Establishment of an Enzyme-Linked Immunosorbent Assay for Detection of Aeromonas hydrophila in Oyster

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Project title

Code/計畫編號

DOH86-TD-107

Translated Name/計畫中文名

建立酵素結合免疫分析法分析牡蠣中病原菌Aeromonas hydrophila

Project Coordinator/計畫主持人

Guo-Jane Tsai

Funding Organization/主管機關

Environmental Protection Administration

Department/Unit

Department of Food Science

Website

https://www.grb.gov.tw/search/planDetail?id=327422

Year

1997

Start date/計畫起

01-07-1996

Expected Completion/計畫迄

01-06-1997

Bugetid/研究經費

600千元

ResearchField/研究領域

食品科技(農)
生物技術(農)

取自病人檢體、食品及菌種中心之Aeromonas hydrophila菌株以及多株非A. hydrophila但血緣相近之菌株,萃取其外膜蛋白,比對其SDS-PAGE圖譜,顯示分子量分別為42及45kD之蛋白為A. hydrophila菌株共有之表面蛋白,將此蛋白以靜脈注射方式打入紐西蘭大白兔,生產相對抗體,所得抗體利用Horseradish peroxidase建立三明治式ELISA (Enzyme-linked immunosorbent assay)。比較各抗體的專一性,以45kD蛋白所得之抗體之專一性較佳,在PBST(Phosphate Buffered salined-Tween)中其對A. hydrophila檢出率為31/59(52.5%),但添加0.05% Teepol,且加熱10min,檢出率達94.9%(56/59)。其對非A. hydrophila菌株之檢出率為19.6%(9/46)。所建立ELISA之抗體與抗體酵素結合體之最適當濃度分別為10及20.mu.g/mL,對外膜蛋白的靈敏度為0.05.mu.g/mL,約相當於10/sup 4/ CFU/mL菌體濃度。將此ELISA應用在魚、蝦及牡蠣中A. hydrophila之分析,顯示牡蠣及蝦成分不會干擾ELISA分析值,靈敏度為10/sup 4/CFU/mL;但魚肉顯著影響ELISA分析。將魚肉在100.degree.C加熱10min,可解決魚肉蛋白的干擾。此ELISA對活的或經加熱而死亡之A. hydrophila菌株之靈敏度相似。由人為添加試驗顯示魚、蝦和牡蠣中即使僅含少量的A. hydrophila(1,10,或100CFU/g),經28.degree.C,12小時前置培養,ELISA即可偵測出來。< The outer membrane proteins (OMP) of Aeromonas hydrophila from various sources were extracted and analyzed. After elution from SDS-PAGE gel, the protein with molecular size of 42 or 45kDa was shown to be the common OMP and was injected individually into New Zealand rabbits intravenously. The antibody against the 45kDa protein was chosen for the following experiments due to its higher specificity. The optimal concentrations of antibody and antibody-peroxidase conjugate for the established enzyme-linked immunosorbent assay (ELISA) were 10 and 20.mu.g/mL, respectively. Of 59 strains of A. hydrophila tested, only 31 (52.5%) were detected in PBST system. However, the detection rate increased to 94.9% (56/59) if 0.05% Teepol was added and the culture was heated at 100.degree.C for 10 min. The false positive rate was 19.6% (9/46). The detection limit of this assay was about 10/sup 4/CFU/mL, which was equivalent to 0.05.mu.g/mL of OMP. The compositions of oyster and shrimp would not interfere with the ELISA results for detection of A. hydrophila. However, fish meat obviously affected ELISA values. This interference could be overcome by heating fish homogenate at 100.degree.C for 10 min. This ELISA could detect both viable and dead A. hydrophila. After 12h pre-incubation at 28.degree.C, the very few amount of A. hydrophila (even 1CFU/g) added in seafood initially could be detected by ELISA.<

Keyword(s)

水生產氣單胞菌
單株抗體
酵素關連免疫吸附法
牡蠣
Aeromonas hydrophila
Monoclonal antibody
Enzyme linked immuno sorbent assay (ELISA)
Oyster

DSpace CRIS

Establishment of an Enzyme-Linked Immunosorbent Assay for Detection of Aeromonas hydrophila in Oyster

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