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A Robust Method for Assaying the Immunoreactive Fraction in Nonequilibrium Systems

The immunoreactive fraction r provides important information on the functional purity of radiolabeled proteins. It is traditionally determined by saturating the radioimmunoconjugate with an increasing excess of antigen, followed by linear extrapolation to infinite antigen excess in a double inverse...

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Autores principales: Denoël, Thibaut, Pedrelli, Luca, Pantaleo, Giuseppe, Prior, John O.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6958493/
https://www.ncbi.nlm.nih.gov/pubmed/31817013
http://dx.doi.org/10.3390/ph12040177
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author Denoël, Thibaut
Pedrelli, Luca
Pantaleo, Giuseppe
Prior, John O.
author_facet Denoël, Thibaut
Pedrelli, Luca
Pantaleo, Giuseppe
Prior, John O.
author_sort Denoël, Thibaut
collection PubMed
description The immunoreactive fraction r provides important information on the functional purity of radiolabeled proteins. It is traditionally determined by saturating the radioimmunoconjugate with an increasing excess of antigen, followed by linear extrapolation to infinite antigen excess in a double inverse “Lindmo plot”. Although several reports have described shortcomings in the Lindmo plot, a systematic examination is lacking. Using an experimental and simulation-based approach, we compared—for accuracy, precision and robustness—the Lindmo plot with the “rectangular hyperbola” extrapolation method based on the Langmuir model. The differences between the theoretical and extrapolated r values demonstrate that nonequilibrium and antigen depletion are important sources of error. The mathematical distortions resulting from the linearization of the data in the Lindmo plot induce fragility towards stochastic errors and make it necessary to exclude low bound fractions. The rectangular hyperbola provides robust and precise r estimates from raw binding data, even for slow kinetics.
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spelling pubmed-69584932020-01-23 A Robust Method for Assaying the Immunoreactive Fraction in Nonequilibrium Systems Denoël, Thibaut Pedrelli, Luca Pantaleo, Giuseppe Prior, John O. Pharmaceuticals (Basel) Article The immunoreactive fraction r provides important information on the functional purity of radiolabeled proteins. It is traditionally determined by saturating the radioimmunoconjugate with an increasing excess of antigen, followed by linear extrapolation to infinite antigen excess in a double inverse “Lindmo plot”. Although several reports have described shortcomings in the Lindmo plot, a systematic examination is lacking. Using an experimental and simulation-based approach, we compared—for accuracy, precision and robustness—the Lindmo plot with the “rectangular hyperbola” extrapolation method based on the Langmuir model. The differences between the theoretical and extrapolated r values demonstrate that nonequilibrium and antigen depletion are important sources of error. The mathematical distortions resulting from the linearization of the data in the Lindmo plot induce fragility towards stochastic errors and make it necessary to exclude low bound fractions. The rectangular hyperbola provides robust and precise r estimates from raw binding data, even for slow kinetics. MDPI 2019-12-03 /pmc/articles/PMC6958493/ /pubmed/31817013 http://dx.doi.org/10.3390/ph12040177 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Denoël, Thibaut
Pedrelli, Luca
Pantaleo, Giuseppe
Prior, John O.
A Robust Method for Assaying the Immunoreactive Fraction in Nonequilibrium Systems
title A Robust Method for Assaying the Immunoreactive Fraction in Nonequilibrium Systems
title_full A Robust Method for Assaying the Immunoreactive Fraction in Nonequilibrium Systems
title_fullStr A Robust Method for Assaying the Immunoreactive Fraction in Nonequilibrium Systems
title_full_unstemmed A Robust Method for Assaying the Immunoreactive Fraction in Nonequilibrium Systems
title_short A Robust Method for Assaying the Immunoreactive Fraction in Nonequilibrium Systems
title_sort robust method for assaying the immunoreactive fraction in nonequilibrium systems
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6958493/
https://www.ncbi.nlm.nih.gov/pubmed/31817013
http://dx.doi.org/10.3390/ph12040177
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