Detection of Organophosphate Bound Butyrylcholinesterase Using a Monoclonal Antibody

Appl Biol Chem (2017) 60(3):233–240 Online ISSN 2468-0842 DOI 10.1007/s13765-017-0272-1 Print ISSN 2468-0834

ARTICLE

Detection of organophosphate bound butyrylcholinesterase using a monoclonal antibody

Sang-Jin Park1,2 . Dong-Hwan Kim1 . Jeongha Yoo1 . Eun Young Hwang3 . Moon-Sik Shin4 . Nam-Taek Lee5 . Il-Rae Cho1 . Hee-Gun Kang1 . Young-Jin Kim1 . Sungman Park1 . Yoon-Won Kim2,3

Received: 14 March 2017 / Accepted: 23 March 2017 / Published online: 5 April 2017 Ó The Korean Society for Applied Biological Chemistry 2017

Abstract In this study, a specific antibody against monoclonal antibody may be used in a competitive ELISA butyrylcholinesterase (BuChE) bound to organophosphate to detect the soman-iBuChE. Taken together, we suggest (OP) nerve agents was developed to be used in enzyme- that the antibody could be applied to evaluate the soman linked immunosorbent assays (ELISA). OP nerve agents presence in blood, serum, and urine. such as sarin, soman, and VX are known to act on the neuromuscular junctions and were synthesized at the Armed Keywords Butyrylcholinesterase Á Diagnostic monoclonal Force Chemical, Biological, and Radiological Defense antibody Á Nerve toxic agents Á Organophosphate Command in Republic of Korea and studied after confirming inactivation of BuChE. Each inactivated OP-BuChE (OP- iBuChE) was used as the antigen for developing monoclonal Introduction antibodies (mAbs) in mice for detection of OP bound BuChE. The fusion was performed after immunization and Nerve agents (NAs) including many organophosphate (OP) then hybridoma cells for the antibodies were generated. As a pesticides continue to pose a health risk and significant result, the partial specific antibody against soman-iBuChE global security concern of terrorism. NAs are alkylphos- was confirmed via ELISA. Thus, the antibody may recog- phonic acid esters. Sarin and soman are methylphospho- nize three-dimensional structure of OP-iBuChE that is nofluoridate containing a fluorine substituent group. VX is changed upon soman and BuChE reaction, not an OP an alkylphosphonothiolate containing sulfur group. These binding pocket of BuChE. Furthermore, this partial specific NAs have a unique C–P bond that is not found in OP pesticides and is very hydrolysis resistant. Moreover, binding reactions of NAs to esterases such as acetyl- Sang-Jin Park, Dong-Hwan Kim and Jeongha Yoo have equally cholinesterase (AChE), cholinesterase (ChE), car- contributed to this paper as first authors. boxylesterases (CarbE), and other proteins occur by & Yoon-Won Kim hydrolysis. Both OP pesticides and NAs lose their acyl [email protected] radicals after their reaction with the esterase [1]. As OP nerve agents enter blood, they cause over stimulation of the 1 Division of Diagnostic Research and Development, ImmuneMed Inc, Chuncheon 24232, Republic of Korea cholinergic nervous system, paralysis, and ultimately death by inactivation of acetylcholinesterases (AChE) and 2 Department of Microbiology, College of Medicine, Hallym University, Chuncheon 24252, Republic of Korea butyrylcholinesterases (BuChE) critical to the central nervous system [2, 3]. In other words, AChE and BuChE 3 Medical Science Institute, College of Medicine, Hallym University, Chuncheon 24252, Republic of Korea inactivates the neurotransmitter acetylcholine (Ach) and is hence viable therapeutic targets in OP nerve agents, which 4 Armed Force Chemical, Biological, and Radiological Defense Command, Seoul, Republic of Korea is characterized by a cholinergic toxidrome. OP nerve agents in air first covalently bind BuChE that is 5 Institute for National BioDefense Research, College of Life Science and Biotechnology, Korea University, Seoul 02841, present in large quantities in plasma, even though AChE is Republic of Korea 1.5 * 4 times higher in affinity to OP than BuChE [4]. 123 234 Appl Biol Chem (2017) 60(3):233–240

