An Elisa assay for avian serum butyrylcholinesterase: A biomarker for organophosphates

Khattab, Ahmed; Walker, C.H.; Johnston, Gail; Siddiqui, M.K.J.; Saphier, P. W.

doi:10.1002/etc.5620131016

Cited by 21 publications

(12 citation statements)

References 19 publications

(2 reference statements)

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“…Sequential sampling of plasma ChE in the same organism is therefore a convenient method of investigating the percentage of inhibition resulting from exposure to OPs. A new approach based on immuno-chemical assays, for example, enzyme-linked immunoadsorbent assay, has also been used to determinate serum B esterase activities [20]. Even so, serum B esterases cannot be used as a biomarker of effect because their inhibition is not directly related to the molecular mechanism of the toxicity of OPs [3].…”

Section: Introductionmentioning

confidence: 99%

Serum B Esterases as a Nondestructive Biomarker in the Lizard Gallotia Galloti Experimentally Treated With Parathion

Sanchez¹,

Fossi²,

Focardi³

1997

Environ Toxicol Chem

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Abstract-Lizards (Gallotia galloti) were given either single or consecutive acute oral treatments of the organophosphorus (OP) insecticide parathion in two different experiments. Brain, serum, and liver microsomal esterase activities and liver microsomal monooxygenase activities were measured 6 and 24 h after the single acute treatment at each of four different doses (Experiment 1) or periodically up to 72 d after a number of consecutive acute treatments at two different doses (Experiment 2). Inhibition of serum butyrylcholinesterase (BChE) and carboxylesterase activities was observed in all treatment groups after 24 h and in the groups treated with 2.5, 5, and 7.5 mg/kg of parathion 6 h after treatment. Brain acetylcholinesterase (AChE) was inhibited at all doses after 6 h but only at the highest dose after 24 h. Highly significant nonlinear correlations, based on a piecewise linear regression model, were obtained between brain AChE activity and serum esterase activities at two sampling times after the single acute treatment. Liver microsomal carboxylesterase was found to be induced at the lower doses 6 and 24 h after treatment. Liver microsomal monooxygenase activity was higher 6 h after treatment than at 24 h, but the difference was not statistically significant. In Experiment 2, serum esterase activities recovered exponentially over a period of weeks. An increase in the recovery time to normal esterase activity was observed after each consecutive acute treatment. Brain AChE activity was inhibited at the end of consecutive administrations of parathion at the higher dose, and liver microsomal monooxygenase activity was inhibited at both doses. Symptoms of poisoning were observed in lizards treated with the higher dose of parathion, but no mortality was recorded. Two main conclusions can be drawn: (1) serum esterase activities recovered extremely slowly after acute treatment with parathion and even more slowly after consecutive acute treatments, and (2) there was a nonlinear correlation between the nondestructive biomarkers, serum ''B'' esterases, and the destructive biomarker, brain AChE, 6 and 24 h after exposure. These results suggest that G. galloti should be an ideal bioindicator organism to assess OP exposure in the Canary Islands instead of the birds commonly used for this purpose.

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Section: Introductionmentioning

confidence: 99%

Serum B Esterases as a Nondestructive Biomarker in the Lizard Gallotia Galloti Experimentally Treated With Parathion

Sanchez¹,

Fossi²,

Focardi³

1997

Environ Toxicol Chem

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“…The ELISA buffers used in the present study are the same as those described in the literature (51). The coating buffer was 0.1 M sodium carbonate͞bicarbonate buffer (pH 9.5).…”

Section: Site-directed Mutagenesis Protein Expression and Bche Actimentioning

confidence: 99%

“…Then, the wells were filled with 100 l of goat anti-mouse IgG horseradish peroxidase conjugate complex diluted to a final 1:3,000 dilution and were incubated at room temperature for 1.5 h, followed by washing four times. The enzyme reactions were started by addition of 100 l of substrate (3,3Ј, 5,5Ј-tetramethylbenzidine) solution (51). The reactions were stopped after 15 min by the addition of 100 l of 2 M sulfuric acid, and the absorbance was read at 460 nm by using a Bio-Rad ELISA plate reader.…”

