified by several specific fragments (e.g. C3H8N+). Finally, the cAb immobilization at different initial solution concentrations was followed by ToF-SIMS, using the technique's molecular sensitivity. We identified some amino acids fragments clearly increasing with the solution concentration (e-9. C3H7O+, C8H10NO+). The cAb immobilization was also measured by the gradual disappearance of the coupling agent. This study is therefore an attempt to quantify the antibody immobilization by using carefully selected molecular ions, different from those emitted from the substrate and/or adventitious organic contaminants. (C) 2004 Elsevier B.V. All rights reserved.