化学物質に関する簡易モニタリング技術 実証試験計画書
Contents
1. 6 1 2 TMB substrate a 10 1004 L 100 500 L HRP 300mL DMSO 450nm 2 RERORE2. PCB D PCB 118 standard 2 118 standard DMSO 1 000 ng mL 250 ng mL 100 ng mL 25 ng mL 10 ng mL Ong mL KAO 2 O 2 8C 30 2 2 DMSO 90 L 90 1 90 L 10 z L 118 1 000 ng mL 100 ng mL 10 ng mL 2 1 STD 5 STD 3 STD 1 E PCB 118 DMSO 7T5uL 90uL 90 L standard 25uL 1 000 ng mL rt C l 1 4 M PCB 118 250 ng mL 25 ng mL 0 ng mL STD 4 STD 2 STD 0 3 HRP BEER AS AGER OD Ae REPT SPRL HRP conjugate d
3. 50 LO TMB substrate 20 28 20 50 L O Stop solution 10 450nm 7 PCB 1 2 DMSO 10 20 10 x 100 x 1 000 1 4 parameter logistic model Four parameter logistic model 4 1 DMSO O OD4so Zero standard Ong mL
4. 10000000600 654 0037 31 27 HHHHHHHH 00000 00000 0000 0009 0000 078 785 6911 FAX 078 735 7817 E mail 000 Yoshinari kobukeQpref hyogo jp 000 HHHHHHHHHHHHHHHHHHHH 185 80738 HHHHHH 245000 2400 4F HHHHHHHH 0000 BD 0000000 0000 03 5531 5235 FAX 03 5531 5236 E mail 00 0 okuyama enbiotec co jp 220000 00000000000000000000000 HHHHHHHHH HHHHHHHHHHHHHHHHHH 00000 000000 HHHHHHHHHHHHHHHH HHHHHHHHH l 1 23000000000000 HEEL ET EIL EE EF EE ELE EIE EE 02 HHHHHHHHHHHH 0000 oe Pa HHnH Em 000000000000000 00000 00 000 Eu sx SOEUR be Serato ree HHHHHHHHHHHHHHHHHHHHH 1000000 HH i uec CREE M MM E S TUTTI EROR 000000000000000000 0000 nn 000000000000000000 00000 00 0000000 000000006 HHHH HH D XX m e TT KNEES
5. PCB 118 PCB 20 PCB 1 A Okuyama Takenaka Nishi H Mizukami S Kozaki M Kirihata Miyatake DEVELOPMENT OF MONOCLONAL ANTIBODIES AND IMMUNOASSAY SYSTEM FOR PRE SCREENING COPLANAR POLYCHLORINATED BIPHENYLS Organohalogen compounds 54 2001 44 47 2 A Okuyama H Takenaka K Nishi H Mizukami S Kozaki M Kirihata K Miyatake H Takigami S Sakai M Morita DEVELOPMENT OF ENZYME LINKED IMMUNOSORBENT ASSAY FOR THE PRE
6. 321000000000 HHHHHHHHHHHHHHHHHH PCB 118HHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHH PCBHH ELISAHHHHHHH ELISAHHHHHHHHHHHHHHPCBHHHHHHHHHHHHHHHHPCBHH HHHHHHHHHHHHHHHHHHHHHHHHH96HHHHHHHHHHHHHHHH 320000000000 0 0000000000000000000000000000000000000 0 31 0000000000 1 L aa PCB EIA System RPN5949 000000000 20 500g 00000 10006000 mnnn ESE EINEN 4 methoxy 3 3 4 trichlorobiphenyl 000000 U 10000000 15 C 35 O00 EE uS 218 00000 pea oes ee a 000 6000 000000000 4200020000 1500000000000 032 0000000000 2
7. ng mL SD CV n 98 4 5 04 5 1 8 55 5 3 90 7 0 8 18 5 1 66 8 9 8 2 8 3 PCB 2 6 Table4 ng mL SD CV 96 n 83 7 9 66 11 5 6 45 4 7 92 17 5 6 17 5 2 88 16 4 6 11 5 Precision profile 2 6 SD CV 10 0 8 0 gt 2 6 0 R 4 0 2 0 0 0 1 10 100 1 000 Conc of PCB 118 in DMSO ng mL 6 Precision profile 12 5 Precision profile 2 6 SD CV 10 0 8 0 gt 2 6 0 R 4 0 2 0 0 0 1 10 100 1 000 Conc of PCB 118 in DMSO ng mL 6 Precision profile 12 Claim 1 Determination of Sensitivity AIM To determine the sensitivity of the assay METHOD The sens
