Increasing the Sensitivity of Immunochromatographic Assay for the Determination of Penicillin in Milk by Oriented Immobilization of Penicillin-Binding Protein on the Colloidal Gold Surface

The sensitivity of competitive immunochromatographic assay (ICA) for the determination of penicillin in milk using colloidal gold (CG) conjugates with recombinant penicillin-binding protein (PBP) prepared by two methods, passive non-oriented immobilization and oriented immobilization with antibodies to the histidine tag, was compared. The detection limit of the optimized ICA with non-oriented PBP immobilization was 5 ng/mL. It was possible to achieve a detection limit of 0.3 ng/mL with visual interpretation due to antibodies to the histidine tag with the use of oriented PBP immobilization. The analysis time was 10 min. The efficiency of two PBP immobilization methods was compared. It was shown that the sensitivity of the assay increased due to an improved PBP orientation and a simultaneous decrease in the number of binding sites on the CG. Thus, targeted immobilization of specific proteins on sensor surfaces using antibodies to the histidine tag can increase the sensitivity of the assay and simultaneously decrease the amount of biospecific reagents used. This approach can be applied to the simple conjugation of recombinant proteins with a histidine tag to nanoparticles.