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1. en 000 PBDE 025p0 0 00000000 Divert 100 000000000000 0000000000000000 000000000000 500 000000 000000000 W040 ec O OO 000000 00 D 000 1000 0 00 Cami 0000000000 1040000000 o0000000000000000000 eppppppppmnmpgggg DD D D ED D D0 D I 0 D DD D I 0000000 0000000000000 EE C00O 00000 00000000000 000000000000000 00000 paie 00 00 Dn SE PBOEDO00O0000000000 0000000 00000000000 EET 000000000 000000000 0000000000000 0000000000000 PBDE ELISA 0 0 00000 0 0000 000000000 0 00m 0000000 000000000 1000 0 00 M5m 0000000000 000000 0 0000 DELSA O D O00 PBDE GE 0000000000000 PBDEQ OOD ee alae 000000000 0000000000 7Z Color Solution 0000 00010000 0000000000000 00000033 55 00000000 engggg 00000000000000000PBDE 0000000000000 enr 00000000000000000 PBI 0001 00000000000000000000 A Ade Noo Foren 000000000 0000000000000000000 JO 00000000200 000000000 Cf 7 1 0 0 Stopping Solution IPBDE Antibody Coupled Pararnagnetic En IDEEN 05 00 PaticesT D 0 0 00000000000 o00000000000000 Pegg 1008000 Deor 00 0000000000000 0000n 27 11 TMesting Solution TI ONES o 00000000 oo un00OOreED 00000000000 SE 000000000000000000 1000 0 00 0250m 0 0 00 DD 001 000000000 Oda 00000000000000000000 00000000 GJ DD Test Tubes 00000000000000000000 0000 PB QO000M00000 CO0000000000 000000000 00 0
2. observations to the analysis of other type of matrices such as food serum milk soil and to perform comparison with other analytical methods Acknowledgements The authors would like to thank Amy McGarvey for technical assistant References 1 IPCS 1994 Environmental Health Criteria 162 Brominated diphenyl ethers International Programme on Chemical Safety World Health Organization Geneva 2 Hardy ML Chemosphere 2002 46 757 777 3 deWitt C A Chemosphere 2002 46 583 624 4 Boon J P Lewis W E Tjoen A Choy MR Allchin C B Law RL De Boer J Hallers Tjabbes C C Zegers B N Environ Sci Technol 2002 46 697 707 5 Anderson O and Blomkvist G Chemosphere 1981 10 1051 1060 6 Klasson Wehler E Hovander L Bergman A Organohalgen Compd 1997 33 420 425 7 Meiroyte D Noren K Bergman A J Toxicol Environ Health 1999 58 329 341 8 Brouwer A Morse D C Lnas M C Schuur A G Murk A J Klasson Wehler E Bergman A and Visser T J 1998 Toxicol Ind Health 14 59 84 9 Hottenstein C S Fleeker J R Herzog D P Rubio F M Lawruk T S Journal of Agricultural and Food Chemistry 1996 44 3576 358 10 Jourdan S W Scutellaro A M Fleeker J R Herzog D P Rubio F M Joumal of Agricultural and Food Chemistry1995 43 2784 2788 11 Lawruk T S Gueco A M Jourdan S W Scutellaro A M Fleeker J R Herzog D P Rubio F M Journal of Wine Rese
3. 00 2000000 0400 Du 0o00 LDD 50 B Bo Compound ppb ppb ENER PBDE Congener 47 0 017 0 135 PBDE Congener 99 0 02 0 15 PBDE Congener 28 0 045 0 9 PBDE Congener 100 0 055 5 5 PBDE Congener 153 0 075 10 PBDE Congener 154 3 5 580 PBDE Congener 183 13 5 2000 PBDE Congener 209 370 3000 2 2 4 5 Tetrabromobiphenyl 6 6 250 2 2 4 4 5 5 Hexabromobiphenyl 130 gt 1000 2 4 5 Tribromobiphenyl 160 2600 2 2 5 Tribromobiphenyl 175 2900 2 4 5 Tribromobiphenyl 240 3200 2 2 4 5 6 Pentabromobiphenyl 320 21000 3 3 4 4 5 5 Hexabromobiphenyl 21000 21000 2 2 4 4 6 6 Hexabromobiphenyl 21000 gt 1000 Decabromobiphenyl 21000 