Production of Antibodies for Detection of Aspergillus parasiticus in Cereals

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Project title

Code/計畫編號

NSC85-2321-B019-009

Translated Name/計畫中文名

Aspergillus parasiticus 抗體的生產及其在穀類黴菌分析上的應用

Project Coordinator/計畫主持人

Guo-Jane Tsai

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Food Science

Website

https://www.grb.gov.tw/search/planDetail?id=209412

Year

1996

Start date/計畫起

01-08-1995

Expected Completion/計畫迄

01-07-1996

Bugetid/研究經費

500千元

ResearchField/研究領域

食品科技(農)

紐西蘭大白兔分別以A. parasiticus菌絲萃取液或其SDS-PAGE純化之4種不同分子大小之單一蛋白行耳靜脈注射,所得之抗體利用Horeseradish peroxidase建立三明治式ELISA分析。比較5種抗體的專一性,顯示由A. parasiticus菌絲萃取液而得之抗體之專一性最佳,其對測試之21株A. parasiticus菌株及11株A. flavus菌株均可偵測,但對其他Aspergillus菌株及包括Penicillium等共18屬之60株真菌,僅2株呈弱陽性反應;另以抑制性ELISA輔助專一性測試,顯示其僅對測試之A. parasiticus及A. flavus有作用。將此ELISA應用在玉米、花生、小麥和米四種穀物之A. parasiticus分析,顯示其靈敏度至少1.mu.g菌絲重/mL,且穀物成分不會干擾ELISA分析值。在人為穀物接菌試驗中,顯示穀物水活性高低明顯影響A. parasiticus黃麴毒素產量,高水活性時(0.952-0.988)黃麴毒素快速產生,且A. parasiticus之ELISA和傳統平板菌落分析法二者之相關性良好,在高水活性(0.952-0.988)之玉米、花生、小麥和稻米之四種穀類中,此二分析法之相關係數分別為0.94、0.86、0.96和0.93。另外,當穀物中A. parasiticus之平板菌落數為10/sup 4/-10/sup 5/cfu/g時,ELISA即可明顯偵測到此菌之存在,此時尚未有黃麴毒素產生。利用YES(Yeast extract sucrose)培養液接菌試驗顯示,由ELISA所測得之菌絲重和由秤重而得者之差異不大,且二者相關性良好,相關係數為0.97。以ELISA及選擇性培養基AFPA分析市售玉米、花生、小麥及稻米各10件,均顯示市售樣品未受A. parasiticus或A. flavus汙染或汙染程度低;若將樣品存在高溼度環境中,經29天儲存,ELISA及AFPA之平板分析均顯示出樣品中A. parasiticus/A. flavus含量明顯增加,小麥及稻米之汙染程度高於花生或玉米,且不同樣品間差異極大。 New Zealand female rabbits were immunized separately with mycelial extract of A. parasitcus or various sizes of proteins isolated by SDS-PAGE of mycelial extract of Aspergillus parasitcus. After three times of boosters, the antibody titers of the sera were above 10/sup 6/. The antibodies were purified and the double-sandwich ELISA were established after the antibody horseradish-peroxidase conjugates were prepared. The antibody against the crude mycelial extract of A. parasitcus had a best specificity among the five antibodies obtained. It only reacted with A. parasitcus and A. flavus and the detection rate was 100% for these toxigenic molds. Among 60 non-A. parasitcus/A. flavus fungi tested, only 2 molds had weak cross-reactions with this antibody. However, no false positive reaction was obtained among these 60 fungi using competitive inhibition ELISA. The sensitivity of this double-sandwich ELISA was 1.mu.g mycelium/mL and the compositions of cereals did not interfere with this assay. When A. parasitcus was inoculated into cereals, it could be detected by ELISA with detection limit of 10/sup 4/-10/sup 5/cfu/g. At this time aflatoxin had not been produced. The correlation coefficients between ELISA and the mold colony count for detecting A. parasitcus in corn, peanut, wheat and rice were 0.94, 0.86, 0.96 and 0.93, respectively. When A. parasitcus was inoculated into YES (yeast extract sucrose) broth, the mycelium amounts calculated from ELISA were similar as those by weighing method and their correlation coefficient was 0.97. The contaminations of A. parasitcus/A. flavus in 10 samples each of corn, peanut, wheat and rice purchased from retail stores were evaluated by ELISA and AFPA plate counts. The results showed that most samples tested were not contaminated. However, after 29d incubation at a humidified condition, the amounts of A. parasitcus/A. flavus increased significantly based on the results from both ELISA and AFPA count. In general, the amounts of A. parasitcus/A. flavus in wheat and rice were higher than those in peanut and corn.

Keyword(s)

黴菌
抗原
酵素免疫分析
ConA結合性
穀類
Mold
Antigen
Enzyme immunoassay
Con A-binding ability
Cereal

DSpace CRIS

Production of Antibodies for Detection of Aspergillus parasiticus in Cereals

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