Determination of polychlorinated biphenyl compounds in electrical insulating oils by enzyme immunoassay

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<ul><li><p>Analytica Chimica Acta 422 (2000) 167177</p><p>Determination of polychlorinated biphenyl compounds inelectrical insulating oils by enzyme immunoassay</p><p>In Soo Kim, Steven J. Setford, Selwayan SainiCranfield Centre for Analytical Science, IBST, Cranfield University, Cranfield, Bedford, MK43 0AL, UK</p><p>Received 7 April 2000; received in revised form 21 June 2000; accepted 7 July 2000</p><p>Abstract</p><p>The development and performance of a competitive indirect immunoassay for the quantitation of polychlorinated biphenylcompounds (PCBs) in insulating oils is described. Reagent preparation and the assay characterisation, optimisation and val-idation steps are described. The dynamic range of the assay for Aroclors 1254 and 1260 in methanol was 301000 ng ml1with 50% signal inhibition values of 217 and 212 ng ml1, respectively. Impending legislation in the UK is likely to de-cree that oils containing &gt;50mg ml1 PCBs be considered contaminated. Assay sensitivity increased with the degree ofPCB chlorination. The assay of structurally related compounds of environmental concern yielded cross-reactivity values of35mg ml1 PCB (neat)but over-estimated PCB levels in diluted oils containing </p></li><li><p>168 I.S. Kim et al. / Analytica Chimica Acta 422 (2000) 167177</p><p>this fact, PCB-contaminated oils are still commonlyencountered partly because some electrical units havenever been refilled with PCB-free insulating oils,whilst those that have may still be contaminated dueto inadequate plant and loading-line cleaning proce-dures. Some PCB contamination is believed to occurdue to the re-use of incompletely reconditioned oil.Since attempts to identify a particular plant as con-taminated have been unsuccessful, the only recourseof action has been to chemically analyse oils.</p><p>The current UK action plan [3] dictates that organi-sations with electrical equipment containing more than5 l of oil, contaminated with more than 50mg ml1PCB will need to reduce these levels to comply withimminent new regulations. Indeed, the most recentEC Directive on the disposal of PCBs (96/59/EC) de-fines a range of chlorinated diphenyl and terphenylcompounds as PCBs. Consequently, this legal defini-tion considers any preparation containing more than50mg ml1 of PCB or PCB equivalent to be treatedas a pure PCB preparation.</p><p>Current PCB measurement methods are either non-specific or utilise complex laboratory-based instru-mental techniques [4]. The former methods measuregeneral properties of the PCB analyte such as totalchlorine content whilst the latter methods are gener-ally more time consuming and expensive, typicallyrequiring sample preparation, chromatographic sepa-ration and detection. Whilst the specific approachesare reproducible and of high sensitivity, the increasingincidence of PCB compounds in the environment hascreated the need for more rapid, simpler and low-costanalytical procedures. Ideally, these techniques shouldhave the durability and flexibility to be automated andapplied either in the laboratory for routine analysis orin the field where assay speed and simplicity are desir-able for the immediate implementation of appropriateremediation procedures.