title: Preparation and characterization of a polyclonal antibody from rabbit for detection of trinitrotoluene by a surface plasmon resonance biosensor.
abstract: A polyclonal antibody against trinitrophenyl (TNP) derivatives was raised in rabbit, and the antibody was applied to detection of trinitrotoluene (TNT) using a surface plasmon resonance (SPR) biosensor. TNP-keyhole limpet hemocyanine (TNP-KLH) conjugate was injected into a rabbit, and a polyclonal anti-TNP antibody was realized after purification of the sera using protein G. Aspects of the anti-TNP antibody against various nitroaromatic compounds, such as cross-reactivities and affinities, were characterized. The temperature dependence of the affinity between the anti-TNP antibody and TNT was also evaluated. The quantification of TNT was based on the principle of indirect competitive immunoassay, in which the immunoreaction between the TNP-beta-alanine-ovalbumin (TNP-beta-ala-OVA) and anti-TNP antibody was inhibited in the presence of free TNT in solution. TNP-beta-ala-OVA was immobilized to the dextran matrix on the Au surface by amine coupling. The addition of a mixture of free TNT to the anti-TNP antibody was found to decrease the incidence angle shift due to the inhibitory effect of TNT. The immunoassay exhibited excellent sensitivity for the detection of TNT in the concentration range of 3x10(-11) to 3x10(-7)g/ml. To increase the sensitivity of the sensor, anti-rabbit IgG antibody was used. After flowing the mixture of free TNT and anti-TNP antibody, anti-rabbit IgG antibody was injected, and the incidence angle shift was measured. Amplification of the signal was observed and the detection limit was improved to 1x10(-11)g/ml.
authors.affiliation: Graduate School of Agriculture, Kyushu University, Fukuoka 812-8581, Japan.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Matsumoto
authors.forename: Akira
authors.initials: A
authors.lastname: Torimaru
authors.forename: Sachiko
authors.initials: S
authors.lastname: Ishitobi
authors.forename: Takatoshi
authors.initials: T
authors.lastname: Sakai
authors.forename: Hiroya
authors.initials: H
authors.lastname: Ishikawa
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Toko
authors.forename: Norio
authors.initials: N
authors.lastname: Miura
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Imato