Preface

In the process of using ELISA kits, ensuring the accuracy of results is of crucial importance. To effectively identify the compatibility between sample formulations and kit components, the spike-and-recovery test— a key validation experiment — is particularly essential. It serves not only as an important tool for evaluating the accuracy of ELISA kits but also as a reliable means to meet the methodological validation requirements for commercial ELISA kits specified in regulatory and pharmacopoeial standards (such as guidelines from ICH or FDA).

What is Spike & Recovery?

Spike & recovery refers to the process of adding a known amount of standard substance to a sample ("spiking"), then evaluating the accuracy of the measured "recovery" value by comparing the test results before and after the addition. This process can reveal whether certain components in the sample and its buffer interfere with the test results. Currently, common factors affecting the quantitative results of ELISA kits include extreme pH, detergents, organic solvents, high protein concentration, and high salt concentration. Therefore, it is vital to ensure the elimination of these interfering factors before conducting the spike-and-recovery experiment.

How to Identify Interfering Factors?

Certain components in the sample or buffer may affect the detection and quantification of HCP (Host Cell Protein) or other impurities, leading to overestimation or underestimation of recovery results — which indicates the presence of interfering factors in the sample. Similarly, the matrices of upstream harvests and process samples may also contain components suspected of interfering with ELISA detection.

In the presence of interference, experimental results typically show low recovery rates. However, if the drug itself or other matrix components interact with the capture or detection antibodies, the recovery rate may be abnormally high.

In addition, non-specific interference (e.g., binding of reagents to the microplate) can also cause an increase in OD (Optical Density) values. Specific interfering factors can be identified through rational control of the experimental process.

When to Conduct the Spike & Recovery Experiment?

Before performing the spike-and-recovery experiment, it is necessary to first complete the dilution linearity analysis of the sample. Only when the Minimum Required Dilution (MRD) is clearly defined does the spike-and-recovery analysis hold significance. (For detailed information on dilution linearity analysis, please refer to "HCP ELISA Method Validation — Dilution Linearity".)

(Tips: Spike-and-recovery analysis must be performed for each type of sample matrix. Meanwhile, if the experimental procedure is optimized, this experiment needs to be repeated to ensure the validity of the detection.)

How to Perform Spike & Recovery Analysis?

Sample Preparation

· Spiked Samples: Add standards of 3-4 different concentrations to different types of test samples to prepare spiked samples. It should be noted that the concentration of spiked samples must be analyzed under the condition of meeting the MRD — that is, the minimum spiked concentration should not be lower than twice the Limit of Quantitation (LOQ).

· Control Samples: Add sample dilution buffer to each type of test sample at the same volume ratio to prepare control samples, which are used to determine the background HCP concentration of the sample.

Experimental Procedure

Use the ELISA kit to detect both spiked samples and control samples simultaneously. It is recommended to set up 3 replicate wells for each sample to calculate the average value.

Result Analysis

Recovery rate calculation formula:

As shown in the example in the table below, we used the E. coli HCP ELISA Kit (F410) to test the spiked sample (100 ng/mL standard mixed with the sample at a volume ratio of 1:4, with a spike concentration of 20 ng/mL) and the control sample (sample dilution buffer mixed with the sample at a volume ratio of 1:4) respectively. The recovery rate was calculated as 95% based on the measured HCP concentration.

In compliance with the analytical method validation guidelines issued by ICH, FDA, EMA, and other relevant regulatory authorities, the acceptable range for Host Cell Protein (HCP) spike-and-recovery rates shall be 75% to 125%.

To help you quickly understand how to prepare spiked samples using the 4-fold dilution method and the detailed procedures for ELISA plate preparation, please watch the following instructional video.

Troubleshooting for Spike-and-Recovery Assays

If the spike-and-recovery data indicates significant product or matrix interference, you may need to obtain accurate detection results by means such as further diluting the sample or replacing the buffer. Ideally, select the same diluent as the standard for sample dilution, as this will minimize deviations to the greatest extent. In some cases, adjusting the detection method may improve the accuracy for specific sample types, but operations must be performed with extreme caution to avoid affecting the reliability of the results.

To fully support customers in conducting spike-and-recovery validation and methodological development, Cygnus provides corresponding standards for each ELISA kit. For details, please refer to the table below.

While spike-and-recovery assays can validate the accuracy of HCP ELISA, this is only one part of the overall methodological validation. A complete validation process must still include analyses such as precision, LOD, LLOQ, ULOQ, and HCP antibody coverage. For each detection kit, Cygnus provides a corresponding QS (Qualification Summary) document. If needed, please click here to fill in the information to request it, or contact XMJ, the exclusive distributor of Cygnus in China, directly. Should you encounter any issues while using Cygnus HCP ELISA kits, our technical support team will be dedicated to providing you with solutions and recommendations.

Cygnus Technologies has specialized in providing products and analytical methods for the biotechnology and biopharmaceutical industries for over 25 years, with the aim of accelerating the R&D phase and enhancing product quality. The bioprocess residue test kits developed and manufactured by Cygnus are used to detect specific impurities across more than 50 different expression systems. As an expert in high-sensitivity analytical technologies focused on immunoassays for biotechnological applications, Cygnus’ products and services are utilized by over 95% of biopharmaceutical companies worldwide and have gained broad recognition from regulatory authorities such as the FDA, NMPA, and EMA.