A Robust Method for Assaying the Immunoreactive Fraction in Nonequilibrium Systems.

Area of Science:

• Biochemistry
• Radiochemistry
• Immunology

Background:

• The immunoreactive fraction (r) is crucial for assessing the functional purity of radiolabeled proteins.
• Traditional methods like the Lindmo plot have known limitations but lack systematic evaluation.

Purpose of the Study:

• To systematically compare the accuracy, precision, and robustness of the Lindmo plot with the rectangular hyperbola method for determining the immunoreactive fraction (r).
• To investigate sources of error in traditional extrapolation methods.

Main Methods:

• Experimental and simulation-based approaches were used for comparison.
• The rectangular hyperbola method, based on the Langmuir model, was evaluated against the Lindmo plot.
• Analysis focused on accuracy, precision, and robustness of extrapolated r values.

Main Results:

• The Lindmo plot is susceptible to errors from nonequilibrium and antigen depletion.
• Linearization in the Lindmo plot introduces mathematical distortions, increasing fragility towards stochastic errors and necessitating exclusion of low bound fractions.
• The rectangular hyperbola method demonstrated robust and precise r estimates from raw binding data, even with slow kinetics.

Conclusions:

• The rectangular hyperbola method is superior to the Lindmo plot for accurate and precise determination of the immunoreactive fraction (r).
• This improved method enhances the assessment of functional purity for radiolabeled proteins, even under challenging kinetic conditions.