The preparation of Colgalt2-ELISA kit

Objective To prepare an Colgalt2 ELISA kit using Colgalt2 N-terminal and C-terminal polyclonal antibody.Methods Colgalt2 partial CDs were amplified from HepG2 cell,and inserted into prokaryotic expression plasmids,and induced the expression of N-terminal recombinant Colgalt2 protein.N-terminal Colgalt2 polyclonal antibody was prepared by immunizing rabbits with purified recombinant protein.Titer and specificity of the antibody was determined by ELISA and Western Blot.Microtiter plate was coated with N-terminal Colgalt2 polyclonal antibody,and other polyclonal antibody of Colgalt2 C-terminal was labeled by HRP.Results We successfully cloned N-terminal of Colgalt2 and obtained the recombinant Colgalt2 protein.ELISA analysis showed that titer of anti-Colgalt2 was higher than 1 ∶ 1 280 000.Western Blot analysis showed that the antibody with a good specificity.We successfully developed an ELISA kit of Colgalt2.The data of the different concentrations of recombinant protein Colgalt2 showed that the ELISA kit had a good sensitivity.From1.25 μg/ml to 25μg/ml,the coefficient of linear correlation was 0.98.Conclusion The Colgalt2-ELISA kit may be used to determine Colgalt2 levels in samples. Key words: Genes ;  Protein engineering;  Antibodies, monoclonal ;  Enzyme-lyinked immunosorbent assay