In situ characterization of antibody grafting on porous monolithic supports.

The efficient immobilization of antibodies on monolithic support is one of the most critical steps when preparing immunoaffinity supports. In this work, the ADECA (amino density estimation by colorimetric assay) method was adapted to tridimensional supports (in a dynamic mode) and proved to be efficient to characterize the antibodies grafting efficiency on 15.3±0.9mg porous glycidyl methacrylate (GMA)-co-ethylene dimethacrylate (EDMA) monolithic columns. The amount of grafted antibodies measured in situ on the monolith by ADECA (8.2±0.2µg of antibodies per milligram of monolith) was consistent with values obtained by bicinchoninic acid assay (BCA) after crushing the monolith. ADECA was shown to be less time-consuming and more versatile than BCA. The ADECA method was further implemented to thoroughly study and optimize the antibody grafting conditions (influence of pH and kinetics of the grafting step) on GMA-based monoliths and to check the covalent nature of the antibody/surface linking and its stability. Using the total amount of grafted antibodies and the amount of recognized antigen, we found that 65±6% of antibodies were able to capture their antigen. Finally, the grafting of Fab and F(ab')(2) fragments demonstrated that no significant improvement of the global binding capacity of the monolith was obtained.

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.

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.