E.coli (K-12 & Alkaline Lysis) HCP ELISA Kit

Different from traditional physical disintegration technique, the HCPs in the pDNA samples are exposed to high alkaline solutions and denatured during the extraction process, leading to a large difference in the detection results measured by immuno-assays. This kit is based on the solid-phase enzyme-linked immunosorbent assay (ELISA) with a double-antibody sandwich technique to detect residual host cell proteins (HCPs) from E.coli K-12 strains. A sheep polyclonal antibody specific to E.coli (K-12 strains) HCPs prepared by alkaline lysis was employed in the assay to capture any remaining HCPs in the sample. The antibody coverage is assessed by the current mainstream method. Both the Calibration standards and test samples were simultaneously added to the microtiter plate coated with the affinity purified capture antibody and followed by incubation and washing. The biotinylated antibody was added to the microtiter plate to bind the HCPs and then reacted with streptavidin labeled with HRP (Horseradish Peroxidase). TMB (3,3',5,5' -tetramethylbenzidine) substrate was added into reaction, HRP catalyzed the oxidation of TMB by H₂O₂ to produce a blue product (maximum absorption peak at 655 nm). Then the stop solution was added to terminate the enzymatic reaction, resulting in a yellow colored product (maximum absorption peak at 450 nm). The absorbance values at 450 nm wavelength were positively correlated with the HCPs concentration in the Calibration standard and the sample. The concentration of HCPs in the sample can be calculated using the dose-response curve.