PROCESS FOR PREPARING THE ACID ENZYMATIC MARKER 3,6-DICHLORO-2-METHOXY-BENZOYL- GLYCYL-ALKALINE PHOSPHATASE ()

摘要

The invention relates to a process for preparing the acid enzymatic marker 3,6-dichloro-2-methoxy-benzoyl-glycyl-alkaline phosphatase meant to be used within the ELISA technique of pesticide analysis, by dissolving 20 mg of 3,6-dichloro-2-methoxy-benzoic acid, 10 mg of N-hydroxysuccinimide and 50 mg of 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide in 1 ml of dimethylformamide at the ambient temperature and stirring the mixture for 4 h, followed by the admixture of 10 mg of glycine in 2 ml of buffer of 50 mM sodium carbonate buffer, at a pH value of 9.6, while stirring the mixture for 24 h at a temperature of 5...10°C, then by centrifugation at 3000 xg in order to separate the precipitated dicamba-glycine which is washed with cold water and is dried, after which 5 mg of the dicamba-glycine derivative, 10 mg of N-hydroxysuccinimide and 40 mg of 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide in 1 ml of dimethylformamide are stirred for 4 h to activate the derivative carboxyl group, 100 μ l of active pesticide is admixed to 1 ml (5 mg/ml) of alkaline phosphatase solution in 50 mM carbonate buffer at pH 9.6, while stirring continuously for 24 h at a temperature of 4°C, the dicamba-glycyl-alkaline phospatase product being then purified by chromatography using Sephadex G25 column eluated with 10 mM phosphate buffer at a pH value of 7.5, the resulting purified marker being stored at a temperature of -25°C.