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Updated: Feb 24, 2026
An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions
Published on: March 14, 2016
Model analysis of bidirectional interference in two-stage labeled-ligand immunoassays
Eleanor R Lewin1, Amitava Dasgupta2, Roland Valdes3
1Department of Pathology, Jefferson University Hospital, Philadelphia, PA, USA.
Insights
Bidirectional interference in immunoassays can occur due to competing ligands. This study models sequential-step immunoassays to understand the conditions that cause both positive and negative interference from interferents.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Immunology
Background:
- Immunoassays are crucial diagnostic tools.
- Sequential-step immunoassays involve sample incubation followed by a wash step.
- These assays can be susceptible to interference from competing ligands, leading to bidirectional interference.
Purpose of the Study:
- To theoretically examine bidirectional interference in sequential-step immunoassays.
- To develop a mathematical model for understanding interference phenomena.
- To identify conditions under which competing ligands cause both positive and negative interference.
Main Methods:
- Modeled competitive binding between analyte and interferent in sequential-step immunoassays.
- Assumed affinity constants reflect dissociation rate constants.
- Analyzed steady-state and non-steady-state sample incubation conditions.
Main Results:
- Determined relationships between analyte and interferent parameters for bidirectional interference.
- Non-steady-state conditions can amplify interferent effects.
- Homogeneous assay formats without wash steps also show bidirectional interference, favoring positive interference.
Conclusions:
- Model calculations confirm the theoretical basis for bidirectional interference in two-stage immunoassays.
- Defined constraints on conditions leading to bidirectional interference.
- Provides insights into optimizing immunoassay design to mitigate interference.
Objectives:
Immunoassays involving sample incubation followed by a wash step prior to introduction of labeled analyte are potentially subject to both positive and negative interference (bidirectional interference) by a competing ligand. We examine this phenomenon from a theoretical standpoint using a mathematical model for sequential-step immunoassays in the presence of interferent.
Design & Methods:
Competitive binding to antibody between analyte and interferent was modeled for sequential-step immunoassays. A primary assumption was that the ratio of affinity constants between the intended analyte and the interferent reflected the ratio of dissociation rate constants, with the higher dissociation rate constant for the lesser affinity ligand.
Results:
Relationships of parameters (relative affinity constants, relative concentrations) for analyte and interferent were determined for conditions in which bidirectional interference can occur, for both steady-state and non-steady-state sample incubation conditions. Non-steady state sample incubation conditions can enhance the effects of an interferent. Homogeneous assay formats utilizing labeled ligand without a wash step can also demonstrate bidirectional interference, but positive interference is favored under such formats.
Conclusions:
Model calculations demonstrate the theoretical basis for bidirectional interference in two-stage immunoassays. Results delineate constraints on conditions in which bidirectional interference can occur.
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