Theory of a sequential addition competitive binding immunoassay based on high-performance immunoaffinity...

Area of Science:

• Analytical Chemistry
• Biochemistry
• Immunochemistry

Background:

• Competitive binding immunoassays are widely used for analyte detection.
• Chromatographic methods offer potential for enhanced immunoassay performance.
• Understanding the theoretical basis of such assays is crucial for optimization.

Purpose of the Study:

• To develop and validate a theoretical model for a sequential injection chromatographic competitive binding immunoassay.
• To establish an equation describing the calibration curve based on assay parameters.
• To investigate the influence of key variables on assay performance.

Main Methods:

• Derivation of a calibration curve equation based on nonlinear chromatographic theory.
• Experimental validation using human serum albumin (HSA) and an anti-HSA antibody column.
• Sequential injection of sample and labeled analyte analog.

Main Results:

• A derived equation accurately described experimental data for HSA binding.
• Assay response was dependent on binding capacity, analyte/analog moles, and flow rate/kinetics.
• Excess labeled analog had minimal impact on the calibration curve.
• Column binding capacity and injection flow rate were primary determinants of assay response.

Conclusions:

• The developed theoretical model provides a framework for understanding and optimizing sequential injection immunoassays.
• Assay performance can be tuned by adjusting flow rate and labeled analog concentration.
• This method offers flexibility for use as either a screening or quantitative assay.