The use of Escherichia coli (E.coli) for expression and amplification in the production of recombinant proteins and plasmid DNA is a relatively simple and cost-effective way of preparing biologics. However, the process of using E. coli inevitably introduces host cell protein (HCP) impurities, which can reduce the efficacy of the product and may lead to adverse toxicity or immune responses, and it is therefore necessary to ensure that the level of HCP residues in the final biologic product is as low as possible.

ELISA is a simple to use, sensitive and objective test for HCPs.This kit uses the principle of double-antibody sandwich enzyme-linked immunosorbent assay (sandwich ELISA) to detect the E.coli HCP residues for plasmids in samples. First, add the E.coli HCP Standard for Plasmids (36721-B) and test sample to the Anti-E.coli HCP coated microtiter strips (36721-A), then add the diluted biotin labeled Anti-E.coli HCP antibody (36721-C), and finally add Streptavidin-HRP (36721-D) to form an antibody + antigen + antibody-Biotin + SA-HRP complex. Subsequently, add TMB substrate (36721-H) into the complex to observe color reaction after washing the complex. TMB was converted from colorless to blue and finally to yellow in the presence of termination solution (36721-I) catalyzed by HRPase. Shades of yellow were positively correlated with the amount of E. coli HCP detected in the sample. This kit can be used for the optimization of the purification process of biologics, the control of impurities in intermediate processes, and the release testing of final products.

The detection range of this kit is 3.125~200 ng/mL; the lower detection limit is 0.5 ng/mL.

Features

• Highly sensitive: Detecting as little as 0.5 ng/mL of E.coli HCP (Plasmid) residuals in in-process and final biological samples.
• Ensures accuracy: The E.coli HCP (Plasmid) residuals can be accurately detected with a recovery of 75%-125%.
• Specificity: Specific detection of E.coli HCP without interference from other exogenous proteins.
• Stable: The difference between lot-to-lot is low, the kit property is not impacted under 37℃ for 7 days.
• Easy signal collection: Using Biotin-Streptavidin system, the signal can be stably enlarged.

Application

• Residual E.coli HCP (Plasmid) test in biological products

Specification

 Product Performance

Components

Storage

This product should be stored at 2°C ~8°C. Unopened product is valid for one year.

^*Upon receipt of the kit, please check whether all components are complete and immediately store them in corresponding condition.

Figures

Precision Display

A) Repeatability

B) Intermediate precision

Table 1. The precision of E.coli HCP ELISA was assayed as the above scheme

10 duplicated were tested at 3 concentrations on the standard curve, and all the CV% were lower than 10% (Table 1A). 3 lot of E.coli HCP ELISA kits were tested and the CV% were lower than 15% (Table 1B).

Accuracy Validation

A) Dilution linearity

The plasmid samples amplified using E.coli cells (concentration 22.6 mg/mL, HCP concentration 55 ng/mL) were gradient diluted with sample diluent, and the HCP residue values were determined using this kit. From the following data, it can be seen that the linearity of the sample dilution determined by this kit is good, the difference between the HCP assayed value and the initial concentration after different times of dilution is small, the linear recovery is in the range of 80%~120%, and the detection value of HCP is linearly correlated with the number of times of dilution.

Table2 Sample dilution linearity

B) Spiking Recovery Rate

The plasmid samples (concentration of 22.6 mg/mL), which had been amplified using E.coli, were diluted 2-fold and mixed with equal volumes of standards of each concentration, and the spiked recoveries ranged from 80% to 120%.

Spiked recovery % = (measured value of spiked samples - measured value of unspiked samples) / theoretical value (amount of spiked standard) x 100%.

Table3 Sample spiking recovery rate

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