Sandwich ELISA for quantitative detection of human collagen prolyl 4-hydroxylase
<p>Abstract</p> <p>Background</p> <p>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...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2010-06-01
|
| Series: | Microbial Cell Factories |
| Online Access: | http://www.microbialcellfactories.com/content/9/1/48 |
| _version_ | 1818061311404146688 |
|---|---|
| author | Myllyharju Johanna Klinzing Katharina Neubauer Antje Osmekhina Ekaterina Neubauer Peter |
| author_facet | Myllyharju Johanna Klinzing Katharina Neubauer Antje Osmekhina Ekaterina Neubauer Peter |
| author_sort | Myllyharju Johanna |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>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 α<sub>2</sub>β<sub>2 </sub>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.</p> <p>Results</p> <p>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 <it>E. coli </it>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 <it>E. coli </it>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<sup>®</sup>) 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.</p> <p>Conclusions</p> <p>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 <it>E. coli </it>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.</p> |
| first_indexed | 2024-12-10T13:46:18Z |
| format | Article |
| id | doaj.art-82626b83043d4dca9aab3165e20064fb |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-12-10T13:46:18Z |
| publishDate | 2010-06-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-82626b83043d4dca9aab3165e20064fb2022-12-22T01:46:26ZengBMCMicrobial Cell Factories1475-28592010-06-01914810.1186/1475-2859-9-48Sandwich ELISA for quantitative detection of human collagen prolyl 4-hydroxylaseMyllyharju JohannaKlinzing KatharinaNeubauer AntjeOsmekhina EkaterinaNeubauer Peter<p>Abstract</p> <p>Background</p> <p>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 α<sub>2</sub>β<sub>2 </sub>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.</p> <p>Results</p> <p>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 <it>E. coli </it>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 <it>E. coli </it>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<sup>®</sup>) 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.</p> <p>Conclusions</p> <p>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 <it>E. coli </it>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.</p>http://www.microbialcellfactories.com/content/9/1/48 |
| spellingShingle | Myllyharju Johanna Klinzing Katharina Neubauer Antje Osmekhina Ekaterina Neubauer Peter Sandwich ELISA for quantitative detection of human collagen prolyl 4-hydroxylase Microbial Cell Factories |
| title | Sandwich ELISA for quantitative detection of human collagen prolyl 4-hydroxylase |
| title_full | Sandwich ELISA for quantitative detection of human collagen prolyl 4-hydroxylase |
| title_fullStr | Sandwich ELISA for quantitative detection of human collagen prolyl 4-hydroxylase |
| title_full_unstemmed | Sandwich ELISA for quantitative detection of human collagen prolyl 4-hydroxylase |
| title_short | Sandwich ELISA for quantitative detection of human collagen prolyl 4-hydroxylase |
| title_sort | sandwich elisa for quantitative detection of human collagen prolyl 4 hydroxylase |
| url | http://www.microbialcellfactories.com/content/9/1/48 |
| work_keys_str_mv | AT myllyharjujohanna sandwichelisaforquantitativedetectionofhumancollagenprolyl4hydroxylase AT klinzingkatharina sandwichelisaforquantitativedetectionofhumancollagenprolyl4hydroxylase AT neubauerantje sandwichelisaforquantitativedetectionofhumancollagenprolyl4hydroxylase AT osmekhinaekaterina sandwichelisaforquantitativedetectionofhumancollagenprolyl4hydroxylase AT neubauerpeter sandwichelisaforquantitativedetectionofhumancollagenprolyl4hydroxylase |