Objective To prepare a monoclonal antibody (mAb) against metallothionein (MT) of freshwater crab (Sinopotamon henanense) and characterize its immunologic properties.Methods Two recombinant MT of S.henanense,namely SUMO-MT and His-MT,were produced by SUMO fusion system and phoA secretion expression system in E.coli.SUMO-MT was used as an antigen to immunize BALB/c mice.By means of the cell fusion technique,multiple cell subcloning,repeated screening with His-MT as detecting antigen,the hybridomas specifically secreting mouse mAb against the MT of S.henanense were generated.The titers of mAbs were measured by indirect ELISA and the specificity of the mAbs was evaluated by Western blotting and Dot-ELISA.Results Two hybridoma cell lines designated mAb-MT2 and mAb-MT3 with the property of secreting mAb against the MT continuously and steadily were successfully obtained.Their immunoglobulin subclass was IgG1.The titers of the ascites fluid were 1 ∶ 500 000 and 1 ∶ 1000 000,respectively.Western blot analysis confirmed that the two mAbs both reacted with recombinant SUMO-MT and His-MT with good sensitivity.The Dot-ELISA demonstrated that the two mAbs reacted specifically not only with recombinant MT but also natural MT.Conclusion The mAbs against MT of S.henanense with high specificity were successfully prepared.
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维普
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