Extremely sensitive and selective antibodies against the explosive 2,4,6-trinitrotoluene by rational design of a structurally optimized hapten
- 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 constantAntibodies 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) × 109l/mol (rabbit R1) and (2.3 ± 0.2) × 109l/mol (rabbit R2). The respective assays show a minimum test midpoint (IC50 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.…
MetadatenAutor*innen: | Steffen Ramin, Michael G. WellerORCiD |
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Dokumenttyp: | Zeitschriftenartikel |
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Veröffentlichungsform: | Verlagsliteratur |
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Sprache: | Englisch |
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Titel des übergeordneten Werkes (Englisch): | Journal of molecular recognition |
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Jahr der Erstveröffentlichung: | 2012 |
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Verlag: | Heyden |
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Verlagsort: | London |
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Jahrgang/Band: | 25 |
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Ausgabe/Heft: | 2 |
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Erste Seite: | 89 |
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Letzte Seite: | 97 |
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Freie Schlagwörter: | ELISA; Immunoassay; Security; Terrorism |
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DOI: | 10.1002/jmr.2162 |
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ISSN: | 0952-3499 |
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ISSN: | 1099-1352 |
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Verfügbarkeit des Dokuments: | Physisches Exemplar in der Bibliothek der BAM vorhanden ("Hardcopy Access") |
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Bibliotheksstandort: | Sonderstandort: Publica-Schrank |
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Datum der Freischaltung: | 19.02.2016 |
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Referierte Publikation: | Ja |
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Datum der Eintragung als referierte Publikation: | 06.02.2012 |
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