ピテロジェニン測定法-最新測定法の解説と開発動向の展望 2.酵素免疫
Contents
1. cm IgG 10ug mL 100uL 4 C 100uL 3 3 F ab 1 ng mL 100uL 3 PBS T 250mL 3 HRP 100uL 1 PBS T 250uL 3 3mg OPD mL 100uL 20 2N 50uL 492nm 490nm Fig 4 ELISA2. MW 1 000 1kDa 2 2007 2008 10 7 2008 10 23 Vg 2 Vg POROS HO HPLC
3. 2 0O 10cm 70 5 6 2 Vg 0 2mL 3 4 2 3 4 2 70
4. 1 2 lt 4 20 4 NaN 0 05 w v 2 3 4 ELISA 1 Fig 1 Vg TBS 1 3 10 30 100ng mL 1 cm 5cm
5. 3 ia F ab PBS BSA T 1 uug mL 100uL 3 IgG Vg F ab HRP ExtrAvidin Peroxidase ELISA PBS BSA T 100uL ExtrAvidin Peroxidase 1 000 1 Okumura et g 1995 1 Vg 0 HRP
6. PBS Biotin 7 NHS Biotin 7 NHS MW 454 DMSO 10 1 10 1750 F ab 100kDa 10mg mLl 0 1IimM Biotin 7 NHS 9 1mg mL 20mM F ab 1 mL 30 EE NMM TR 350 2 4 F ab PBS
7. 2002 2006 10 1 5 000 20 000 2
8. 11 Vg 10 10 Vw 50mM 14 6mg mL EDTA 10mM PMSF 900KIU mL PBS TBS 4 C 15 000 X g 10 Vg Vg
9. 39 20C 1 Vg Vg 170
10. IgG IgG 96 1 0 2msg IgG F ab IgG Fc F ab F ab fig 3 IgG 10DG 100mM 100mM
11. 1 cm 1 100ug mL 1uL 9 cm 1 g 20mL TBS P he 1 20uL Tween 20 10uL 1 0 19 Tween 20 TBS TBS T 20mL 5 TBS T 2 10mL T BSSNONAKS SN HRP IgG Vg 1L 59 TBS20mL 1 Tween 20 20uL 10 1 TBS T20mL 5 3 HRP IgG TBS T10mL
12. 23G 1 mL 1 2 1 5mL 1 4 30mL
13. 100uL 20 2N 30 HL 492nm 490nm Vg Vg
14. 1 Yamanaka et al 1998 Pagrus major Vg Yamanaka et al 1998 TC0 Ohkubo er al 2003 Acanthogobius flavimanus 530kDa Vg 530 320kDa Vg 320 2 Vg 178 Es 1mg mL 1 kg 1mL 1mg Ez kg 739 0452 2 17 5 E mail kazfujii affrc go jp 6 4 C Vg 10mM 1 74mg mL PMSF 10
15. 23G 18G 0 3 0 8mm E PMSF TBS Vg 2 KIU Kallikrein Inhibitor Unit 1 KIU pHS 2 2 1 300KIU 1 TIU 3 MW 174 MW 136 100mM 17 4g L 13 6g L
16. 2 0 1 000pg R 0 996 Fig 5A 7 8pg Fig 6 2 0 16pg Fig 3B R 0 990 17 Fig 6 2 0pg 0 086 0 0004 0 082 0 0006 3 5 400units mg HRP 20 16pg 2 2 0 1 000pg 8 Vg
17. 0 3mL min TBS 30kDa Vg 530 Gel Filtration Calibration Kit HMW GE Vg 320 20 4 13 000 Xg 0 100mM PP Superose 6 320kDa TBS Mono Q HR 5 5 5X50mm GE TBS Superose 6 Vg 320
18. pH7 3 3 pH 4 MW 121 20mM 2 4g 900mL pH gt 0 pH 1L MW S8 150mM 8 7g pH 5 1cm 1 0 1ImL 1
19. 12 31 40 2009 Rep Mar Ecol Res Inst No 12 31 40 2009 2 New Methodology for the Determination of Fish Vitellogenin and Future Problems 2 Enzyme Linked Immunosorbent Assay ELISA for Quantification of Fish Vitellogenin Kazunori Fujii 1 ELISA Enzyme Linked Immunosorbent Assay IgG ELISA ELISA
20. 0 2mL TiterMax Gold Vg 3 5 21G TiterMax Gold
21. eeerrerereewdrrrrgrruraeropenwmworrre2rrPgrtg2eetaoa rear y 2 63x 0 486 Ll y 1 99x 0 30 0 039 0078 016 031 063 1 3 25 50 Vg ng mL Fig 8 Relative errors of vitellogenin concentrations calculated by regression equations in Fig 7A black column and 7B white column Error nominal value calculated valuel nominal value x 100 HRP OPD ng mL 1992 ImmunoPure Avidin Alkaline Phosphatase Conjugated HRP SuperSignal ELISA Femto Chemiluminescent Substrate DELFIA Eu labeled streptavidin
22. 350KIU mL 0 9 10 4 C 3 000 Xg 10 E 80 10mM PMSF 100mM PP pH7 3 15X40mm E 0 3mL 1 mL mm 280nm 500mM PP 1 mL BSA Lowry Lowry et al 1951 150mM 20mM pH8 0 TBS Superose 6 HR 10730 GE
23. 8 MW 82 100mM 8 2g 900mL pH pH 1L 100mM 5 8g pH 9 8 0g 0 20g 1 15g 0 20g 1L 10 MW 106 MW 84 50mM 3 3g L 4 2g L pH9 6 2 pH
24. 0 020 80ng mL eligi eigaetal 0 020ng mL 2 400 2000 A 600 a 1 200 i 800 400 0 0 0 1 0 2 0 3 0 40 B 331X 19 5 R 0 996 15 10 HRP pg 0 08 0 09 0 10 Git 1 0D 490nm Fig 5 Relationship between quantity of horseradish peroxidase HRP and color intensity optical density OD of o phenylenediamine in the range of 2 0 1 000pg A and 2 0 16pg B Each point represents the mean of quadruplicate determinations standard deviation of the mean 0 039ng mL 0 174 0 003 0 154 0 005 3 0 039 5 0ng mL R 0 996 Fig 7A 0 078ngnL Pig 8 y 331x 19 5 y 526x 43 7 m 16 31 63 130 250 500 1 000 HRP pg Fig 6 Relative errors of quantity of horseradish peroxidase HRP calculated by regression equatio
