Highly sensitive enzyme immunoassays for the detection of β-lactam antibiotics

Abstract

Specific and sensitive antibodies against β-lactam antibiotics are difficult to raise due to the chemical reactivity of the β-lactam ring. The antibiotic-protein conjugates used as the immunogen can easily react with primary amino groups as for instance the ε-amino group of lysine side chains in proteins. This leads to the degradation of the immunogen to a complex mixture and, therefore, to an unpredictable immune response of the host animal. We produced antisera against the hydrolyzed form of β-lactam antibiotics by immunizing rabbits with stable conjugates mimicking this form. Addition of penicillinase in the immunoassay leads to hydrolysis of the β-lactam antibiotic, which is recognized by the antiserum. In a competitive enzyme-immunoassay, benzylpenicillin could be detected at levels of 0.05 ng ml-1 and cloxacillin at levels of 0.1 ng ml-1 in pasteurized milk. The sensitivity and also the selectivity of these EIAs are remarkably high and offer a wide range of different applications. The procedure should be applicable for other β-lactam antibiotics, and may therefore in future play an important role in food quality control and assurance.