Sharma Rashmi, Tuteja Urmil, Khushiramani Rekha, Shukla Jyoti, Batra H V
Division of Microbiology, Defence Research and Development Establishment, Gwalior, India.
Hybridoma (Larchmt). 2008 Apr;27(2):113-21. doi: 10.1089/hyb.2007.0546.
Monoclonal antibodies (MAbs) were produced by fusing SP2/0 myeloma cells with spleen cells of BALB/c mice that were immunized with live whole cells of the four most prevalent Leptospira serovars--namely, autumnalis, australis, grippotyphosa, and icterohaemorrhagiae. A total of 26 MAbs (10 autumnalis, 5 australis, 4 grippotyphosa, and 7 icterohaemorrhagiae) were produced that showed specific, restricted, or broad cross-reactivity when tested with 19 standard pathogenic and 3 standard saprophytic serovars by MAT and dot-ELISA. Monoclonal antibodies like AT4 and AT5 against serovar autumnalis; AS1 and AS2 against serovar australis; GR1, GR3, and GR4 raised against serovar grippotyphosa; and also the MAbs IC3 to IC7 against serovar icterohaemorrhagiae were all usable as typing reagents in a rapid dot-ELISA. Selected MAbs were subsequently utilized in a rapid sandwich dot-ELISA for identification of Leptospira serovars as well as for antigen detection in experimentally infected mice and guinea pigs. Results of rapid sandwich dot-ELISA were compared with dark field microscopy, culture, and PCR in experimentally infected animals and sandwich dot-ELISA detected the presence of Leptospira antigen during the bacteremia stage in all experimental animals. Besides detecting antigens in animals infected with homologous serovars, the sandwich dot-ELISA employing pooled capture and revealing antibodies also detected Leptospira in the group of animals infected separately with the serovars australis and icterohaemorrhagiae. Results showed PCR to be a reliable and rapid test for demonstration of Leptospira in the plasma samples. The rapid sandwich dot-ELISA appeared more advantageous over PCR in being simple, rapid, field based, and economical. This method shows better promise of being used as a bedside test for routine diagnostic purposes.
通过将SP2/0骨髓瘤细胞与用四种最常见的钩端螺旋体血清型（即秋季热、澳洲型、爪哇型和出血性黄疸型）的活全细胞免疫的BALB/c小鼠的脾细胞融合，制备了单克隆抗体（MAb）。共产生了26种单克隆抗体（10种针对秋季热、5种针对澳洲型、4种针对爪哇型和7种针对出血性黄疸型），在用微量凝集试验（MAT）和斑点酶联免疫吸附测定（dot-ELISA）与19种标准致病血清型和3种标准腐生血清型进行测试时，显示出特异性、有限或广泛的交叉反应性。针对秋季热血清型的单克隆抗体如AT4和AT5；针对澳洲型血清型的AS1和AS2；针对爪哇型血清型产生的GR1、GR3和GR4；以及针对出血性黄疸型血清型的单克隆抗体IC3至IC7，均可在快速斑点酶联免疫吸附测定中用作分型试剂。随后，将选定的单克隆抗体用于快速夹心斑点酶联免疫吸附测定，以鉴定钩端螺旋体血清型，并用于检测实验感染小鼠和豚鼠中的抗原。将快速夹心斑点酶联免疫吸附测定的结果与暗视野显微镜检查、培养和聚合酶链反应（PCR）在实验感染动物中的结果进行比较，夹心斑点酶联免疫吸附测定在所有实验动物的菌血症阶段均检测到钩端螺旋体抗原的存在。除了检测感染同源血清型动物中的抗原外，采用混合捕获和显色抗体的夹心斑点酶联免疫吸附测定还在分别感染澳洲型和出血性黄疸型血清型的动物组中检测到了钩端螺旋体。结果表明，PCR是检测血浆样本中钩端螺旋体的可靠且快速的试验。快速夹心斑点酶联免疫吸附测定在简单、快速、基于现场和经济方面比PCR更具优势。该方法更有希望用作常规诊断目的的床边检测方法。
