Multi-site analytical validation of an assay to detect anti-JCV antibodies in human serum and plasma

doi:10.1016/j.jcv.2011.10.003

Journal of Clinical Virology

Volume 53, Issue 1, January 2012, Pages 65-71

https://doi.org/10.1016/j.jcv.2011.10.003 Get rights and content

Abstract

Background

JC virus (JCV) infection is a prerequisite for development of progressive multifocal leukoencephalopathy (PML). We previously described the development of a novel, two-step enzyme-linked immunosorbent assay (ELISA) that detects anti-JCV antibodies in human serum or plasma, and the potential clinical utility of anti-JCV antibody status for PML risk stratification.

Objectives

To validate the anti-JCV antibody ELISA at multiple clinical laboratories in order to demonstrate the robustness of the method.

Study design

Analytical validation of the ELISA was performed at four laboratories by evaluation of intra- and inter-assay precision, analytical specificity and sensitivity, matrix interference, robustness, sample and reagent stability.

Results

Analytical validation demonstrated that the assay is sensitive, specific, and precise. The assay sensitivity was estimated at 1.7 ng/mL using a humanized anti-JCV monoclonal antibody control. The sensitivity to detect JCV infection was estimated to be 97.5%. The specificity of the assay to discriminate JCV-specific antibodies from antibodies directed to BK virus, a related polyomavirus, was also demonstrated. The inter- and intra-assay precision was ≤6.0% and 9.8% for the screening step and 2.6% and 11.3% for the confirmation step. Results obtained for plasma and serum were highly congruent, and assay robustness was demonstrated by the highly concordant results generated by four laboratories testing a common panel of 100 blinded samples.

Conclusions

The novel, two-step ELISA to detect anti-JCV antibodies in human serum and plasma is robust, and assay performance is consistent and reproducible in multiple laboratories, making it suitable to support testing for global clinical studies.

Section snippets

Background

Progressive multifocal leukoencephalopathy (PML) is a rare demyelinating disease of the central nervous system associated with certain immunomodulatory agents such as efalizumab, natalizumab, and rituximab.¹ While multiple factors contribute to PML development, infection with JC virus (JCV), a common polyomavirus, is a known prerequisite.² Therefore, evaluating infection by JCV may help elucidate the risk of PML. We earlier reported the development of a novel enzyme-linked immunosorbent assay

Objectives

The goal of this study was to analytically validate the anti-JCV antibody assay at four clinical laboratories to demonstrate the robustness and other analytical parameters to support sample evaluation.

Samples

Serum and plasma samples from healthy volunteers and multiple sclerosis (MS) patients were purchased from Bioreclamation, Inc. (Hicksville, NY). Additionally, serum and urine samples from MS patients enrolled in the open-label, single-arm, multinational Safety of TYSABRI^® Redosing and Treatment (STRATA) studies 101MS321 and 101MS322 were used to establish assay cut points and assess analytical specificity and diagnostic sensitivity of the assay.

Anti-JCV antibody assay

The two-step anti-JCV antibody assay was performed

Assay cut points and diagnostic sensitivity

Assay cut points, seropositivity, and sensitivity (analytical false-negative rate) were statistically determined by evaluating serum and urine samples from >800 natalizumab-treated MS patients from the STRATA study. The anti-JCV seropositivity rate in the STRATA population was estimated as 53.6%. The sensitivity of the assay to identify those infected with JCV was estimated to be 97.5% (1–2.5% analytical false-negative rate) based on the percentage of serum samples from urinary JCV DNA positive

Discussion

Our findings demonstrate the successful analytical validation of a novel, 2-step assay to detect JCV-specific antibodies in human serum and plasma across four independent laboratories. The assay has been previously shown to have high sensitivity to detect JCV-specific antibodies on the basis of the low false-negative rate of 2.5%.³ In this manuscript, the specificity of the assay to detect anti-JCV antibodies, as well as excellent precision and robustness in four different laboratories has been

Funding

Funding for the assay validation study was provided by Biogen Idec Inc.

Competing interest

Tatiana Plavina is currently employed by and possesses shares of Biogen Idec. Melissa Berman, Moses Njenga, and Mary Crossman are currently employed by Biogen Idec. Michaela Lerner was employed by and possesses shares of Biogen Idec. Leonid Gorelik and Kenneth Simon are currently employed by and possess shares of Biogen Idec. Brian Schlain is currently employed by Biogen Idec. Meena Subramanyam is currently employed by and possesses shares of Biogen Idec.

Ethical approval

Not applicable

Acknowledgements

The authors thank laboratory staff at Focus Diagnostics, Quintiles Laboratories and Unilabs for performing validation experiments, and Dr. Geoffrey Kuesters for assistance with characterization experiments. The authors also thank Drs. Paula Hochman and Bill Aschenbach for scientific discussions and valuable suggestions for the manuscript. Copyediting assistance was provided by Infusion Communications and funded by Biogen Idec Inc.

