S.cerevisiae HCP ELISA kit

Sample

Recombinant products

Species Reactivity

Yeast

Intended Use

This kit is intended for use in determining the presence of Saccharomyces cerevisiae protein contamination in products manufactured by recombinant expression in this yeast cell line. The kit is for Research and Manufacturing Use Only and is not intended for diagnostic use in humans or animals.

Contents of Kit

Anti-S. cerevisiae, biotinylated
Affinity purified goat antibody conjugated to biotin in a protein matrix with preservative. 1x12mL
Anti-S. cerevisiae coated microtiter strips, 12x8 well strips in a bag with desiccant
S. cerevisiae Standards
Solubilized S. cerevisiae HCPs in bovine albumin with preservative. Standards at 0, 2, 8, 25, 75, and 200ng/mL. 1 mL/vial
Streptavidin: Alkaline Phosphatase
In a protein matrix with preservative. 1x12mL.
PNPP Substrate
p-nitrophenyl phosphate in a Diethanolamine buffer with preservative. 1x12mL
Wash Concentrate (20X)
Tris buffered saline with preservative. 1x50mL

Storage

All reagents should be stored at 2°C to 8°C for stability until the expiration date printed on the kit.
The substrate reagent should not be used if its absorbance at 405nm is greater than 0.4.
Reconstituted wash solution is stable until the expiration date of the kit.
After prolonged storage, you may notice a salt precipitate and/or yellowing of the wash concentrate. These changes will not impact assay performance. To dissolve the precipitate, mix the wash concentrate thoroughly and dilute as directed in the "Reagent Preparation" section.

Performance Characteristics

Hook Capacity
Increasing concentrations of HCPs> 200 ng/mL were assayed as unknowns. The hook capacity, defined as that concentration which will give an absorbance reading less than the 200 ng/mL standard was>50 μg/mL.

Precision

Both intra (n=20 replicates) and inter-assay (n=5 assays) precision were determined on 3 pools with low (2ng/mL), medium (75ng/mL), and high concentrations (200ng/mL). The % CV is the standard deviation divided by the mean and multiplied by 100.

Detection Limit

The lower limit of detection (LOD) is defined as that concentration corresponding to a signal two standard deviations above the mean of the zero standard. LOD is 0.5ng/mL.
The lower limit of quantitation (LOQ) is defined as the lowest concentration, where concentration coefficients of variation (CVs) are <20%. The LOQ is 0.9ng/mL.

General Description

Recombinant expression of proteins by S.cerevisiae is an efficient method to obtain cost effective quantities of a desired protein. Many of these recombinantly produced proteins 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 from S. cerevisiae. Such impurities can result in adverse toxic or immunological reactions and thus it is desirable to reduce host cell impurity 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 a 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. Western Blot may be able to detect HCPs in samples from upstream in the purification process but 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 semiquantitative 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 mild lysate washed of S. cerevisiae cells to obtain HCPs typically encountered in your initial product recovery step. 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 Creative Diagnostics recommends a method we find superior to 2D Western blot. We term this method 2D HPLC-ELISA. 2D HPLCELISA 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 impurities 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 impurities 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 qualified for testing of final product HCPs in many different products regardless of growth and purification process. When the kit can be satisfactorily qualified for your samples, the application of a more process specific assay is probably not necessary in that such an assay would only provide information redundant to this generic assay. However, if your qualification 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 impurities 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 qualified experimentally by each laboratory. The use of a process specific assay with more defined antigens and antibodies in theory may yield better sensitivity however Saccharomyces cerevisiae HCP ELISA Product Insert 2 such an assay runs the risk of being too specific in that it may fail to detect new or atypical impurities 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, Creative Diagnostics is available to apply its proven technologies to develop such antibodies and assays on custom basis.

Standard Curve