Nuevos métodos para la inmovilización orientada de anticuerpos sobre soportes sólidos

Resumen

Immobilized antibodies have a broad variety of uses and a great potential in areas as immunoassays, biosensors and immunoaffinity chromatography, due to their high sensitivity and high specificity. However, it is very important to know how the antibodies are attached to the support¿s surface in order to determine if the antigen binding sites of the antibody are accessible to the antigen. The influence of an antibody¿s orientation on its activity has been broadly studied, so it has been proved that a random antibody orientation diminishes its activity. Usually it is desired to have the antibody bound to the surface through the Fc region instead Fab, where the antigen binding site is. Nevertheless, there is not an ideal method for coupling the antibody. We can find a wide variety of methods for antibody coupling, and we should choose the method that fits better to our desired application. For these reasons we have developed several methodologies that allow us to bind the antibody through different areas and to different kinds of surfaces: agarose-amine-aspartic, boronate-glyoxyl, IDA-Cu 2+ -glyoxyl, ANEA-epoxy, CM-epoxy, CM-glyoxyl, glyoxyl-agarose. With all these supports we have studied the kinetics of the immobilization, the amount of bound antibody, the orientation of the antibody, and the activity that the antibody present after immobilization ¿ that is, how much antigen a molecule of antibody can bind. So, depending of the conjugate, we could have between 1.78 to 12.5 nanomols bound antibody/g support while the activity, expressed as mol antigen/mol antibody, between 0.6 to 1.6. Moreover, we wanted to be sure that the supports that we were going to work with were unable to react non-specifically with the proteins that we wanted to detect. Non-specific adsorption can affect the sensitivity of an immunoaffinity column and they can give false positives when using the immobilized antibodies as a biosensor. So, once the antibody is immobilized, we have designed also different strategies to block the conjugates, preventing the non-desired protein adsorptions. It has been also proposed a new approach to bind antibodies to high hydrophobic surfaces, via protein A. This protein could bound to modified lipases that were adsorbed onto hydrophobic surfaces, and afterwards the antibody would react with the protein A. The adsorption mechanism of the lipase permitted us to desorpt the bioconjugate (lipase+protein A+antibody) whenever we wanted without disrupting the conjugate. Later, this chimeric protein could bound to another hydrophobic support ¿for example, magnetic nanoparticles- through the same mechanism. Finally, it has been described the different applications of the immobilization strategies depending of the orientation of the antibody. For example, some supports will be more adequate for immunoassays, while others can be better applied in biosensors.