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Updated: Aug 8, 2026
Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody (mAb) by Neutralizing TNF Using an In Vitro Bioanalytical Method
Published on: September 16, 2017
A one-step, competitive electrochemiluminescence-based immunoassay method for the quantification of a fully human
D Horninger1, E Eirikis, C Pendley
1Department of Clinical Pharmacology and Experimental Medicine, Centocor, Inc., 145 King of Prussia Road, Radnor, PA 19087, USA.
Insights
A novel competitive electrochemiluminescence immunoassay was developed to quantify anti-TNFalpha monoclonal antibodies in human serum. This assay is accurate and precise, supporting pharmacokinetic studies.
Area of Science:
- Biotechnology
- Immunochemistry
- Analytical Chemistry
Background:
- Tumor Necrosis Factor-alpha (TNFα) plays a critical role in inflammatory diseases.
- Monoclonal antibodies targeting TNFα are crucial therapeutics.
- Accurate quantification of these antibodies in biological matrices is essential for clinical monitoring.
Purpose of the Study:
- To develop a quantitative, one-step, competitive electrochemiluminescence (ECL)-based immunoassay.
- To determine a fully human, anti-TNFα monoclonal antibody in human serum.
- To provide a robust method for supporting pharmacokinetic studies.
Main Methods:
- Utilized a competitive immunoassay format.
- Employed a biotinylated mouse anti-variable region-specific antibody and a ruthenium-labeled anti-TNFα antibody.
- Implemented a single 2-hour incubation step followed by ECL detection.
Main Results:
- Developed a sensitive assay for anti-TNFα monoclonal antibody in neat human serum.
- Achieved a measurement range of approximately 1600-fold, with higher concentrations manageable via dilution.
- Demonstrated suitable assay accuracy, precision, and reproducibility.
Conclusions:
- The developed competitive ECL immunoassay is suitable for quantifying anti-TNFα monoclonal antibodies in human serum.
- This assay provides a reliable tool for pharmacokinetic studies of therapeutic antibodies.
- The competitive assay format offers a viable alternative for measuring macromolecules in complex biological samples.
Abstract:
A quantitative, one-step, competitive electrochemiluminescence (ECL)-based immunoassay for the determination of a fully human, anti-TNFalpha monoclonal antibody in human serum has been developed. A biotinylated, mouse anti-variable region-specific antibody and a ruthenium-labeled anti-TNFalpha antibody were the only specific reagents needed to develop the assay. A single incubation step of 2 h followed by ECL detection was used. The assay was capable of measuring the analyte in neat serum over approximately a 1600-fold range with higher concentrations measured following a single dilution. Assay accuracy, precision, and reproducibility were suitable to support pharmacokinetic studies of the analyte. This competitive assay format offers an alternative approach to the development of immunoassays for the measurement of macromolecules in complex matrices to support preclinical and clinical studies.
