Preparation of Anti-dinitrotoluene Polyclonal Antibody and Effect of the Hapten Spacer Length in Coating Antigen on Immunoassay Sensitivity

Environmental pollution of soil, river water, and underground water by explosive–related compounds is a serious problem in the areas of buried landmines and/or landmine factories (Hilmi et al., 1999). These compounds are suspected to have human toxicity, mutagenicity, and carcinogenicity (Kennel et al., 2000; Sayama et al., 1998; Tchounwou et al., 2001; Banerjee et al., 1999). Therefore, there is a pressing need to determine the concentrations and distributions of these compounds in the environment. The main component of landmines is 2,4,6–trinitrotoluene (TNT). TNT released into the environment is decomposed and converted to dinitrotoluenes (DNTs) and aminodinitrotoluenes (ADNTs), among other products, by microorganisms, light, and heat. These compounds have been measured by using HPLC or other instrumental analytical devices (Borch and Gerlach, 2004; Ahmad and Roberts; 1995). These instrumental analyses, however, require tedious pre–treatments such as extraction from samples (Halasz et al., 2002). On the other hand, detection methods using an antigen–antibody reaction, such as enzyme–linked immunosorbent assay (ELISA) and fluorescent and chemiluminescent immunosensors, are more useful and are able to detect ppb levels of explosive compounds (Goldman et al., 2003; Green et al., 2002; Wilson et al., 2003). We have been engaged in development of an antibody with high avidity to explosive–related compounds. In recent years, we have focused on the development of highly sensitive surface plasmon resonance (SPR) sensing methods for the detection of TNT using an antigen– antibody reaction (Sakai et al., 2003; Shankaran et al., 2004; Shankaran et al., 2005a; Shankaran et al., 2005b; Matsumoto et al., 2005; Shankaran et al., 2006). According to the reports of Jenkins et al. (2000, 2001), the concentrations of 2,4–DNT and other decomposed nitroaromatic compounds around and over buried landmines are higher than the concentration of TNT. In the present study, a polyclonal antibody against 2,4–DNT was raised in a rabbit, and the antibody was applied to the detection of 2,4–DNT using ELISA. The aim was to prepare anti–DNPh–KLH antibody from a rabbit and to clarify the characteristics for the detection of 2,4–DNT based on the principle of indirect competitive ELISA. In the course of the detection of free 2,4–DNT, we recognized that the combination of the structure of the coating antigen–protein, free 2,4–DNT, and the anti– DNPh–KLH antibody are an important factor in sensitivity using an indirect competitive immunoassay. Therefore, the effect of the structures of the coating antigen–protein conjugates on the sensitivity of the detection of free 2,4–DNT was investigated in detail. Preparation of Anti–dinitrotoluene Polyclonal Antibody and Effect of the Hapten Spacer Length in Coating Antigen on Immunoassay Sensitivity