Development Of Sandwith ELISA For Detecting Residual Proteins From Biological Products In Quality Control

Traditional biological products include vaccines, antiserum, allergenic extracts, blood, blood products, and blood derivatives. Recent advances in biotechnology have resulted in improvements to traditional biological. The more important result is the availability of many novel biological therapeutic products including cytokines, monoclonal antibodies for therapy and diagnosis, somatic cell and gene therapy products, and human and xenogeneic tissues. Biological products, for the most part, are complex materials often derived from living materials from living donors capable of transmitting infectious agents, thus must be closely evaluated and monitored during the production process and in post approval surveillance to ensure the continued safety and efficacy of the product. Evaluations include quality control (QC), that is, a system for verifying and maintaining a desired level of quality in a product or process by careful planning, use of proper equipment, continued inspection, and corrective action as required. QC includes the review of source and safety of the starting material, purification, reagents, protein residuum and contaminants. The protein residuum in biological products include many components, such as eukaryotic, prokaryotic (Escherichia coli)host cell proteins (HCP), embryonated egg proteins, and serum proteins in the culture medium(e.g bovine serum albumin). In abroad, so many methods are taken to reduce this impurity in the product QC, and highly sensitive and reliable analytical methods are also taken in QC process. In our country, upper limits for the content of these proteins have been established these years, but correlated researches are lagged compared to abroad, currently used assays are hard to satisfy the needs of scientific research and market development.In this study, we lysised the Escherichia coli cells and immunized rabbits with E.coli cell proteins.The polyclonal antibody against HCP was developed. pAb serum was primarily purified by ammonium sulfate precipitation, further by anion exchange chromatography and then by affinity chromatograph. The purified pAb was labeled with biotin, and then avidin magnifying system was put into use. The biotin-avidin sandwich (BAS) ELISA system to analyse the HCP with rabbit pAb purified by affinity chromatograph was successful established. The detection limit reached to 0.32μg/L and this assay was highly sensitive and quantitative in the range of HCP concentration from 1 to 100μg/L. Intra- and interassay coefficient variations were 7.7% and 6.2%. The sensitivity, precision, and accuracy of the kit were determined and satisfied. Comparing with commercialized ELISA kit of foreign country, no significant distinction was found. In addition, we immunized mouse with E.coli cell proteins for the development of monoclonal antibodies (mAbs) to HCP of E.coli. 33 hybridoma cell lines serecting mAbs to HCP of E.coli were developed. One of the mAbs recognizing chaperonin GroEL molecule was obtained, verified by immunoprecipitation and mass spectrometry. This mAb may provide a useful tool for studying the structure and function of chaperonin GroEL.The second part of this study was to establish sandwich ELISA for detecting the concentration of bovine serum albumin (BSA). The routine murine B cell hybridoma technique was employed and 11 hybridomas serecting mAbs against BSA were selected. After purification of specific mAbs by ammonium sulfate precipitation, and anion exchange chromatography, the purified mAbs were labeled with HRP by sodium metaperiodate. Two mAbs coated and detected were selected for quantitative analysis. Following optimization of the working concentrations of coated and detecting mAbs, we successfully established ELISA kit for detecting BSA. The detection limit was 0.38μg/L and quantitative in the range of BSA concentration from 0.5 to 40μg/L. Intra- and interassay coefficient variations were all lower than 10% at three concentrations. The data showed that the sandwich ELISA kit we established was found to be 10 times more sensitive than the mAb ELISA assays reported before, and not less sensitive than currently used pAb ELISA assays in abroad.In conclusion, based on the development of developed monoclonal antibodies against BSA and polyclonal antibody against E.coli cell proteins, we established sandwich ELISA kit for detecting BSA and E.coli cell proteins with high specificity and sencitivity which would be useful for the application in biological products QC and further investigation.