8. 420000000 HHHHHHHHHUHHH 500000000 50000000 11000000 ennt HHHHHHHHHHH 9000000 HHHHHHHHHHHHHHH 0 0000 0000000000 HHHHHHHH 000000 VRP O0000 201000000 ennt 0000 up urat 4300000000000000 100000000 ELISAHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH ELISAHH HHHHHHH1HHHHHHHHHHHHHHHHH e 000 ELISAHHHHHHHHHHHHHHHHHHH sELISAHHHHHHHHHHHHHHHHHHHHH sELISAHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH 0 20 sELISAHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH 0000 sELISAHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH 0000000000000000000000000000000000 ELISAHHHHHHHHH 10 OOOO E
9. 50 PCB PCB Un nn 00450 0 1 10 100 pe 0 29 0 128 X 28 1 0 128 Conc of PCB 118 in DMSO ng mL SOFTmax Molecular Devices Corp 2 2 PCB gt 20g PCB 100 LM DMSO KER 1 000 DMSO 50 EIA X CXx amp Y Y D 1B OD4so 0 621 28 1 1 29 0 621 0 621 0 128 1 1 46 34 6 ng mL 34 6 50 200 8 7 ng g 2 PCB EIA 4118 100 PCB products CR KC 300 3 87 1 26 KC 400 7 36 1 14 KC 500 9 14 1 11 KC 600 1 69 1 59 EIA 11 KC 500
10. 2 8 A E Antibody coated microtiter plate PCB 118 8 wells strip plate seal 2 8 30 PCB 118 standard 3 3 4 trichloro 4 methoxybiphenyl 1 000 ng mL 118 8 DMSO DMSO CHR p 4 96 well 12 strip Competitor HRP conjugate concentrate HRP HRP dilution buffer p 5 Wash buffer concentrate 10 p 5 TMB substrate T 3 3 5 5 tetramethylbenzidine
11. OOOO 0 000000000000000000000000000000000 30000000 HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH e HHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHH1HHH8SHHHHH3HHHHHHHH HHHHHHH 8HHHHHHHHHHHHHSDHHHHHHHHH SDHHHHH 35200 10SDHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH z1Tz e 00000000 0 0000000000000000000000 1000 30000 8000003000 0000000000000 800000000000000000000000000 e 0000000 000000001000000 30000000000000000000000000 A l 0 00000000000003000000000000000000000000 HHHHHHH 10000000000000 20000000000000000000 90000000 0000000000000000000000000000000000000000000 0 0000000000000000000000000000000000000000 e HHHHHHHHHH 000060 20000000000006 100000 30000000000000000 HHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH 3000000000000000000000000000000000000000000 ag EL EE ELEP EE LET EEEEE EFEEH o 0000000 3000000 0 0000000000000000000000 5900 500000000000 50HHHHHHx100HHHHHHHHHHHHHHHHHHHHHH 00000 0000000000000000000 500 0 0 0 05 0 0 000000000000000000000000000000000000000000 0 000000000000000000 20000 1900000000000
12. PCB 118 PCB 18 15 PCB 1 PCB 118 PCB 118 3 3 4 trichloro 4 methoxy biphenyl PCB 118 o 96 Fo 1 40 2 EIA PCB 118 EIA PCB 96 PCB DMSO HR
13. 12 02100000000 0 0000000000000000000000000000000000000000 HHHHHHHHHHHH 000000 HHHHHHHHHHHHHHH1IHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH 00000 700000000 0 000000000000000300000000000000000000000000 0000000000000000000000 7 0000000 HHHHHHHHHHH HHHHHHH PCB 0 025 EEE BEDET EE EDI _ 10 50 e 000000 0003000000000000000000000 00 0000000000000 PCBHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH ELISA 0 000000000000000000000000000000000000000000 0000000000000000000 135 100000000 100000000 0000000 50000000000000000000000 4000000000000000000 0 000000000000000000 02000000000 100000000000000000000000000000000000000000 000 pbHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHH elHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH 1000000000000 6100000 000000000000000000 0000000000000000 000000 0 00000000000000000 6200000000 0 000000000000000000000000000000000000000000 0 0000000000000000000 0000 0 00000000000000000000000000000000000 00000 0 000000000000000000000000000000000000000000 000000000000000000000000 14
