臺灣博碩士論文加值系統

赭麴毒素A (Ochratoxin A, OTA) 主要由 Aspergillus ochraceus及Penicillium verrucosum 所產生之二級代謝產物，會對動物造成腎毒性，肝毒性及致癌性。在穀物或其他產品，例如咖啡豆、堅果、葡萄酒及動物組織都曾經發現OTA的汙染。針對全球性市場之食品安全，有必要對OTA污染進行監測。本實驗室致力於發展能簡單、快速，並且與色層分析技術具有一致性之免疫分析方法用來偵測OTA的含量，但其前提需要有具有高敏感度、專一性之anti-OTA抗體。
將OTA接在keyhole limpet hemocyanin（KLH）上用來免疫老鼠，取小鼠之脾臟細胞與小鼠骨髓瘤細胞株FO細胞進行細胞融合，挑選有表現較高效價之融合瘤細胞進行篩選及limiting dilution，最後以1-D2-5G hybirdoma cell line所分泌之單株抗體(mAb)進行分析。在corss reaction分析中，mAb對OTA有專一性，對OVA, KLH及BSA無反應。於coating OTA-OVA 0.5 �慊/well，進行indirect competitive ELISA (icELISA)，其偵測極限為10ng/well，IC50為26 ng/well。

Ochratoxin A (OTA) is a secondary metabolite naturally produced primarily by Aspergillus ochraceus and Penicillium verrucosum, causes nephrotoxicity, hepatotoxicity, and carcinogenicity in animal. It has been found as a common contaminant of cereals or in other products such as coffee beans, nuts, wine, and animal organs. Monitoring of OTA is essential for food safety in the worldwide market. We were made to develop an immunoassay-based method that is usually simple and rapid compared to chromatographic techniques for the detection of OTA, but it has need higher sensitivity and specifies of anti-OTA antibody.
Monoclonal antibodies (mAb) against Ochratoxin A were produced from the hybridoma cell line 1-D2-5G. The hybridoma cell line 1-D2-5G was established after selection and limiting dilution by the fusion of FO myeloma cells with spleen cells isolated from a BALB/c mouse immunized with the OTA-KLH conjugate. After the assay of cross-reaction, anti-OTA mAb was specified with OTA, no cross-reactive with OVA, KLH and BSA. The level of 50% inhibiting concentration coating with 0.5 �慊 OTA-OVA per well was 26 ng/well in a competitive indirect enzyme-linked immunosorbent assay (ciELISA), and the detection limit was less than 10 ng/well.

ENGLISH ABSTRACT i
CHINESE ABSTRACT v
TABLE OF CONTENT vi
LIST OF ABBREVIATIONS viii
LIST OF TABLES ix
LIST OF FIGURES x
CHAPTER
I INTRODUCTION 1
1.1 Ochratoxin A 1
1.2 Monoclonal Antibody Production 4
1.3 Analytical Methods for the Detection of Ochratoxin A 8
1.4 Object in the Research 10
II MATERIALS AND METHODS 12
2.1 Source of Materials and Chemical Compounds 12
2.2 Buffer and Solution 14
2.3 Medium 16
2.4 Apparatus 17
2.5 Preparation of Various OTA Conjugates 18
2.6 Production of Polyclonal Antibody against Ochratoxin A 22
2.7 Production of Monoclonal Antibody against Ochratoxin A 22
2.8 Enzyme-linked immunosorbant analyses 28
2.9 Production and Extraction of Ochratoxin A 29
2.10 High Performance Liquid Chromatographic Analysis 31
III RESULTS 32
3.1 Native PAGE of OTA-KLH and OTA-OVA conjugate 32
3.2 Production of anti-OTA polyclonal antibody 32
3.3 Production of anti-OTA monoclonal antibody 37
3.4 Optimum OTA-OVA Concentration for Coating ELISA plates and Antibody Dilution for ELISA. 42
3.5 Specificity of monoclonal antibody 45
3.6 Competitive indirect ELISA 45
3.7 Ascites Production of Monoclonal Antibodies 48
3.8 Analysis of OTA 52
IV DISCUSSION 54
V REFERENCES 59

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