Co-immobilization of cholesterol esterase, cholesterol oxidase and peroxidase onto alkylamine glass beads for measurement of total cholesterol in serum

Abstract

Commercially available cholesterol esterase, cholesterol oxidase and peroxidase have been co-immobilized onto alkylamine glass beads (pore diameter 55 nm) through glutaraldehyde coupling with a conjugation yield of 2.3 mg/g of support and 76% retention of initial activity. The co-immobilized enzyme system showed maximum activity at pH 7.0, when incubated at 37 °C for 12 min. A method was developed for total serum cholesterol determination employing co-immobilized enzymes. There was a linear relationship between A520 and cholesteryl acetate concentration ranging from 5 mg to 50 mg/dl reaction mixture. The minimum detection limit of the method is 50 mg/dl. Within day and between day coefficient of variation were <1.0% and <6%, respectively. A good correlation (r=0.83) was found between the total serum cholesterol obtained by the present method and commercial Enzo-kit method employing free enzymes. Among the various serum substances tested at their physiological concentrations; Testosterone, vit D and progesterone caused 59%, 41% and 39% inhibition, while NaCl, KCl, CuSO4, creatinine, NaHCO3, albumin and estrogen had practically no effect.