Hygromycin B antibody production and characterization by a surface plasmon resonance biosensor

Sensitive and accurate methods are needed for the detection of hygromycin B antibiotic in fluids and tissues of farm animals. Sheep antisera were produced from hygromycin B-keyhole limpet hemocyanin and were screened with immunodiffusion, ELISA, and fluorescent latex assays. The antisera were evaluated with the BIAcore, a surface plasmon resonance biosensor, for their binding properties without using signal-generating labels. Hygromycin B was immobilized on the sensor chip, and the capture (binding) of the antibody resulted in a proportional increase in mass. Evaluation of the association (k(a)) and dissociation rate (k(d)) contants showed that one antibody had an affinity constant (k(a)/k(d)) of 1.64E+10. The binding capacities and antisera specificity were determined using a competitive binding of the added drug and hygromycin sensor, detecting hygromycin B from 2.5 ng/mL to 5 mg/mL. Neomycin, gentamicin, spectinomycin, dihydrostreptomycin, and streptomycin (1000 times above safe levels) had negligible binding with the antisera. The BIAcore analysis was more rapid and accurate than the immunochemical assays and allow rapid development of methods of hygromycin B analysis in biological samples.