Preparation of Anti-dinitrotoluene Polyclonal Antibody and Effect of the Hapten Spacer Length in Coating Antigen on Immunoassay Sensitivity
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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.
収録刊行物
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- Journal of the Faculty of Agriculture, Kyushu University
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Journal of the Faculty of Agriculture, Kyushu University 54 (1), 165-171, 2009-02-27
九州大学大学院農学研究院
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詳細情報 詳細情報について
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- CRID
- 1390853649615004288
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- NII論文ID
- 120001204031
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- NII書誌ID
- AA00247166
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- DOI
- 10.5109/14054
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- HANDLE
- 2324/14054
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- ISSN
- 00236152
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- 本文言語コード
- en
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- データソース種別
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- IRDB
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