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

Kazutaka Nagatomo, Kiyoshi Matsumoto, J. Sachiko Ishitobi, Mao Koga, Kiyoshi Toko, Norio Miura

Research output: Contribution to journalArticle

2 Citations (Scopus)

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 languageEnglish
Pages (from-to)165-171
Number of pages7
JournalJournal of the Faculty of Agriculture, Kyushu University
Volume54
Issue number1
Publication statusPublished - Feb 2009

Fingerprint

2,4-dinitrotoluene
haptens
Haptens
immunoassays
polyclonal antibodies
Immunoassay
coatings
antigens
Antigens
antibodies
Antibodies
rabbits
Rabbits
enzyme-linked immunosorbent assay
Enzyme-Linked Immunosorbent Assay
Fissurellidae
cross reaction
absorbance
blood proteins
alkaline phosphatase

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 journalArticle

Nagatomo, Kazutaka ; Matsumoto, Kiyoshi ; Ishitobi, J. Sachiko ; Koga, Mao ; Toko, Kiyoshi ; Miura, Norio. / Preparation of anti-dinitrotoluene polyclonal antibody and effect of the hapten spacer length in coating antigen on immunoassay sensitivity. In: Journal of the Faculty of Agriculture, Kyushu University. 2009 ; Vol. 54, No. 1. pp. 165-171.
@article{61d4bfb8cda14a6b993f96f81aa0c1d3,
title = "Preparation of anti-dinitrotoluene polyclonal antibody and effect of the hapten spacer length in coating antigen on immunoassay sensitivity",
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.",
author = "Kazutaka Nagatomo and Kiyoshi Matsumoto and Ishitobi, {J. Sachiko} and Mao Koga and Kiyoshi Toko and Norio Miura",
year = "2009",
month = "2",
language = "English",
volume = "54",
pages = "165--171",
journal = "Journal of the Faculty of Agriculture, Kyushu University",
issn = "0023-6152",
publisher = "Faculty of Agriculture, Kyushu University",
number = "1",

}

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.

UR - http://www.scopus.com/inward/record.url?scp=63649121024&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=63649121024&partnerID=8YFLogxK

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 -