Medical Microbiology Laboratory, Department of Microbiology, Centre of Excellence in Life Sciences, Bharathidasan University, Tiruchirappalli, TN, 620024, India.
Center for Translational Medicine, School of Medicine, Temple University, Philadelphia, PA, 19140, USA.
Sci Rep. 2017 Jun 12;7(1):3250. doi: 10.1038/s41598-017-03658-0.
Leptospirosis is underdiagnosed due to low sensitivity, need of specialised equipment, and expensive reagents for serological and molecular diagnosis respectively. Considering the sensitivity, rapidity, inexpensive reagents and collection of clinical samples, the monoclonal antibody based antigen detection method from urine samples has been developed and evaluated. LigA (LK90) based B-cell specific epitopes were predicted and synthesised as peptides for the production of monoclonal antibody. LK90: SNAQKNQGNA (amino acids: 543 to 552), and LK90: DHHTQSSYTP (amino acids: 1110 to 1119) with VaxiJen score of 1.3719 and 1.2215, respectively were used. Thirty two and 28 urine samples from confirmed and seronegative healthy human subjects, respectively were included for the evaluation of MAb-based dot blot ELISA. The specificity of the evaluated MAbs, P1B1 and P4W2 were found to be in the range of ~93-96%. Moreover, the MAbs did not show cross-reactivity with other bacterial antigens as confirmed by IgG ELISA, further validating its specificity for leptospiral antigens. These findings suggest that the developed MAb based dot blot ELISA is a simple, rapid performed in less than 8 h, inexpensive with a ICER of $8.7/QALY, and affordable in developing countries and area where laboratory facilities are limited.
钩端螺旋体病的诊断率较低，这是由于其敏感性低，分别需要专门的设备和昂贵的血清学和分子诊断试剂。考虑到敏感性、快速性、试剂成本低廉以及临床样本的采集，已经开发并评估了基于单克隆抗体的尿液抗原检测方法。针对尿液样本，预测并合成了针对 LigA（LK90）的 B 细胞特异性表位肽，用于生产单克隆抗体。LK90：SNAQKNQGNA（氨基酸：543 至 552）和 LK90：DHHTQSSYTP（氨基酸：1110 至 1119）的 VaxiJen 评分分别为 1.3719 和 1.2215。分别纳入了 32 份和 28 份来自确诊和血清阴性健康人类受试者的尿液样本，用于评估基于单克隆抗体的斑点印迹 ELISA。评估的单克隆抗体 P1B1 和 P4W2 的特异性在 93-96%左右。此外，通过 IgG ELISA 证实这些单克隆抗体与其他细菌抗原没有交叉反应，进一步验证了其对钩端螺旋体抗原的特异性。这些发现表明，开发的基于单克隆抗体的斑点印迹 ELISA 是一种简单、快速（在 8 小时内完成）、廉价（增量成本效益比为 8.7 美元/QALY）的方法，适用于发展中国家和实验室设施有限的地区。