</p><p>Immunochemical techniques offer a simple, low-cost means of routinely and specifically measur-ing compounds in decentralised locations and haveconsequently been used for PCB analysis [513].These methods have primarily been developed forthe determination of PCBs in predominantly polarmatrices such as soil, water and milk. Indeed, twocommercially available enzyme-linked immunosor-bent assay (ELISA) test kits for PCBs based on acompetitive-format tube type assay are also widely</p><p>available (RaPID assay and Envirogard, SDI Eu-rope Ltd., Alton, UK). However, both of these sys-tems have been designed to work in essentially polarenvironments and are only semi-quantitative, yieldingonly a concentration range as opposed to a discreetnumerical value. None of the above approaches hasconsidered the analysis of oils for PCBs. Conse-quently, this paper describes the development of arapid extraction and ELISA-based method for thequantitation of PCBs in electrical insulating oils. Akey objective of the work has been to develop a simpleprotocol, amenable to both field-based and automatedlaboratory-based high-throughput analysis. Whilst thelower chlorinated Aroclors are usually predominantin transformer oils, the assay was optimised usingAroclors 1254 and 1260. The optimised procedurewas compared against a commercial PCB ELISA kit.</p><p>2. Materials and methods</p><p>2.1. Reagents</p><p>General chemical and biological reagents were ofanalytical grade and were purchased from Sigma-Aldrich or Merck (both Poole, Dorset, UK). Deionised-reverse osmosis water was purified using an Elgas-tat system (Elga, High Wycombe, UK). Phosphatebuffered saline-Tween (PBST) solutions were pre-pared from 10 mM buffer salts, pH 7.4, 0.15 M NaCland 0.05% (v/v) Tween 20. The carbonate buffer usedwas 0.2 M at pH 9.6. Aroclors 1242, 1248, 1254, 1260and 1262 (Ultra Scientific, North Kingstown, RI) weresupplied as 100mg ml1 solutions in methanol anddiluted in methanol as required. For cross-reactivitystudies, the structurally similar non-PCB compoundspentachlorophenol (PCP), 2,4-dichlorophenoxyaceticacid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (TC-PAA) and 2-(2,4,5-trichlorophenoxy)propionic acid(TCPPA) from Sigma-Aldrich were used. The Na-tional Grid Co. (Leatherhead, UK) donated an unusedPCB-free transformer oil and five used oils containingAroclors. The used oils had the following gas chro-matographically determined PCB concentrations inmg ml1: A, 4; B, 35; C, 51; D, 10; E, 6. Oil clean-upwas facilitated using C18 solid phase extraction (SPE)columns containing 500 mg of resin in 3 ml capacitycolumns, from IST Ltd. (Hengoed, Clwyd, UK).</p></li><li><p>I.S. Kim et al. / Analytica Chimica Acta 422 (2000) 167177 169</p><p>Anti-PCB monoclonal antibody (MAb, ResearchDiagnostics Inc., Flanders, NJ) was diluted 10-fold to500mg ml1 antibody protein, aliquoted and storedat 20C with no significant loss in activity over 1year. Goat anti-mouse antibody-horseradish perox-idase (GAM-HRP) conjugate (Sigma-Aldrich) wassimilarly aliquoted and stored at 20C and diluted in10% (v/v) foetal bovine serum (FBS, Sigma-Aldrich)with PBST as required and was stable for 1 month at4C. Coating antigen was synthesised by conjugat-ing bovine serum albumin (BSA) to TCPPA (Fluka)using 1-ethyl-3,3-dimethylaminopropylcarbodiimidehydrochloride (EDC) and adsorbed onto microtitrewell walls. Residual adsorption sites were blockedusing SuperBlock PBS buffer (Pierce, Chester, UK).TCPPA and BSA solutions were prepared in 0.1 M2-[N-morpholino] ethane sulphonic acid (MES) buffer,pH 4.5, containing 50% (v/v) N,N0-dimethylformamide(DMF). Conjugate protein concentrations were deter-mined using the Coomassie Plus protein assay (Mod-ified Bradford method, Pierce). o-Phenylenediamine(OPD) substrate was purchased as 5 mg tablets fromSigma-Aldrich and dissolved in 0.15 M citrate buffer,pH 5 (citric acid 7.3 g, Na2HPO42H2O 11.86 g).1-Step TURBO TMB was purchased from Pierce andused as received. Hydrogen peroxide stock solution(30% w/w) was from Sigma-Aldrich.</p><p>2.2. Instrumentation</p><p>Two types of microtitre plate were evaluated:Polysorb 96-well (A/S Nunc, Roskilde, Denmark)and ImmunoWare 8-well EIA Strip Plates (Pierce).During incubation, the plates were mechanicallyshaken with an iEMS incubator/Shaker HT (Lab-Systems, Finland) before being washed using aneight-channel manual washer (Nunc). The extent ofcolour development in the plates was determined us-ing a Titertek Multiscan MCC Microwell plate reader(LabSystems). Coating antigen conjugate was recov-ered using a Sephadex G-25 size exclusion column(PD-10, Pharmacia, Sweden).