No abstract

SUMMARY.We developed an improved enzymatic assay of D-sorbitol in human erythrocytes by employing highly speci®c D-sorbitol dehydrogenase from Pseudomonas sp. (EC 1.1.1.14) and replacing perchloric acid (HClO 4 ) and potassium carbonate (K 2 CO 3 ), generally used for deproteinization, with sodium hydroxide (NaOH) and zinc sulphate (ZnSO 4 ). In this assay, erythrocytes were separated from plasma by centrifugation and washed once with physiological saline. Subsequently, the erythrocytes were lysed with distilled water and proteins precipitated with NaOH and ZnSO 4 . After centrifugation, the resulting colourless supernatant was mixed with a glycine buffer (pH 9´0) containing NAD + and D-sorbitol dehydrogenase. After incubation for 30 min at 37°C, the NADH produced was measured uorimetrically. The¯uorescence intensities were corrected for sample blanks, and the values of D-sorbitol were normalized for haemoglobin content.The method had an analytical range of 1±180 mmol/L. The intra-and inter-assay precisions were < 3´3% and < 5´8%, respectively. The detection limit was 0´65 mmol/ L. In terms of the linearity, precision and sensitivity, the improved method using NaOH and ZnSO 4 was superior to the conventional method using HClO 4 and K 2 CO 3 .

We designed a rapid, quantitative turbidimetric immunoassay for serum antistreptolysin-O (ASO), based on the immunoprecipitating velocity with purified streptolysin-O (SLO), which can be measured turbidimetrically at 340 nm. The assay system involves polyoxyethylene mono-p-nonylphenyl ether to accelerate and enhance the immunoprecipitation reaction. No antibody excess phenomenon is observed, up to 1,700 U/ml. Grossly icteric sera can be assayed. Patient sera gave acceptable results. Free cholesterol offers no interference. The standard curve is linear for values up to 800 U/ml. Results measured by the proposed method and those by the conventional microtitration method correlated well. Analytical recovery averaged 90.1% (80.3%-102.5%). Within-run and day-to-day CVs ranged from 2.1% to 3.5% and from 2.5% to 6.9%, respectively. Working SLO is stable for at least 15 days at 4 degrees C. The proposed method has proven to be superior to other available methods for measuring serum ASO.