Moreover, the diagnostic test using AChE is out of detection retrospective detection of exposure [13–15]. There are range for OP because there is a very low amount of AChE in about four approaches to detect exposure to NAs: AChE plasma. Therefore, it is useful to analyze BuChE covalently activity measurement, determination of OP hydrolysis bound to OP nerve agents to know the kind of nerve agents. products in plasma and urine, fluoride reactivation of Furthermore, Manoharan et al. reported that BuChE was phosphonylated binding sites, and mass spectrometric about 700 times higher in activity than AChE in plasma determination of cholinesterase adducts [3]. Of the when the result of comparison with esterase activity of approaches listed above, the colorimetric assay lacks sen- BuChE and AChE was 5.6 ± 1.2 and 0.008 ± 0.001 U/g, sitivity and specificity, while gas chromatography/tandem respectively [5]. In general, plasma of adult human is about mass spectrophotometer (GC/MS–MS) methods are cur- 3500 g, total BuChE units in plasma are 19,600 (about rently limited to laboratory analyses, are time-consuming, 196 nmol), and total AChE units in plasma is 20 (about and require highly trained personnel. Therefore, rapid, 0.05 nmol). Finally, BuChE is 4000 times more than AChE sensitive, and field-deployable methods are still needed. In in plasma and is superior to detect the infiltration and con- other words, new or improved diagnostic techniques or tamination of OP nerve agents in blood. After NAs bind to tools can provide rapid and reliable evaluation of OP-agent AChE and BuChE, there is a non-enzymatic time-dependent exposure, and will enhance our ability to respond quickly intramolecular rearrangement which leads to loss of one to an emergency and thus will improve our medical capa- alkyl group bound to the phosphorus, known as ‘‘aging bility against OP exposure. Recently, immunochromato- reaction’’ which means the time between NA exposure and graphic test strips using anti-OP-HSA antibody against OP- irreversible phosphonylation [3]. Phosphonylated AChE and HSA peptide [16] and anti-AChE antibody against AChE BuChE may be subject to be spontaneously changed [17] have been developed as a rapid diagnostic test kit. dealkylation (aging) and dephosphonylation (reactivation) However, these antibodies as detection reagents of OP have [6]. Herein, aging proceeds through P–O bond scission and low sensitivity and specificity because they are not the this acid-catalyzed process results in formation of a nega- antibodies specific enough to recognize specific binding tively charged phosphonyl-cholinesterase adduct [7, 8]. sites between OP and HSA or OP and AChE. Based on Thus, early diagnosis of OP nerve agent exposure is essen- these findings, we present the first report on development tial to do therapy prior to onset of life threatening signs. of the monoclonal antibody recognizing the three-dimen- Therefore, use of OP-BuChE as a biomarker to detect sional structure of inactivated soman-BuChE adduct for exposure to nerve agents, there are several advantages as detection of soman in intoxicated persons. In this study, the follows: (1) BuChE is present in large quantities in human monoclonal antibody recognizing the three dimensionally plasma and tissue that is easy to obtain, (2) BuChE reacts changed structure of BuChE by soman was developed. This rapidly with low doses of OP esters, (3) OP makes an monoclonal antibody can be used for diagnosis the soman irreversible covalent bond with BuChE, and it is stable in contaminated person more rapidly and easily. the circulation as well as in stored plasma [9], (4) the half- life of BuChE is 12 days in the circulation; it means that blood can be drawn several days after an incident to detect Materials and Methods OP-BuChE adducts [10], (5) methods to extract BuChE from plasma in a single step by binding to immobilized Synthesis of organophosphate nerve agents monoclonal antibody have been developed [11], (6) digestion of BuChE with pepsin yields the active site- Human butyrylcholinesterase (BuChE) was purchased containing peptide modified on Ser198 by OP. A small- from Mybiosource, Inc (San Diego, CA, USA). BuChE sized peptide has an advantage in mass spectral analysis was dissolved in phosphate buffered saline (PBS) at pH 7.4 where small peptides ionize more readily than large pep- containing 0.02% of sodium azide(NaN3). Organophos- tides [11, 12], and (7) mass spectrometry methods have phate (OP) nerve agents (NAs) were synthesized and been developed by the TNO laboratory in Rijswijk and approved for binding out from the Armed Force Chemical, adopted worldwide to diagnose OP exposure by analyzing Biological, and Radiological (CBR) Defense Command in OP adducts on BuChE [11]. Republic of Korea. The NAs were dissolved in PBS at pH Most research on diagnostic methods of NA exposure 7.4 to make 2.2 mM solutions and used immediately. has been directed at the most available samples of exposed person such as the exposed blood (serum, plasma, whole Preparation of organophosphate- blood, or red cells) and urine. Intact NAs are available in butyrylcholinesterase