An enhanced butyrylcholinesterase method to measure organophosphorus nerve agent exposure in humans

Pantazides, Brooke G.; Watson, Caroline M.; Carter, Melissa D.; Crow, Brian S.; Perez, Jonas W.; Blake, Thomas A.; Thomas, Jerry D.; Johnson, Rudolph C.

doi:10.1007/s00216-014-7718-7

Cited by 47 publications

(67 citation statements)

References 26 publications

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“…The selective capture of HuBChE from human plasma or serum by immobilized monoclonal antibodies has been developed by the CDC as a first step in the diagnosis of exposure to chemical nerve agents [10; 11; 12; 24]. To date the commercially available 3E8 monoclonal has been used for immunomagnetic purification of HuBChE.…”

Section: Discussionmentioning

confidence: 99%

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Comparison of 5 monoclonal antibodies for immunopurification of human butyrylcholinesterase on Dynabeads: KD values, binding pairs, and amino acid sequences

Peng

Brimijoin

Hrabovská

et al. 2015

Chemico-Biological Interactions

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Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger of nerve agents and organophosphorus pesticides. Mass spectrometry methods detect stable nerve agent adducts on the active site serine of HuBChE. The first step in sample preparation is immunopurification of HuBChE from plasma. Our goal was to identify monoclonal antibodies that could be used to immunopurify HuBChE on Dynabeads Protein G. Mouse anti-HuBChE monoclonal antibodies were obtained in the form of ascites fluid, dead hybridoma cells stored frozen at −80°C for 30 years, or recently frozen hybridoma cells. RNA from 4 hybridoma cell lines was amplified by PCR for determination of their nucleotide and amino acid sequences. Full-length light and heavy chains were expressed, and the antibodies purified from culture medium. A fifth monoclonal was purchased. The 5 monoclonal antibodies were compared for ability to capture HuBChE from human plasma on Dynabeads Protein G. In addition, they were evaluated for binding affinity by Biacore and ELISA. Epitope mapping by pairing analysis was performed on the Octet Red96 instrument. The 5 monoclonal antibodies, B2 12-1, B2 18-5, 3E8, mAb2, and 11D8, had similar KD values of 10−9 M for HuBChE. Monoclonal B2 18-5 outperformed the others in the Dynabeads Protein G assay where it captured 97% of the HuBChE in 0.5 ml plasma. Pairing analysis showed that 3E8 and B2 12-1 share the same epitope, 11D8 and B2 18-5 share the same epitope, but mAb2 and B2 12-1 or mAb2 and 3E8 bind to different epitopes on HuBChE. B2 18-5 was selected for establishment of a stable CHO cell line for production of mouse anti-HuBChE monoclonal.

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Section: Discussionmentioning

confidence: 99%

“…HuBChE makes stable adducts with nerve agents and organophosphorus pesticides which makes it possible to detect exposure in blood samples taken from patients days after poisoning [8; 9]. Mass spectrometry methods can detect the FGESAGAAS peptide covalently bound to nerve agents using as little as 75 μl of human serum [10; 11]. …”

Section: Introductionmentioning

confidence: 99%

Comparison of 5 monoclonal antibodies for immunopurification of human butyrylcholinesterase on Dynabeads: KD values, binding pairs, and amino acid sequences

Peng

Brimijoin

Hrabovská

et al. 2015

Chemico-Biological Interactions

Self Cite

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“…QCs, processed concurrently with samples, and the coefficient of determination of each standard curve were examined to verify the run was within quality specifications established in previous work. 25 …”

Section: Methodsmentioning

confidence: 99%

Enhanced Stability of Blood Matrices Using a Dried Sample Spot Assay To Measure Human Butyrylcholinesterase Activity and Nerve Agent Adducts

Perez

Pantazides

Watson³

et al. 2015

Anal. Chem.

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Dried matrix spots are safer to handle and easier to store than wet blood products, but factors such as intra-spot variability and unknown sample volumes have limited their appeal as a sampling format for quantitative analyses. In this work, we introduce a dried spot activity assay for quantifying butyrylcholinesterase (BChE) specific activity which is BChE activity normalized to the total protein content in a sample spot. The method was demonstrated with blood, serum, and plasma spotted on specimen collection devices (cards) which were extracted to measure total protein and BChE activity using a modified Ellman assay. Activity recovered from dried spots was ∼80% of the initial spotted activity for blood and >90% for plasma and serum. Measuring total protein in the sample and calculating specific activity substantially improved quantification and reduced intra-spot variability. Analyte stability of nerve agent adducts was also evaluated, and the results obtained via BChE-specific activity measurements were confirmed by quantification of BChE adducts using a previously established LC-MS/MS method. The spotted samples were up to 10-times more resistant to degradation compared to unspotted control samples when measuring BChE inhibition by the nerve agents sarin and VX. Using this method, both BChE activity and adducts can be accurately measured from a dried sample spot. This use of a dried sample spot with normalization to total protein is robust, demonstrates decreased intra-spot variability without the need to control for initial sample volume, and enhances analyte stability.

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“…Calibrators for GB-BChE, and VX-BChE were prepared from the synthetic peptides (TNO, Rijswik, the Netherlands) at concentrations ranging from 1–250 ng/mL 14, 16 .…”

Section: Methodsmentioning

confidence: 99%

Bridging the gap between sample collection and laboratory analysis: using dried blood spots to identify human exposure to chemical agents

et al. 2016

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Public health response to large scale chemical emergencies presents logistical challenges for sample collection, transport, and analysis. Diagnostic methods used to identify and determine exposure to chemical warfare agents, toxins, and poisons traditionally involve blood collection by phlebotomists, cold transport of biomedical samples, and costly sample preparation techniques. Use of dried blood spots, which consist of dried blood on an FDA-approved substrate, can increase analyte stability, decrease infection hazard for those handling samples, greatly reduce the cost of shipping/storing samples by removing the need for refrigeration and cold chain transportation, and be self-prepared by potentially exposed individuals using a simple finger prick and blood spot compatible paper. Our laboratory has developed clinical assays to detect human exposures to nerve agents through the analysis of specific protein adducts and metabolites, for which a simple extraction from a dried blood spot is sufficient for removing matrix interferents and attaining sensitivities on par with traditional sampling methods. The use of dried blood spots can bridge the gap between the laboratory and the field allowing for large scale sample collection with minimal impact on hospital resources while maintaining sensitivity, specificity, traceability, and quality requirements for both clinical and forensic applications.

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An enhanced butyrylcholinesterase method to measure organophosphorus nerve agent exposure in humans

Cited by 47 publications

References 26 publications

Comparison of 5 monoclonal antibodies for immunopurification of human butyrylcholinesterase on Dynabeads: KD values, binding pairs, and amino acid sequences

Comparison of 5 monoclonal antibodies for immunopurification of human butyrylcholinesterase on Dynabeads: KD values, binding pairs, and amino acid sequences

Enhanced Stability of Blood Matrices Using a Dried Sample Spot Assay To Measure Human Butyrylcholinesterase Activity and Nerve Agent Adducts

Bridging the gap between sample collection and laboratory analysis: using dried blood spots to identify human exposure to chemical agents

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