25. Amano H Fujita T Hiramatsu N Shimizu M Sawaguchi S Matsubara T Kagawa H Nagae M Sullivan C G and Hara A 2007 Egg yolk proteins in gray mullet Mugil cephalus Purification and classification of multiple lipovitellins and other vitellogenin derived yolk proteins and molecular cloning of the parent vitellogenin genes Exp Zool 307A 324 341 2002 Pleuronectes yo og7jge 33 41 2006 pp 88 102 http search funakoshi co jp data datasheet I CYT G l pdf 1992 37 144 150 Lowry O H Rosebrough N J Farr A L Randall R J 1951 Protein measurement with the Foin phenol reagent J Biol Chem 193 265 272 Ohkubo N Mochida K Adachi S Hara A Hotta K Nakamura Y and Matsubara T 2003 Development of immunosorbent assays for two forms of enzyme linked vitellogenin in Jap
26. pH4 5 10kDa 10mgmL IgG 96 1 0 1mg 0 4mg mL 37C 15 20 F ab 100kDa Fc 0kDa 3SkDa 10 100kDa Superose 12 HR 10 30 GE IgG 10mg mL 0 4mg mL 37C 15 lt 20 PBS F ab F ab 2 10 Biotin 7 NHS 2 4 F ab Fig 3 Procedures for fragmentation of IgG to F ab and labeling of F ab with biotin
27. 6 ELISA ELISA HRP OPD 30mg OPD 10mL 100mM pHS 0 2uL HRP OPD HRP OPD HRP 7 MW 192 MW 294 100mM 19 2g L 29 4g L pHS 0 2 3 pH
28. lgG 4 2 0 1 Tween 20 PBS PBS T 230nL PBS T 3 lsG 8 12
29. F ab 0 05 NaN 4 C 2 3 20C 6 ELISA Fig 4 ELISA IgG 50mM pH9 6 IgG ng cWm IgG Vg 10uug mL 100nL
30. R 2 R 0 99 Vg X3 Vg 100 5 3 2 2 3 7 0 20 200ng mL HRP 400units mg 4 10uL 2 0 2 000pg Wallac1420 ARVOsx 490nm
31. 1 TBS T20mL 5 3 Fig 1 Dot blotting procedure for screening antibody titer of antiserum 3 3uL 1 TBS T 3 10m L OPD 10ng mL Vg Fig 2 4 Wy 100 30 10 3 1 pg mL Fig 2 Dot blotting of the purified vitellogenin Vg for screening antibody titer of anti vitellogenin antiserum JE
32. Amano et g 2007 Mugil cephalus POROS HO 3 3 Vg 3 Pleuronectes yo o7g7ge Vg Vg E POROS HQ Vg 1 mgmL 0 2mL
33. E Vg E2 Vg SPG 320L 5 A G HiTrap Protein A FF HiTrap Protem G HP GE
34. procedure for marbled sole vitellogenin 1 BSA PBS PBS BSA I 4 C MR PBS BSA 1 0 01 0 05 NaN NaN HRP NaN PBS BSA HRP PBS BSA T Vg 0 19 Tween 20 PBS BSA PBS BSA T 100uL Vg PBS BSA 9 2 i 3 IgG Vg 12 8
35. anese common goby Acanthogobius flavimanus Gen Comp Endocrinol 131 353 364 Okumura H Hara A Saeki F Todo T Adachi S and Yamauchi K 1995 Development of a sensitive sandwich enzyme linked immunosorvent assay ELISA for vitellogenin in the Japanese eel Anguilla Japonica Fish Sci 61 283 289 2007 127 71 80 Yamanaka S Arizono K Matsuda Y Soyano K Urushitani H Iguchi T and Sakakibara R 1998 Development and application of an effective detection method for fish plasma vitellogenin induced by environmental estrogens Biosci Biotechnol Biochem 62 1196 1200
36. ns in Fig SA black column and SB white column Error nominal value calculated valuel nominal value Xx 100 63x 0 486 Vg ng mL Vg ng mL 0 15 0 20 0 258 OD 490nm Fig 7 Standard curves in the range of 0 039 S 0ng mL A and 0 039 0 16ng mL B in ELISA of marbled sole vitellogenin Vg Each point represents the mean of quadruplicate determinations standard deviation of the mean 0 039 0 16ng mL Fig 7B R 0 998 5 Fig 8 0 020ng mL 5 0 16ng mL 2 0 039 5 0ng mL Vg 9 ELISA ELISA 6
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