21000 PCB Aroclor 1254 3 180 PCB Congener 37 160 2000 PCB Congener 77 21000 21000 2 2 4 4 Tetrachlorobiphenyl 16 360 2 2 4 4 5 Pentachlorobiphenyl 54 1500 PCP 3300 210 000 2 4 D 210 000 210 000 24 OOOO PBDE O en 00000000 000000000 UU 0000 D APEC O O00000 0 un Hd 0 00 00 UD Anotop 25 Plus E 3 L3 Co O O Oo L3 O OO D opp a wpa EZE FE LIES LTE L3 L3 LJILLJ Ooo LIT LIII Lr LECH EE ICH O oO CI 1 c4 c3 53 oo CO r3 l Lr On L3 r3 oog EJ nooo nooo oO a e oO oo l EA A TETEA Onooo Pro ioo Foo Oo
4. 00000000 0 0 0003 55 0000000000 1000 0 00 0360 0 000 0 00 00 00000 0 OColor Solution 0 O EES WEE EC 0 0000 00PB0E0 000000000 9000000000 ao ooo 0000000000000 000 OOO000zFQoO00MO0MO E EE 00000000000000 o0m00000m00ooo0000 200 IStopping Solution D D 00000000 nndumnunnnnunnnnuuu 00000000000 000000000 J0000000000000000000 000000000000000000 000000000000000000 0000000000000000 O00000000000000000000 0000000000000 00000000 PB N000000000000000 00000000 NO 000000000 0 0 00000 0 00 000000000000 PBEDDDO naonna 10000 00 0 10 0000000000000000000 000000000000 00000000 00000000000000000000 sm 000000000000000 DODO 0000000000rsoesa SOO RUC OO COU OO OOO 999909000 00 0 00000 Meeti DOOM 0 0 000000 0000000 ood honongonomooggg Sept echt 20000000000 0000000000000 H 0000000000000000000 eg PBDE E OO 00000 0 GPBDE BE 00000000000000000000 Antibody Coupled Pararragnetic 00000 000250 50040 00000 eins O 0 00 T et aa L U0 O Particles unu 0000000000 BOO BB 000000000000000 0000000000000 000000 0 p Frenn IH HL HD DD D op DL e 0000000000 000000 00 oooooocomoomoodo 8000000 0 BQ00000000000000000 1000000000 000000 Vegetic 000000000000 00000000 000000000000 H 000000000000000 1000 0 00 060 Separation Rack Ba agdgagannapnpnuduagadauu munnmpsempnnuunuugnugs 00000000000000 00000 0700000000 GPBDE Enzyme x0 000 00000000000 pago 00000000000 Comoe g ugnnuugnnnuuunnnunn 000000000000000mM0 ennuunnn dr EMEN TENET IR DM 00000000000000 DD DD D D DD DD D ED D D
5. 25 50 100 500 and 1 000 parts per trillion ppt of BDE 47 Results and Discussion Dose Response Curve and Sensitivity Figure 1 illustrates the mean standard curve for the BDE 47 calibrators collected over 24 assays error bars represent one standard deviation SD n 24 The displacement at the 25 ppt level is significant B496 B Bo where B Bo is the absorbance at 450 nm observed for a sample or standard divided by the absorbance at the zero standard The assay sensitivity in water based on 9096 B Bo is 17 ppt Stii Precision Table 1 shows the results from a precision study in which surface and groundwater samples fortified with BDE 47 at 3 concentrations were gt each measured 5 times per assay and on five different days The within and between day variation was estimated by the method of Bookbinder and Panosian Coefficients of variation below 5 were observed Absorbance 450 nm i 8 Table 1 Tabie 2 Precision of BDE 47 Measurements in Water BDE 47 Recovery in Water es Recovery Mean SD Added pp epp ppn 62 5 56 4 2 9 90 Accuracy Known amounts Replicates of BDE 47 were added to Days four groundwater samples obtained from Warminster PA The samples included a municipal water source a reservoir a lake a pond 1315 53 105 250 270 6 128 108 500 310 4 283 102 101 4 Nuit 96 CV within assay 32 96 CV between ase 44 l and a creek The accuracy recovery was assessed by a