adducts the organism for a few hours; therefore, blood sampling should be obtained within a few hours after OP exposure. To make OP-BuChE adduct, 1.68 mL of BuChE (49.4 Thus, intact agents do not seem to be a good target of nmole) in PBS (pH 7.4) was reacted with 0.84 mL 123 Appl Biol Chem (2017) 60(3):233–240 235 organophosphate nerve agents (2.2 lmol) at 25 °C for 4 or 100 ng of BuChE in 100 ll was used to coat each well for 24 h. BuChE and NAs were mixed at 1:45 molar ratio to 2 h at room temperature. After washing, the plates were produce OP-BuChE adducts. The molar ratio is enough to blocked with 200 ll of blocking buffer (0.015 M PBS achieve 100% inactivation of BuChE. containing 0.1% tween 20, 0.5% Bovine serum albumin) for 1 h. For screening of monoclonal antibodies, the Measurement of butyrylcholinesterase enzymatic supernatant from each clone of hybridoma cells was activities incubated for 2 h at room temperature. After washing as above, 100 ll of diluted goat anti-mouse IgG-HRP conju- The enzymatic activity of OP-iBuChE mixture was mea- gate antibody was added for 1 h at room temperature. After sured using the Ellman’s method [18, 19]. Briefly, 0.5 llof washing as described, 100 ll of TMB substrate was added, the OP and BuChE mixture was treated with 0.5 mM 5,5- and incubated for 7 min at RT. The reaction was stopped dithiobis (2-nitrobenzoic acid) (DTNB) and 1 mM using equal volume of 2 N H2SO4. Then the absorbance of butyrylthiocholine (Sigma-Aldrich, MI, USA) in 1 mL of reactant was measured using UV spectrophotometer at 0.1 M potassium phosphate buffer at pH 7.0 for 10 min at 450 nm. room temperature (RT). The inactivation was measured by absorbance of the mixture at 412 nm wavelength using the Purification of monoclonal antibody Spectra Max M5 microplates reader (Molecular Device, from hybridoma supernatant LLC., CA, USA). To measure the inhibitory proportion of iBuChE conjugated to nerve agents, the free nerve agents, The hybridoma cells were cultured in a tissue culture plates not conjugated to BuChE, were eliminated by using with DMEM containing 10% FBS and 1:100 penicillin/ molecular weight cutoff centrifugal filter (10 kDa cutoff) streptomycin, and then, the culture medium was replaced to and centrifuged for 5 min at 10,000 rpm, 4 °C, total six a serum-free DMEM. The cultured hybridoma was col- times. The residual OP in the filtrated solution was mea- lected and centrifuged at 2500 rpm for 5 min, 4 °C. After sured and assayed by GC/MS–MS. collected supernatant was filtered with the 0.22 lm filter top, supernatant was applied to equilibrated protein A Preparation of hybridoma cells producing agarose column at 4 °C. After washing, antibodies were monoclonal antibody eluted with 100 mM glycine, pH 2.5, and then neutralized with 1 M Tris, pH 9.5. The obtained fractions were dia- Six-wk-old female BALB/C mice were included in the lyzed in PBS overnight, and the antibody concentrations study, and four female BALB/c mice were immunized with were measured. BuChE containing aluminum chloride and incomplete Freund’s adjuvants at a dosage of 20 lg protein per mouse through footpad manner. After 2 weeks, serum was col- Results lected from four mice and then antibody titer was measured with ELISA. After antibody titration, the mice were In this study, we demonstrated the detection of inactivated boosted with the same protein dose every 2 weeks. If the BuChE mediated by OP nerve agents using monoclonal serum antibody titer is over 1.5 OD at 1:1000 dilution as a antibodies. To evaluate the binding between iBuChE and result of ELISA, the mouse was killed and splenocytes OPs, we modeled the structures of BuChE binding nerve were isolated. The cells were fused with Sp2/0-Ag14 agents including sarin, soman, or VX. myeloma cells using polyethylene glycol. The fused cells Before immunization for production of mAbs against were selected and proliferated in HAT medium to eliminate OP-iBuChE, the binding site between BuChE and OPs unfused cells. The selected fused cells were transferred in were investigated by using 3D molecular docking and DEME medium. The hybridoma cells producing mono- virtual screening program (AutoDockVina). As shown in clonal antibodies (mAbs) were subcloned through the Fig. 1, the structure of the human BuChE active site gorge limiting dilution method followed by direct ELISA is schematized [20]. The toxic mechanism of OP is based measurement. on the interaction and irreversible inhibition of BuChE by phosphorylation and phosphonylation of the active site Screening of monoclonal antibody against BuChE Ser198 [21, 22]. The comparison of aged and non-aged and OP-iBuChE using ELISA forms of BuChE is shown in Fig. 2A and yellow and purple structures indicate non-aged form and aged form, respec- The plates were incubated with 100 ll of coating buffer tively. These structures have a little difference at 0.215 (0.2 M sodium carbonated, pH 9.2) overnight at 4 °C. angstroms (A˚ ) RMSD (root mean square deviation). After three washes of 0.015 M PBS with 0.1% tween 20, Therefore, it may be difficult to produce specific mAbs to 123 236 Appl Biol Chem (2017) 60(3):233–240

