Modifide Direct Competitive Enzyme-Linked Immunosorbent Assay for Aflatoxin B1 in rice - PowerPoint PPT Presentation

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Modifide Direct Competitive Enzyme-Linked Immunosorbent Assay for Aflatoxin B1 in rice

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Title: Modifide Direct Competitive Enzyme-Linked Immunosorbent Assay for Aflatoxin B1 in rice


1
Modifide Direct Competitive Enzyme-Linked
Immunosorbent Assay for Aflatoxin B1 in rice
?????????????? Aflatoxin B1 ????????????????
Direct Competitive Enzyme-Linked Immunosorbent
Assay
2
  • ????????????
  • ?.?. ??????? ?????????
  • D4810056
  • ?.?. ????? ?????????????
  • m4810131

3
????????????
  • ?????????????????????????? alflatoxin B1 ??????

4
??????????????????
  • ??????????????????? alflatoxin B1
    ????????????????????? ????????????????????????????
    ?????????????????????????????????????

5
???????
  • ??????????????????????? Chu (1989)
    ?????????????????? coat ????? well ??? microtiter
    plate ???? aflatoxin B1-oxime-BSA ??? incubate
    ??? ??????????????????? ??????????????????????????
    ????? aflatoxin B1 ??????? ??? anti-aflatoxin B1-
    BSA- HRP conjugate ?????????? ??? ???? incubate
    ??? ????????????????????????????????? aflatoxin
    B1 ??????? ???????????? antigen
    ??????????????????? microtiter plate

6
  • ???????????????????? antibody ???????
    ????????????????? ???????????????????? antibody
    ?????????????????? microtiter plate ??????
    incubate ??????????? substrate ???????????????????
    ??????????????????????? spectrophotometer
    ????????? aflatoxin B1 ???????????????????????????
    ??????????????? aflatoxin B1 ???????

7
????
  • Aflatoxin ???????????????????????? Aspergillus
    flavus ??????????????????????????????????????
    ?????????????????????????????????????????

8
????????????????????
  • ??????????????? AR grade
  • ???????????????????? (Sample extraction solvent )
  • Carbonate coating buffer
  • Phosphate buffered saline (PBS) pH 7.4 ??????? 10
    ????

9
  • PBS-Tween
  • Conjugate buffer
  • Substrate buffer
  • Enzyme Substrate

10
  • Aflatoxin B1-BSA ??????? 500 ????
  • Anti-Aflatoxin (B1) Peroxidase conjugate
  • Blocking 1
  • Colour stop reagent 0.5 N

11
  • ??????? ???????????????? ??????
  • ?????????

12
??????????????
  • coat plate ???? Aflatoxin B1-BSA (sigma A6655)
    100 ????????? ??? well ?????????? 2
    ?????????????????????
  • Incubate ?? moist chamber ??? 4C ???????
  • ???????? PBS-T 3 ?????
  • block ???? BSA 1 200 ???????????? well ??? 37C
    ???????? 1 ???????

13
  • ???????? PBS-T 3 ?????
  • ???????????????????? 50 ???????????? well
    ??????????? Anti-AFB1- peroxidase conjugate 50
    ???????????? well
  • Incubate ?? moist chamber ??? 37C 45 ????
  • ???????? PBS-T 3 ?????

14
  • ???? Enzyme Substrate 100 ???????????? well
  • Incubate ???????? 10-15 ????
  • ????????????????? Colour stop reagent 25
    ???????????? well
  • ??????????????????????? 492 ????????

15
??????????
16
???????? 1 ??????????????????????? standard
aflatoxin B1
???????????(ug/kg) ??????????????????? 492 nm
7.5 0.215
15 0.218
30 0.220
60 0.212
120 0.213
240 0.215
480 0.196
960 0.207
1920 0.189
3840 0.137
7610 0.103
17
???????? 2 ??????????????????????????????????? 17
????????
???????????? ???????????? ??????????????????? 492 nm
1 ?????   0.387
2 ????????   0.406
3 ????   0.376
4 ??????????? 4 ??????????? 0.382
5 ???????? ???? 5 ???????? ???? 0.402
5 ???????? ???????? 5 ???????? ???????? 0.380
6 ?????   0.359
7 ?????   0.415
8 ????????   0.384
9 ??????   0.368
10 ?????? 10 ?????? 0.374
11 ??????? 11 ??????? 0.370
12 ???   0.348
13 ???   0.361
14 ????   0.369
15 ?????   0.347
16 ??????? 16 ??????? 0.361
18
  • ?????? 2 ????????????????? standard aflatoxin ??
    microtiter 96 well
  • plate

19
??????????????
  • ????? aflatoxin ????????????????????
    ??????????????????? 17 ????

20
?????????????
  • Anonymous. 1991. Aflatoxin analysis in groundnuts
    and groundnut products. Pages 195-278 in. The
    groundnut Aflatoxin problem review and database
    (Mehan, V.K., Mcdonald, D., Haravu, L.J., and
    Jayanthi, S. eds.). Patancheru, AP. 502324, India
    International Crops Research Institute for the
    Semi-Arid Tropics.
  • Fan, T.S.L., and Chu, F.S. 1984. An indirect
    ELISA (Enzyme-Linked Immunosorbent Assay) for
    detection of aflatoxin B1 in corn and penut
    butter. Journal of Food Protection 47 263-266

21
  • Chu, F.S. 1989. Current immunochemical methods
    for analysis of Aflatoxin in groundnuts and
    groundnut products. Pages 101-172. In Aflatoxin
    contamination of groundnuts proceedings of the
    International workshop, 6-9 October 1987, ICRISAT
    Center, India. Patancheru, AP. 502324, India
    International Crops Research Institute for the
    Semi-Arid Tropics.
  • Wilson, D.M. 1989. Aflatoxin analytical methods
    for groundnut. Pages 191-199. Ibid
  • Anajaiah, V., Mehan, V.K., Jayanthi, S., Reddy,
    D.V.R. and Mcdonald, D. 1989. Enzyme-Linked
    Immunosorbent Assay (ELISA) for alflatoxin B1
    estimation in groundnuts. Pages 1983-189. Ibid

22
FIN !!
??????????? WEB ????
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