14. gt 2 20
15. H H 11000000 6 50 250 DMSO 118 000000 00000 000 20 00000000000 00000 6 5 6 5 rk 00000 1 70 5 0000000 2 10 8 9 n 800 300000000 00000 2 90 9 70 00000 11 60 16 5 HHHHHHHHHHHHHHH6HH 300000000 00000 3 29 10 70 00000 10 60 12 5 9010000 9000300068000 nennnnnnnn ae 00000 1 90 3600 00 00 7 20 9 1 HHH HHHHHHHHHHHHHHH2HHHHHHHH 00000 0 889 15 2 PCB 28 31 33 66 709 1059 1100 ennn 21000000 nnnn 000000000 78 40 112 000 0000000 820 0 960 n 20 0000 0000000 820 0 990 000000 Ra 0 94 Gnnn 00000 140000 160 300 0000000000000 00000000 10 0000000000000 20 0000000000000000000 30 44000000000 410000000 EET E EE EI 17 00000 04 000000000000000 110 10 4 50 120 41 50 0000000 0 0000000000000 EK 00000000000 o 0000000 0000000 oe us
16. Stop solution mL 1 N Plate seal 5 PCB PCB
17. BRE EET EE 0 0000000000000000000 li 00000000000000000000 000 ELISAHHHHHHHHHH PCB EIA System O O 160 11 290 HHHHHHHHHHHHHHHHHHHHHHHHHHHHH 000000000 160 80 00000000000 0000000000000000000000000000 0 000000000000000000000 20000000000000000000000 00000000 000000 HHHHHHHHHHHHHHHH 00 HH 00 0 000000000 00000000000000000000 00 unu MM MN EP 1 S ANNA TET 1 1 EE ETE o roo t 2 2 1 E EL EET EJ a e aE A ER 2 SNB 2 HP BE OR EI 3 3HHHHHHHHHHHHHHHHHHHHHHHHHHHH 4 CRI 4 l B lt 4 C ENN Ri 6 4 EP EE ELE lt lt 6 4 2 EEEDEP EI E E E ccs ets eee heo 7 4 9 a Ee ERI he theo 8 4 4 E ED toh eee oo res 10 B ALLP 11 ELIE 11 11 SHTA 12 en SED e bv EXPE EE 12 TE ed 12 EL dep satia 12 D
18. aE _ HHHHHHH 000000000 OO ________ 00000 OOOO 00000000 0000 0000000 OOO 00000000000 440000000 0 00000000000000000000000000000000000 00000000000000000000000000000000000000000 00000 000000000 00 400000000000000000000000 b 0000000000000 000000 000000 a EU TELE HHHHHHHHHHHHHH 000 6800 000000 000000 0 0000000000000000000000000000000000000000 000300000000000000000000000000000 0000000 910000000000000000000000000000000000000 10 407 0 0000000000000000000000000000000000000000 000000000000000000000000000000000 0000000000 000000 PCB 1189 ACCU Standard 2 3 4 4 5 PeCB 0 0 DMSO 00 HHHHHHHHHHHHHHHHHHH lID 06 HHHHHHHHHHHHHHHHHH 0000 HHI HHHHHHHHHHH ug L 0000 118 100 250 1000 2500 1000 118 100 250 1000 2500 1000 PCB 118 10 I PCB 118 25 PCB 77 10 25 100 250 1000 PCB 105 10 25 100 250 1000 2 2 4 4 5 100 250 1000 2500 1000 5 HeBB
19. 300mL 300ml D 2 8C 30 6 strip 8wells strip 2 HRP 50 14 4 HRP 50 20 28 C 30 5 Wash buffer 3
20. PCB 14 KC 400 PCB 8 1 DMSO Soxhlet extraction n hexane 16hrs DMSO exchange 1 2 GC ECD 2 GC ECD Polluted soil Decontaminated soil 0 444 0 039 0 009 0 031 2 0 99 R 0 94 PCB concentration by EnBio PCB EIA system ug g dry soil 0 1 1 10 100 1000 PCB concentration by GC ECD ng g dry soil 2 GC ECD From Proceedings for 9th meeting of countermeasure f
