Sol−Gel-Based Immunoaffinity Chromatography: Application to Nitroaromatic Compounds

Abstract

A sol−gel-based method for immunoaffinity purification using sol−gel-entrapped anti-dinitrophenyl (DNP) antibodies (Abs) was developed. Polyclonal antiserum (whole antiserum) and purified immunoglobulines (IgGs, isolated from the whole antiserum), which recognize nanogram quantities of a variety of di- and trinitroaromatic compounds, were entrapped in SiO2 sol−gel-derived matrixes, and their binding properties were examined with 2,4-dinitrophenylhydrazine (DNPH) used as an analyte. Binding properties of the entrapped Abs were determined by the evaluation of the optimal sol−gel composition for entrapment and the optimal conditions for binding and elution of the analyte. We found that a hydrophilic, flexible “wet” gel with a tetramethoxysilane:aqueous ratio of 1:8, enriched with 10% PEG exhibited high binding capacities with low nonspecific binding. Under the tested conditions the sol−gel-entrapped Abs bound the analyte in a dose-dependent, highly reproducible manner (antibody- and antigen-wise), and binding was equally effective with either polyclonal whole antiserum or protein A purified IgGs (eliminating the need to purify IgGs from the whole antiserum). The analyte could easily be eluted at high recoveries (90%) and the Abs were well-retained in the sol−gel matrix and did not leach out even at extreme pH conditions or in organic solvents. The sol−gel immunoaffinity columns exhibited binding capacities that were either significantly higher or did not differ significantly from those of protein A−agarose covalently coupled Abs over a wide range of IgG (0.5−15 μL corresponding to 1−30 μg protein) and analyte amounts (20−320 ng).