Methods:
The assay quantitates Human Interferon beta (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 validated sample concentration was ≤ 20% (≤ 25% for LLOQ and ULOQ) for teh 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.
Methods:
The assay quantitates Human Interferon beta (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 validated sample concentration was ≤ 20% (≤ 25% for LLOQ and ULOQ) for teh 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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