| Summary: | 碩士 === 嘉南藥理科技大學 === 生物科技系暨研究所 === 92 === The objective of this study is to develop an immunoassay strip (immunochromatographic strip) for cholera toxin (CT) using polyclonal and monoclonal antibodies against CT produced. In this study, the enzyme-linked immunosorbent assay (ELISA) for CT was set up using anti-cholera toxin polyclonal antibodies (anti-CT pAb), and the CT immunoassay strip using anti-cholera toxin monoclonal antibodies (anti-CT mAb) was developed. Conventional methods for determining this CT is to use reverse passive latex agglutination (RPLA) to determine CT protein and use polymerase chain reaction (PCR) to determine CT gene. Although RPLA can determine 1-2 ng/mL of CT protein, it can effect its accuracy by other enterotoxin of samples and took 20~24 hours. PCR has more sensitive and rapid than other immunoassays, but it is easy to mistake the non-toxin gene in other V. cholerae.
Firstly, this study was to produce and purify polyclonal and monoclonal antibodies against CT. The experiment result was that mouse anti-CT pAb from mouse ascites purified by Hitrap™ rProtein A Column. This anti-CT pAb could determine 0.05 µg/mL of CT and low cross-reaction. The five high-titer anti-CT mAb-producing hybridoma cell lines selected and designated CT-3A, CT-4C, CT-11D, CT-11H, and CT-9A. The isotypes of the five anti-CT mAbs were identified as IgM heavy chain and λ light chain. For the purification of anti-CT mAb, Hitrap™ IgM Purification Column was used. The highest titer of anti-CT mAb (CT-3A and CT-9A) determined by indirect ELISA was 1:15,625. Secondly, we made the colloidal gold from the chloroauric acid (HAuCl4) and labeled anti-CT mAb with the 20 nm colloidal gold. This anti-CT mAb immunoassay strip set up could determine in 40-100 µg/mL CT and without cross-reaction. Moreover, this strip could be applied to test samples in fact, and detection time was approximately 15-20 minutes.
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