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

Peng, Hong; Brimijoin, Stephen; Hrabovská, Anna; Targosova, Katarina; Krejci, Éric; Blake, Thomas A.; Johnson, Rudolph C.; Masson, Patrick; Lockridge, Oksana

doi:10.1016/j.cbi.2015.08.024

Cited by 18 publications

(33 citation statements)

References 25 publications

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“…7 The heavy and light chain nucleotides of B2 18-5 were sequenced, cloned into expression vectors, expressed in a stable Chinese hamster ovary cell line, and the antibody purified by Syd Labs, Natick, MA. 8 B2 18-5 efficiently immunopurifies HuBChE from plasma stored at 4 or −20 °C. 8 Antimouse IgG conjugated to horse radish peroxidase was from Cell Signaling, cat# 7076.…”

Section: Materials and Methodsmentioning

confidence: 99%

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Monoclonal Antibodies That Recognize Various Folding States of Pure Human Butyrylcholinesterase Can Immunopurify Butyrylcholinesterase from Human Plasma Stored at Elevated Temperatures

et al. 2016

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Human plasma to be analyzed for exposure to cholinesterase inhibitors is stored at 4 °C or lower to prevent denaturation of human butyrylcholinesterase (HuBChE), the biomarker of exposure. Currently published protocols immunopurify HuBChE using antibodies that bind native HuBChE before analysis by mass spectrometry. It is anticipated that the plasma collected from human casualties may be stored nonideally at elevated temperatures of up to 45 °C for days or maybe weeks. At 45 °C, the plasma loses 50% of its HuBChE activity in 8 days and 95% in 40 days. Our goal was to identify a set of monoclonal antibodies that could be used to immunopurify HuBChE from plasma stored at 45 °C. The folding states of pure human HuBChE stored at 4 and 45 °C and boiled at 100 °C were visualized on nondenaturing gels stained with Coomassie blue. Fully active pure HuBChE tetramers had a single band, but pure HuBChE stored at 45 °C had four bands, representing native, partly unfolded, aggregated, and completely denatured, boiled tetramers. The previously described monoclonal B2 18-5 captured native, partly unfolded, and aggregated HuBChE tetramers, whereas a new monoclonal, C191 developed in our laboratory, was found to selectively capture completely denatured, boiled HuBChE. The highest quantity of HuBChE protein was extracted from 45 °C heat-denatured human plasma when HuBChE was immunopurified with a combination of monoclonals B2 18-5 and C191. Using a mixture of these two antibodies in future emergency response assays may increase the capability to confirm exposure to cholinesterase inhibitors.

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Section: Materials and Methodsmentioning

confidence: 99%

“…8 B2 18-5 efficiently immunopurifies HuBChE from plasma stored at 4 or −20 °C. 8 Antimouse IgG conjugated to horse radish peroxidase was from Cell Signaling, cat# 7076.…”

Section: Materials and Methodsmentioning

confidence: 99%

Monoclonal Antibodies That Recognize Various Folding States of Pure Human Butyrylcholinesterase Can Immunopurify Butyrylcholinesterase from Human Plasma Stored at Elevated Temperatures

et al. 2016

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“…Similar to B2 18-5, monoclonal mAb2 has broad species specificity. Pairing analysis on the Octet Red96 system showed that mAb2 and B2 18-5 bind to overlapping but not identical epitopes on HuBChE [16]. B2 18-5 paired with mAb2 when B2 18-5 was the capture antibody, but not in the reverse format when mAb2 was the capture antibody.…”

Section: Discussionmentioning

confidence: 99%

“…Use of these monoclonal antibodies for immunoextraction of BChE from human plasma is described in [16]. …”

Section: Methodsmentioning

confidence: 99%

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Monoclonal antibodies to human butyrylcholinesterase reactive with butyrylcholinesterase in animal plasma

