Preparation and full characterization of a micro-immunoaffinity monolithic column and its in-line coupling with capillary zone electrophoresis with Ochratoxin A as model solute.
Résumé
A micro-immunoaffinity monolithic column (μIAC) was developed and in-line coupled with capillary zone electrophoresis in a fully automated way with Ochratoxin A as test solute. The in-line micro-immunoaffinity columns based on monolithic methacrylate polymers (EDMA-GMA) were prepared in situ at the inlet end of a PTFE coated fused silica capillary by UV initiated polymerization and subsequently grafted with antibodies. These μIACs were thoroughly characterized. The synthesis of the polymeric support was first demonstrated to be reproducible in terms of permeability, surface properties and efficiency. The antibodies immobilization was then studied by a new original hydrodynamic method (ADECA) allowing the in situ quantitative determination (at a miniaturized scale) of the total amount of immobilized antibodies. The combination of this measurement with the binding capacity of the μIAC allowed, for the first time, the in situ determination of immobilized antibody activity. A total of 260 ± 15 ng (1.6 ± 0.1 pmol) of IgG antibodies/cm in 75 μm i.d. monolithic column (i.e. 18 μgmg(-1)) was obtained with (anti-Ochratoxin A/Ochratoxin A) as antibody/antigen model. 40% of the immobilized antibodies remain active corresponding to a binding capacity of 1.2 ± 0.2 pmol antigen/cm (i.e. 600 pg/cm of our test solute OTA), a very high capacity when dealing with trace analysis and with regard to the detection limits (30 pg and 0.5 pg with UV and LIF detection, respectively). The recovery yields were quantitative with negligible non-specific adsorption and allow analysis of diluted samples (1 ngmL(-1)) for a percolated volume of 10 μL. It was also demonstrated that despite the progressive denaturation of antibodies consecutive to the elution step, the binding capacity of the μIAC remained high enough to implement at least 15 consecutive analyses with the same column and in a fully automated way.