The OP nerve agents react with BuChE in plasma. The reactants inhibit the enzymatic activities of BuChE. The inactivation of BuChE by sarin, soman, and VX was measured by Ellman’s assay [18, 19]. Active BuChE was diluted in twofold series and used as the criteria of standard activation. The inactivation of BuChE was measured when the value of BuChE by OP is over 0.99. After the 4-hour reaction, BuChE retains 0.9% activity. In the 24-hour reaction, BuChE was completely inactivated (data not shown). Therefore, all experiments were performed after the complete inactivation of the OPs. Moreover, OP nerve agents were mostly toxic compounds in mammals and insects. Hence, it is important that OP nerve agent must be eliminated to make specific antibodies using animal model from the reaction solutions. To remove the residual of OP nerve agents, the reacted solution was filtrated by molecular weight cutoff filter. After that, the concentration of free OP nerve agents was confirmed using GC/MS–MS whether the residual of OP nerve agents exists or not. As shown in Table 1, the residuals of sarin, soman, and VX were confirmed to be under 1 part per billion (ppb), 1 and Fig. 1 Schematic representation of the cholinesterase active site of butyrylcholinesterase monomer. This figure was adapted from an 100 ppb, respectively. Although free OP nerve agents were image in the butyrylcholinesterase study [20] and modified by authors not completely eliminated from reaction compared with the of this work. The average depth of BuChE gorge is 20 A˚ , and the standard, the OP nerve agents were injected to mice at ˚ average entrance radius is 2.75 A with the maximum radius of 0.1 ng/ll with the injection volume under LD without 4.00 A˚ . Antibody (Ab) size is 52 A˚ in diameter and 150 A˚ in length 50 clinical symptom. As a result, the amount of native BuChE, sarin-iBuChE, soman-iBuChE, and VX-iBuChE as anti- be able to distinguish aged form from non-aged form due to gens for immunization was produced with 7.80, 2.88, 2.59, little difference of 3D model for two structures. Figure 2B and 1.79 mg, respectively. is magnified from red circle of Fig. 2A and shows the The affinity of each antibody against OP-iBuChE was binding site for OP nerve agents. The prediction image in assessed by ELISA using the sera which is gathered from Fig. 2C shows binding between Ser198 and OP through mouse after second or third boosting for monoclonal anti- aging pathway and the red circle indicates the phosphory- body against OP-iBuChE. The ratio of serial dilution is lation between OP and BuChE residue Ser198. Herein, the 1:100, 1:1000, 1:5000, 1:10,000, 1:50,000, and 1:100,000 prediction image for binding Ser198 and OP was drawn with 1 9 PBS. The fusion was processed when the titer of with PBD ID 1p0i of non-aged form among the known 1:1000 diluent is over 1.5 OD at 450 nm. Each anti-OP- BuChE structures. As shown in Fig. 2D–G, these dimen- inactivated BuChE (anti-OP-iBuChE) monoclonal anti- sional structures represent binding and phosphorylation of bodies were evaluated for its affinity (specificity) by using inactivated sarin-BuChE, soman-BuChE, VX-BuChE, and different concentration of antigen and antibodies. Among diazinon-BuChE, respectively. them, binding affinity of the anti-soman-iBuChE mono- OP nerve agents such as sarin, soman, and VX were clonal antibody was confirmed only in soman-BuChE synthesized in the Armed Force CBR Defense Command in adduct. Three of the confirmed antibodies were presented Republic of Korea. In order to determine the purity and with highly OD value against antigen protein and each amount of the synthesized OP nerve agents including sarin, antibody showed for similar level of OD. For ELISA, soman, and VX, the synthesized OPs were measured using active forms of BuChE or soman-BuChE were used to coat GC/MS–MS and the chromatograms are shown in Fig. 3. 96-well microplates at 100 ng/well. Each antibody was The purity of the synthesized OPs was determined to be added in twofold dilution series and absorbance was 97.8% for sarin, 99.5% for soman, and 62.6% for VX, and measured by ELISA. The result of binding capacity of anti- their concentrations were 41.2, 55.2, and 86.3 mg, soman-iBuChE monoclonal antibodies for BuChE and respectively (Table 1). Although lower purity (62.6%) for soman-iBuChE is shown in Fig. 4. The concentration of nerve agent VX indicated its impaired structure, actually anti-soman-iBuChE monoclonal antibody was saturated at the VX inactivated the enzymatic activities of human 500 ng/mL of concentration of BuChE and soman- BuChE (data not shown). iBuChE, and then, soman-iBuChE was two times higher 123 Appl Biol Chem (2017) 60(3):233–240 237

