Sandwich ELISA for quantitative detection of human collagen prolyl 4-hydroxylase
dc.contributor.author | Osmekhina, Ekaterina | |
dc.contributor.author | Neubauer, Antje | |
dc.contributor.author | Klinzing, Katharina | |
dc.contributor.author | Myllyharju, Johanna | |
dc.contributor.author | Neubauer, Peter | |
dc.date.accessioned | 2020-11-09T12:58:30Z | |
dc.date.available | 2020-11-09T12:58:30Z | |
dc.date.issued | 2010-06-17 | |
dc.description.abstract | Background: We 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. Results: We 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. Conclusions: Here 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. | en |
dc.identifier.eissn | 1475-2859 | |
dc.identifier.uri | https://depositonce.tu-berlin.de/handle/11303/11866 | |
dc.identifier.uri | http://dx.doi.org/10.14279/depositonce-10756 | |
dc.language.iso | en | en |
dc.relation.ispartof | 10.14279/depositonce-8959 | |
dc.rights.uri | https://creativecommons.org/licenses/by/2.0/ | en |
dc.subject.ddc | 570 Biowissenschaften; Biologie | de |
dc.subject.other | Shake Flask | en |
dc.subject.other | Sandwich ELISA | en |
dc.subject.other | Oxygen Transfer Rate | en |
dc.subject.other | Crude Cell Extract | en |
dc.subject.other | Protein Disulphide Isomerase | en |
dc.title | Sandwich ELISA for quantitative detection of human collagen prolyl 4-hydroxylase | en |
dc.type | Article | en |
dc.type.version | publishedVersion | en |
dcterms.bibliographicCitation.articlenumber | 48 | en |
dcterms.bibliographicCitation.doi | 10.1186/1475-2859-9-48 | en |
dcterms.bibliographicCitation.journaltitle | Microbial cell factories | en |
dcterms.bibliographicCitation.originalpublishername | BioMed Central | en |
dcterms.bibliographicCitation.originalpublisherplace | London | en |
dcterms.bibliographicCitation.volume | 9 | en |
tub.accessrights.dnb | free | en |
tub.affiliation | Fak. 3 Prozesswissenschaften::Inst. Biotechnologie | de |
tub.affiliation.faculty | Fak. 3 Prozesswissenschaften | de |
tub.affiliation.institute | Inst. Biotechnologie | de |
tub.publisher.universityorinstitution | Technische Universität Berlin | en |