Enzyme Immunoassays of Kanamycin Group Antibiotics with High Sensitivities Using Anti-Kanamycin as a Common Antiserum: Reasoning and Selection of a Heterologous Enzyme Label1

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Abstract
An antiserum against kanamycin (anti-KM) was elicited in rabbits immunized with a kanamycin immunogen prepared by a three-step procedure using N-(m-maleimido-benzoyloxy) succinimide as a cross-linker. KM and tobramycin (TOB) were labeled with β-D-galactosidase utilizing another cross-linker, N-(gamma-maleimidobutyryloxy)succinimid. The labeled KM showed very strong affinity to anti-KM antiserum and that of TOB had an adequate affinity to anti-KM. Increases in the assay sensitivities at the B/B0, value of 50% of KM and dibekacin were 183- and 191,000-times, respectively, on changing the enzyme label from KM to TOB. The optimal conditions for highly sensitive enzyme immunoassay (EIA) of KM using anti-KM and the enzyme labeled with TOB with satisfactory accuracy and precision were determined. Highly sensitive ETAs of four KM analogs with measurement ranges of 1 to 100 ng/tube were also developed using the labeled TOB and anti-KM as common reagents. Various commonly used drugs were found to have little reactivity in this immunoassay, indicating that the EIA is specific to KM and its analogs. The reasoning and the selection of TOB as the label are also discussed.

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