The main objective is to demonstrate the feasibility of qualitative and semi-quantitative determination of anti-HIV antibodies, based on the allosteric response of an immobilized engineered Escherichia coli P-galactosidase. The suitability of this enzyme and the conditions for homogeneous assay had been previously demonstrated. In the present work, the operational conditions for engineered E. coli P-galactosidase immobilization on activated agarose supports have been determined. A solid biocatalyst has been prepared and characterized in order to determine the activation degree of the immobilized enzyme in the presence of anti-HIV antibodies. The solid-phase biocatalyst has been shown to be able to modulate its activity and produce an unequivocal signal in presence of anti-HIV containing sera from different patients. Finally, the conceptual design of a possible assay based on the above methodology is proposed. (c) 2007 Elsevier Inc. All rights reserved.