Comparison of an antibody and its recombinant derivative for the detection of the small molecule explosive 2,4,6-trinitrotoluene

[Display omitted] ► We describe a monoclonal antibody that recognizes 2,4,6-trinitrotoluene (TNT). ► An effective scFv was developed from the anti-TNT monoclonal antibody. ► The scFv provided enhanced limits of detection relative to the monoclonal. Antibodies are commonly used as recognition element...

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Bibliographic Details
Published in:Analytica chimica acta 2013-01, Vol.759, p.100-104
Main Authors: Liu, Jinny L., Zabetakis, Dan, Acevedo-Vélez, Glendalys, Goldman, Ellen R., Anderson, George P.
Format: Article
Language:English
Subjects:
Affinity
Amino Acid Sequence
Analytical chemistry
Antibodies
Antibodies, Monoclonal - chemistry
Antibodies, Monoclonal - genetics
Assaying
Cell Line
Chemistry
Circular dichroism
Circular Dichroism - methods
Cloning, Molecular
Competitive immunoassay
Escherichia coli - genetics
Exact sciences and technology
Explosions
Explosive detection
Format
Immunoassay - methods
Limit of Detection
MAGPIX
Magplex microspheres
Melting
Miscellaneous
Molecular Sequence Data
Recognition
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Single chain antibody
Single-Chain Antibodies - chemistry
Single-Chain Antibodies - genetics
Surface plasmon resonance
TNT
Trinitrotoluene - analysis
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Summary:[Display omitted] ► We describe a monoclonal antibody that recognizes 2,4,6-trinitrotoluene (TNT). ► An effective scFv was developed from the anti-TNT monoclonal antibody. ► The scFv provided enhanced limits of detection relative to the monoclonal. Antibodies are commonly used as recognition elements in immunoassays because of their high specificity and affinity, and have seen extensive use in competitive assays for the detection of small molecules. However, these complex molecules require production either in animals or by mammalian cell cultures, and are not easily tailored through genetic manipulation. Single chain antibodies (scFv), recombinantly expressed molecules consisting of only the antibody's binding region joined via a linking peptide, can provide an alternative to intact antibodies. We describe the characterization of a new monoclonal antibody (mAb), 2G5B5, able to detect the small molecule explosive 2,4,6-trinitrotoluene (TNT) and the scFv derived from its variable regions. The mAb and scFv were tested by surface plasmon resonance to determine their affinity for an immobilized TNT surrogate; dissociation constants were determined to be 1.5×10−13M and 4.8×10−10M respectively. Circular dichroism was used to determine their melting temperatures. The mAb is more stable melting at ∼75°C while the scFv melts at ∼65°C. The recognition elements were incorporated into a competitive assay format using a bead-based multiplexing platform to examine their sensitivity and specificity. The scFv was able to detect TNT ∼10-fold more sensitively than the mAb in this assay format, allowing detection of TNT concentrations down to at least 1μgL−1. The 2G5B gave similar detection limits to a commercial anti-TNT mAb, but was less specific, recognizing 1,3,5-trinitrobenzene (TNB) equally well as TNT.
ISSN:0003-2670
1873-4324
DOI:10.1016/j.aca.2012.10.051