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Anti-JC virus antibodies: implications for PML risk stratification
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Patients are closely monitored clinically and by serial MRI. Recent data suggest that JCV seroconversion or a rising JCV index may predate the development of PML,54 but further confirmatory studies are required. JCV antibody testing has not been examined in patients with rheumatic disease and is unlikely to be cost-effective in this population with a much lower incidence of PML.
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The plates were dried at 37 °C for 3.0–3.5 h and then stored with desiccant at 2–8 °C. Surface chemistry and other components were evaluated using assay controls from the original two-step JCV antibody ELISA and a panel of patient serum samples with varying normalized optical density (nOD) values.3,4 Signal-to-noise ratios were used to select optimal components and conditions.
JC virus (JCV) infection is a prerequisite for development of progressive multifocal leukoencephalopathy (PML). The development and validation of a two-step enzyme-linked immunosorbent assay (ELISA) that detects JCV antibodies in human serum or plasma and its clinical utility for stratification of PML risk have been described.
To develop a second-generation JCV antibody ELISA kit with improved assay performance characteristics.
The assay design was optimized by pre-coating the JC virus-like particles (VLP) on microtiter plates. Assay cut-points were statistically established using sera from >1300 multiple sclerosis patients from natalizumab clinical studies. The assay was analytically validated and then used to determine the presence of JCV antibodies in both treatment-naïve and natalizumab-treated MS patients, as well as in natalizumab-treated PML patients.
An improved assay for detection of JCV antibodies in human serum and plasma was developed. Key enhancements included improved delineation and reproducibility of low JCV antibody responses and assay ease of use. The assay was validated, demonstrating good agreement with the original two-step JCV antibody ELISA, and similar seroprevalence of 50%–60%. Samples from 63 natalizumab-treated PML patients collected 6–180 months prior to PML diagnosis tested JCV antibody positive. One patient tested JCV antibody negative 15 months prior to PML diagnosis but JCV antibody positive 2 months prior to PML diagnosis.
The validated second-generation JCV antibody ELISA offers improved assay design as a kit and enhanced performance characteristics that advance routine clinical use of the assay as a PML risk stratification tool.
Anti-JC virus antibodies in a large German natalizumab-treated multiple sclerosis cohort
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For the 4.7% of patients who became seronegative, our data suggest that this may be a natural fluctuation of antibody levels near the lower cut point of the assay (nOD450 0.1).6 Together with the typical, albeit low, analytical variability of the assay,11 this may result in alternating positive and negative results. Similarly, most of the patients who converted from seronegative to seropositive status over the relatively short interval of this study also had antibody levels that appeared to fluctuate around the upper cut point of the assay (nOD450 0.25).
To investigate the rate of seropositivity of anti-JC virus (JCV) antibodies in a German multiple sclerosis (MS) cohort treated with natalizumab in the postmarketing setting and to assess anti-JCV serostatus in samples obtained before diagnosis of progressive multifocal leukoencephalopathy (PML).
This was a blinded, retrospective cross-sectional and longitudinal analysis for anti-JCV antibodies using a confirmatory 2-step ELISA on 2,782 blood samples obtained from 2,253 patients nationwide for routine testing for anti-natalizumab antibodies during open-label treatment between 2007 and 2010.
Of the natalizumab-treated patients with MS, 58.8% tested positive for anti-JCV antibodies. The rate of seropositivity was higher in males and increased with age, with a plateau between age intervals 20–29 and 30–39 years. In longitudinal analyses, 19 of 194 (9.8%) patients converted from anti-JCV antibody-negative to seropositive status over 7.7 months; 4.7% reverted from antibody-positive to seronegative status over 7.9 months. Antibody levels, especially in the latter group, were low, indicating fluctuations around the lower cut point of the assay. Neither anti-JCV serostatus nor antibody levels were associated with immunosuppressive pretreatment, duration of natalizumab treatment, or anti-natalizumab antibodies. All samples obtained from 10 patients who developed PML were seropositive (13 samples before PML diagnosis [2.0–37.6 months]; 2 samples at diagnosis). Antibody levels in these samples were higher than those in samples from seropositive patients who did not develop PML.
These data argue for the potential clinical utility of JCV serology for PML risk stratification. However, further investigations of fluctuations in serostatus and of antibody levels for a more precise understanding of the predictive value are warranted.
CI confidence interval; IgG immunoglobulin G; JCV JC virus; MS multiple sclerosis; nOD₄₅₀ normalized optical density; PML progressive multifocal leukoencephalopathy RR relative risk.
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View full text

Multi-site analytical validation of an assay to detect anti-JCV antibodies in human serum and plasma

Abstract

Background

Objectives

Study design

Results

Conclusions

Section snippets

Background

Objectives

Samples

Anti-JCV antibody assay

Assay cut points and diagnostic sensitivity

Discussion

Funding

Competing interest

Ethical approval

Acknowledgements

Virology

Progressive multifocal leukoencephalopathy in patients on immunomodulatory therapies

Annu Rev Med

Progressive multifocal leukoencephalopathy and newer biological agents

Drug Saf

Anti-JC virus antibodies: implications for PML risk stratification

Ann Neurol

Recommendations for the bioanalytical method validation of ligand-binding assays to support pharmacokinetic assessments of macromolecules

Pharm Res

Prevalence of polyomavirus BK and JC infection and replication in 400 healthy blood donors

J Infect Dis

Cross-reactivity of T lymphocytes recognizing a human cytotoxic T-lymphocyte epitope within BK and JC virus VP1 polypeptides

J Virol