Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 165-171 |
| Number of pages | 7 |
| Journal | Journal of the Faculty of Agriculture, Kyushu University |
| Volume | 54 |
| Issue number | 1 |
| Publication status | Published - Feb 2009 |
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All Science Journal Classification (ASJC) codes
- Agronomy and Crop Science
- Biotechnology
Cite this
Preparation of anti-dinitrotoluene polyclonal antibody and effect of the hapten spacer length in coating antigen on immunoassay sensitivity. / Nagatomo, Kazutaka; Matsumoto, Kiyoshi; Ishitobi, J. Sachiko; Koga, Mao; Toko, Kiyoshi; Miura, Norio.
In: Journal of the Faculty of Agriculture, Kyushu University, Vol. 54, No. 1, 02.2009, p. 165-171.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Preparation of anti-dinitrotoluene polyclonal antibody and effect of the hapten spacer length in coating antigen on immunoassay sensitivity
AU - Nagatomo, Kazutaka
AU - Matsumoto, Kiyoshi
AU - Ishitobi, J. Sachiko
AU - Koga, Mao
AU - Toko, Kiyoshi
AU - Miura, Norio
PY - 2009/2
Y1 - 2009/2
N2 - 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.
AB - 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.
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M3 - Article
VL - 54
SP - 165
EP - 171
JO - Journal of the Faculty of Agriculture, Kyushu University
JF - Journal of the Faculty of Agriculture, Kyushu University
SN - 0023-6152
IS - 1
ER -