Preparation of anti-dinitrotoluene polyclonal antibody and effect of the hapten spacer length in coating antigen on immunoassay sensitivity

A polyclonal antibody against 2,4-dinitrotoluene (2,4-DNT) has been raised in rabbit, and the antibody was used to detect 2,4-DNT using an enzyme-linked immunosorbent assay (ELISA) method. A 2,4-dinitro-phenyl-keyhole limpet hemocyanine (DNPh-KLH) conjugate was injected into a rabbit, and a polyclonal anti-DNPh-KLH antibody was realized after purification of the serum using protein G column. Aspects of the anti-DNPh-KLH antibody to various nitroaromatic compounds, such as cross-reactivities and avidity, were characterized. The temperature dependence of the avidity between the anti-DNPh-KLH antibody and 2,4-DNT was also evaluated. The quantification of 2,4-DNT was based on the principle of indirect competitive ELISA, in which the immunoreaction between the coating antigen-protein conjugates and the anti-DNPh-KLH antibody were inhibited in the presence of free 2,4-DNT in solution. The detection was performed using alkaline phosphatase-labeled anti-rabbit IgG with p-nitrophenylphosphate as a substrate. The addition of a mixture of free 2,4-DNT to the anti-DNPh-KLH antibody was found to decrease the absorbance at 405 nm due to the competitive effect of 2,4-DNT. The effect of the structure of the coating antigen-protein conjugate on the competition of free 2,4-DNT or coating antigen toward anti-DNPh-KLH antibody was also investigated. The immunoassay exhibited excellent sensitivity for the detection of 2,4-DNT in the concentration range of 1 ng mL ^-1 to 10μg mL ^-1.