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Title: Engineered Antibodies for Monitoring of Polynuclear Aromatic Hydrocarbons

Abstract

Polynuclear aromatic hydrocarbons (PAHs) are a large class of structurally similar pollutants. Rapid, inexpensive, and high-throughput methods to identify and monitor PAHs are needed in several DOE focus areas, including human and ecosystem health effects, risk and exposure assessment, decontamination and decommissioning, and remediation. DOE has sponsored and participated in several demonstration projects in which commercial immunoassay kits proved useful and cost-effective for detection of PAHs and other pollutants. The emerging generation of sensors and residue recovery methods will require panels of antibodies with relatively subtle differences in cross-reactivity. This project is based on the premise that genetic engineering should be much more successful than conventional polyclonal and monoclonal antibody methods for developing these antibody panels. One objective of this project has been to define the structural basis and mechanisms by which antibodies bind and cross-react with various PAHs. A second objective has been to use this information to produce recombinant antibodies with improved performance in analytical procedures that DOE can use. A third objective has been development of PAH residue recovery and cleanup methods that will be compatible with immunoassays, and make instrumental analysis faster, more accurate, and less expensive.

Authors:
; ; ;
Publication Date:
Research Org.:
University of California at Berkeley; Scripps Research Institute; University of Hawaii (US)
Sponsoring Org.:
USDOE Office of Environmental Management (EM) (US)
OSTI Identifier:
825746
Report Number(s):
EMSP-54546-2000
R&D Project: EMSP 54546; TRN: US200423%%396
DOE Contract Number:
FG07-96ER62316
Resource Type:
Technical Report
Resource Relation:
Other Information: PBD: 6 Mar 2000
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL AND ANALYTICAL CHEMISTRY; 54 ENVIRONMENTAL SCIENCES; 59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; ANTIBODIES; DECOMMISSIONING; DECONTAMINATION; DETECTION; ECOSYSTEMS; GENETIC ENGINEERING; IMMUNOASSAY; MONITORING; MONITORS; PERFORMANCE; POLLUTANTS; POLYCYCLIC AROMATIC HYDROCARBONS; RESIDUES

Citation Formats

Karu, Alexander E., Roberts, Victoria A., and Li, Qing X. Engineered Antibodies for Monitoring of Polynuclear Aromatic Hydrocarbons. United States: N. p., 2000. Web. doi:10.2172/825746.
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Karu, Alexander E., Roberts, Victoria A., & Li, Qing X. Engineered Antibodies for Monitoring of Polynuclear Aromatic Hydrocarbons. United States. doi:10.2172/825746.
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Karu, Alexander E., Roberts, Victoria A., and Li, Qing X. Mon . "Engineered Antibodies for Monitoring of Polynuclear Aromatic Hydrocarbons". United States. doi:10.2172/825746. https://www.osti.gov/servlets/purl/825746.
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@article{osti_825746,
title = {Engineered Antibodies for Monitoring of Polynuclear Aromatic Hydrocarbons},
author = {Karu and Alexander E. and Roberts, Victoria A. and Li, Qing X.},
abstractNote = {Polynuclear aromatic hydrocarbons (PAHs) are a large class of structurally similar pollutants. Rapid, inexpensive, and high-throughput methods to identify and monitor PAHs are needed in several DOE focus areas, including human and ecosystem health effects, risk and exposure assessment, decontamination and decommissioning, and remediation. DOE has sponsored and participated in several demonstration projects in which commercial immunoassay kits proved useful and cost-effective for detection of PAHs and other pollutants. The emerging generation of sensors and residue recovery methods will require panels of antibodies with relatively subtle differences in cross-reactivity. This project is based on the premise that genetic engineering should be much more successful than conventional polyclonal and monoclonal antibody methods for developing these antibody panels. One objective of this project has been to define the structural basis and mechanisms by which antibodies bind and cross-react with various PAHs. A second objective has been to use this information to produce recombinant antibodies with improved performance in analytical procedures that DOE can use. A third objective has been development of PAH residue recovery and cleanup methods that will be compatible with immunoassays, and make instrumental analysis faster, more accurate, and less expensive.},
doi = {10.2172/825746},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Mon Mar 06 00:00:00 EST 2000},
month = {Mon Mar 06 00:00:00 EST 2000}
}
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Technical Report:
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DOI:  10.2172/825746
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  • ; ;
    'The objective of this multidisciplinary project is to use molecular biological techniques to derive a set of antibodies with useful affinities and selectivities for recovery and detection of polynuclear aromatic hydrocarbons (PAHs) in environmental and biological samples. The long-term goal is to develop immunodetection methods that will be useful in biomarker research and regulatory monitoring of PAHs. APPROACH The aims and approaches remain the same as in the original proposal. My laboratory cloned and characterized two PAL-I-specific recombinant Fab antibodies (rFabs). The authors are deriving new affinities and specificities for PAHs by mutagenesis of these rFabs, and by selection ofmore » new rFabs from combinatorial phage display libraries. Dr. Qing Li''s group designed and synthesized PAH haptens that were essential for my laboratory''s work. Dr. Victoria Roberts''s group developed molecular models that suggested the mechanism of PAH binding and predicted mutations to alter it. Dr. Li''s laboratory is using the recombinant antibodies they produce to develop immunoaffinity and immunoassay methods to quantify PAHs in environmental samples.'« less

