| Enzyme-linked immunosorbent assay (ELISA) was established in 1972 applied to Medical Research. ELISA analysis of microcystins was earlier applied abroad. It's also applied at home, but it wasn't as matuar as abroad. In this paper, We study mainly in ELISA analysis of microcystins. It include extracting and purification of MC-LR, making incomplete antigen to complete antigen of MC-LR, screening and cuiluring monoclonal antibody cell line of MC-LR.(1)We got enough cyanobacteria from DianchiLake by sieving andlyophilization. Algae cell walls was damaged by acetic acid, and microcycstins overflowed from cell into acetic acid. Enrichment the acetic acid by Sep-pak C18 column, then elution the column by 80% methanol solution and gather the eluent. Using Liquid Chromatography Assay analyzed the Algae toxin extracts, discovering the retention time is 30th min to 31th min.Collect the separation liquid from LC within 30th min to 31th min. Enrichment the separation liquid by Sep-pak C18 column again, then elution the column by 80% methanol solution and gather the eluent. The eluent is crude extracts of MC-LR. Coating the crude extracts on Fluorescent silica gel thin layer plate, and by this further precise separation, we finally got the refined MC-LR. And its purity quotient is 96.88% by LC-MS identifiing and the external standard method. We totally got 4.6 mg refined MC-LR.(2) Molecular weight of MC-LR is 995.2D. MC-LR is a kinds of typical incompl- -ete antigen. It has reactiveness but not immunogenicity during immunological response. So we adopted Protein-coupled technology to make MC-LR has immun- -ogenicity and have an effective immune response during animal immunization. In this paper, we activated MC-LR by cysteamine. The resault is an amino was linked in double bond of the seventh amino acid of MC-LR. Then,we choosed Bovine Serum Albumin(BSA) molecular weight is 67000D as vector. Made MC-LR and BSA connected by Two-step glutaraldehyde.We identified the molicular weight of H2N-etMC-LR by LC-MS, and it confirmed our activation was succesful. The qualitative identification and quantitative identification of MC-LR-BSA conjugate was separately by Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS). The results showed that coupling ratio is 3 to 18:1, the average coupling ratio is 8:1.(3) Using the identified MC-LR-BSA to immune Balb/c mice by intraperitoneal injection, and the dose is 100ug per each mice. It need 3 to 5 booster immunizations, and every two weeks it need one.We got serum titer was practically up to 1:256000. Fetch spleen cells of immuned mice and myeloma cells SP2 / 0 to fusion by Hybridoma technology. The fused cells was cultured by HAT selective medium. The results of indirect ELISA indicated that four McAbs could cross reacted with MC-LR. Cultured the cell lines under Large-scale cultivation. The results of indirect ELISA showed that the most appropriate concentration of coating antigen is between 10 and 20 nanogram per hole.
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