Background:The StratifyJCV® test is a qualitative assay to classify MS patients as anti-JC virus (JCV) antibody positive or negative. Quantification of anti-JCV antibody levels in serum and cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients might add to the progressive multifocal leukoencephalopathy (PML) risk assessment.Objective:The objective of this study is to test sera of patients in a quantitative anti-JCV antibody assay, and to compare the results with preexisting data from the StratifyJCV® test.Methods:Sera of a total of 175 MS patients and matched non-MS-controls were tested for anti-JCV antibodies using glutathione S-transferase-tagged-VP1 as antigen. Antibody reactivity was quantified in arbitrary units using human immunoglobulin as standard.Results:The comparison of our assay with StratifyJCV® showed good inter-assay agreement (kappa 0.6), and strong correlation for antibody reactivity ( r²= 0.94). Discordant samples had low-reactive positivity, and a higher proportion (13% vs. 4%) tested positive in the StratifyJCV® test only.Conclusions:The method presented is a tool for the reliable quantification of anti-JCV antibodies, which demonstrates good agreement with results from StratifyJCV®. In contrast to StratifyJCV®, we pre-adsorbed all of the sera with BK virus (BKV) VP1 protein to reduce cross-reactivity. This step may account for a higher species-specificity of our assay. As such, our assay might be a promising additional tool for PML risk assessment.