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JC polyomavirus (JCPyV) can cause progressive multifocal leukoencephalopathy (PML), a debilitating, often fatal brain disease in immunocompromised patients. JCPyV-seropositive multiple sclerosis (MS) patients treated with natalizumab have a 2- to 10-fold increased risk of developing PML. Therefore, JCPyV serology has been recommended for PML risk stratification. However, different antibody tests may not be equivalent. To study intra- and interlaboratory variability, sera from 398 healthy blood donors were compared in 4 independent enzyme-linked immunoassay (ELISA) measurements generating &gt;1,592 data points. Three data sets (Basel1, Basel2, and Basel3) used the same basic protocol but different JCPyV virus-like particle (VLP) preparations and introduced normalization to a reference serum. The data sets were also compared with an independent method using biotinylated VLPs (Helsinki1). VLP preadsorption reducing ≥35% activity was used to identify seropositive sera. The results indicated that Basel1, Basel2, Basel3, and Helsinki1 were similar regarding overall data distribution (P = 0.79) and seroprevalence (58.0, 54.5, 54.8, and 53.5%, respectively;P = 0.95). However, intra-assay intralaboratory comparison yielded 3.7% to 12% discordant results, most of which were close to the cutoff (0.080 &lt; optical density [OD] &lt; 0.250) according to Bland-Altman analysis. Introduction of normalization improved overall performance and reduced discordance. The interlaboratory interassay comparison between Basel3 and Helsinki1 revealed only 15 discordant results, 14 (93%) of which were close to the cutoff. Preadsorption identified specificities of 99.44% and 97.78% and sensitivities of 99.54% and 95.87% for Basel3 and Helsinki1, respectively. Thus, normalization to a preferably WHO-approved reference serum, duplicate testing, and preadsorption for samples around the cutoff may be necessary for reliable JCPyV serology and PML risk stratification.

Inter- and Intralaboratory Comparison of JC Polyomavirus Antibody Testing Using Two Different Virus-Like Particle-Based Assays