title: Extremely sensitive and selective antibodies against the explosive 2,4,6-trinitrotoluene by rational design of a structurally optimized hapten.
abstract: Antibodies are a promising tool for the fast and selective trace detection of explosives. Unfortunately, the production of high-quality antibodies is not trivial and often expensive. Therefore, excellent antibodies are a rare and limiting resource in fields such as biosensing, environmental analysis, diagnostics, cancer therapy, and proteomics. Here, we report the synthesis, bioconjugation, and application of the structurally optimized hapten 6-(2,4,6-trinitro)-phenylhexanoic acid to improve the selectivity and sensitivity of antibodies for the detection of one of the most important explosives, trinitrotoluene. With a conjugate of bovine serum albumin and a highly purified N-hydroxy-succinimide (NHS)-activated hapten, two rabbits were immunized to obtain polyclonal antibodies. The immunization process was monitored by enzyme-linked immunosorbent assay to gain information about the progress of antibody titer and affinity. Finally, the polyclonal antibodies reached an affinity constant of (5.1 ± 0.6) × 10(9) l/mol (rabbit R1) and (2.3 ± 0.2) × 10(9) l/mol (rabbit R2). The respective assays show a minimum test midpoint (IC(50) value) of 0.1 ± 0.01 µg/l (R1) and 0.2 ± 0.02 µg/l (R2) and a working range of 0.005 to 150 µg/l (R1) and 0.007 to 200 µg/l (R2), which corresponds to more than four orders of magnitude for both. This is quite remarkable for a competitive immunoassay, which is often believed to have a narrow dynamic range. The limit of detection was calculated to 0.6 ng/l (R1) and 1.5 ng/l (R2), which is up to 100 times improvement in relation to the assay of Zeck et al. (1999) on the basis of a monoclonal antibody. The excellent selectivity of the polyclonal antibodies was comprehensively examined by determining the cross-reactivity to common explosives and other nitroaromatics including nitro musk components. The widely held belief that polyclonal antibodies generally display higher cross-reactivities than monoclonals could be disproved.
authors.affiliation: BAM Federal Institute for Materials Research and Testing, Division 1.5, Protein Analysis, Richard-Willstätter-Strasse 11, 12489 Berlin, Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Ramin
authors.forename: Michael G
authors.initials: MG
authors.lastname: Weller
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000915:Antibody Affinity / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology*; D002138:Calibration; D002417:Cattle; D003429:Cross Reactions / Q000276:immunology; D015195:Drug Design; D053834:Explosive Agents / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology*; D006241:Haptens / Q000737:chemistry* / Q000276:immunology*; D007114:Immunization; D015538:Nitrosation; D011817:Rabbits; D012710:Serum Albumin, Bovine / Q000276:immunology; D017186:Titrimetry; D014303:Trinitrotoluene / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology*
chemical_list: D000906:Antibodies; D053834:Explosive Agents; D006241:Haptens; D014303:Trinitrotoluene; D012710:Serum Albumin, Bovine