Sample prep is one of the earliest steps in the ELISA workflow and should always be carefully optimized to ensure accurate and reproducible results. This editorial looks at some of the different types of samples being analyzed by ELISA and shares practical tips for preparing them.
Optimizing sample prep is a key part of ELISA development
ELISA sample prep is part of the pre-analytical process. It encompasses everything from sample collection, storage, and transport, to clarifying and (in many cases) diluting the sample material prior to testing. Each of these steps has the potential to influence experimental results.
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“Optimizing ELISA sample prep should involve systematically testing the impact of different processing steps on the analyte of interest,” reports Dr. Tobias Polifke, Managing Director at CANDOR Bioscience. “Factors to consider include the type of collection vessel used, the temperature during storage and transport, and whether additional measures are required to preserve critical or unstable analytes.”
“Ultimately, an optimized sample prep ensures that the integrity of the target of interest is maintained throughout handling—from sample collection to detection,” adds Chelcie Eller, Head of ELISA Assay Development at Abcam. “This includes preserving target stability, minimizing biological matrix effects, and determining the appropriate sample dilution for the assay range.”
Failure to optimize ELISA sample prep can manifest as inconsistent data or, in a worst-case scenario, false positive or false negative results. “If you take this risk, you may as well simply guess experimental outcomes instead of performing measurements,” says Polifke. “Optimizing sample prep should always be considered a central part of standard ELISA development.”
General best practice recommendations
General best practice recommendations for ELISA sample prep include minimizing freeze-thaw cycles, evaluating potential matrix effects, and optimizing the sample dilution. “To minimize freeze-thaw, we usually recommend that samples are prepared as single-use aliquots and stored at -20°C for short term (<6 months) or at -80°C for long term (>6 months),” notes Eller. “We also advise adding protease inhibitors to sample buffers, particularly when working with extracts.”
To evaluate matrix effects, Anne Sloan, Technical Scientist at Cell Sciences, suggests performing spiking experiments, whereby a known concentration of the analyte is added into both the sample matrix and a standard diluent and the percent recovery calculated. “Recovery should be in the region of 80–120%,” she says. “If this is not the case, it indicates that components within the sample matrix are interfering with analyte detection.”
According to CJ Xia, VP of Marketing and Science at Boster Bio, optimizing the sample dilution to maximize the signal-to-noise ratio may sometimes require running a full plate of serially diluted samples to calculate multiple EC50 values. “This is because samples may have varying levels of target expression, even within the same treatment group, which means you cannot use the same dilution ratio for all samples,” he explains.
Another recommendation is to consider using specialized buffers and additives when preparing samples. “Interference reducing diluents such as LowCross-Buffer® can minimize matrix effects, including epitope masking, while additives like CANDOR’s Antibody Stabilizer conserve the structure of proteins and antibodies to prevent undesirable changes during storage,” comments Polifke.
Sample type-specific optimization
Depending on the sample, different preparation methods may be required. The following tips cover four of the most common sample types analyzed by ELISA:
While non-adherent cells can be collected by centrifugation, adherent cells must first be detached from the culture flask. “We typically recommend scraping to collect adherent cells prior to centrifuging at 500 x g for 5 minutes at 4oC,” explains Eller. “The cells should then be rinsed twice with PBS and the pellet solubilized at 2x107 cell/mL in a suitable extraction buffer by incubating on ice for 20 minutes. Following this, the samples should be clarified by centrifugation at 18,000 x g for 20 minutes at 4°C, then the supernatants can be transferred into clean tubes and the pellets discarded.”
Tissue extracts are more technically challenging to prepare as they require thorough rinsing with PBS to remove any blood prior to enzymatic digestion and/or mechanical homogenization. “When preparing tissue extracts, the samples should either be freshly harvested or have been stored at -80°C prior to processing, and the sample buffer should always contain protease inhibitors to prevent target degradation by endogenous enzymes.” advises Xia. “It is also important to note that the presence of endogenous biotin in tissue samples prohibits ELISA analysis with kits that use biotin as part of the detection system.”
Serum samples are prepared by collecting whole blood into a serum separator tube, allowing clot formation to occur, and then centrifuging. However, Polifke reports that the type of blood collection tube can impact the measurement of certain analytes and cautions that samples from different patients may exhibit different levels of masking or aggregation. “When optimizing sample prep for serum, you ideally need very high sample numbers from patients with many different physiological conditions,” he says. “Unfortunately, this is often difficult to achieve for validation purposes. In such cases, using LowCross-Buffer for sample dilution can help to mitigate patient-specific effects and ensure assay quality.”
Plasma samples are prepared in a similar manner to serum samples but necessitate the use of an anti-coagulant such as citrate, EDTA, or heparin. When choosing an anti-coagulant, it is important to consider its compatibility with the analyte being studied. “Using the wrong anti-coagulant can result in chelating the target protein on the wrong epitope, which can interfere with antibody binding,” says Xia. “If there is no data on which anticoagulant to use, such as when developing a new assay, this would be something to keep in mind and optimize for.”
Recommended reading
For further insights into the importance of pre-analytical sample handling, Eller recommends a recent publication co-authored by Abcam’s Vice President of Assay Development Platforms, Andrew Ball. This identifies several critical pre‐analytical variables, including the collection tube type and time to centrifugation, when measuring blood-based Alzheimer’s disease biomarkers.
Next steps
Once the ELISA sample prep method has been established, downstream processing steps must also be optimized. Here, Sloan stresses the importance of using the same buffer for both sample dilution and preparing the standard curve, as well as including positive and negative controls to validate assay performance. “It is also important that each step of the assay is performed consistently to ensure accurate and reproducible results,” she says.