Peng

Brimijoin

Hrabovská

et al. 2016

Chemico-Biological Interactions

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Five mouse anti-human butyrylcholinesterase (BChE) monoclonal antibodies bind tightly to native human BChE with nanomolar dissociation constants. Pairing analysis in the Octet system identified the monoclonal antibodies that bind to overlapping and independent epitopes on human BChE. The nucleotide and amino acid sequences of 4 monoclonal antibodies are deposited in GenBank. Our goal was to determine which of the 5 monoclonal antibodies recognize BChE in the plasma of animals. Binding of monoclonal antibodies 11D8, B2 18-5, B2 12-1, mAb2 and 3E8 to BChE in animal plasma was measured using antibody immobilized on Pansorbin cells and on Dynabeads Protein G. A third method visualized binding by the shift of BChE activity bands on nondenaturing gels stained for BChE activity. Gels were counterstained for carboxylesterase activity. The three methods agreed that B2 18-5 and mAb2 have broad species specificity, but the other monoclonal antibodies interacted only with human BChE, the exception being 3E8, which also bound chicken BChE. B2 18-5 and mAb2 recognized BChE in human, rhesus monkey, horse, cat, and tiger plasma. A weak response was found with rabbit BChE. Monoclonal mAb2, but not B2 18-5, bound pig and bovine BChE. Gels stained for carboxylesterase activity confirmed that plasma from humans, monkey, pig, chicken, and cow does not contain carboxylesterase, but plasma from horse, cat, tiger, rabbit, guinea pig, mouse, and rat has carboxylesterase. Rabbit plasma carboxylesterase hydrolyzes butyrylthiocholine. In conclusion monoclonal antibodies B2 18-5 and mAb2 can be used to immunoextract BChE from the plasma of humans, monkey and other animals.

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Online coupling of immunoextraction, digestion, and microliquid chromatography-tandem mass spectrometry for the analysis of sarin and soman-butyrylcholinesterase adducts in human plasma

et al. 2017

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Organophosphorus nerve agent (OPNA) adducts formed with human butyrylcholinesterase (HuBuChE) can be used as biomarker of OPNA exposure. Indeed, intoxication by OPNAs can be confirmed by the LC/MS analysis of a specific HuBuChE nonapeptide on which OPNAs covalently bind. A fast, selective, and highly sensitive online method was developed to detect sarin and soman adducts in plasma, including immunoextraction by anti-HuBuChE antibodies, pepsin digestion on immobilized enzyme reactors (IMER), and microLC/MS analysis of the OPNA adducts. The potential of three different monoclonal antibodies, covalently grafted on sepharose, was compared for the extraction of HuBuChE. The online method developed with the most promising antibodies allowed the extraction of up to 100% of HuBuChE contained in plasma and the digestion of 45% of it in less than 40 min. Moreover, OPNA-HuBuChE adducts, aged OPNA adducts, and unadducted HuBuChE could be detected (with S/N > 2000), even in plasma spiked with a low concentration of OPNA (10 ng mL). Finally, the potential of this method was compared to approaches involving other affinity sorbents, already described for HuBuChE extraction. Graphical abstract Online coupling of immunoextraction, digestion, and microliquid chromatography-tandem mass spectrometry for the analysis of organophosphorous nerve agent adducts formed with human butyrylcholinesterase.

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

Cited by 18 publications

References 25 publications

Monoclonal Antibodies That Recognize Various Folding States of Pure Human Butyrylcholinesterase Can Immunopurify Butyrylcholinesterase from Human Plasma Stored at Elevated Temperatures

Monoclonal Antibodies That Recognize Various Folding States of Pure Human Butyrylcholinesterase Can Immunopurify Butyrylcholinesterase from Human Plasma Stored at Elevated Temperatures

Monoclonal antibodies to human butyrylcholinesterase reactive with butyrylcholinesterase in animal plasma

Online coupling of immunoextraction, digestion, and microliquid chromatography-tandem mass spectrometry for the analysis of sarin and soman-butyrylcholinesterase adducts in human plasma

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