Immunoassays for avian butyrylcholinesterase: Implications for ecotoxicological testing and clinical biomonitoring

Khattab, Ahmed; Ali, Ibtisam S

doi:10.1016/j.etap.2007.06.006

Cited by 7 publications

(5 citation statements)

References 25 publications

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“…A protocol of competitive ELISA indicated a detection limit of 0.15 ng/ml when a type of polyclonal antibody was used for detection of butyrylcholinesterase content in avian blood (Khattab and Ali, 2007). Another protocol of competitive ELISA indicated a detection limit of 5 ng/ml when a type of monoclonal antibody was used for detection of ChE content in human serum (Kondo et al, 1995).…”

Section: Sensitivity Of Elisamentioning

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%

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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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Section: Sensitivity Of Elisamentioning

confidence: 99%

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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“…They exert their toxicity by phosphorylation of the serine hydroxyl group in the active site of acetylcholinesterase (AChE), leading to the inactivation of this enzyme which regulates the neurotransmitter acetylcholine. − Exposure to even small amounts of OPs is fatal. At present, four approaches including detection of unbound OPs, , measurement of metabolites, , assay of enzyme activity , and quantification of phosphorylated enzyme adducts , have been developed for evaluation of OPs exposure. Although measurement of unbound OPs and metabolites is a sensitive and accurate method, it is generally performed at laboratories using large equipment such as liquid or gas chromatography coupled with mass spectrometry (HPLC/GC/MS). − …”

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confidence: 99%

Electrochemical Detection of Dual Exposure Biomarkers of Organophosphorus Agents Based on Reactivation of Inhibited Cholinesterase

Yuan

Zhang

et al. 2013

Anal. Chem.

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Considering inter- or intra-individual variation in the normal levels of acetylcholinesterase (AChE), real-time measurement of AChE via the reactivation from a postexposure sample was used, and thus a baseline-free and reliable approach was proposed for detecting/screening low-dose organophosphorus pesticides (OPs) poisons. The principle of this technology is on the basis of parallel measurements of AChE activity before and after reactivation from a postexposure to simultaneously provide the content of dual biomarkers of both enzyme inhibition and enzyme adducts. It is more accurate and reliable compared with only one biomarker (inhibition or adduct). Reactivation from a postexposure sample is a better individual enzyme baseline compared to pre-exposure from the population average level in currently available approaches. AChE activity was measured with an electrochemical method. Greatly enhanced sensitivity was achieved by using Fe3O4/Au nanocomposites to enrich thiocholine, the hydrolysis product of active AChE, followed by electrochemical oxidative desorption of the adsorbed thiocholine. The validation of this method for measurement of OP exposures was further explored with in vitro paraoxon inhibited human red blood cells (RBCs) samples and demonstrated its practicability.

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“…Following exposure, OP pesticides and nerve agents readily interact with enzymes and proteins within the biological matrix to produce a number of relevant biomarkers, including the following: (i) OP adducts by phosphorylation of proteins, including enzymes, resulting in the loss of enzyme (e.g., cholinesterase) activity; (ii) metabolites by hydrolysis; (iii) unbound free OP in fluids . All are typical biomarkers of exposure to OP agents; therefore, some methods such as enzyme activity assays, immunoassays of OP adducts − and metabolites, and GC/LC MS analysis of OP adducts, metabolites, and free OPs have been developed for biomonitoring of exposure to OP agents. , However, for simple and rapid diagnosis/screening, especially in emergency cases, laboratory-based analytical methods (GC/LC MS) are not ideal because of lack of portability and lack of real time results. , Immunoassays of OP adducts or metabolites for diagnosis of OP exposure are challenged because of unavailability of OP-specific antibodies. Hence, the most utilized detection method is to measure enzyme activity, since it is a rapid and simple method for OP exposure evaluation and risk assessment. , Some methods, including the colorimetric Ellman assay, fluorescence assays, the Michel (ΔpH) ChE assay, radioactive assays, and the Walter Reed Army Institute of Research (WRAIR) assay, , have been developed.…”

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confidence: 99%

Magnetic Electrochemical Sensing Platform for Biomonitoring of Exposure to Organophosphorus Pesticides and Nerve Agents Based on Simultaneous Measurement of Total Enzyme Amount and Enzyme Activity

Wang

et al. 2011

Anal. Chem.

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We report a new approach for electrochemical quantification of enzymatic inhibition and phosphorylation for biomonitoring of exposure to organophosphorus (OP) pesticides and nerve agents based on a magnetic bead (MB) immunosensing platform. The principle of this approach is based on the combination of MB immunocapture-based enzyme activity assay and competitive immunoassay of the total amount of enzyme for simultaneous detection of enzyme inhibition and phosphorylation in biological fluids. Butyrylcholinesterase (BChE) was chosen as a model enzyme. In competitive immunoassay, the target BChE in a sample competes with the BChE immobilized on the MBs to bind to the limited sites of anti-BChE antibody labeled with quantum dots (QD-anti-BChE), followed by stripping voltammetric analysis of the bound QD conjugate on the MBs. This assay shows a linear response over the total BChE concentration range of 0.1-20 nM. Simultaneous real time BChE activity was measured on an electrochemical carbon nanotube-based sensor coupled with a microflow injection system after immunocapture by the MB-anti-BChE conjugate. Therefore, the formed phosphorylated BChE adduct (OP-BChE) can be estimated by the difference values of the total amount of BChE (including active and OP-inhibited) and active BChE from established calibration curves. This approach not only eliminates the difficulty in screening of low-dose OP exposure (less than 20% inhibition of BChE) because of individual variation of BChE values but also avoids the drawback of the scarce availability of OP-BChE antibody. It is sensitive enough to detect 0.5 nM OP-BChE, which is less than 2% BChE inhibition. This method offers a new method for rapid, accurate, selective, and inexpensive quantification of OP-BChE and enzyme inhibition for biomonitoring of OP and nerve agent exposures.

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Immunoassays for avian butyrylcholinesterase: Implications for ecotoxicological testing and clinical biomonitoring

Cited by 7 publications

References 25 publications

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

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

Electrochemical Detection of Dual Exposure Biomarkers of Organophosphorus Agents Based on Reactivation of Inhibited Cholinesterase

Magnetic Electrochemical Sensing Platform for Biomonitoring of Exposure to Organophosphorus Pesticides and Nerve Agents Based on Simultaneous Measurement of Total Enzyme Amount and Enzyme Activity

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