Validation of a Microsphere Immunoassay for Serological Leptospirosis Diagnosis in Human Serum by Comparison to the Current Gold Standard

A microsphere immunoassay (MIA) utilising Luminex xMap technology that is capable of determining leptospirosis IgG and IgM independently was developed. The MIA was validated using 200 human samples submitted for routine leptospirosis serology testing. The traditional microscopic agglutination (MAT) method (now 100 years old) suffers from a significant range of technical problems including a dependence on antisera which is difficult to source and produce, false positive reactions due to auto-agglutination and an inability to differentiate between IgG and IgM antibodies. A comparative validation method of the MIA against the MAT was performed and used to determine the ability of the MIA to detect leptospiral antibodies when compared with the MAT. The assay was able to determine samples in the reactive, equivocal and non-reactive ranges when compared to the MAT and was able to differentiate leptospiral IgG antibodies from leptospiral IgM antibodies. The MIA is more sensitive than the MAT and in true infections was able to detect low levels of antibody in the later stages of the acute phase as well as detect higher levels of IgM antibody earlier in the immune phase of the infection. The relatively low cost, high throughput platform and significantly reduced dependency on large volumes of rabbit antisera make this assay worthy of consideration for any microbiological assay that currently uses agglutination assays.

本研究开发了一种基于Luminex xMap技术的微球免疫测定法（microsphere immunoassay, MIA），可独立检测钩端螺旋体病IgG与IgM抗体。该微球免疫测定法采用200份用于常规钩端螺旋体病血清学检测的人类样本完成验证。拥有百年历史的传统显微镜凝集试验（microscopic agglutination test, MAT）存在一系列显著技术缺陷：依赖难以获取与制备的抗血清、因自动凝集引发假阳性反应，且无法区分IgG与IgM抗体。本研究通过将MIA与MAT进行对比验证，以评估MIA相较于MAT检测钩端螺旋体抗体的性能。结果表明，该检测方法可区分反应性、可疑性与非反应性样本，且能够有效区分钩端螺旋体IgG与IgM抗体。相较于MAT，MIA的灵敏度更高：在真实感染病例中，其可在急性期后期检测到低水平抗体，同时在感染免疫阶段早期检测到更高水平的IgM抗体。该检测平台成本相对较低、通量较高，且大幅降低了对大量兔抗血清的依赖，因此对于当前采用凝集试验的各类微生物学检测而言，该方法具备极高的应用考量价值。