Sandwich ELISA for quantitative detection of human collagen prolyl 4-hydroxylase

Microbial Cell Factories

Technical Notes

Ekaterina Osmekhina¹  Katharina Klinzing¹  Peter Neubauer²  Johanna Myllyharju³  Antje Neubauer³ 

[1] Bioprocess Engineering Laboratory, Department of Process and Environmental Engineering, University of Oulu, P.O.Box 4300, FIN-90014, Finland;Bioprocess Engineering Laboratory, Department of Process and Environmental Engineering, University of Oulu, P.O.Box 4300, FIN-90014, Finland;Laboratory of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Ackerstr. 71-76, D-13355, Berlin, Germany;Oulu Center for Cell-Matrix Research, Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, University of Oulu, P.O.Box 5000, FIN-90014, Finland;

关键词: Shake Flask;    Sandwich ELISA;    Oxygen Transfer Rate;    Crude Cell Extract;    Protein Disulphide Isomerase;   

DOI  :  10.1186/1475-2859-9-48

 received in 2010-03-31, accepted in 2010-06-17,  发布年份 2010

来源: Springer

【 摘 要 】

BackgroundWe describe a method for specific, quantitative and quick detection of human collagen prolyl 4-hydroxylase (C-P4H), the key enzyme for collagen prolyl-4 hydroxylation, in crude samples based on a sandwich ELISA principle. The method is relevant to active C-P4H level monitoring during recombinant C-P4H and collagen production in different expression systems. The assay proves to be specific for the active C-P4H α2β2 tetramer due to the use of antibodies against its both subunits. Thus in keeping with the method C-P4H is captured by coupled to an anti-α subunit antibody magnetic beads and an anti-β subunit antibody binds to the PDI/β subunit of the protein. Then the following holoenzyme detection is accomplished by a goat anti-rabbit IgG labeled with alkaline phosphatase which AP catalyzes the reaction of a substrate transformation with fluorescent signal generation.ResultsWe applied an experimental design approach for the optimization of the antibody concentrations used in the sandwich ELISA. The assay sensitivity was 0.1 ng of C-P4H. The method was utilized for the analysis of C-P4H accumulation in crude cell extracts of E. coli overexpressing C-P4H. The sandwich ELISA signals obtained demonstrated a very good correlation with the detected protein activity levels measured with the standard radioactive assay. The developed assay was applied to optimize C-P4H production in E. coli Origami in a system where the C-P4H subunits expression acted under control by different promoters. The experiments performed in a shake flask fed-batch system (EnBase®) verified earlier observations that cell density and oxygen supply are critical factors for the use of the inducer anhydrotetracycline and thus for the soluble C-P4H yield.ConclusionsHere we show an example of sandwich ELISA usage for quantifying multimeric proteins. The method was developed for monitoring the amount of recombinant C-P4H tetramer in crude E. coli extracts. Due to the specificity of the antibodies used in the assay against the different C-P4H subunits, the method detects the entire holoenzyme, and the signal is not disturbed by background expression of the separate subunits.

【 授权许可】

CC BY   
© Osmekhina et al; licensee BioMed Central Ltd. 2010