Monoclonal Antibody-Based Enzyme Immunoassay for Mercury(II) Determination

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<ul><li><p>Mon Imfor M</p><p>Alexa</p><p>DepartmAlte Aka</p><p>A moand appdetermilimit offormat inoncombest demeasuriwere diquentlywas bet</p><p>Theand thbiologiand caplace inBesidemost tbe founHg, Hgthe higtoxic, fment it is consequently not only interesting to deter-mine the total amount of mercury but also the differentspecies.</p><p>Thelyzedspectrospeciatimmunmetal</p><p>enes.hetwl</p><p>nsthensoggn</p><p>the hb)</p><p>wme</p><p>ER</p><p>ria</p><p>ers</p><p>rbPhosphate-buffered saline (PBS), 40 mmol/L, pH 7.2</p><p>(10.15 mol/L NaCl).PBS washing buffer: PBS diluted 1:10, containing</p><p>0.5 ml/L Tween 20.</p><p>1 Tohock@we</p><p>METHODS 22, 4952 (2000)doi:10.1006/meth.2000.1035, available online at http://www.idealibrary.com on</p><p>1046-2023CopyrightAll rightstotal amount of mercury is conventionally ana-by physical methods, e.g., atomic absorptionscopy. Most of these methods are not suited forion of mercury at a low-cost level. In contrastoassays are simple and inexpensive tools forspeciation. Large numbers of samples can be</p><p>Substrate solutions for horseradish peroxidase(POD): (I) tetramethylbenzidine (TMB) 1.2 mmol/L,H3PO4 8.0 mmol/L, penicillin G 12.0 mg/L, dimethylsulfoxide (DMSO) 10.0% (v/v); (II) hydrogen peroxide 3mmol/L, Na2HPO4 z 12 H2O 3.5 mmol/L, NaH2PO4 z1H2O 132.0 mmol/L; one part of solution (I) and twoparts of solution (II) were mixed shortly before use.</p><p>Stopping solution for POD: 1 mol/L H2SO4.whom correspondence should be addressed. E-mail:ihenstephan.de.</p><p>49/00 $35.00 2000 by Academic Pressof reproduction in any form reserved.oclonal Antibody-Based Enzymeercury(II) Determination</p><p>nder Marx and Bertold Hock1</p><p>ent of Botany, Technische Universitat Munchen at Weihenstephan,demie 12, D-85350 Freising, Germany</p><p>noclonal antibody (K3C6) was developed against Hg(II)lied in different enzyme immunoassay (EIA) formats to</p><p>ne the test system with the highest sensitivity. A detection1.0 mg/L Hg(II) could be achieved with a competitiven contrast to a detection limit of 2.1 mg/L Hg(II) with apetitive EIA. A competitive displacement EIA yielded thetection limit of 0.4 mg/L Hg(II) and was well suited tong real samples. For this purpose different water samplesluted at least 1:10 to avoid matrix effects and subse-spiked with 1 mg/L HgCl2. Recovery of the spiked samplesween 80 and 120%. 2000 Academic Press</p><p>toxicity of mercury depends on the metal speciese route of uptake into the body (1). It passescal membranes very easily, can bind to enzymes,n disrupt vital functions. Accumulation takesthe body because of its highly lipophilic nature.</p><p>s organic mercury compounds, which are theoxic, there are three inorganic species that cand as environmental contaminants: elementary(I), and Hg(II). Vapor of elementary Hg showshest lipophilic properties and is therefore veryollowed by Hg(II) and Hg(I). For toxicity assess-</p><p>screhourfor taremetaagaibutagaimunrecoas in</p><p>W(MAMAbthe</p><p>MAT</p><p>Mate</p><p>Buff</p><p>Camunoassay</p><p>d for the occurrence of a metal species withinSeveral antibodies (Abs) were already developeddetection of heavy metals (27). Basically thereo approaches to generating Abs: (1) The heavyis bound to a chelator, e.g., EDTA; Abs raisedt this complex do not recognize the metal itself</p><p>entire structure. (2) Abs are produced directlyt the heavy metal attached to a suitable im-en; the advantage of this method is that the Abizes the free metal and not a cagelike structure</p><p>e first approach.ave recently developed the monoclonal antibodyK3C6, which is directed against Hg(II) (8). Thisas used to improve assay conditions suited forasurement of water samples.</p><p>IALS AND METHODS</p><p>ls</p><p>and Solutions</p><p>onate buffer, 50 mmol/L, pH 9.6.