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

dc.contributor.authorOsmekhina, Ekaterina
dc.contributor.authorNeubauer, Antje
dc.contributor.authorKlinzing, Katharina
dc.contributor.authorMyllyharju, Johanna
dc.contributor.authorNeubauer, Peter
dc.date.accessioned2020-11-09T12:58:30Z
dc.date.available2020-11-09T12:58:30Z
dc.date.issued2010-06-17
dc.description.abstractBackground: 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.eissn1475-2859
dc.identifier.urihttps://depositonce.tu-berlin.de/handle/11303/11866
dc.identifier.urihttp://dx.doi.org/10.14279/depositonce-10756
dc.language.isoenen
dc.relation.ispartof10.14279/depositonce-8959
dc.rights.urihttps://creativecommons.org/licenses/by/2.0/en
dc.subject.ddc570 Biowissenschaften; Biologiede
dc.subject.otherShake Flasken
dc.subject.otherSandwich ELISAen
dc.subject.otherOxygen Transfer Rateen
dc.subject.otherCrude Cell Extracten
dc.subject.otherProtein Disulphide Isomeraseen
dc.titleSandwich ELISA for quantitative detection of human collagen prolyl 4-hydroxylaseen
dc.typeArticleen
dc.type.versionpublishedVersionen
dcterms.bibliographicCitation.articlenumber48en
dcterms.bibliographicCitation.doi10.1186/1475-2859-9-48en
dcterms.bibliographicCitation.journaltitleMicrobial cell factoriesen
dcterms.bibliographicCitation.originalpublishernameBioMed Centralen
dcterms.bibliographicCitation.originalpublisherplaceLondonen
dcterms.bibliographicCitation.volume9en
tub.accessrights.dnbfreeen
tub.affiliationFak. 3 Prozesswissenschaften::Inst. Biotechnologiede
tub.affiliation.facultyFak. 3 Prozesswissenschaftende
tub.affiliation.instituteInst. Biotechnologiede
tub.publisher.universityorinstitutionTechnische Universität Berlinen

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