Saengjaruk Patcharin, Chaicumpa Wanpen, Watt George, Bunyaraksyotin Gaysorn, Wuthiekanun Vanaporn, Tapchaisri Pramuan, Sittinont Chuanpit, Panaphut Thanachai, Tomanakan Kanchana, Sakolvaree Yuwaporn, Chongsa-Nguan Manas, Mahakunkijcharoen Yuvadee, Kalambaheti Thareerat, Naigowit Pimjai, Wambangco Michael Angelo L, Kurazono Hisao, Hayashi Hideo
Department of Microbiology and Immunology, Mahidol University, Bangkok, Thailand.
J Clin Microbiol. 2002 Feb;40(2):480-9. doi: 10.1128/JCM.40.3.480-489.2002.
Hybridomas secreting specific monoclonal antibodies (MAb) to all members of the genus Leptospira (clone LF9) and those that are specific only to the pathogenic species (clones LD5 and LE1) were produced. MAb LF9, which was immunoglobulin G1 (IgG1), reacted to a 38-kDa component of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated whole-cell lysates of all Leptospira spp., while MAb LD5 and MAb LE1, which were IgG1 and IgG2a, respectively, reacted to the 35- to 36-kDa components of all serogroups of the pathogenic species of LEPTOSPIRA: The MAb LD5 was used in a dot blot-enzyme-linked immunosorbent assay (dot-ELISA) for detecting Leptospira antigen in urine samples serially collected from two groups of patients diagnosed with leptospirosis, i.e., 36 clinically diagnosed patients and 25 Leptospira culture confirmed patients. Their serum samples were tested serologically by IgM Dipstick assay, indirect immunofluorescence assay (IFA), and/or microscopic agglutination test (MAT). Urine samples of 26 patients diagnosed with other illnesses and 120 healthy individuals served as controls. For the first group of patients, who had been ill for an average of 3.4 days before hospitalization, the IgM Dipstick test, IFA, and MAT were positive for 69.4, 70.0, and 85.7% of patients, while the Leptospira antigenuria tested by the MAb-based dot-ELISA was positive for 75.0, 88.9, 97.2, 97.2, and 100% of patients on days 1, 2, 3, 7, and 14 of hospitalization, respectively. All but 1 of 11 patients whose serum samples collected on the first day of hospitalization were IgM seronegative, were positive by urine antigen test on day 1. This is strong evidence that detection of antigen in urine can provide diagnostic information that could be useful in directing early therapeutic intervention. The MAT was positive in 10 of 12 patients (83.3%) of the 25 culture-positive Leptospira patients who had been ill for an average of 5.04 days before hospitalization, and the Leptospira antigen was found in 64.0, 84.0, 96.0, 100, 100, 100, and 100% of the patients' urine samples collected on days 1, 2, 3, 4, 5, 6, and 7 of hospitalization, respectively. Leptospira antigenuria was found in 3 of the 26 patients diagnosed with other illnesses and 1 of the 120 healthy controls. The reasons for this positivity are discussed. The detection of antigen in urine by the monoclonal antibody-based dot-ELISA has high potential for rapid, sensitive, and specific diagnosis of leptospirosis at a low cost.
制备了分泌针对钩端螺旋体属所有成员的特异性单克隆抗体（MAb）的杂交瘤（克隆LF9）以及仅对致病菌种具有特异性的杂交瘤（克隆LD5和LE1）。MAb LF9为免疫球蛋白G1（IgG1），与十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离的所有钩端螺旋体属菌种全细胞裂解物的38-kDa成分发生反应，而MAb LD5和MAb LE1分别为IgG1和IgG2a，与钩端螺旋体致病菌种所有血清群的35至36-kDa成分发生反应。MAb LD5用于斑点印迹-酶联免疫吸附测定（斑点ELISA），以检测从两组诊断为钩端螺旋体病的患者（即36例临床诊断患者和25例钩端螺旋体培养确诊患者）连续采集的尿液样本中的钩端螺旋体抗原。通过IgM试纸条检测、间接免疫荧光测定（IFA）和/或显微镜凝集试验（MAT）对他们的血清样本进行血清学检测。26例诊断为其他疾病的患者和120名健康个体的尿液样本作为对照。对于第一组患者，他们在住院前平均患病3.4天，IgM试纸条检测、IFA和MAT的阳性率分别为69.4%、70.0%和85.7%，而基于MAb的斑点ELISA检测的钩端螺旋体抗原尿在住院第1、2、3、7和14天的阳性率分别为75.0%、88.9%、97.2%、97.2%和100%。住院第一天采集血清样本IgM血清学阴性的11例患者中，除1例之外，其余患者在第1天尿液抗原检测均为阳性。这有力地证明尿液中抗原检测可提供有助于指导早期治疗干预的诊断信息。25例培养阳性的钩端螺旋体患者中，平均住院前患病5.04天，12例患者中有10例（83.3%）MAT呈阳性，住院第1、2、3、4、5、6和7天采集的患者尿液样本中钩端螺旋体抗原的检出率分别为64.0%、84.0%、96.0%、100%、100%、100%和100%。26例诊断为其他疾病的患者中有3例以及120名健康对照中有1例发现钩端螺旋体抗原尿。讨论了这种阳性的原因。基于单克隆抗体的斑点ELISA检测尿液中的抗原在低成本快速、灵敏且特异性诊断钩端螺旋体病方面具有很高潜力。