6. ANA Bioanalytical Approaches for POPs Detection preserved buffer During the assay procedure magnetic separation was performed using a magnetic separation rack Abraxis Warminster PA The device consists of a magnetic separation rack and a test tube holder which fits over a magnetic separation rack containing permanent rare earth magnets The two piece design allows for up to sixty tubes to be set up incubated and magnetically separated without removing the tubes from the tube holder Water samples were analyzed directly without any sample extraction or pre concentration After mixing 1 1 with methanol 250 mL of the sample and ant PBDE coupled magnetic particles 500 mL were added to a disposable glass test tube and incubated for 20 minutes at room temperature An aliquot of PBDE HRP solution 250 mL was added and the reaction was incubated for 20 minutes at room temperature A magnetic field was applied to the magnetic solid phase to facilitate washing and removal of unbound PBDE HRP and to eliminate any potential interfering substances The enzyme substrate and chromogen peroxide TMB were then added 500 mL and incubated for 20 minutes The reaction is stopped with the addition of 2 N H5SO 500 mL and the final color was read with analyzed using a photometer by determining the absorbance at 450 nm The observed absorbance data were compared to a linear regression line using a log log standard curve prepared from calibrators containing 0
7. D puni 00 00 D cono O00 COO D I DD Pa 1000 0 00 030ml 0000000000 EE ARO En S G PBDE 7 PBDE Standards gnuununmnmugugauunuunnn 000000000 0000000000 0 HOO 0025 005 01 05 10 DO 6NOOOOCOUDDOOOOO000 0000000 00000000000 PBOE 7 0000 0 0 000000 0000000 0000000000000000 0000 100000000000000 08 ppp sten nn 1w0ooop C000000000000 0 00000 T O Seele GE 10000 00 0 2m 0000080000 0000000000000000 00000 CEET EEN EE e 600000 Control O O O O ODDiluent Zero Standard Sande TS UT 23 ETA ES LA Bono Oooo o0 DII ICH j ICH Pb rue eo ooo ooo Ono 02557 ooo V o Aaa CH N oo Om Oo oo OA z 9 t o0000 So0259 290000 CH wNroVawneo Ip COOC 3 250 uL PBDE Standards T 6 00 020000000 PBDE D OO ibody Coupled uada O Ee ke o o a Lr D D Ta o0 4A O EO LL O 20 Un uL O me Conjugate O O O 2 0000 0000 200 0 Zo B oma 20 L3 mr Ooo ICH 22 g noon ooo Ono loo oO Ooo oaa 00000 000000 OPBDE Control UO O D ada O O O O
8. OPBDE netic Particles 0 0 00500 ul 0 0000 0000 OPBDE 0 0000 EE Co CIE Lr OG M o 131 000 D mu au Solution 0 I Hd uut 500 uL 00 Oo OO 160 0000000000 000000000 10 20000000000 00000000000 19000pbt D D 1 000 2A000000000000000 00 Etisag 000000000000000 a dtu 0000 18 5004L 0 OD D D 0 O EStoppingSolution 9902500000000 0 0235 0 00000000000000 00000000 0 00 0000 00000 19 0 000000 im0eg00 T M shing Solution TO 0 D 00 0 00 200 pad m0 000010 000000000 0000 2000000000000 5000000 GE ER 0000 450 n a AA IUS S AAA enOO000000 TEL 03 5444 9891 FAX 03 5444 9860 Zi E mail ecoGechemco jp 10000000000000000000 VRL httpy wwjechemcojp poo 2pp4000 0000000000000 000 08 B40 0 0 00 00 00 0 3 0000 000000000 B BdYU TI pomo 00000 0x0 00 0 00000 02000000 00000000 4 0000 00000 8 B 0000000 n44 4000000000 00 0 000000 00AbrexisO D D D D oo 000 0000000000 0OPBDE ELISA 0 OO O aaaaaa 00000 00000 0 017p b0 O 08 8 90 0 D 0 O0 LU LLL LU LE LE LU LT 000 000000 00 00 DPBDEJ ELISA 0 pad UD 000CM 00000 00000 Dodd 0n 00d nd 000000000 B B 978 OO 800000000000 g soap 00000