21. 00 0000 0000000000 00000 1 00000 2 90 000000000 imo PCB EIA SystemHHHHHHHHHHHHHHHHHHHHHH PCBHHHHHHH 4100000 gt HHHHHHHHHHHHHTurboVapll 2 0 0 0 5 125mm ADVANTEC HHHHPCBHHHHH 000 HHHHPCBHHHHH DMSO 000000000 0000 0000000 0000000 HHHHHHH gt 10 507 0000000000000 200000000000000 HHHHHHHHHHHHHHHH m 000000000000000000000 0 000000000000000000000000000 40 00000000 0000000000 00000000000 49 0000000000000 DMSO0 5mL 0 00 40100 0000 0 00000000000000000000000000000000000000 000000000 HHHHDMSOHHHHH 1mLHHHEIAHHHHHHHHH 22 EI BE EY EJ 1 2 00 0000 4100000 gt 1L 00000 4 1L HHHHH m 0000000000000000000000 50mL 1000 1000 00000 00000000 200mL 0 00000 0000 1000000000 mL 0000000000000000000000 50mL 1000 lt 1000 OU HHHHHH 00000 0000 200mLO 00000 lt 000000000 E 40 00000 1m 0000000000000 1005 L 1 1 L1 D D H H H 49010 00 lt 05mL DMSO 0 0000000000000 40 000000000000000 30000 DMSOHHHH Im 0000000000000 2 2 1 EnBio PCB EIA system EnBio PCB EIA system Code RPNJ412 96 wells STORAGE Store at 2 8T EXPIRY The expiry date is stated on the package and will be at least 4 weeks from
22. 100 2 2 5 TriCB 18 0 1 KC 600 1 69 18 5 2 3 3 20 lt 0 1 KC 500 2 4 4 TriCB 28 3 5 100 2 4 5 TriCB 31 12 9 2 3 4 TriCB 33 2 6 Coplanar PCBs IUPAC 9 CR 2 2 3 5 44 01 9 4 3 4 TeCB 77 17 8 2 2 5 5 TeCB 52 lt 0 1 3 4 4 5 TeCB 81 lt 3 0 2 3 4 4 66 15 2 3 3 4 4 5 126 lt 3 0 2 3 4 5 70 14 9 3 3 4 4 5 5 169 lt 0 1 2 2 3 5 6 95 lt 0 1 2 3 3 4 4 PeCB 105 2 5 2 2 4 5 5 101 lt 0 1 2 3 4 4 5 114 3 4 2 8 3 4 4 PeCB 105 2 5 2 3 4 4 5 PeCB 118 100 2 3 3 4 6 PeCB 110 0 88 2 3 4 4 5 PeCB 123 lt 0 1 2 8 4 4 5 PeCB 118 100 2 3 3 4 4 5 HexCB 156 2 2 2 2 83 4 4 5 HexCB 138 0 1 2 3 3 4 4 5 157 0 1 2 2 83 4 5 6 HexCB 149 0 1 2 3 4 4 5 5 HexCB 167 0 1 2 2 4 4 5 5 HexCB 158 0 1 2 3 3 4 4 5 5 189 0 1 2 2 3 3 4 4 5 HepCB 170 lt 0 1 2 2 3 3 4 5 6 174 lt 0 1 PAHs CR 2 2 8 4 4 5 5 HepCB 180 0 1 Acenaphtene 0 1 2 2 3 4 5 5 6 187 0 1 Acenaphtherene 0 1 2 2 9 9 4 4 5 5 OctCB 194 0 1 Anthracene 0 1 2 2 3 3 4 4 5 6 OctCB 196 0 1 Benzo a anthracene 0 1 2 2 3 3 4 5 5 6 OctCB 199 0 1 Benzo a pyrene 0 1 2 2 3 4 4 5 5 6 OctCB 203 0 1 Benzo b fluoranthene 0 1 Benzo ghi perylene 0 1 Other related compounds CR Benzo k fluoranthene 0 1 Biph
23. 4 5 TriCB 31 12 9 lt 0 1 2 3 4 TriCB 33 2 6 lt 0 1 2 2 3 5 44 lt 0 1 2 27 5 57 52 0 1 2 3 4 4 TeCB 66 15 2 Other related compounds CR 2 3 4 5 70 14 9 Biphenyl 0 1 2 2 3 5 6 95 0 1 1 2 dichlorobenzene 0 1 2 2 4 5 5 PeCB 101 0 1 3 4 dichloroaniline 0 1 2 3 3 4 4 PeCB 105 2 5 3 4 dichloroanisole 0 1 2 3 3 4 6 PeCB 110 0 88 3 4 dichloronitro benzene 0 1 2 3 4 4 5 PeCB 118 100 3 4 dichlorophenol lt 0 1 2 2 3 4 4 5 HexCB 138 0 1 3 4 dichlorotoluene 0 1 2 2 3 4 5 6 HexCB 149 0 1 1 2 3 trichlorobenzene lt 0 1 2 2 4 4 5 5 HexCB 153 0 1 3 4 5 trichloroaniline 0 1 2 2 3 3 4 4 5 HepCB 170 0 1 3 4 5 trichlorophenol 0 1 2 2 3 3 4 5 6 HepCB 174 0 1 2 3 7 8 TCDD lt 0 1 2 2 3 4 4 5 5 180 0 1 2 3 7 8 TCDF lt 0 1 Un 2 2 3 4 5 5 6 187 0 1 2 2 3 3 4 4 5 5 OcICB 194 0 1 2 2 3 3 4 4 5 6 OctCB 196 lt 0 1 2 2 3 3 4 5 5 6 OctCB 199 lt 0 1 2 2 3 4 4 5 5 6 OctCB 203 0 1 CONCLUSIONS The assay is highly selective to PCB 118 among predominant PCB congener in PCB products There is no significant cross reactivity with other related compounds such as PAHs and polychlorobenzen derivatives Claim 7 Demonstration of the correlation between GC ECD and EIA in fish samples AI To demonstrate the applicability to fish samples METHOD Fish samples are pretreated a