</p></li><li><p>Mercury(II) StandardsHgC</p><p>solutio10). Thfurtherstandawere e</p><p>BSAGBovi</p><p>conjugaScarpapared a</p><p>AntiboThe</p><p>has beproteincell culwere p</p><p>MethodNoncom</p><p>MercBSA</p><p>surfacenight,90 minwere amin indryl gK3C6 (was admL PO(1:10,0bufferexceptnally t</p><p>resulting in the development of a blue color propor-l t</p><p>petiortwaagede.</p><p>peA1</p><p>ba00inusifics b</p><p>peicrblogC</p><p>e Ml2tetioter</p><p>enupe</p><p>effiugaI)-d walsa</p><p>FIG. 1.HgCl2. A7.2 to 10</p><p>2.wh</p><p>ons. Th</p><p>50 MARX AND HOCKl2 1 g/L was dissolved in 2% (v/v) HNO3 (stockn) to prevent loss of mercury during storage (9,e stock solution was stable for 1 month. It wasdiluted with PBS for the preparation of Hg(II)</p><p>rds. Safety precautions, e.g., wearing gloves,mployed in handling mercury(II).</p><p>SH Conjugatene serum albuminglutathione (BSAGSH)tes were provided by Professors A. Rigo and M.(University of Padova, Padua, Italy) and pre-s described (8).</p><p>diesMAb K3C6 (IgG1), directed against mercury(II),en described recently (8). It was purified by</p><p>A affinity chromatography from protein-freeture supernatant. Labeled goat anti-mouse Absurchased from Pierce and Sigma.</p><p>spetitive Enzyme Immunoassay against</p><p>ury(II)GSH conjugate (10 mg/mL) was adsorbed to the</p><p>of a 96-well microtiter plate (200 ml/well, over-4C). After blocking with 300 mL 1% gelatin for, Hg(II) standards ranging from 0.1 to 1000 mg/Ldded to the wells (200 ml/well). During the 30-cubation period Hg(II) was bound to the sulfhy-roups of the BSAGSH conjugate. The MAb200 ml/well, diluted 1:5000, 1-h incubation time)ded to each well, followed by incubation of 200D-labeled second anti-mouse Ab for 1 h</p><p>00). The plates were washed with washingbetween steps. All dilutions were made in PBSfor the coating reaction (carbonate buffer). Fi-he enzymesubstrate reaction was carried out,</p><p>tionstopsorp(Titeysispackpeatrang</p><p>ComBS</p><p>withincu(1:25werewasspectime</p><p>ComM</p><p>andmL Hof thHgCcubamencroti</p><p>InfluCoTh</p><p>theconjHg(Itilleent sthe</p><p>Influence of pH on the absorption in the EIA at 100 mg/Lbsorptions were normalized by setting the absorption at pH0%.</p><p>FIG.tions,soluti100%o the amount of bound Hg(II). The reaction wasd with H2SO4, yielding a yellow color, and ab-n was measured at 450 nm with an EIA readerek Multiscan II, Flow Laboratories). Data anal-s carried out with a commercial EIA softwaree (EIA3, Flow Laboratories). The assay was re-at least three times to calculate the mean test</p><p>titive EIAGSH-coated plates (10 mg/mL) were blocked% gelatin. Then 200 mL HgCl2 (100 mg/L) wasted for 30 min. Subsequently 100 mL of K3C6) and 100 mL HgCl2 in the range 0.1100 mg/Lcubated for 1 h. A second Ab labeled with PODed in the last step for the detection of boundAb (see above). The plates were washed threeetween incubation steps.</p><p>titive Displacement EIA for Hg(II)otiter plates coated with BSAGSH (10 mg/L)cked with 1% gelatin were incubated with 200l2 (100 mg/L) for 30 min. Subsequently, 200 mLAb K3C6 (1:5000) was incubated for 1 h. Then</p><p>standards (0.01100 mg/L) were added and in-d for 30 min followed by a labeled second Ab asned above. After each incubation step the mi-plates were washed three times.</p><p>ce of pH and Salt Concentration on Hg(II)ling Efficiency</p><p>influence of the pH and salinity of the buffer onciency of coupling of Hg(II) to the BSAGSHtes was checked with the noncompetitive</p><p>EIA. Instead of PBS (40 mmol/L, pH 7.2) dis-ater or a phosphate buffer (pH 7.2) with differ-</p><p>ine concentration (10200 mmol/L) was used forlinity experiments. For the pH experiments</p><p>Influence of different salinities on the Hg(II) EIA. Absorp-ich were achieved with 100 mg/L HgCl2 in different PBS, were normalized. Absorption at 40 mmol/L PBS was set toe coefficient of variation was below 11%.a</p></li><li><p>phosphate buffer (40 mmol/L) with different pH values(211)pared wGSH cplate. Acompetmalizeequatio</p><p>whereconcencertainabsorpand AHgCl2.