Section: Site-directed Mutagenesis Protein Expression and Bche Actimentioning

confidence: 99%

Computational redesign of human butyrylcholinesterase for anticocaine medication

Pan

Gao

Yang

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

173

312

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Molecular dynamics was used to simulate the transition state for the first chemical reaction step (TS1) of cocaine hydrolysis catalyzed by human butyrylcholinesterase (BChE) and its mutants. The simulated results demonstrate that the overall hydrogen bonding between the carbonyl oxygen of (؊)-cocaine benzoyl ester and the oxyanion hole of BChE in the TS1 structure for (؊)-cocaine hydrolysis catalyzed by A199S͞S287G͞A328W͞Y332G BChE should be significantly stronger than that in the TS1 structure for (؊)-cocaine hydrolysis catalyzed by the WT BChE and other simulated BChE mutants. Thus, the transition-state simulations predict that A199S͞ S287G͞A328W͞Y332G mutant of BChE should have a significantly lower energy barrier for the reaction process and, therefore, a significantly higher catalytic efficiency for (؊)-cocaine hydrolysis. The theoretical prediction has been confirmed by wet experimental tests showing an Ϸ(456 ؎ 41)-fold improved catalytic efficiency of A199S͞S287G͞A328W͞Y332G BChE against (؊)-cocaine. This is a unique study to design an enzyme mutant based on transitionstate simulation. The designed BChE mutant has the highest catalytic efficiency against cocaine of all of the reported BChE mutants, demonstrating that the unique design approach based on transition-state simulation is promising for rational enzyme redesign and drug discovery. molecular dynamics ͉ rational design ͉ transition-state stabilization ͉ cocaine ͉ enzyme-substrate binding C ocaine is recognized as the most reinforcing of all drugs of abuse (1-3). The disastrous medical and social consequences of cocaine addiction have made the development of an effective pharmacological treatment a high priority (4-6). However, cocaine mediates its reinforcing and toxic effects by blocking neurotransmitter reuptake, and the classical pharmacodynamic approach has failed to yield small-molecule receptor antagonists because of the difficulties inherent in blocking a blocker (1-5). An alternative to receptor-based approaches is to interfere with the delivery of cocaine to its receptors or accelerate its metabolism in the body (5,(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). An ideal molecule for this purpose should be a potent enzyme catalyzing the hydrolysis of cocaine into biologically inactive metabolites. The dominant pathway for cocaine metabolism in primates is butyrylcholinesterase (BChE)-catalyzed hydrolysis at the benzoyl ester group (Fig. 3, which is published as supporting information on the PNAS web site), and the metabolites are all biologically inactive (5, 18). Clearly, BChE-catalyzed hydrolysis of cocaine at the benzoyl ester is the metabolic pathway most suitable for amplification. However, the catalytic activity of this plasma enzyme is Ϸ1,000-fold lower against the naturally occurring (Ϫ)-cocaine than that against the biologically inactive (ϩ)-cocaine enantiomer (19)(20)(21)(22). (ϩ)-cocaine can be cleared from plasma in seconds, before partitioning into the CNS, whereas (Ϫ)-cocaine has a plasma half-life of Ϸ45-90 min, long enough for ...

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“…This process may compromise the determination of the actual inhibition of ChE. As indicated by Khattab et al (1994) and Li et al (2005), the problem could be solved by predetermination of ChE-IR levels using antibodies. The results of the present study suggest that the polyclonal antibodies developed against the recombinant protein ChE are as efficient as those developed against the native ChE in detecting ChE content in Daphnia magna exposed to anticholinesterase.…”

Section: Applicability Of Antiserummentioning

confidence: 99%

“…To ascertain the "normal" activity of ChE in samples, a technique is required that is capable of quantifying the content of the enzyme. Due to its quick, sensitive, and cost-effective nature, an immunoassay, such as enzyme-linked immunosorbent assay (ELISA), is well suited for fulfilling this task (Khattab et al, 1994;Li et al, 2005;Khattab and Ali, 2007).…”

Section: Introductionmentioning

confidence: 99%

Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna

Liu

Yuan

2016

J. Zhejiang Univ. Sci. B

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Abstract:To yield cholinesterase (ChE) from prokaryotic expression, the ChE gene that belongs to Daphnia magna was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using forward primer 5'-CCCYGGNGCSAT GATGTG-3' and reverse primer 5'-GYAAGTTRGCCCAATATCT-3'. To express the gene, one sequence of the amplified DNA, which was able to encode a putative protein containing two conserved carboxylesterase domains, was connected to the prokaryotic expression vector PET-29a(+). The recombinant vector was transformed into Escherichia coil BL21 (DE3). Protein expression was induced by isopropy-D-thiogalactoside. The expressed ChE was used as an immunogen to immunize BALB/c mice. The obtained antibodies were tested for their specificity towards crude enzymes from species such as Alona milleri, Macrobrachium nipponense, Bombyx mori, Chironomus kiiensis, Apis mellifera, Eisenia foetida, Brachydanio rerio, and Xenopus laevis. Results indicated that the antibodies had specificity suitable for detecting ChE in Daphnia magna. A type of indirect and non-competitive enzyme-linked immunosorbent assay (IN-ELISA) was used to test the immunoreactive content of ChE (ChE-IR) in Daphina magna. The detection limit of the IN-ELISA was found to be 14.5 ng/ml at an antiserum dilution of 1:22 000. Results from tests on Daphnia magna exposed to sublethal concentrations of triazophos indicated a maximal induction of 57.2% in terms of ChE-IR on the second day after the animals were exposed to a concentration of 2.10 μg/L triazophos. Testing on animals acclimatized to a temperature of 16 °C indicated that ChE-IR was induced by 16.9% compared with the ChE-IR content detected at 21 °C, and the rate of induction was 25.6% at 10 °C. The IN-ELISA was also used to test the stability of ChE-IR in collected samples. Repeated freezing and thawing had no influence on the outcome of the test. All these results suggest that the polyclonal antibodies developed against the recombinant ChE are as efficient as those developed against the native ChE in detecting ChE content in Daphnia magna.

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An Elisa assay for avian serum butyrylcholinesterase: A biomarker for organophosphates

Cited by 21 publications

References 19 publications

Serum B Esterases as a Nondestructive Biomarker in the Lizard Gallotia Galloti Experimentally Treated With Parathion

Serum B Esterases as a Nondestructive Biomarker in the Lizard Gallotia Galloti Experimentally Treated With Parathion

Computational redesign of human butyrylcholinesterase for anticocaine medication

Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna

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