Alkaline phosphatase capture test for the rapid identification of Escherichia coli and Shigella species based on a specific monoclonal antibody

Abstract

A specific monoclonal antibody for Escherichia coli and Shigella sp. alkaline phosphatase was used in an immunocapture assay and allowed identification of E. coli either in culture isolates or directly in clinical specimens. The assay was easy and required only four steps: (i) alkaline phosphatase was released within 10 min by using a gentle lysis procedure, (ii) cell lysates were transferred to antibody-coated tubes for 45 min, (iii) p-nitrophenyl phosphate substrate was added, and (iv) alkaline phosphatase activity was detected in a microsample spectrophotometer at 410 nm. This immunocapture assay was highly specific: only one false-positive reaction was observed with a Klebsiella pneumoniae lysate among the 205 non-E. coli strains tested. The assay was sensitive, detecting 10(7) CFU/ml from culture isolates or 10(5) CFU/ml from urine specimens which had first been grown in phosphate-limiting medium for 2 h. At these bacterial concentrations, the percentages of detected E. coli were high: 91% for blood cultures, 95.4% for culture isolates, and 96.8% for urine specimens.