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Production of an anti-ochratoxin A monoclonal antibody and development of immunochromatographic test : Ochratoxin A에 특이적인 단클론항체의 생산과 면역크로마토그래피 시험의 개발

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Authors

조영진

Advisor
서진호
Issue Date
2003
Publisher
서울대학교 대학원
Keywords
오크라톡신 에이 (OTA)Ochratoxin A (ota)오크라톡신 에이-BSA conjugate (OTA-BSA)Ochratoxin A-BSA conjugate (OTA-BSA)단클론항체 (mAb)Monoclonal antibody (mab)하이브리도마HybridomaEnzyme-Linked Immunosorbent Assay (ELISA)Test line (detection zone)Immunochromatographic strip test (ICT)Control line (control zone)Colloidal gold시험선비교선Nitrocellulose membrane
Description
Thesis (master`s)--서울대학교 대학원 :농생명공학부,2003.
Abstract
Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium
species, and causes nephrotoxicity, hepatotoxicity, and carcinogenicity in
animals. With the amounts of imported agricultural commodities increasing and
natural occurrence of OTA in agricultural commodities in E.U. have been reported
by many groups, monitoring of OTA is essential for food safety. For the
development of a detection method for OTA, immunoassay-based method can be used
because of its rapidity and simplicity. To use those of advantages, a monoclonal
antibody (mAb) was produced and the cell line that secreted anti-OTA-mAb was
established through hybridoma technology.
This study was performed by a fusion of myeloma cells (Sp2/0-Ag14) and spleen
cells from the immunized mice, and then hybridoma cells were screened by the
modified competitive direct ELISA (cdELISA). After the screening, we selected
one positive hybridoma cell (C7G25) that produced an anti-OTA monoclonal
antibody which was of the IgG2a subclass with a ¥ê type light chain. Also the
IC50 and detection limit for OTA were found to be 1.2 ng/§¢ and 0.12 ng/§¢,
respectively. Cross reactivities of the monoclonal antibody for ochratoxin B,
aflatoxin B1, and fumonisin B1, BSA were 31.7, 0, 0, and 0 %, respectively.
An immunochromatographic strip test assay format was applied and developed for
simple and rapid detection of OTA as another alternative. This assay was based
on the competitive inhibition reaction scheme due to the small size of OTA. For
this study, the OTA-BSA conjugate was immobilized in a defined detection zone
(test line) on a porous nitrocellulose membrane. Furthermore, protein A/G was
immobilized in a control zone (control line), the anti-OTA-mAb (C7G25) was
conjugated to colloidal gold particles which served as a detection reagent, and
then a standard OTA solution was added to the detector reagent (mAb-gold
conjugate) on microplate wells. A few minutes later, the nitrocellulose membrane
was dipped into the sample-detector reagent mixture. The mixture was then passed
along the nitrocellulose membrane by capillary action past the OTA-BSA in the
detection zone, which would not bind with the particles that already had OTA
bound to their surface without red line. In the absence of OTA, the immunogold
(detector reagent) was bound to the OTA-BSA within the detection zone and
resulting in red line, which was in inverse proportion to OTA concentrations.
This competitive format test was able to detect up to 500 ppb of OTA
Language
English
URI
http://library.snu.ac.kr/search/DetailView.ax?sid=1&cid=0001083927

https://hdl.handle.net/10371/67619
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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Food and Animal Biotechnology (식품·동물생명공학부)Theses (Master's Degree_식품·동물생명공학부)
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