Highly Sensitive Enzyme Immunoassays for the Detection of β-Lactam Antibiotics

Specific and sensitive antibodies against g -lactam antibiotics are difficult to raise due to the chemical reactivity of the g -lactam ring. The antibiotic-protein conjugates used as the immunogen can easily react with primary amino groups as for instance the k -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 g -lactam antibiotics by immunizing rabbits with stable conjugates mimicking this form. Addition of penicillinase in the immunoassay leads to hydrolysis of the g -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 g; -lactam antibiotics, and may therefore in future play an important role in food quality control and assurance.