Back to Basics: Generating Spike & Recovery Analysis Data
The importance of spike and recovery
Every sample matrix is unique and has the potential to interfere with your ELISA assay. Conducting a spike and recovery assay is an essential step to test the compatibility of your test sample formulation with the immunoassay reagents, so you can have confidence in the accuracy of your results and qualify your assay in accordance with regulatory guidelines.
What is spike and recovery?
Spike and recovery involve introducing (“spiking”) known amounts of analyte into your various sample types and measuring whether “recovery” measurements are accurate. In some cases, this test may reveal that the product protein itself, or certain components in the product formulation buffer interfere with the ability of the assay to detect HCPs or other contaminants. Factors such as high or low pH, high protein or salt concentration, or presence of detergents or organic solvents are all known to interfere with ELISA quantification. For each sample type to be tested, you should demonstrate that the assay can accurately recover analyte spiked into that sample matrix. This includes both in-process samples and your final drug product.
Types of interference
Interference occurs when your product or certain components in the product formulation buffer cause inaccuracies in the ability of the assay to detect and quantify HCPs or other impurities, leading to either overestimating (“over-recovery”) or underestimating (“under-recovery”) of impurities. Similarly, upstream harvest and in-process samples may contain components in their matrices that can interfere with ELISA detection. In the cases where interference is detected, it typically manifests as under-recovery of the spiked analyte. However, if the drug substance or other matrix component interacts with the capture or detection antibody, this could result in over-recovery values.
Other types of non-specific interference can also occur with ELISA assays, such as higher-than-expected ODs due to non-specific binding of reagents to the plate. These types of interference are revealed with the inclusion of appropriate controls. When optimizing a new assay, it is important to follow the kit protocol as closely as possible to minimize the potential for such issues that can impact the dynamic range of the assay, and thus your results.
When should spike and recovery analysis be performed?
Before performing spike and recovery analysis, it is first recommended to conduct dilution linearity studies with your samples to establish that conditions of antibody excess are met and to confirm the working concentration is in an acceptable range. This experiment will determine the Minimum Required Dilution (MRD) of your sample to then set up your spike and recovery assay.
As mentioned above, it is necessary to perform spike and recovery analysis for each type of sample matrix you will evaluate. Moreover, if your manufacturing process changes at a later time, this experiment needs to be repeated to ensure the assay remains valid.
Interested in learning more about the concept of Dilution Linearity? Check out our previous post and whiteboard video:
How is spike and recovery analysis performed?
Spike and recovery analysis should be performed by spiking 3-4 concentration levels covering the analytical range of the assay into your various sample types at the MRD, and then testing in the assay. When selecting these concentrations, be sure that the lowest spiked concentration is at least 2 times the Limit of Quantitation (LOQ) of the assay, which is reported in the product documentation.
In the example presented below, we spiked 1 part of the 100 ng/mL standard from our E. coli HCP ELISA kit (F410) into 4 parts “neat” (undiluted) sample (e.g. spike 100 μL of 100 ng/mL standard into 400 μL sample). This resulted in a final sample concentration of 20 ng/mL. In parallel, a control dilution of 1 part of assay diluent (zero standard) to 4 parts of sample was also performed, to determine the contribution of endogenous HCP in the sample prior to spiking. In this way, both the spiked and diluted neat/negative samples are assayed, and the endogenous contribution of HCP from the sample prior to spiking can be determined by subtracting the contribution of HCP in the zero standard sample (in this case, 6 ng/mL) from the total HCP measured in the spiked sample (in this case, 25 ng/mL).
| ICH, FDA and EMA guidelines on analytical procedure validation consider recovery values within 75% to 125% of the spiked HCP concentration to be in the acceptable range. |
The table below shows the example data, and illustrates how the spike recovery value of 95% was calculated.
Spike Concentration (ng/mL) | Total HCP Measured (ng/mL) | % Spike Recovery | |
4 parts final product + 1 part “zero standard” | 0 | 6 | NA |
4 parts final product + 1 part "100ng/mL standard" | 20 | 25 | 95% [(25-6)/20] |
For a step-by-step guide to sample plate set-up with four dilutions, watch our training video:
Troubleshooting spike & recovery values
If your spike and recovery data suggest significant product or matrix interference, it may be necessary to further process the sample by alternative methods, such as further dilution. The same diluent used to prepare the kit standards is ideally the preferred material for dilution of your samples. In other cases, modification of the assay protocol can affect accuracy in some sample types. Our Technical Services experts can provide case-by-case guidance on how best to solve poor spike and recovery problems experienced with your samples.
While spike and recovery analysis should be performed to ensure the accuracy of your assay, it is only one step toward establishing that your HCP ELISA is fit-for-purpose. Further studies, including precision, LOD, LLOQ and ULOQ, and HCP antibody coverage analysis, are needed to qualify your assay for use.
More questions? Our technical support team is ready to help you.