CST BLOG: Lab Expectations

The official blog of Cell Signaling Technology (CST), where we discuss what to expect from your time at the bench, share tips, tricks, and information.

Safeguard the Quality of Your ELISA Data: Ensuring ELISA Lot-to-Lot Reproducibility

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Ready-to-use ELISA kits can streamline your research and development workflows by removing the need for optimization. However, it is essential that the ELISA kits you choose exhibit high lot-to-lot reproducibility for your projects to stay on track. If the ELISA kit lot number changes halfway through a long-term experiment, you need to be confident that the new lot will perform comparably to the old lot, producing similar signal/blank ratios and generating percent coefficient of variation (%CV) values that show any inter-assay variability to fall within an acceptable range.

Antibodies are core components of ELISA kits that have been heavily implicated in the reproducibility crisis—the growing realization that the results of many scientific experiments cannot be repeated. Addressing this issue requires researchers to be careful with product selection, and antibody manufacturers to ensure that only the highest quality, most rigorously validated products reach the market. Critically, antibodies should demonstrate high specificity for their targets and be proven to work in the applications stated on the product datasheet.

At CST, we adhere to the Hallmarks of Antibody Validation when developing and releasing our antibodies to assure you of data you can trust. By carefully tailoring which of these six complementary validation strategies are applied to each product, we are able to determine the specificity, sensitivity, and functionality of an antibody in any given assay. This means that, when you choose a CST® antibody, you have the assurance that it is covered by our product performance guarantee.

Our antibodies form the basis of our ready-to-use FastScan, PathScan®, and PathScan® Rapid Protocol (RP) ELISA kits, all of which are configured in a sandwich assay format for superior target specificity compared to direct and indirect ELISA. By identifying optimal antibody pairs and carrying out all of the assay development and validation up front when designing and producing our ELISA kits, we safeguard the quality of your data throughout the entire duration of your project.


Over the past six years working as a Product Scientist at CST, a big part of my role has been to test our ELISA kits for lot-to-lot variation to ensure consistency. Scientific reproducibility is a core CST value, which is evident in the way we apply our stringent validation standards. Ultimately, we want our customers’ research to remain uninterrupted and for them to trust their results, regardless of the ELISA kit lot they receive. By producing every new lot in accordance with our established SOPs, we ensure all our ELISA kits demonstrate the consistent, reliable performance researchers associate with CST.

ELISA Kit Lot Testing Requirements

Before making any new ELISA kit lot available to researchers, we run a comprehensive series of tests. These serve to confirm that the new lot has high specificity for the target of interest (low background signal), comparable sensitivity to the current lot (similar limit of detection), and the capacity to produce a sensitivity curve with a broad dynamic range.

The types of tests that we perform include the following:

Testing with Positive and Negative Controls 

Depending on the ELISA, the controls used for validation purposes could be biologically relevant samples or samples with known (high and low) endogenous levels of the target protein. By generating side-by-side titration curves with the current and new ELISA kit lots that show how the absorbance signal changes at different protein concentrations, we can confirm that the datasets correlate. Because every model system is different, we recommend customers use our titration curves as a guide for determining their own sample loading protein concentrations.

Since our FastScan, PathScan, and PathScan RP ELISA kits are validated for relative (not absolute) quantitation, they do not include standards. Instead, we advise that samples are normalized by either protein amount or cell number. We do, however, provide a lyophilized positive control with our FastScan kits to verify assay performance.

Optical  Density (OD) Signal Thresholds

For an ELISA kit lot to pass validation, the positive control sample must have an absolute optical density (OD) value greater than 1.5, while the blank sample should fall below a signal threshold of 0.3. When testing our PTM-specific ELISA kits, we also set appropriate induction thresholds to ensure the phospho control shows significant signal above the negative, non-phospho control sample.

ELISA Kit lot-to-lot negative and positive control testing dataELISA Kit lot-to-lot negative and positive control testing data

Figure 1. Representative positive (top) and negative (bottom) control lot testing data for FastScan™ Cas9 (S. pyogenes) ELISA Kit #29666.

In addition, each new ELISA kit lot must have a signal/blank ratio of greater than 5.0 for the highest titration point. Importantly, this value must fall within a similar range to that of the current lot for the new lot to pass our reproducibility standards.

Signal/Blank Ratio Data Summary for PathScan® Phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) Sandwich ELISA Kit #12001

Concentration (mg/ml) #12001 Lot 34 #12001 Lot 35
0.075 18.3 16.9
0.0375 11.9 11.9
0.01875 7.8 7.7
0.009375 4.8 4.8

Figure 2. Representative passing signal/blank ratio data from lot validation of PathScan® Phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) Sandwich ELISA Kit #12001.

Percent Coefficient of Variation (%CV) Values

Another critical testing parameter when verifying a new ELISA kit lot is the %CV. To generate this value, we run a titration curve on three randomly selected strips of the new lot to determine the mean (µ) and standard deviation (SD, σ) for each replicate, before applying the formula:

%CV = (σ / µ) x 100

The inter-assay variance between the current lot and the new lot must be less than 15% for the new lot to be released.

Percent Coefficient of Variation (%CV) Data Summary for PathScan® Phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) Sandwich ELISA Kit #12001

Concentration (mg/ml)  #12001 Lot 34 #12001 Lot 35 Standard Deviation  %CV
0.075 2.734 3.115 0.040 1.28
0.0375 1.781 2.201 0.030 1.34
0.01875 1.166 1.425 0.028 1.93
0.009375 0.710 0.894 0.015 1.66

Figure 3. Representative passing %CV data from lot validation of PathScan® Phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) Sandwich ELISA Kit #12001.

To ensure that our ELISA kits demonstrate consistent performance over time, with no signal degradation, we routinely perform a follow-up quality control (QC) test at half the shelf life.

Quality Results for Your Next ELISA Experiment

Ready-to-use ELISA kits can offer many benefits for your research, provided they are sourced from a reliable manufacturer. Your assay is only as good as the antibodies used to make it. At CST, we use our high-quality, rigorously validated antibodies as the basis of our FastScan, PathScan, and PathScan RP ELISA kits, with each new lot being subjected to comprehensive testing before being made available to our customers. By verifying that every new ELISA kit lot exhibits comparable performance to the previous lot, we aim to keep your project on track without any unplanned interruptions.

To determine which of our ELISA kits best meets your needs, visit Which CST® ELISA (Enzyme-Linked Immunosorbent Assay) Kit Is Best for Me?

Need to eliminate potential supply problems or reserve a single lot of any of our products? Consider CST your partner for bulk  ELISA kit orders and lot reservations. When you purchase in bulk, volume-based discounts may also be available. Submit a Custom Formulations Request to learn more.

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Emily Eastabrook
Emily Eastabrook
Emily Estabrook graduated with a degree in biology from Boston University and was particularly obsessed with immunology and infectious diseases. Now a Product Scientist at CST, she works to ensure customers are provided with reproducible, high-quality assay kits, and expert technical support. When she is not working she enjoys writing, traveling, and snuggling with her cat.

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