Enzyme Immunoassay of The Antibiotic Viomycin

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A novel crosslinking reagent N-(m-maleimidobenzoyloxy) succinimide (MBS) was introduced to enzyme immunoassay of peptide hormons such as angiotensin I, insulin and humanchorionic gonadotropin. Ten of the crosslinking reagents analogous to MBS were synthesized and examined by their stabilities and reactivities. The most stable pH of these reagents were approximately 6.0 and the best condition of their N-acylation was chosen as 30° for fourty minutes in phosphate buffer pH 7.5. For enzyme immunoassay of viomycin, specific antiserum to viomycinhad to be obtained. For this, the strategy of preparing viomycin protein conjugate was first the selective protection of?β-amino group of β-lysine residue in viomycin with acetyl group by three steps. The second process consist of hemisuccinylation of the N<SUP>θ</SUP>-amino group followed byamide bond formations of the introduced carboxylic acid of hemisuccinyl function with amine groups of bovine serum albumin (BSA) by mixed anhydride method. The obtained viomycin-BSA conjugate gave a single band on its sodium dodecylsulfate disk electrophoresis and from itselectophoretic distance, nine of viomycin residues were calculated to be introduced per moleculeof BSA. The serum obtained from femail rabbits injected with the conjugate was kept at -20° until used. Viomycin was coupled with β-D-galactosidase easily by using the crosslinking reagent MBS and the obtained conjugate retained the enzyme activity and immuno reactivity of their components. Competitive binding test between the conjugate and viomycin to the antiserum to viomycin resulted that viomycin could be detected from 100pg to 4ng.

Enzyme Immunoassay of The Antibiotic Viomycin

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