Title: Modifide Direct Competitive Enzyme-Linked Immunosorbent Assay for Aflatoxin B1 in rice
1Modifide 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
22FIN !!
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