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

Abstract

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.

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Detecting Collagen Molecules at Picogram Level through Electric Field-Induced Accumulation

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Microfluidic-Based Detection of AML-Specific Biomarkers Using the Example of Promyelocyte Leukemia

“…The method of a sandwich enzyme-linked immunosorbent assay (ELISA) is suitable for this purpose. In a sandwich ELISA a capture antibody on the surface of a matrix is used to bind the analyte specifically [ 17 ]. A second antibody labelled with an enzyme which is specific for another epitope of the analyte is added and binds to the antibody–antigen complex [ 17 ].…”

“…The result of the binding of the analyte to the antibodies can now be quantified with a substrate for the enzyme on the detection antibody [ 17 ]. This method is highly specific, because only a positive signal is obtained when both antibodies have specifically bound the antigen to different epitopes [ 17 , 18 ]. Non-specific binding is reduced by an optimized number of washing steps.…”

“…Non-specific binding is reduced by an optimized number of washing steps. A sandwich ELISA can be used to detect a target protein in unpurified samples with antibodies against different epitopes [ 17 ].…”