24. BLU HRP BE 450nm lal lal PCB PCB 8 wells strip 6 5 250 ng ml PCB 118 PCB PCB 4
25. PCB Sf ELISA 3
26. Sample Mean ng mL SD CV n High 49 4 3 6 9 1 6 Low 21 5 1 9 6 CONCLUSIONS The between plate precision was shown to be within acceptable limits Claim 6 Cross reactivitiy of the Assay AIM To investigate the degree of cross reactivity of the predominant PCB congener in PCB products e g Aroclor and Kanechlor METHOD Standard solution of zener were prepared in DMSO RESULTS The cross reactivities CR are described as ratios between IC50 value of each compound and that of PCB 118 This was done for significant cross reactants only PCB products CR PAHs CR Fanechior 300 3 87 423 Acenaphtene 0 1 equivalent to Aroclor 1242 Acenaphtherene 0 1 Kanechlor 400 736 80 5 Anthracene lt 0 1 Aroclor 1248 Benzo a anthracene 0 1 Kanechlor 500 9 14 100 Benzo a pyrene lt 0 1 Aroclor 1254 Benzo b fluoranthene 0 1 Kanechlor 600 1 69 18 5 Benzo ghi perylene lt 0 1 Aroclor 1260 Benzo k fluoranthene 0 1 Ratios between value of each PCB product and that Chrysene 0 1 of Kanechlor 500 are described in parenthesis Dibenzo ah anthracene 0 1 Fluoranthene lt 0 1 Predominant PCBs IUPAC f CR Hexachlorobenze 0 1 2 3 DiCB 5 0 1 Indeno 123cd pyrene 0 1 2 4 DiCB 8 0 1 Naphthalene 0 1 2 2 5 TriCB 18 0 1 Phenanthrene 0 1 2 3 3 TriCB 20 0 1 Pyrene 0 1 2 4 4 TriCB 28 33 Hexachlorobenze 0 1 2
27. EL EE 12 AL DE 13 RR 13 DUIS P a ptu dn 13 ME SE re LS 14 or EE ie e ihr ore iot 4 14 6 10 TEA ELE oe SR eoe Ee tor ro yere ns 14 esee ee E ere oe d e otn n n to Reiten 14 1000000 2000000000000 30000000000000000 110000000000 110000000000 HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHUHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH 000000000 160 80 310 0000000000000000000000000 0000000000000000000000000000000000000000000 HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH EET EE FI ET EET EL EL ELEE 000000000000000000000 HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHHELISAHHHHHHHHHHHH 000 000000000 1120000000 0 0000000000000000000000000 e 00000000000000 e ED Oo BULL EE 2100000000 POO DEORE EOP oo EIE ELE ee EL ET HI 01 00000000
28. SCREENING OF COPLANAR POLYCHLORINATED BIPHENYLS Organohalogen compounds 58 2002 333 336 ELISA 3 H Takigami T Etoh T Nishio S Sakai APPLICATION OF SOLVENT EXTRACTION TECHNOLOGY TO PCB CONTAMINATED SOIL AND CHEMICAL BIOASSAY MONITORING Organohalogen compounds 66 2004 1221 4 T Tsutsumi Y Amakura A Okuyama H Mizukami Y Tanioka Ueda Sakata K Sasaki T Maitani Applicability of ELISA to screen for dioxin like PCBs in retail fish Organohalogen compounds 66 2004 603 4 A PCB Takigami et al 2004 PCB PCB A Tsutsumi et aL 2004
