Bronshtein, A., Institute of Plant Protection, Volcani Center, Bet Dagan, 50250, Israel
Aharonson, N., Institute of Plant Protection, Volcani Center, Bet Dagan, 50250, Israel
Turniansky, A., Institute of Plant Protection, Volcani Center, Bet Dagan, 50250, Israel
Altstein, M., Institute of Plant Protection, Volcani Center, Bet Dagan, 50250, Israel

A sol-gel-based method for immunoaffinity purification using sol-gel-entrapped antidinitrophenyl (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).