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Specificity of two-site immunoassays.
L M Boscato1, G M Egan, M C Stuart
1Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, NSW, Australia.
Journal of Immunological Methods
|February 24, 1989
Summary
Two-site immunoassays may not always improve specificity, as cross-reactions can occur unexpectedly. Computer simulations and experiments reveal complexities in antibody binding, impacting assay accuracy for human chorionic gonadotrophin.
Area of Science:
- Immunochemistry
- Biochemical Assays
- Analytical Chemistry
Background:
- Two-site immunoassays are widely used for analyte detection.
- Specificity is a critical parameter for immunoassay performance.
- Radioimmunoassays (RIAs) are a common benchmark for immunoassay specificity.
Purpose of the Study:
- To investigate the theoretical and experimental cross-reaction in two-site immunoassays.
- To compare cross-reactivity in two-site immunoassays versus radioimmunoassays using identical monoclonal antibodies.
- To elucidate the factors contributing to unexpected cross-reactivity in two-site immunoassay systems.
Main Methods:
- Theoretical investigation using computer simulations.
- Experimental validation of cross-reactivity in two distinct two-site immunoassays.
- Comparison with radioimmunoassays utilizing the same monoclonal antibodies for human chorionic gonadotrophin (hCG).
Main Results:
- Substances with negligible cross-reactivity in RIAs can yield identical assay responses in two-site immunoassays, especially with excess antibody.
- Unexpected cross-reactivity was observed in one two-site immunoassay, not predictable from antibody specificity or RIA cross-reactivity.
- The beta subunit of hCG exhibited unexpected cross-reactivity due to an apparent alteration in antibody specificity after binding to the second antibody.
Conclusions:
- Two-site immunoassays do not inherently guarantee enhanced specificity compared to RIAs.
- Complex antibody-binding interactions in two-site immunoassays can lead to unpredictable cross-reactivity.
- Understanding these binding complexities is crucial for accurate immunoassay development and interpretation.