</p><p>2.3. Preparation and immobilisation of coatingantigen</p><p>Coating antigen was prepared by reacting EDCwith the carboxyl group on the PCB analogue TCPPA.</p><p>The amine-reactive o-acylisourea intermediate wasthen reacted with amine groups on BSA to form astable carrier-proteinanalogue conjugate. First, 1 mlof 5 mg ml1 TCPPA and 400ml of 10 mg ml1 BSAin MES buffer, pH 4.5, were stirred with 200ml offreshly prepared 10 mg ml1 EDC in water for 2 h at25C. The Sephadex G-25 column was pre-washedwith 0.2 M carbonate buffer, pH 9.6, and loaded withthe reaction mixture. Conjugated material was elutedusing 0.2 M carbonate buffer and collected as 0.5 mlfractions. The protein concentration of each fractionwas determined using the Coomassie protein assayaccording to the manufacturers instructions usingBSA as the calibration standard. Fractions 48 con-tained the greatest amount of protein and were pooled;the combined protein concentration was 1.3 mg ml1.The conjugate was diluted to 1 mg ml1 in 0.2 Mcarbonate buffer and stored at 4C until required.The TCPPA:BSA conjugation ratio was determinedphotometrically to be approximately 20:1.</p><p>Coating antigen stock solution was appropriatelydiluted in carbonate buffer and 150ml volumes dis-pensed into microtitre plate wells. The wells weresealed and incubated at 25C for 4 h to allow adsorp-tion of coating antigen onto the well walls, washedthree times with PBST and excess fluid removed byinversion and rapping on an absorbent towel. Wellswere blocked with SuperBlock PBS buffer for 30 minat 25C, then washed and dried as before. The plateswere sealed and stored at 4C until required.</p><p>2.4. ELISA procedure</p><p>A 90ml volume of appropriately diluted anti-PCBMAb in PBST and 10ml of sample or standard inmethanol was added to each TCPPA-coated well. Theplates were then incubated at 37C (primary incu-bation) for a given time, then washed and rap-driedas before. Next, 100ml volumes of 1:4000 dilutedGAM-HRP conjugate in FBS-PBST were added toeach well before incubation at 37C (secondary incu-bation) for a given time, washing and rap-drying.</p><p>HRP activity was determined using either 1-StepTURBO TMB or OPD. The former substrate wassupplied ready for use, whilst the latter was preparedimmediately before required by adding 15ml of 30%(v/v) H2O2 to 15 ml of 1 mg ml1 OPD in 0.15 Mcitrate buffer. In both cases, 100ml of substrate was</p></li><li><p>170 I.S. Kim et al. / Analytica Chimica Acta 422 (2000) 167177</p><p>dispensed into each well. The plates were incubatedfor 30 min or until the absorbance (optical den-sity, OD) of the standard zero concentration blankreached 1.0 after which the reaction was stopped byadding 100ml of 2 M H2SO4. The plate was read at492/630 nm. All tests were performed a minimum ofthree times.</p><p>2.5. Data evaluation</p><p>The logit-log model, the most widely used proce-dure for immunoassay data evaluation [14], was usedto analyse the data. The model represents a continu-ous sigmoidal function with a single inflection point,described by the equation</p><p>y D a d1 C .x=c/b C d</p><p>where a represents the maximum current at zero ana-lyte (upper asymptote), b the slope of the linear portionof the sigmoidal curve, c the analyte concentration atmid-point (50% signal reduction), and d the residualcurrent at infinite dose (lower asymptote; backgroundcurrent C non-specific binding). These constant val-ues were calculated using the curve fit (math) func-tion of Sigma plot for Windows (Jandel Scientific).The practical quantitative range of the ELISA wasdefined as the linear portion of the sigmoidal curve,which was also calculated using Sigma plot. The assaylimit of detection (LOD) was defined as the analyteconcentration corresponding to ODzero analyte .2 S:D:zero analyte/. Accuracy is a measure of the system-atic error in the assay and was reported as % bias,calculated as 100% .