Development of a theoretical model for chromatographic-based competitive binding immunoassays with simultaneous

Injection of sample and label

D. S. Hage, D. H. Thomas, A. R. Chowdhuri, William Clarke

Research output: Contribution to journalArticle

Abstract

This study examined the theory and behavior of an HPLC-based chromatographic competitive binding immunoassay with the simultaneous injection of sample and a labeled analyte analogue. Equations based on nonlinear chromatographic theory were derived to describe the calibration curve for this assay in a system with adsorption-limited kinetics and homogeneous binding sites. These equations related the assay response (B/B(o)) to the column's binding capacity, the moles of injected analyte or labeled analogue, and the flow rate/adsorption kinetics of the system. There was good agreement between the predicted theoretical response and experimental data obtained for the binding of human serum albumin (HSA) to an immobilized anti-HSA antibody column. This theory was also successful in describing the changes that occurred in the calibration curve when the flow rate or amount of labeled analogue applied to the column was varied. A comparison was made between the results of this study and previous theoretical work that examined the behavior of a related, sequential injection competitive binding method. On the basis of the results reported in this work, several general guidelines were developed for the design and optimization of simultaneous injection methods for use in such areas as clinical testing, pharmaceutical analysis, and environmental monitoring.

Original languageEnglish (US)
Pages (from-to)2965-2975
Number of pages11
JournalAnalytical Chemistry
Volume71
Issue number15
DOIs
StatePublished - 1999
Externally publishedYes

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Serum Albumin
Labels
Assays
Flow rate
Calibration
Adsorption
Kinetics
Binding Sites
Antibodies
Monitoring
Testing
Pharmaceutical Preparations

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Development of a theoretical model for chromatographic-based competitive binding immunoassays with simultaneous : Injection of sample and label. / Hage, D. S.; Thomas, D. H.; Chowdhuri, A. R.; Clarke, William.

In: Analytical Chemistry, Vol. 71, No. 15, 1999, p. 2965-2975.

Research output: Contribution to journalArticle

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