Process-Related Impurity ELISA Kits
BSA ELISA Kit
This kit is intended for quantitating bovine serum albumin (BSA). The use of serum-free defined media greatly reduces the number of potential impurities, but it may still be necessary to determine the levels of trace impurities, such as proteins and growth factors used in these media. Most commercial formulations of serum-free media contain significant amounts of albumin and transferrin, either of bovine or human origin, and insulin from various species.
This highly sensitive assay for BSA has a detection limit of <250 pg/ml. It can be used to optimize purification processes and in routine quality control of in-process streams, as well as in testing the final product. Please use Sample Diluent, Item No. F031A, with these kits; available separately.
该试剂盒用于定量牛血清白蛋白 (BSA)。 使用无血清成分确定的培养基大大减少了潜在杂质的数量，但仍可能需要确定微量杂质的水平，例如这些培养基中使用的蛋白质和生长因子。 大多数商业化的无血清培养基配方含有大量的白蛋白和转铁蛋白，无论是来自牛的还是来自人的，以及来自不同物种的胰岛素。
这种高灵敏度的 BSA 检测方法的检测限 <250 pg/ml。 它可用于优化纯化过程和过程中流的常规质量控制，以及测试最终产品。 请在这些套件中使用样品稀释剂，货号 F031A； 可单独购买。
F030 PI BSA ELISA
Technical Bulletin_Avoiding Kit Contamination
- Can the ELISA Protocol in the Product Insert be Modified?
The assay protocol recommended in the kits’ product insert have been developed with consideration given to typical limits of detection required in bioprocess impurity analysis. The assays are very robust with respect to protocol options and thus it is possible to modify the protocol to achieve performance parameters more optimal for your analytical needs. Sample volume, incubation times, and use of various sequential (forward or reverse sequential) schemes can lead to significant changes in sensitivity, reduction of non-specific sample matrix affects, or greater upper analytical range. If you change the protocol, it will be necessary to qualify that those changes achieve acceptable accuracy, specificity, and precision. Please refer to the section below on “How to Qualify HCP & Impurity Assays.” For advice on how best to modify an assay to meet your objectives you are encouraged to contact our Technical Services Department.
- How do I Maintain Quality Control of ELISA?
We manufacture our kits for lot-to-lot consistency and restrict and evaluate changes in any components or procedures that could impact accuracy in the customers’ laboratory. However, for generic kits, Cygnus performs QC on a limited range of parameters that may or may not be sensitive to your product specific issues. The routine run-to-run quality control of ELISA is best accomplished by assaying control samples across the important analytical range of the assay. We recommend 3 controls, a low control in the range of about 2 to 4 times the assay Limit of Quantitation (LOQ), a medium control, and a high control. Ideally, these controls should be made using your source of analyte (e.g. HCPs from your process). Furthermore, the controls should be in the same matrix as your critical samples or after dilution to MRD for your final drug substance. By using your source of analyte in your sample matrices, you will be best able to identify any problems within the run or between runs and kit lots. Cygnus offers Multi-Analyte Controls (MAC™); this set of low, medium and high controls is compatible with CHO 3G HCP ELISA (F550), HEK 293 HCP ELISA (F650R), E. coli HCP ELISA (F410) and Protein A Mix-N-Go ELISA (F610). MAC™ is designed to serve as a set of controls that span the analytical range of four popular Cygnus assays, and can also be used to trend the performance of each of these four assays over time. For more information on MAC– (F465R) or advice on how to make and establish controls specific to your needs please contact technical support.
Use of laboratory-specific controls is the only way to assure total quality control of the assay for your needs. Controls should be made in bulk, aliquoted for single use, and frozen at -80°C until stability studies indicate some other storage conditions are adequate. Once you have statistically established a range for these samples, they will become your most sensitive and specific tool to assure quality control of the assay. Do not rely on curve fit parameters as quality control specifications in the absence of true analyte controls. Curve fit parameters such as R square, slope, y-intercept, and upper and lower asymptotes are not sensitive or specific enough to reliably detect assay problems. Use of such parameters in the absence of true analyte controls, will frequently yield erroneous results. Contact our Technical Services Department for advice on how to make and establish controls specific to your needs.
Numbers of Replicates – When precision is very good (average replicate %CV on ODs are less than 5%) we feel duplicate analysis is adequate and the most effective approach. In the case of duplicate analysis, we do not allow for editing of any apparent outliers since there is no statistical basis for establishing which of the duplicates is inappropriate. Thus, in duplicate analysis we suggest repeating any sample that yields a %CV greater than 20%.