Fig. 2 Position of Ser198 at the three-dimensional structure of BuChE that is possible to be phosphorylated by OP (A, B). Three-dimensional structure of BuChE Ser198 phosphorylated (C) and the OP-BuChE structures for sarin-iBuChE (D), soman-iBuChE (E), VX- iBuChE (F), and diazinon- iBuChE (G)

than native BuChE for binding afﬁnity of anti-soman- deep and narrow active site gorge consisting of approxi- iBuChE antibodies. In other words, anti-soman-iBuChE mately 55 residues [20]. The OP binding site of BuChE monoclonal antibody shows higher afﬁnity to soman- containing Ser198 catalytic triad of esteratic site is located iBuChE than BuChE although it shows cross-reactivity. at the center of the subunit at the end of a deep and narrow gorge. Furthermore, Asp70 and Tyr332 residues of peripheral anionic site (PAS) at the mouth of the gorge are Discussion involved in activation control and in the initial binding of positively charged substrates such as choline esters. These In conclusion, the developed anti-soman-iBuChE antibody two residues control the functional architecture of the was secreted from hybridoma cells and could be used to BuChE active site gorge, and they have a hydrogen bond evaluate the intoxication of soman among OP nerve agents. [23]. Furthermore, the average entrance radius of the BChE As shown in Fig. 1, the monomer of BuChE has a 20 A˚ monomer is 2.75 A˚ with the maximum radius of 4.00 A˚ , 123 238 Appl Biol Chem (2017) 60(3):233–240

Fig. 3 Synthesis of organophosphate (OP) nerve agents including sarin (A), soman (B), and VX (C). The synthetic OPs were measured by GC/MS–MS and chemical structures generated using ChemDraw software

which is larger than that of the AChE [24, 25]. In general, to the B cell surface, and it may be more inaccessible to the antibody (Ab) size is 52 A˚ in diameter, 150 A˚ in length. gorge by hindrance of B cell surface antibody. In other Thus, the antibody against the OP binding site of BuChE words, the speciﬁc antibody could not get in the binding may be difﬁcult to interact with Ser198 of BuChE. More- pockets of Ser198. In addition, the progressive inhibition of over, during the development, the antibodies are attached cholinesterase by OPs is due to phosphorylation or

123 Appl Biol Chem (2017) 60(3):233–240 239

Table 1 Reaction conditions between BuChE and OP, and the residual amount of OP after ﬁltration Native BuChE (14.4 mg) Sarin (41.2 mg) Soman (55.2 mg) VX (86.3 mg)

1 mol 68,000 g 140.09 g 182.18 g 267.36 g 1:45a 1 g 0.09 g 0.12 g 0.18 g Residual amount of OP – \1 ppbb \1 ppbb \100 ppbb Produced amount of OP-iBuChE 7.80 mg 2.88 mg 2.59 mg 1.79 mg a Treatment of organophosphate nerve agent (sarin, soman, or VX) to BuChE can lead to 100% deactivation of BuChE when it was treated less than 45 times based on the molar concentration of BuChE. The ratio of 1:45 is when OP was treated 0.09 g of sarin, 0.12 g of soman, and 0.18 g of VX per g of each BuChE b ppb (part per billion)

rapidly. And it is required to further develop the speciﬁc antibodies for discriminating between BuChE and OP-inactivated BuChE to further screen the intoxicated persons.

Acknowledgments This work was supported by the Institute of Civil Military Technology Cooperation (15-SN-SS-04), South Korea.

References

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