Optimization and Validation of an ELISA kit for the Quantification of Four Interferon-Beta (IFN-β) Marketed Compounds in Human Serum

Authors: K. Abuarjah, E. Gonzalez, A. Mancino, K. O'Toole, H. Desai, C. Beaver

Author Affiliation: inVentiv Health Clinical

Presentation: 2012 AAPS poster

Purpose

Interferons (IFNs) are a group of cytokines used to treat Multiple Sclerosis. Four trademarked compounds (Rebif®, Avonex®, Betaseron®, and Extavia®) were independently validated using a commercial ELISA kit over a range of 5-200 pg/ml. Assay parameters were evaluated and optimized to improve the performance for the individual compounds.

Methods

The assay quantitates Human (IFN-beta) in serum by sandwich enzyme linked immunosorbent assay (ELISA) and has been developed to measure low/basal levels of IFN-beta. The Interferon binds to plates coated with antibody and the detection is accomplished using a biotinylated secondary antibody followed by streptavidin conjugated to horseradish peroxidase (HRP). Tetramethy-benzidine (TMB) is the substrate. Prior to preparing the standards and QCs of the fortified solutions in human serum, it was necessary to screen the matrix in order to identify the matrices with below lower limit of quantitation (BQ) levels of endogenous analyte.

Results

The validation parameters of accuracy, precision, robutness, freeze-thaw stability, long-term stability, and bench-top stability were evaluated for each compound. The intra-run and inter-run (pooled) precision (%CV) and accuracy (%RE) for each validation sample concentration was ≤ 20% (≤ 25% for LLOQ and ULOQ) for the four compounds. The inter-run total error (|%CV| + |%RE|) was < 30% (< 40% for LLOQ)

Conclusion

Four sensitive assays for the detection of Rebif®, Avonex®, Betaseron®, and Extavia® were developed over a range of 5-200 pg/ml. The methods were reliable and robust, and considered suitable for the analyses of pharmacokinetic studies in human serum.

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