29. enyl 0 1 Chrysene 0 1 1 2 dichlorobenzene 0 1 Dibenzo ah anthracene 0 1 3 4 dichloroaniline 0 1 Fluoranthene 0 1 3 4 dichloroanisole 0 1 Hexachlorobenze 0 1 3 4 dichloronitro benzene lt 0 1 Indeno 123cd pyrene lt 0 1 3 4 dichlorophenol lt 0 1 Naphthalene 0 1 3 4 dichlorotoluene lt 0 1 Phenanthrene lt 0 1 1 2 3 trichlorobenzene lt 01 lt 0 1 3 4 5 trichloroaniline 0 1 Hexachlorobenze 0 1 3 4 5 trichlorophenol 0 1 2 3 7 8 TCDD lt 0 1 2 3 7 8 TCDF lt 0 1 10 2 PCB 3 3 4 trichloro 4 methoxy biphenyl PCB 118 100 80 X PCB 118 native A E a 1 10 0 100 1 000 Conc of Analyte in DMSO ng mL 60 X B Bo 40 20 0 4 5 PCB 118 native 3 6 5 ng ml Zero standard DMSO n 8 SD 3x SD 4 3 PCB 8
30. ilution buffer Competitor HRP conjugate concentrate 60 1 BJ 4 2m HRP conjugate dilution buffer 20 L O Competitor HRP conjugate concentrate 4 HRP D 3 HRP 1 3 2 30p L 90 u L 2 5 Wash buffer D Wash buffer concentrate 10
31. itivity defined as three standard deviations below the mean optical density of 8 zero standard replicates was determined The corresponding concentration was calculated from a standard curve ranging between 1 to1 000 ng ml set up in 8 replicates The grand mean zero and standard values were then used to calculate the sensitivity The data was plotted as mean SD as a 4 parameter plot and calculated values by an attached calculation soft wear ofa micro plate reader The sensitivity defined as the concentration on the standard curve equivalent to 3 standard deviation below zero standard n 10 was determined to be 6 5 ng ml of PCB 118 in DMSO RESULTS This was determined as 6 5 ng ml No cross ranging was found between the absorbance of zero standard below 3SD Table 2 Intra assay of the absorbancies OD450 for PCB 118 n 8 0 8 OD iso 0 6 0 4 0 2 0 0 e 1 10 100 1 000 Conc of 118 ng mL 2 Claim 2 Demonstration of Within Assay Precision AI lt To determine the within assay precision of measurement of controls in the assay METHOD Low medium and high controls were prepared from a stock standard The results were obtained by using one batch of performance trial kit SULTS Sample Mean ng mL SD CV n High 98 4 5 04 5 1 8 Med 55 5 3 90 7 0 8 Low 18 5 1 66 8 9 8 CONCLUSIONS The within assay precision was shown to be within acceptable limits Claim 3 Demon
32. or soil and groundwater contaminations 450 453 2003 Japanese language 1 DMSO DMSO n hexane extraction Silica gel H2S04 reflux 44 treatment DMSO exchange 2 Rd 4 PCB 10 000 1 000 100 10 PCB concentration by EnBio PCB EIA ug kg 0 1 10 100 PCB concentration mg kg 4 PCB Drawn from Organohalogen compounds 58 397 400 2002 10 1 Cross reactivity CR PCB 118 100 Table 1 PCB products CR Predominant congeners IUPAC CR KC 300 3 87 42 3 2 3 DICB 5 0 1 KC 400 7 36 80 5 2 4 DiCB 8 lt 0 1 KC 500 9 14
