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Calibration free concentration analysis by surface plasmon resonance in a capture mode
Jonathan Visentin1, Laetitia Minder2, Jar-How Lee3
1CHU de Bordeaux, Laboratoire d'Immunologie et Immunogénétique, Hôpital Pellegrin, Place Amélie Raba Léon, Bordeaux, France; Université de Bordeaux, UMR CNRS 5164, 146 rue Léo Saignat, Bordeaux, France.
Calibration Free Concentration Analysis (CFCA) now offers a capture format (CCFCA) for precise active concentration measurements. This method accurately quantizes analyte binding, even for sensitive molecules, enhancing biomolecular interaction studies.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Immunology
Background:
- Surface Plasmon Resonance (SPR) is crucial for quantifying biomolecular interactions.
- Accurate analyte concentration is vital for reliable SPR data.
- Traditional methods can overestimate active protein concentration.
Purpose of the Study:
- To introduce and validate Capture CFCA (CCFCA) for active concentration measurement.
- To demonstrate CCFCA's utility with sensitive analytes and immobilized ligands.
- To determine kinetic and equilibrium constants using CCFCA.
Main Methods:
- Implementation of CCFCA for active concentration determination.
- Validation using HLA-B*44:02 antigen and W6/32 antibody.
- Kinetic and equilibrium analysis of W6/32-HLA complexes.
Main Results:
- CCFCA provided active concentration measurements comparable to covalent CFCA.
- CCFCA successfully measured W6/32 antibody concentration on various captured HLA molecules.
- Consistent CCFCA results were observed across different capture levels, including low levels.
Conclusions:
- CCFCA is a versatile tool for measuring active concentrations, especially for analytes with ligands unsuitable for covalent immobilization.
- The capture format enhances throughput and reduces sensor chip usage for kinetic analysis of multiple complexes.