(measured valuetrue value)/true value). Cross-reactivity was calculated as 100%.IC50-value Aroclor 1254 standard/IC50-value cross-reacting sample). Extraction efficiencies were calcu-lated as 100% .measured value/expected value).</p><p>2.6. Transformer oil preparation</p><p>2.6.1. Direct dilutionOil samples were diluted 10-fold in 2-propanol and</p><p>50-fold in methanol and assayed.</p><p>2.6.2. Liquidliquid extractionOil samples (100ml) were added to 1 ml of extrac-</p><p>tion solvent, either methanol, acetonitrile or dimethyl</p><p>sulphoxide (DMSO) and vigorously agitated using abench top vortex mixer for 1 min. After phase sepa-ration, 100ml of the upper phase was further diluted50-fold in methanol and immediately assayed.</p><p>2.6.3. KOHethanol/sulphuric acid extractionOil samples (100ml) were vortexed with 1 ml of 1 M</p><p>KOH in ethanol for 5 min. Next, 1 ml of n-hexane wasadded and the mixture vortexed for a further minute.After phase separation, the upper hexane phase wastransferred to a fresh glass tube, 1 ml of concentratedsulphuric acid added and the mixture vortexed for1 min. After phase separation, the hexane phase wasremoved and evaporated under nitrogen. The residualmaterial was re-dissolved in 1 ml of 2-propanol, thenfurther diluted 50-, 100- and 200-fold in methanolprior to assay.</p><p>2.6.4. Solid phase extractionA C18 SPE column was pre-wetted with 2 ml of</p><p>2-propanol followed by 1 ml of 10% (v/v) transformeroil in 2-propanol. Retained materials were eluted with10 ml of 2-propanol then 10 ml of n-hexane. Each frac-tion was collected separately, mixed well and 100mlfractions evaporated under nitrogen, re-dissolved anddiluted 500- or 1000-fold in methanol and assayed.</p><p>2.7. Commercial PCB assay kit</p><p>The optimised assay was tested in parallel with asemi-quantitative PCB kit, utilising antibody coatedmicrobeads in tube format (RaPID assay, SDI Eu-rope Ltd.). The kit uses an indirect competitiveassay format with anti-PCB antibody covalentlyimmobilised on paramagnetic particles and usedaccording to the manufactures instructions. Thefive PCB-containing transformer oil samples and a100mg ml1 Aroclor 1260 standard were prepared bythe KOHethanol/sulphuric acid method and diluted10,000-fold for the oil samples and 40,000-fold for theAroclor 1260 standard in methanol. Extracts (200ml)were incubated with 250ml PCBenzyme conjugateat room temperature for 15 min prior to bead recov-ery using a magnetic tray (SDI). The tubes werewashed twice with wash buffer, filled with 500ml ofsubstrate/chromogen and incubated for 20 min. Thereaction was halted using 500ml of 2 M sulphuricacid and the absorbance measured at 450 nm.</p></li><li><p>I.S. Kim et al. / Analytica Chimica Acta 422 (2000) 167177 171</p><p>3. Results and discussion</p><p>3.1. ELISA optimisation</p><p>3.1.1. Optimisation of coating antigen and primaryantibody concentration</p><p>Optimisation was achieved using the checkerboardtitration method in which serial dilutions of bothcoating antigen and primary antibody were incubatedtogether. Since no sample was present, 100ml ofprimary antibody was used. Primary and secondaryincubation times of 1 h and OPD substrate (incuba-tion temperature 25C) were used in all tests. Thehighest OD readings were recorded at a coatingantigen and primary antibody concentration of 10and 1.25mg ml1, respectively. These concentrationswere correspondingly employed in subsequent assaysunless otherwise stated.</p><p>Both the Polysorb and Immunoware plates werecoated with 10mg ml1 TCPPA-BSA and incubatedwith 5, 2.5,...</p></li></ul>

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