Enzyme-linked immunosorbent assay for the detection of fermentation metabolites: aminoglycoside antibiotics.

A heterogeneous microplate enzyme immunoassay with a sensitivity of 10 pg/ml (1 pg/assay) toward gentamicin has been developed for the detection of aminoglycoside antibiotics in fermentation broths. Purified gentamicin antibody was coated onto the surface of the wells of microtiter plates and incubated with gentamicin-alkaline phosphatase conjugate. The amount of enzyme bound to the antibody was quantified by measuring the change in absorbance at 410 nm after the addition of the substrate, p-nitrophenylphosphate. Competitive assays performed by incubating the antibody and enzyme conjugate with various aminoglycosides showed that the antibody probe cross-reacted with all aminoglycosides tested, except neomycins B and C. No cross-reaction was detected with non-aminoglycoside antibiotics. Complex fermentation broths did not interfere with this assay. Cultures known to produce aminoglycosides were detected at levels well below the concentrations required for antimicrobial activity. This technique is useful in the identification, quantification and screening of fermentation metabolites.