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/ Каталог / Реагенты для научных исследований / Реагенты Cygnus Technology

Набор для иммуноферментного анализа для измерения белков клеток-хозяев E. coli (Immunoenzymetric Assay Kitforthe Measurementof E. coli HostCellProteins)

E-coli-HRP HCP ELISA (+4 C)

E.coli HCP ELISA kit (Cygnus Technologies, Inc.)

This kit was developed using broadly reactive polyclonal antibodies to the hundreds of different HCPs and is intended for use in determining the presence of E.coli host cell protein contamination in products manufactured by recombinant expression. Our assay validation has determined that the LOD for this kit is ~0.2ng/mL.

Интсрукция (PDF)

Recombinant expression in E. coli is a relatively simple and cost effective method for production of certain proteins and pDNA. Many of these products are intended for use as therapeutic agents in humans and animals, and as such, must be highly purified. The manufacturing and purification process of these products leaves the potential for contamination by host cell proteins (HCPs) from E. coli. Such contamination can reduce the efficacy of the therapeutic agent and result in adverse toxic or immunological reactions and thus it is desirable to reduce HCP contamination to the lowest levels practical. Immunological methods using antibodies to HCPs such as Western Blot and ELISA are conventionally accepted. While Western blot is a useful method aiding in the identity of HCPs, it suffers from a number of limitations.
Western blot is a complex and technique dependent procedure requiring subjective interpretation of results. Furthermore, it is essentially a qualitative method and does not lend itself to obtaining quantitative answers. The sensitivity of Western blot is severely limited by the volume of sample that can be tested and by interference from the presence of high concentrations of the intended product. While Western Blot may be able to detect HCPs in samples from upstream in the purification process, it often lacks adequate
sensitivity and specificity to detect HCPs in purified downstream and final product. The microtiter plate immunoenzymetric assay (ELISA) method employed in this kit overcomes the limitations of Western blots providing on the order of 100 fold better sensitivity. This simple to use, highly sensitive, objective, and semi-quantitative ELISA is a powerful method to aid in optimal purification process development, process control, routine quality control, and product release testing. This kit is “generic” in the sense that it is intended to
react with essentially all of the HCPs that could contaminate the product independent of the purification process. The antibodies have been generated against and affinity purified using a blended lysate of E. coli from a variety of strains including the commonly used DH5α and BL21 strains. This relatively mild lysing procedure is intended to obtain HCPs typically encountered in initial product recovery steps, such as clarification of conditioned media when the product is secreted, or after osmotic shock or mild detergent and mechanical disruption, to obtain inclusion bodies and other intracellular proteins. Western blot was used as a preliminary method and established
that the antibodies reacted to the majority of HCP bands resolved by the PAGE separation. If you have need of a more sensitive and specific method to demonstrate reactivity to individual HCPs in your samples Cygnus Technologies recommends a method we find superior to 2D Western blot. We term this method 2D HPLC-ELISA. 2D HPLC-ELISA can yield much better sensitivity and specificity as compared to 2D Western blot. For more information on this 2D HPLC-ELISA analysis please contact our Technical Services department.
Special procedures were utilized in the generation of these antibodies to insure that low molecular weight and less immunogenic contaminants as well as high molecular weight components would be represented. As such, this kit can be used as a process development tool to monitor the optimal removal of host cell contaminants as well as in routine final product release testing. 

Because of the high sensitivity and broad reactivity of the antibodies, this generic kit has been successfully validated for testing of final product HCPs in many different products regardless of growth and purification process. When the kit can be satisfactorily validated for your samples, the application of a more process specific assay may not be necessary, in that such an assay would only provide information redundant to this generic assay. However, if your validation studies indicate the antibodies in this kit are not sufficiently
reactive with your process specific HCPs it may be desirable to also develop a more process specific ELISA. This later generation assay may require the use of a more specific and defined antisera. Alternatively, if the polyclonal antibody used in this kit provides sufficient sensitivity and broad antigen reactivity, it may be possible to substitute the standards used in this kit for ones made from the contaminants that typically co-purify through your purification process and thus achieve better accuracy for process specific
HCPs. The suitability of this kit for a given sample type and product must be determined and validated experimentally by each laboratory. The use of a process specific assay with more defined antigens and antibodies in theory may yield better sensitivity however such an assay runs the risk of being too specific in that it may fail to detect new or atypical contaminants that might result from some process irregularity or change. For this reason it is recommended that a broadly reactive “generic” host cell protein assay be
used as part of the final product purity analysis even when a process specific assay is available. If you deem a more process specific assay is necessary, Cygnus Technologies is available to apply its proven technologies to develop such antibodies and assays on custom basis.

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Область использования:Производство:Cygnus Technology
Кат. номер:F410
Цена (с НДС 20%):по запросуВ корзину
Наименование: Набор для иммуноферментного анализа для измерения белков клеток-хозяев E. coli (Immunoenzymetric Assay Kitforthe Measurementof E. coli HostCellProteins) / E-coli-HRP HCP ELISA (+4 C).
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info@LD.ru   тел.: +7 495 369-20-43