33. s shown in the following figure Treat 20 g of homogenized fish sample with 2 mol L KOH for ovemight at RT Alkali digestion Extract by shaking with methanol n hexane 3 2 Sulfuric acid treatment Treat with concentrated sulfuric acid till extracts become colorless Multi layer silica gel column Apply to multi layer silica gel column and elute with n hexane Apply to alumina column and elute with 5 vol DMSO exchange Exchange to 100uL of dimethylsulfoxide by purge EIA analysis RESULTS Total PCBs conc 20 000 y 0 15x 1 71 r 0 98 n 20 D amp lt 10 000 3 5 000 0 0 90 100 150 GC ECD ng g CONCLUSION A good correlation between GC MS data and EIA measurements in fish samples was demonstrated Claim 8 Demonstration of the correlation between GC ECD and EIA in soil samples AI To demonstrate the applicability to PCB contaminated soil samples METHOD PCB polluted and decontaminated soil samples are pretreated as shown in the following figure DMSO exchange m hexane 16hrs RESULT m D Es Polluted soil Decontaminated soil 9 9 y7044440039x 0 009 0031 E R 0 90 R 0 94 gt 53 8 lt n 0 1 1 10 100 1000 PCB concentration by GC ECD pg g dry soil CONCLUSION A good correlation between GC ECD data and EIA measurements in polluted and decontaminated soil samples was demonstrated
34. stration of Between Assay Precision AI To demonstrate the between assay precision of measurement of controls in the assay METHOD Low medium and high controls were prepared from a stock standard The results were obtained by using one batch of performance trial kit RESULTS Sample Mean ng mL SD CV n High 83 7 9 66 11 5 6 Med 45 4 7 92 17 5 6 Low 17 5 2 88 16 4 6 CONCLUSIONS The between assay precision was shown to be within acceptable limits Claim 4 Determination of Stability AIM To demonstrate the stability of reagents when stored at the recommended temperature of 4 C METHOD The standard curves were obtained using reagents from the 4 C stability trial at time points at 0 3 and 6 months using one batch of kit Low and high controls were prepared from a stock standard RESULTS Typical assay data for a standard curve of 0 1 000ng ml surro gate standard OD450 6 months Sample Mean ng mL SD CV 96 High 101 10 7 10 6 3 Low 25 8 3 24 12 5 CONCLUSIONS The reagents give a good response after 6 months storage at 4 C The 6 months period precision was shown to be within acceptable limits Claim 5 Demonstration of Between plate Precision Al To demonstrate the between plate precision of measurement of controls in the assay METHOD Low and high controls were prepared from a stock standard The results were obtained by using six batch of performance trial kit RESULTS
35. the date of dispatch Warning For research use only Not recommended or intended for diagnosis of disease in humans or animals Do not use internally or externally in humans or animals Amersham EI Biosciences 1 2 3 4 5 6 1 2 1 2 1 2 10 1 2 3 4 5 11 1 2 3 2 2 Ro 2 PET VRE RD 3 1 U U UJ ett dL 3 DE MEIE TT ERR CIR EP 4 tA l2 00 17 md Y QH 4 i E a lT E T O 4 AAAA AAO RISE 5 HRP LLL 5 a ONE iie some tatis td HE E EE a 5 6 6 6 7 EE e innt tic Et qasqu q 7 IGE 7 GC ECD 8 GERRI 8 e a a SI N NEEE ONONE 8 9 10 10 11 M REA 11 11 0 12 BB 13 13 RIGA amp 5 zero standard 13 DURE OTHE FERALAS lt fu 13 1 PCB
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