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Sensors and Materials
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Sensors and Materials, Volume 15, Number 8 (2003)
Copyright(C) MYU K.K.
pp. 439-452
S&M541 Research Paper of Special Issue
Published: 2003

Preparation of a Polyclonal Antibody and a Bioassay for Nitroaromatic Compounds by an Enzyme-Linked Immunosorbent Assay Technique and a Surface Plasmon Resonance Biosensor [PDF]

Takatoshi Sakai, Akira Torimaru, Katsura Shinahara, Norio Miura, Toshihiko Imato, Kiyoshi Toko and Kiyoshi Matsumoto

(Received November 14, 2003; Accepted December 3, 2003)

Keywords: surface plasmon resonance, immunosensor, nitroaromatic compounds, trinitrotoluene, antigen-antibody interaction

Sensitive and selective detection of 2, 4, 6-trinitrophenyl derivatives (TNP derivatives) based on surface plasmon resonance (SPR) was performed using polyclonal anti-TNP antibody and N-(TNP)-ovalbumin (TNP-OVA) conjugate. TNP-bovine serum albumin (TNP-BSA) conjugate was injected into the mice, and polyclonal anti-TNP antibody was gained after purification of the sera using protein G. TNP-OVA was immobilized on the Au film of the SPR sensor chip by physical adsorption. The incidence angle shift of the TNP-OVA immobilized sensor increased steeply with increasing concentration of anti- TNP antibody up to 20 g/ml and increased only slightly above this concentration. The additions of TNP derivatives into the anti-TNP antibody solution were found to decrease the incidence angle shift because of the inhibition effects of TNP derivatives. The lowest detection limit for trinitrotoluene (TNT) by SPR was 1 10–7 g/ml, whereas for TNP-6- aminohexanoic acid (TNP-aha) it was 3 10–9 g/ml. Evaluations of affinity constants of anti-TNP antibody were performed. Analyses of SPR data were carried out by assump- tions of the Langmuir isotherm and equilibrium state of immunoreaction. The value of association constant between anti-TNP antibody and immobilized TNP-OVA (K1) was 6.4 106 M–1. The values of association constants between antibody and TNP-aha (K2) were 2.7 and 8.5 106 M–1 when 20 and 10 g/ml antibody were used, respectively.

Corresponding author: Kiyoshi Matsumoto


Cite this article
Takatoshi Sakai, Akira Torimaru, Katsura Shinahara, Norio Miura, Toshihiko Imato, Kiyoshi Toko and Kiyoshi Matsumoto, Preparation of a Polyclonal Antibody and a Bioassay for Nitroaromatic Compounds by an Enzyme-Linked Immunosorbent Assay Technique and a Surface Plasmon Resonance Biosensor, Sens. Mater., Vol. 15, No. 8, 2003, p. 439-452.



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