A Surface Plasmon Resonance-based Immunosensor for Sensitive Detection of Bisphenol A

A method for the determination of bisphenol A (BPA), a representative endocrine-disrupting chamicals, was developed usnig the surface plasmon resonance (SPR) sensor. BPA-ovalbumin (BPA-OVA) conjugate was immobolized on an Au thin film of the SPR sensor chip by physixal absorption, and BPA determinations were performed by an indirect competitive immounoassay, where a BPA sample containing an anti-BPA antibody is intriduced into the SPR sendor system. The adddition of BPA into the anti-BPA antibody solution (0.8μg/ml, final concentration) was found to decrease the incidence angle shift sharply because of an inhibition effect of BPA. The RSDs (n=3) of each point were less than 4%. An evaluation of the affinity constant (K1) between anti-BPA antibody and BPA-OVA conjugate on the chip was found to be 2.26*10[6]M[-1], and that (K2) between anti-BPA antibody and free BPA (analyte) was 5.72*10[4]M[-1]. The lowest detection limit for BPA by SPR was almost the same as that by ELISA, 19[9]g/ml(1ppb)

On the Relationship Between the Structure of Immunogen and Affinity of Raised Antibody for Nonylphenol

An antibody for 4-nonylphenol (NP), which is suspected to be an endocrine disrupting compound, was perpared. First, an amino group was introduced at the ortho-position of NP (mixture of branched alkyl chain compounds), and the NP-protein conjugates were prepared. When NP was measured using antibody raised with the conjugate as an immunogen by an indirect competitive ELISA, the antibody showed an affinity with NP, but the magnitude of the affinity was weak. The antibody showed stronger affinity with NP having branched alkylgroup than with NP having straight chain. Next, an attention was paid to the position of hydroxyl- and alkyl-group on NP, and new immunogens were prepared. The antibodies raised with newly prepared immunogens, however, showed only very weak affinity with NP. These results sggested that the antibody have stronger affinity with the solid phase antigen than with free NP, resulting weak competition. In order to elucidate the recognition property of the antibody against the antigen, the cross-reactivity of the antibody against various kinds of antigens were investigated. As the results, the antibody did recognize an oxygen atom or a hydroxyl-group on the benzene ring. Based on these results, a solid phase antigen (NP-O-hexanoate-ovalbumin conjugate) having the structure including oxygen atom on the benzene ring was prepared. By the measuring system using NP-O-hexanoate-ovalbumin conjugate as a solid phase antigen, the antibody showed affinity with free NP. For sensitive measurement, the combination of a solid phase antigen, free antigen and an antibody is the most important factors in an indirect competitive immunoassay

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-dinitrophenyl. 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 10 μg mL^-1