</p><p>RESUL</p><p>TheEIA fowas adfree thHg(II)Then tamounenzymrange bthe tes</p><p>To imof pHBSAGtions (1ent pHassay w</p><p>40 mmol/L). The highest absorption was achieved withupH</p><p>hol cpsnt</p><p>eien. Hho20horpl/Lh</p><p>toe</p><p>rlyrd</p><p>nceg</p><p>bye</p><p>r fivI)of</p><p>labGonerificram</p><p>sedI)</p><p>FIG. 3.sorption</p><p>4.eden</p><p>2.</p><p>51Hg(II) DETERMINATION BY EIAwas applied. Hg(II) standard solutions were pre-ith the different buffers and added to the BSA</p><p>onjugates, which were adsorbed to a microtiterll other steps were done according to the non-</p><p>itive EIA. Absorption of all variants was nor-d by transformation to %A according to then</p><p>%A 5A 2 A0</p><p>AC 2 AC0z 100 [1]</p><p>A is the absorption at a certain pH or salttration with 100 mg/L HgCl2; A 0, absorption at a</p><p>pH or salt concentration without HgCl2; AC,tion at pH 7.2, 40 mmol/L with 100 mg/L HgCl2;C0, absorption at pH 7.2, 40 mmol/L without</p><p>TS AND DISCUSSION</p><p>MAb K3C6 was first applied in a noncompetitiver the detection of Hg(II). BSAGSH conjugatesorbed to the surface of a microtiter plate. Theiol groups of the conjugate were able to bindvery tightly from Hg standard solutions (11, 12).he MAb K3C6 was bound to the conjugate. Thet of bound MAb was detected with a seconde-labeled MAb. This assay format yielded a testetween 2.1 and 21.9 mg/L Hg(II) and a middle oft (IC50) of 7.3 6 2.96 mg/L Hg(II).prove the sensitivity of the assay, the influence</p><p>and salinity on the coupling of Hg(II) to theSH conjugate was investigated. Standard solu-00 mg/L HgCl2) were prepared in PBS of differ-and applied in the EIA. All other steps of theere carried out with the standard PBS (pH 7.2,</p><p>a cothe pas sturagroumayafter</p><p>Wefficgatein p(10As sabsommowithbut</p><p>Thcleatowaa cothe Hplesjustm</p><p>FopetitHg(IsitesavaiBSAa strIf thspectest0.65creabounHg(I</p><p>Competitive and competitive displacement Hg(II) EIA (ab-range, 0.21.6).</p><p>FIG.distilltreatmHgClling buffer at pH 7.2. Decreasing or increasingresulted in a strong reduction of the absorptionn in Fig. 1. This effect could be due to struc-</p><p>hanges in the BSAGSH conjugate (13). Thiol, which are responsible for the binding of Hg(II),o longer be available for the coupling reactionis conversion.</p><p>also observed an influence of salinity on thecy of coupling of Hg(II) to the BSAGSH conju-g(II) standards (100 mg/L HgCl2) were preparedsphate buffer at different salt concentrations0 mmol/L PBS) or distilled water, respectively.wn in Fig. 2 there was a significant decrease intion with salt concentrations in the range 020</p><p>in comparison to the control (40 mmol/L). Also,igh salinity there was a decrease in absorptiona much lesser extent than with low salinity.results of both the pH and salinity experimentsdemonstrated that the Hg EIA is very sensitivechanges in assay conditions. PBS at pH 7.2 andntration of 40 mmol/L proved to be optimal for(II) binding reaction. Measurement of real sam-the noncompetitive EIA therefore requires ad-</p><p>nt of pH and salinity.urther improvement of assay sensitivity a com-e EIA (14) was applied. In this assay format, freeand immobilized Hg(II) compete for free bindingthe MAb K3C6. In the absence of free Hg(II) thele Ab binds to the mercury immobilized on theSH conjugate. After incubation of the second Abg absorption signal can be detected in this case.e is an excess of free Hg(II) it will block the</p><p>Ab binding sites and no signal is expected. Ange of 1.04.9 mg/L Hg(II) and an IC50 of 2.1 6g/L Hg(II) were achieved (Fig. 3). Further in-in sensitivity was achieved when the MAb wasto the BSAGSHHg conjugate before freewas added (14). In this competitive displace-</p><p>Determination of Hg(II) in water of different sources: (1)water, (2) tap water, (3) Lake Mensa, (4) effluent of sewaget plant. Each sample was diluted and spiked with 1 mg/Lw</p><p>h</p></li><li><p>ment EIA free Hg(II) was added after a washing stepand displaced the bound Ab. If there was an excess ofHg(II) the Ab was completely displaced. This EIA for-mat yielded a test range of 0.42.4 mg/L Hg(II) and anIC50 of 0.8 6 0.32 mg/L Hg(II) as shown in Fig. 3. It isobvious that the highest sensitivity was obtained withthe competitive displacement EIA. It yielded a twofoldenhancitive Esample</p><p>Differiver wwere aEach ssubseqthe addistilleFig. 