The use of E. coli for recombinant expression in the production of some proteins and plasmid DNA is a relatively simple and cost-effective way. The processes that use E. coli for product production are likely to contaminate host cell proteins (HCPs) from E. coli, and this contamination may reduce the efficacy of therapeutic drugs and lead to undesirable toxic or immune responses, so that the level of this contaminant needs to be minimized during purification of therapeutic drugs for use in humans or animals.

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 residual E.coli HCP in samples. First, add the E.coli HCP standard and test sample to the Anti-E.coli HCP coated microtiter strips (36712-A), then add the diluted biotin labeled Anti-E.coli HCP antibody (36712-C), and finally add Streptavidin-HRP (36712-D) to form an antibody + antigen + antibody-Biotin + SA-HRP complex. Subsequently, add TMB substrate (36712-G) 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 (36712-H) 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 2.22 ng/mL.

Feature

• Highly sensitive: Detecting as little as 2.22 ng/mL of E.coli HCP residuals in in-process and final biological samples.
• Ensures accuracy: The E.coli HCP 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 test in biological products

Specification

Product Performance

Components

^*The Standard (36712-B) is Lyophilized powder.

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

Specificity Demonstration

Figure 1. The specificity of E.coli HCP ELISA kit was shown in the figure above

9 samples were estimated for the non-specific reaction with the antibodies in the kit. The concentration of the 9 samples were all below the Limit of Quantitation (LOQ) (2.22 ng/mL).

Precision Display

A) Repeatability

B) Intermediate precision

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

8 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

Three concentrations of E.coli HCP standards, high, medium and low, were spiked to 10 μg/mL of E.coli expressed purified protein (several residue-free HCP) in equal proportions, while the sample spiked at the high value was made a multiplicative dilution.

HCP in the doubly diluted samples, as well as in samples of different spiked concentrations, and in unspiked samples was detected with linear and spiked recoveries ranging from 80% to 120%.

Spiked recovery = measured value / (spiked standard x 50% + spiked specimen value x 50%)

Table 2. Accuracy Validation

The linear and spiked recoveries of E.coli HCP in the doubly diluted samples, as well as the samples with different spiked concentrations, and in the unspiked samples, ranged from 80% to 125% (Table 2).

Documents:

Safety Data Sheet

36712_MSDS_HB20250310_EN.PDF

Manuals

36712_Manual_Ver.EN20250310.pdf