  • ; ;
    'The long-term goal of this project is to develop antibodies and antibody-based methods for detection and recovery of polynuclear aromatic hydrocarbons (PAHs) and PAH adducts that are potential biomarkers in environmental and biological samples. The inherent cross-reactivity will be exploited by pattern recognition methods. Dr. Karu''s laboratory uses new haptens representing key PAHs to derive recombinant Fab (rFab) and single-chain Fv (scFv) antibodies from hybridoma lines and combinatorial phage display libraries. Computational models of the haptens and combining sites made by Dr. Roberts''s group are used to guide antibody engineering by mutagenesis. Dr. Li''s laboratory develops enzyme immunoassays (EIAs), sensors,more » and immunoaffinity methods that make use of the novel haptens and antibodies for practical analytical applications in support of DOE''s mission. This report summarizes work completed in one and one-half years of a 3-year project, with close collaboration between the three research groups. Dr. Alexander Karu''s laboratory: the authors proceeded with the two strategies described in the original proposal. Site-directed mutagenesis was used to correct differences in the rFab N-terminal amino acids that were introduced by the degenerate PCR primers used for gene amplification. The binding constants of the rFabs with the corrected sequences will be compared with those of the parent MAbs, and should be very similar. The 4D5 and 10C10 heavy and light chain sequences are being moved to the pCOMB3H phagemid vector to facilitate selection of new engineered mutants.'« less

  • ; ;
    This project was undertaken to fill needs in ODE's human and ecosystem health effects research, site remediation, rapid emergency response, and regulatory compliance monitoring programs. Doe has greatly stimulated development and validation of antibody-based, rapid, field-portable detection systems for small hazardous compounds. These range from simple dipsticks, microplate enzyme-linked immunosorbent assays (ELISAs), and hand-held colorimeters, to ultrasensitive microfluidic reactors, fiber-optic sensors and microarrays that can identify multiple analytes from patterns of cross-reactivity. Unfortunately, the technology to produce antibodies with the most desirable properties did not keep pace. Lack of antibodies remains a limiting factor in production and practical use ofmore » such devices. The goals of our project were to determine the chemical and structural bases for the antibody-analyte binding interactions using advanced computational chemistry, and to use this information to create useful new binding properties through in vitro genetic engineering and combinatorial library methods.« less

  • ; ;
    The objective is to develop improved antibody-based methods for detection of multiple polynuclear aromatic hydrocarbons (PAHs), to fill several needs in DOE's remediation, regulatory monitoring, ecotoxicology, and human health effects missions. Present-generation immunochemical detection methods have already proven to be useful and cost-effective in DOE applications. The problem being addressed is that the unique properties of PAHs make it impractical to generate antibodies with the required diversity, specificity and selectivity, by the previous techniques. The scientific goals are to determine the mechanisms by which antibodies bind PAHs, use genetic engineering and computational chemistry techniques to construct improved antibodies, and tomore » devise methods for making immunochemical and instrumental analysis more compatible. The potential relevance is that our results should provide a rational basis by which immunochemical and other molecular recognition systems for PAHs and other large classes of toxic pollutants such as PCBs could be produced and deployed with substantially less cost, labor, and development time.« less

  • ; ;
    A method for concentration of trace quantities of six representatives of the polynuclear aromatic hydrocarbon (PAH) family (fluoranthene, benzo(k)flouranthene, benzo(j)flouranthene, benzo(a)pyrene, benzo(ghi)perylene, and indeno(1,1,3-cd)pyrene) has been developed and successfully applied to PAH monitoring in finished drinking water and their raw water sources in 10 selected water supplies in the eastern United States. PAH is collected by passing water through polyurethane foam plugs. Water is heated to 62 plus or minus 2 degrees Centigrade prior to passage, and flow rate is maintained at approximately 250 ml/minute to obtain quantitative recoveries. The collection is followed by elution of foam plugs with organicmore » solvent, purification by partitioning with solvents and column chromatography on Florisil, and analysis by two dimensional thin layer chromatography-fluorometry and gas liquid chromatography-flame ionization detection.« less