2, 4-Dinitrophenyl (DNP) is a widely used hapten in molecular biology and immunoassay fields. Considering that 2, 4-dintrophenylhydrazine (DNPH) could be used as DNA probe and bind with protein carbonyl to form a stable 2 4-dinitrophenyl (DNP) hydrazone product, on which the level of oxidative stress could be validated with a sensitive noncompetitive ELISA, we prepared DNP-aminocaproic acid and NHS-aminocaproic acid-dinitrobenzene and the conjugates between DNP and carrier proteins such as bovine thyroglobulin (BTG) and bovine serum albumin (BSA). High titer antibody producing spleen cells were removed and fused with myeloma cells of SP2/0 origin. Using a conventional immunization protocol, twenty stable murine monoclonal antibodies (MAbs) producing cell lines to DNP were generated. The donor mouse produced antiserum with a high titer of 1/1,280,000. Five MAbs were selected for further characterization as class and subclass. After four successive limiting dilutions, antibodies were produced by five clones with high affinities ranging from 10(10) to 10(11) M(-1). These clones were found to be of IgG(1) subclass with kappa and lambda light chain. Competitive ELISA and SPR-based sensing system for the detection of DNPH are both used to confirm the specificity of MAb (4D(9)A(9)C(2)C(2)).