4.nificansmalldilutioa dilutwith thThe indue tosalinitytimatiodependpH orantigencan inflrapidlyin the cter. InHg(II)</p><p>CONCL</p><p>Antisiderabies. Thferentcombin</p><p>Thedetectithe medilutedresult fity. If tthe det</p><p>is 4 mg/L Hg(II). Recovery of spiked samples was be-tween 80 and 120%.</p><p>Further work is required to check in which mannerextreme conditions (e.g., pH, salt concentration) influ-ence the displacement reaction in the competitive dis-placement EIA.</p><p>NO</p><p>arersgatass.</p><p>ER</p><p>aisl C</p><p>prilakes</p><p>. Bind77earavirintewenkyli</p><p>chuSAake444aroyn096o, Jasa91ark. Par</p><p>661arm. Mhilef</p><p>amabc05.</p><p>52 MARX AND HOCKement of sensitivity compared with the compet-IA. This effect can be explained by the highervolumes containing Hg(II).</p><p>rent water samples (tap water, distilled water,ater, and effluent of a sewage treatment plant)</p><p>nalyzed with the competitive displacement EIA.ample was diluted 1:10, 1:100, and 1:1000 anduently spiked with 1 mg/L HgCl2. Recovery ofded Hg(II) was high for tap water (119%) andd water (81%) at a dilution of 1:10, as shown inHigher dilution (1:100 or 1:1000) does not sig-tly improve the results. The water sample of alake, however, yielded recoveries of 46% at an of 1:10, 85% at a dilution of 1:100, and 102% ation of 1:1000. Similar results could be obtainede effluent of a sewage treatment plant (Fig. 4).</p><p>creasing recovery with higher dilutions may bematrix effects. For example, unsuitable pH orof the samples can be responsible for underes-</p><p>n of the Hg EIA. Usually antigenAb reactionsstrongly on physiological conditions. Extreme</p><p>salinity has been reported to interfere with theAb reaction (15, 16). Also, organic substancesuence EIA performance as they bind Hg(II) very(17). Spiking of samples with Hg(II) can resultomplete binding of mercury to the organic mat-this case Hg(II) is no longer available for the</p><p>EIA.</p><p>USION</p><p>bodies directed against heavy metals are of con-le interest for speciation and availability stud-e MAb K3C6 against Hg(II) was applied in dif-assay formats to achieve high sensitivity</p><p>ed with suitable assay conditions.competitive displacement EIA yielded the beston limit [0.4 mg/L Hg(II)] and was well suited toasurement of real samples. Samples have to be</p><p>at least 1:10 to avoid matrix effects, whichrom organic substances or extreme pH or salin-he dilution of the samples is taken into accountection limit of the competitive displacement EIA</p><p>ACK</p><p>We(Univconjunical0357)</p><p>REF</p><p>1. KtaS</p><p>2. BWJ</p><p>3. L2</p><p>4. RDF</p><p>5. SB</p><p>6. WSU</p><p>7. B8</p><p>8. M9. C</p><p>110. L11. C</p><p>912. S13. T14. M</p><p>115. Y</p><p>D16. P</p><p>(Hd</p><p>17. B2WLEDGMENTS</p><p>e grateful to Professors Adelio Rigo and Marina Scarpaity of Padova, Padua, Italy) who prepared several coatinges. We also thank Sabine Schapermeier for her skillful tech-istance. The work was supported by the EC (EV5V-CT94-</p><p>ENCES</p><p>er, G., and Tolg, G. (1980) in The Handbook of Environmen-hemistry (Hutzinger, O., Ed.), Vol. 3, Part A, pp. 158,</p><p>nger-Verlag, Berlin.e, D. A., Chakrabarti, P., Khosraviani, M., Hatcher, F. M.,thoff, C. M., Goebel, P., Wylie, D. E., and Blake, R. C. (1996)iol. Chem. 271, 2767727685.garde, F., and Zettervall, O. (1974) Scand. J. Immunol. 3,285.dan, D. T., Meares, C. F., Goodwin, D. A., McTigue, M.,d, G. S., Stone, M. R., Leung, J. P., Bartholomew, R. M., andcke, J. M. (1985) Nature 316, 265268.art, J. D., Roberts, V. A., Crowder, M. W., Getzoff, E. D., andovic, S. J. (1994) J. Am. Chem. Soc. 116, 415416.e, D. E., Lu, D., Carlson, L. D., Carlson, R., Babacan, K. F.,ster, S. M., and Wagner, F. W. (1992) Proc. Natl. Acad. 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