- What About Automation of Immunoassays?
Many of our customers have expressed an interest in automating our ELISAs to improve throughput and assay performance. Cygnus Technologies has much experience with various automation platforms. These systems include automated microtiter plate instruments and other liquid handling systems as well as several “biosensor” platforms. Using our proven and well-qualified antibodies and reagents in a biosensor platform still presents your lab with significant challenges in re-qualifying and optimizing the reagents. Before considering adapting our reagents to an automated system, you are encouraged to contact our Technical Service Department for guidance on how to approach assay development, qualification, and validation.
The promises of automation are higher throughput, faster turnaround of results, and improved performance in terms of parameters like sensitivity and precision. The practical ability of the many systems now being marketed to meet these promises is variable. Before embarking on a quest for automation, we urge you to contact our Technical Services Department. After evaluating your testing needs we can guide you to the system that best meets your requirements.
- What is the Storage and Stability of ELISA Kits?
Cygnus kits and reagents have been formulated to withstand shipment and/or temporary storage under ambient temperature conditions without any significant deterioration. Orders within North America are shipped next day delivery service without cold packs. International orders are shipped with cold packs to protect from extreme heat. Although stability studies have demonstrated that Cygnus kits can be held several weeks at ambient temperatures without any significant deterioration, we recommend storage at 2-8°C upon receipt of the products. Our components have been challenged at elevated temperature excursions, addressing concerns over extreme summertime temperatures, delays in shipping, custom clearances and/or refrigeration problems at the customer locations.
Should you have concerns regarding the performance of kits that have been subject to temperature deviations, we recommend an analysis of your control samples and historical specifications that determine the integrity of the kit. If the curve absorbances, sensitivity, and controls are within range, then the kit is acceptable for use.
- What Issues to Consider When Diluting Samples?
Some samples, particularly those from upstream in your purification process will have impurity analyte concentrations above the analytical range of our very sensitive ELISA kits. Such samples may require very large dilutions in order to overcome “Hook Effect” and to achieve acceptable “sample dilution linearity”. In addition to the “Hook Effect,” the matrix of some samples may interfere non-specifically with the assay and also result in under recovery of the true analyte levels. Simple dilution of those samples is often adequate to buffer out such interference provided the dilution does not reduce analyte concentrations below the Limit of Quantitation (LOQ) of the assay. In cases where dilution of your samples is not an option contact our Technical Service Department for advice on how best to overcome sample matrix interference.
Cygnus offers assay specific diluents for each of its kits. The catalog numbers for these diluents can be found in the kit product insert or by contacting Customer Service. We strongly recommend use of those diluents because they are the same formulation as the matrix used for the kit standards. Thus, as you dilute your samples in our diluent, your sample matrix begins to approach that of the standards and in this way greatly minimizes any dilution artifacts that could occur if you were to use another diluent.
If you elect to use another diluent you must validate that it provides accurate results. We recommend two critical experiments for validation:
- First, you must assay the diluent alone to determine that it does not yield absorbance values significantly above or below the absorbance for the kit zero standard. OD values above the zero standard indicate that the diluent may have low levels of the analyte or that the diluent causes an increase non-specific binding, relative to the kit standards matrix. OD values less than the kit standard may indicate that the proposed diluent is lowering non-specific binding and may also be inhibiting specific binding. In any event use of a diluent that does not match the standards risks significant errors when the sample concentration gets multiplied by the dilution factor.
- Second, you must perform a spike & recovery experiment into your proposed diluent at several levels across the analytical range of the assay. For a diluent to be deemed acceptable we suggest a recovery specification of 95% to 105%. In general, the formulation of diluents should be a neutral pH with some carrier protein added to block non-specific adsorptive losses of the analyte. Use of just PBS or TBS without a carrier protein can be problematic because the analyte diluted in the range of the assay (ng/mL) can very significantly adsorb to the dilution tube resulting in low recovery!
Your diluents should not have sodium azide as a preservative nor significant detergent concentrations as these will reduce assay accuracy.
- Why Perform Dual Wavelength Analysis?
Catalog #: F033-12
Catalog #: F290
Catalog #: F070
Catalog #: F031A
Catalog #: F004
Catalog #: F120
Catalog #: F031
Catalog #: F031E-1
Catalog #: F031G
Catalog #: F006-12