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The Study on Microcystin-LR’s Development of Enzyme-linked Immunosorbent Assay

Author: ZhangAi
Tutor: YangLiang
School: Yunnan University
Course: Ecology
Keywords: Microcystin -LR Extract Purification Complete antigen Hybridoma technique Enzyme-linked immunosorbent assay (ELISA)
CLC: R115
Type: Master's thesis
Year: 2010
Downloads: 63
Quote: 0
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Abstract


ELISA (Enzyme-linked immunosorbent assay, ELISA) was established in 1971 and used for medical research. ELISA analysis of microcystin was earlier foreign application, the country also has applications, but not as mature foreign. In this paper, the microcystin-LR ELISA as explore objects, including toxin microcystin-LR extraction and purification of microcystin-LR of complete antigen, as well as microcystin-LR single clonal antibody screening cultured cell lines. (1) Through the algae from Dianchi Lake to fish for fresh sieved sample selection, freeze-dried to obtain cyanobacteria algae powder; destroy algae cell wall with acetic acid solution, so that microcystin overflow cells; through C18 column enrichment enrichment, with 80% eluted with methanol and collect microcystin extract; against microcystin extract by liquid chromatography analysis to determine the MC-LR retention time of 30min ~ 31min, collect MC-LR liquid separation, concentration, purification through MC-C18 column LR, MC-LR obtained crude extract, the fluorescent silica gel TLC plate separation further precision obtained under MC-LR was purified sample; by liquid chromatography - mass spectrometry (LC-MS) identification, using the external standard method to obtain purity of 98.79% of MC-LR purified sample. The total test sample extract MC-LR refined 6.4mg. (2) MC-LR molecular weight of 995.2D, a typical hapten. In the course of the immune response, not only the reactive immunogenicity of the immunogenic MC-LR, we use the protein coupling techniques, so that in the course of immunization of animals produce an effective immune response. Firstly, activated with two cysteamine MC-LR, of MC-LR in the seventh position of the amino acid dehydroalanine introduce a group of double bonds, and a molecular weight of 67000D used bovine serum albumin (BSA) as a carrier protein by-step glutaraldehyde make MC-LR get coupled to BSA. Biological simple method by intermediate H2N-etMC-LR molecular weight were identified, determining the activation of MC-LR very successful; respectively, through sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) technique, matrix-assisted laser desorption ionization time of flight Funky (MALDI-TOF-MS) detection, conjugates MC-LR-BSA qualitative and quantitative identification. The results show that the coupling ratio of from 3 to 18:1, the average coupling ratio is 8:1. (3) selection of the identified MC-LR-BSA immunized Balb / c mice, according to 100ug / only intraperitoneal injection, booster 3 to 5 times, once every two weeks to strengthen the immune obtained titer of near 1 : 25600 serum. Hybridoma technique, spleen cells with myeloma cells SP2 / 0 cell fusion with HAT selective medium for selection culture, the use of indirect ELISA screening positive cells Kong, will be screened positive cells To obtain stable cell clones cell lines, expanding culture. Using indirect ELISA MC-LR optimal coating concentration of 10ng ~ 20ng / holes.

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