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. 

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 2 to 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 Control Sets for several ELISA kits including; CHO, HEK, E.coli, PG13, and Protein A. For more information on Control Sets 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%.

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.

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.

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. 

Cygnus Technologies now offers several ELISA kits for detection of natural Protein A as well as most modified recombinant constructs. Our original kit, F050H has proven to be valid for use in detecting both natural and structurally conserved recombinant Protein A. Catalog F400 and F400Z kits were offered to better detect unnatural recombinant constructs of Protein A such as GE Healthcare’s MabSelect SuRe™ ligand. In addition, we offer kits to other unnatural constructs.

For first time users we recommend trying our F600 or F610 kits as these are the most robust and easy to use kits on the market. The F600 and F610 kits utilize a proprietary Mix-N-Go™ protocol that eliminates the heating and centrifugation steps. These assays use the same antibodies as our F400 and F400Z kits. The F600 uses standards calibrated to natural and conserved Protein A, while the F610 uses standards calibrated to unnatural constructs such as MabSelect SuRe™.

The simple sample dilution step employed in these kits overcomes the product antibody interference seen in other methods and as such has been successfully validated for a large number of therapeutic antibodies.

The Cygnus Protein A Kit, Catalog F050H uses antibodies made to natural Protein A with qualification performed against only the natural and the highly conserved recombinant Protein A. With the introduction of MabSelect SuRe™ we have evaluated its reactivity in these kits. Our data has shown that it has only about a 20% weight to weight reactivity relative to the Protein A standards in these kits. This 20% reactivity may be greater or less depending upon other factors such as the product antibody being purified, which may contribute to further interference or non-specificity. 

The F400 and F400Z assays address the low reactivity to MabSelect SuRe™ seen in our other kits, while incorporating several other improvements in the methodology. The biotinylated antibody used in our original kits has been replaced with a different antibody directly labeled with HRP. Improvements include more sensitivity, simplified assay protocol, more standards, and less probability of product matrix interference. The F400 and F400Z kits detect natural, conserved recombinant, and MabSelect SuRe™ Protein A ligands essentially 1:1. Similar to the F050H assay, these assays use a sample denaturation step to overcome sample/product antibody interferences. The F400Z kit uses the same antibodies, standards and sample treatment procedure as the F400 kit. The only difference is that F400Z has a "recovery enhancer" reagent that improves analytical recovery in antibody: fusion proteins and antibody fragments that are not completely removed during the sample treatment step. 

The protocols in our ELISA kits recommend the use of dual wavelength analysis for microtiter plates readers appropriately equipped. For alkaline phosphatase – PNPP based assays the two wavelengths are 405 nm and 492 nm. For horse radish peroxidase – TMB based assays the wavelengths are 450 nm and 630 nm. In both assay types the first wavelength listed, also termed the "test wavelength", is critical and thus your microtiter plate reader should be equipped with the appropriate 405 nm and 450 nm filters. It is not absolutely necessary to use dual wavelength analysis with any of our assays and thus the second wavelength (492 nm and 630 nm - also called the "reference wavelengths") are optional. In theory, the use of dual wavelength analysis should provide better precision. Microtiter plate readers with the appropriate software will automatically subtract the reference wavelength absorbance from the test wavelength absorbance. In theory dual wavelength analysis can help to overcome any non-wavelength specific imperfections in the plate. In practice, because the optical quality of our microtiter plates is excellent, there is usually no significant statistical improvement in the data by subtracting out the reference absorbance from the test absorbance. Most plate readers are equipped to perform dual wavelength analysis. Since the reference wavelength is not critical, microtiter plate readers with reference filters close to those we recommend can be substituted. For example, you may use a 490 nm or a 500 nm filter in place of the 492 nm or a 600 nm or 650 nm filter in place of the 630 nm filter.