9. arch 1994 5 205 214 12 Shelver W L Keum Y S Kim H J Rutherford D Hakk H Bergman D and Li Q X J Agri Food 907 21 ANA Bioanalytical Approaches for POPs Detection Chem 2005 web release April 15 2005 13 Bookbinder M J Panosian K J Correct and Incorrect Estimation of Within and Between day Variation Clin Chem 1986 32 1734 1737 22 000 00000 000 0 00000 0000 OPB ELSA 0000 000000 00 D 0 00 D HEIL BEL DL D DI
10. nalyzing the samples before and after the addition of BDE 47 and then subtracting the estimated concentration of PBDE before spiking None of the samples had significant levels of PBDEs Added amounts were accurately recovered Table 2 An average assay recovery of 101 was obtained 906 20 ANA Bioanalytical Approaches for POPs Detection Specificity Table 3 summarizes the cross reactivity data of the PBDE assay for various PBDE congeners as well as other environmental contaminants such as PCBs PCP and 2 4 D The percent cross reactivity was determined by estimating the amount of analogue required to displace 50 of the enzyme conjugate and comparing to the 50 displacement of the BDE 47 standard curve The results showed that the antibody recognizes BDE 47 and BDE 99 equally well the higher the brominate substitution the less cross reactivity was observed The antibodies have less than 0 1 cross reactivity for Aroclor 1254 Table 3 Specificity Cross Reactivity Conclusions This work describes the performance characteristics of a magnetic particle based ELISA for the detection of PBDEs in groundwater samples The assay is fast and eliminates the need for expensive instrumentation and solvent disposal The ELISA exhibits good precision and accuracy which can provide consistent and cost effective monitoring of water samples Using this ELISA fifty results can be obtained in about one hour Future efforts will be to extend these
11. o La e0000000000 UL LI oooi oo a H p3 H geo L3 15 h po OOo Oo O D o D i 13 oo l Lar Oonoo Oo COOC R 000000 11000000000000 poo 0000000002000 Mean ppb UD 0000 0 000000 0000000 Data Reduct Xformation Read Mode Wavelength Units HR gt Blk Calibrators of Cals of Reps 0000000000000 2000000000000 Concentrations 41 2 B 4 35 366 Range Correlation Rep CV e0000000Mbaisgo 000 Ult B Bo 90 0000 D 0000000 17ppt Do 017ppHTT O O ULL 000000 0000000000 00000000 PBDETLU LU DU 20000 1100000000000 000000000000 Amountof Recovery PBDE Mean S D Added ppt ppt ppt 62 5 56 4 2 87 125 131 5 5 30 250 210 6 12 80 500 510 4 28 32 Average Ult 00d 020000000000 Control 1 2 Replicates 25 5 5 Days 5 5 0 00 25 50 100 500 1000 Lin Regression Ln Ln Absorbance 450 nm PPT 0 PPT PPT PPT PPT PPT PPT 20 1000 0 990 10 n 25 25 25 0 047 0 095 0 514 CV TTT 316 217 1 98 CV UD 435 478 397 enpoo dpad0d 008000000 uult 105 0023 DO000O0O0C000 au 10 0000 O NO 9F TEL 03 5444 9891 FAX 03 5444 9860 E mail eco echemco jp URL http wwwjechemco jp

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