Fluoroimmunosensing system using antigens immobilized on cellulose–paraffin matrix for Brucella melitensis antibody assay

An immunosensing system for Brucella melitensis antibody (BrAb) assay which is useful for the diagnosis of brucellosis has been developed. To circumvent the difficulty of regeneration of immunocomposite surface, the natural material of microcellulose–paraffin was adopted, and it resulted in a considerable retention of the activity of the absorbed and encapsulated B. melitensis antigen (BrAg). The surface of the immunocomposite layer prepared was easily renewed by simple polishing. The renewed surface served as a platform for the competitive immuno-reaction of horseradish peroxidase (HRP)–BrAb and BrAb with BrAg bound to the surface. By using 3,3′,5,5′-tetramethylbenzidine (TMB) as the substrate, the amount of HRP–BrAb bound is quantified fluorimetrically, which is in turn related to the BrAb (analyte) content in samples. The amplification effect was obtained by using the enzymatic reaction, and an improved detection limit of 2.7 ng/ml was thus realized. The optimum analytical conditions such as pH, the amount of the labeled antibody and the flow rates of the substrate carrier solution were studied. The proposed method shows a pseudo linear response ranging from 2.7 to 90 ng/ml. The method has been employed to determine BrAb in serum samples.