A second-generation ELISA (STRATIFY JCV DxSelect™) for detection of JC virus antibodies in human serum and plasma to support progressive multifocal leukoencephalopathy risk stratification

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Abstract

Background

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.

Objective

To develop a second-generation JCV antibody ELISA kit with improved assay performance characteristics.

Study design

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.

Results

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.

Conclusions

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.

Section snippets

Background

Progressive multifocal leukoencephalopathy (PML) is a rare demyelinating disease of the central nervous system associated with the use of certain immunomodulatory agents.1 While multiple host and viral factors contribute to the development of PML, infection with a common polyomavirus, JC virus (JCV), is a known prerequisite.2 We previously reported the development and validation of a novel two-step enzyme-linked immunosorbent assay (ELISA) to detect JCV antibodies in human serum or plasma and

Objectives

Our objectives were to enhance the robustness and performance characteristics of the two-step JCV antibody ELISA, translate it into a kit format, validate the enhanced assay, and demonstrate its potential utility as a tool for stratifying multiple sclerosis (MS) patients by risk of developing PML.

Samples

Assay cut-points were determined using serum samples from 228 urinary JCV DNA-positive patients (a subset of the 875 MS patients enrolled in the Safety of Tysabri Re-dosing and Treatment [STRATA] study [NCT00297232] whose data were used for development of the original two-step JCV antibody assay)3 and serum samples from 1091 MS patients collected at baseline in the STRATIFY-1 study (NCT01070823). Serum samples from 792 MS patients enrolled in the STRATIFY-2 study (NCT01070836) and 538 patients

ELISA plate surface chemistry and coating buffer selection

Signal-to-noise ratios were calculated for both positive control 1 (PC1) and positive control 2 (PC2) relative to NC for all surface chemistries and coating buffer conditions, with the PC2/NC ratio being the primary parameter, as it reflects the signal-to-noise ratio of low levels of JCV antibodies. MediSorp and MaxiSorp plates with Focus's proprietary PBS yielded the two highest PC2/NC ratios, which were 3.8 and 3.6, respectively (Supplementary Table A). MediSorp plates with Focus's

Discussion

We previously described the development and validation of a two-step JCV antibody ELISA. Here, we reported the development of a second-generation JCV antibody ELISA that offered two major improvements. First, stabilization of JC VLP onto the microtiter plates and the development of a ready-to-use kit improved the reproducibility and ease of use of the assay. Immobilization of JC VLP on the plates minimized variability resulting from dilution and coating of JC VLP. Second, the dynamic range of

Funding

Funding for the assay development and validation studies was provided by Biogen Idec Inc.

Competing interests

Peter Lee, Albert Castro, Dipeshkumar Jaiswal, and Suzanne Rivas are currently employed by Focus Diagnostics. Tatiana Plavina, Melissa Berman, Brian Schlain, and Meena Subramanyam are currently employed by and own stock in Biogen Idec.

Ethical approval

Not required.

Acknowledgments

The authors thank the laboratory staff at Focus Diagnostics, Dr. Vijaya Nagabhushanam and the laboratory staff at National Jewish Health, and Dr. Nang Nguyen and the laboratory staff at University of Rochester Medical Center for performing the validation experiments. The authors also thank Dr. Wayne Hogrefe for scientific discussions on assay development. Editorial support was provided by Joshua Safran of Infusion Communications and was funded by Biogen Idec Inc. and Elan Pharmaceuticals, Inc.

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Co-lead authors who contributed equally to this work.

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