A bead-enzyme linked immunosorbent assay (bead-ELISA) for detection and quantification of cholera toxin (CT) in broth cultures of Vibrio cholerae O1 has been developed. Under optimal buffer and pH conditions the bead-ELISA could consistently detect 40 pg/ml of CT. None of the ingredients of commonly used media for in vitro culture of V. cholerae O1 hindered the performance of the bead-ELISA. Evaluation of the sensitivity and specificity of the bead-ELISA against the commonly used reversed passive latex agglutination (RPLA) test for detection of CT was performed using a collection of 239 strains of V. cholerae O1 (including both biotypes and serotypes) which were examined by a gene probe encoding for the A1 subunit of CT. Although both the assays were highly specific, the bead-ELISA was more sensitive than the RPLA. Quantification of CT by the bead-ELISA revealed that the concentration of CT produced by the strains of V.which were negative by the RPLA was lower than 1 ng/ml and therefore below the minimum detection ability of the RPLA. The bead-ELISA is a simple, specific and highly sensitive assay for routine detection of CT and is recommended for routine use in clinical microbiology laboratories.
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 .
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