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KMID : 0386819920020010053
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Journal of the Institute of Health Environmental Sciences
1992 Volume.2 No. 1 p.53 ~ p.88
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Production of Polyclonal Antibody to Aflatoxin B1 and its Application to ELISA of Aflatoxin B1 Contamination in Rice
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Abstract
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This study was performed to investigate the contamination route of aflatoxin in rice. In order to attain the object, isolation of aflatoxin-producing strains in agricultural products produced in Korea were empolyed for the detection of aflatoxin
B₁in
samples. A survey of natural occurrence of aflatoxin B₁in rice produced in Korea, pot and field experiments for accumulation and translocation of aflatoxin B₁in rice plant, and an experiment for aflatoxin production by Asp. Flavus in rice
medium
during storage were carried out.
@ES The results were as follows:
@EN 1) To isolate the aflatoxin-producing strains in agricultural products, a total of 342 samples, including 209 from cereals, 70 from beans, 30 from peanut, and 33 from soil were collected from Youngnam district of Korea from March to July in
1989.
From the 342 samples 280 strains of Aspergillus sp. were isolated. As the results of screening by TLC, 29 strains expressed fluorescence spot and 4 strains (1.4%) had the same Rf value of standard aflatoxin. The 4 strains were classified as Asp.
Flavus
group by the examination of physical properties and morphology.
2) For the production of antibody to aflatoxin B₁, antigen was prepared by conjugation of aflatoxin B₁-(0-carboxymethy1) oxime to bovine serum albumin(BSA). The recovery rate of aflatoxin B₁-oxime was 87. Antibody to aflatoxin B₁-oxime was
87%.
Antibody to aflatoxin B₁having a titer a titer of 1.180 was produced in 13 weeks by New Zealand white female rabbits immunized with aflatoxin B₁-oxime-BSA conjugate. The antibody cross-reacted more actively with aflatoxin B₂(118%) and less
extent
with other aflatoxin analogues. Detection limit for the antibody in a competitive direct enzyme-linked immunosorbent assay was 1ng/ml for aflatoxin B₁. The new polyclonal antibody was applicable to the ELISA detection of aflatoxin B₁in rice
after
a
simple extraction of samples in methanol-water(70:30)
3) Natural occurrence of aflatoxin B₁in rice was investigated in 88 samples obtained from whole country of Korea from March to September in 1988 and 65 samples from Youngnam district of Korea from March to July in 1989. The results showed that
3.4% of
the 1988's samples and 4.6% of the 1989's were positive, respectively. However the positive samples were still within 10 ppb, the maximum residual level of aflatoxin B₁for food of Korea.
4) Aflatoxin contamination of rice before harvest was investigated in pot and field experiments. In the pot experiment, pots were treated with aflatoxin B₁at the level of 10, 100, and 1,000 ppb at ear formation stage of rice plant. Aflatoxin
B₁was
detected only in root of rice pant after 72 hours. There was no sign for the translocation of aflatoxin B₁from soil and water to rice plant by immunohistochemical staining.
5) In the field experiment, 1, 5, and 10 ㎍ of aflatoxin B₁and spore suspension of Asp. Flavus(10³, 10E6, and 10E9conida/ml) were treated with various conditions. Kernels from the treated plant showed slightly lower ripening rate and 100-grain
weights than did those of controls. The nutrient and fatty acids composition of unpolished rice of treated group were similar to those of controls. At harvest, a wide range of aflatoxin B₁content was observed in the samples among cultivars.
Content of
aflatoxin B₁of unhulled rice samples from the Asp. Flavus inoculated kernel exhibited significantly difference(p<0.05) among inoculation methods including brush, knife, and needle. However no tendency of toxin levels among treatments by
inoculation
time, inoculum size, inoculation point was observed. By immunohistochemical staining, aflatoxin-detected rice embryo clearly showed black color.
6) Aflatoxin production by Asp. Flavus in rice medium was influenced by moisture content and temperature, especially with the temperature cycling at 28℃ for 18 hours and 15℃ for 6 hours. In case of 15 days' incubation by the temperature
cycling,
aflatoxin B₁content of the autoclaved and Asp. Flavus inoculated rice was 56.4mg/kg. However the rice inoculated with the used strain contained 62.8-71.6mg/kg of aflatoxin by the incubation for 3 months at room temperature regardless of initial
condition.
With the above results, it could be concluded that aflatoxin contamination in rice generally occurs during the storage after harvest.
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