Husson M O, Mielcarek C, Izard D, Leclerc H
Laboratoire de Bactériologie, Faculté de Médecine, Lille, France.
J Clin Microbiol. 1989 Jul;27(7):1518-21. doi: 10.1128/jcm.27.7.1518-1521.1989.
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.
一种针对大肠杆菌和志贺氏菌属碱性磷酸酶的特异性单克隆抗体被用于免疫捕获测定，该测定可用于鉴定培养分离物中的大肠杆菌或直接在临床标本中鉴定大肠杆菌。该测定方法简便，仅需四个步骤：（i）通过温和的裂解程序在10分钟内释放碱性磷酸酶；（ii）将细胞裂解物转移至包被抗体的试管中45分钟；（iii）加入对硝基苯磷酸底物；（iv）在410nm处用微量样品分光光度计检测碱性磷酸酶活性。这种免疫捕获测定具有高度特异性：在测试的205株非大肠杆菌菌株中，仅观察到肺炎克雷伯菌裂解物有一次假阳性反应。该测定方法灵敏，可从培养分离物中检测到10⁷CFU/ml，或从首先在限磷培养基中培养2小时的尿液标本中检测到10⁵CFU/ml。在这些细菌浓度下，检测到的大肠杆菌百分比很高：血培养为91%，培养分离物为95.4%，尿液标本为96.8%。
