Detection of Polyclonal Antibody Against Any Area of the Protein-Antigen Using Immobilized Protein-Antigens: The Critical Role of the Immobilization Protocol

Laboratorio de Tecnologi´a Enzima´tica, Departamento de Biocata´lisis, Instituto de Cata´lisis yPetroleoqui´mica-CSIC, Campus UAM, Cantoblanco 28049 Madrid, SpainReceived October 25, 2005; Revised Manuscript Received December 7, 2005Antigens immobilized on solid supports may be used to detect or purify their corresponding antibodies (Ab) fromserum. Direct immobilization of antigens on support surfaces (through short spacer arms) may promote interestingstabilizing effects on the immobilized antigen. However, the proximity of the support may prevent the interactionof some fractions of polyclonal Ab with some regions of the antigen (those placed in close contact with thesupport surface). Horseradish peroxidase (HRP) was immobilized on agarose by different protocols of multipointcovalent immobilization involving different regions of the antigen surface. Glyoxyl-agarose, BrCN-agarose, andglutaraldehyde-agarose were used as activated supports. Each HRP-immobilized preparation was much morestable than the soluble enzyme, but it was only able to adsorb up to 60-70% of a mixture of polyclonal anti-HRPantibodies. On the other hand, HRP was also immobilized on agarose through a very long, flexible, and hydrophilicspacer arm (dextran). This immobilized HRP was hardly stabilized, but it was able to adsorb 100% of the polyclonalanti-HRP. The absence of steric hindrances seems to play a critical role favoring the complete recognition of allclasses of polyclonal Ab. Another solution to achieve a complete adsorption of polyclonal Ab on immobilized-stabilized antigens has been also reached by using a mixture of the differently immobilized and stabilized HRP-agarose preparations. In this case, an improved storage and operational stabilities of the immobilized antigenscan be combined with the complete adsorption of any class of antibody.