ELISA Controls Guide
Understanding ELISA Controls
A Guide to Optimal Accuracy and Reproducibility
ELISA (Enzyme-Linked Immunosorbent Assay) is a powerful tool used in diagnostics, research, and drug development. However, the accuracy and reliability of your ELISA results depend heavily on how well you design your plate layout and incorporate proper controls.
In this guide, you'll learn how to set up essential ELISA controls, including standards, blanks, spiked matrices, and total activity wells. We'll walk you through how to organize your samples in duplicates, minimize variability, and interpret the data with confidence.
Whether you're new to ELISA or looking to fine-tune your current workflow, this page will help you achieve high assay precision, reduce background noise, and ensure your results are statistically valid.
Overview
Example ELISA Plate Layout
All ELISA Controls
Use mandatory controls every time to ensure your assay is functional and data is valid. Add project-dependent controls when working with difficult samples, seeking publication-quality data, or validating a new assay.
- Standards: Required to generate the standard curve for quantifying unknowns.
- Zero Standard (S0) / NSB (Non-Specific Binding) Controls: Contains all reagents except the analyte and helps assess background signal from reagents of matrix. Also referred as Sample Blank.
- Positive Control: Confirms the assay is functioning correctly by producing a known positive signal. Although ideal for all ELISA experiments, this approach is primarily applied during assay validation or when troubleshooting specific issues.
These controls enhance data quality or troubleshoot complex variables but may not be necessary in all settings. These could include Wavelength Control, Negative Matrix Control, Total Activity Control and more.
The use of additional controls is determined by the researcher and should be guided by the specific needs of the project and the characteristics of the samples being tested.
For further information about all the possible controls, please see Table 1.
| Control | Blank | S0 (Zero Standard) | Non-specific binding (NSB) | Secondary Antibody | Wavelength Control | Negative Matrix Control | Standards | B0 Control | Total Activity Control | Positive matrix Control | Spiked Matrix Control | Endo-genous protein control |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Type | Negative | Negative | Negative | Negative | Negative | Negative | Positive | Positive | Positive | Positive | Positive | Positive |
| ELISA Format | All | Sandwich, Direct, Indirect | Competi-tive with enzyme-conjugated analyte | Indirect & Sandwich | All | All | All, specially complex sample matrix | Competi-tive | All | All, specially complex sample matrix | All | Only for DIY ELISA |
| Definition | Empty well or with dilution buffer | Everytime when performed an ELISA | Everytime when performed competitive ELISA | Indirect or Sandwich ELISA in absence of detection Ab | Absorbance measured at different wavelength than substrate | Run with sample matrix free from analyte | Known concentra-tions of purified antigen | Same as S0 but applied in competitive ELISA | Small amount of enzyme-conjugated protein and substrate incubation | Same matrix as sample with known endoge-neous analyte | Sample matrix spiked with a known quantity of purified antigen | Known quantity of endoge-nous protein |
| Purpose | Substract absorbance from plastic plate and buffer | Identifies back-ground due to non-specific binding | Identifies back-ground due to non-specific | Identifies back-ground caused by NSB of labeled secondary Ab | Identifies background caused by plastic plate artefacts | Determines contribution of analyte to final absorbance | Test functionality and allows quantitative detection of analyte | Tests for functionality in competitive ELISA, achieves max colour | Tests for enzymatic activity | Helps to identify matrix interference with known analyte when comparing to known standards | Helps to identify matrix interference comparing to diluted standards | To confirm standards behave like endoge-nous protein |
| Notes | Often skipped. S0, NSB or wavelength controls as alternatives | Often used to correct measurements values, or as lowest point on standard curve | Sometimes also used as general term to refer to S0 controls | Sometimes grouped in as an NSB control | Usually performed by the micropate reader | Difficult to obtain real sample free from analyte | Always run in all quantitative ELISA | Used as a reference against which to compare test values | Used for qualitative control only | Spiked control often preferred | % Recovery should be 100%, deviation > 20% suggests matrix interference | Not needed in most ELISA experi-ments |
Common ELISA Issues Caused by Poor Control Strategy
If standards are improperly prepared, contaminated, degraded, or pipetted inconsistently, the resulting standard curve will be unreliable. This leads to inaccurate quantification of analytes in samples.
Failure to include a Positive Control (such as a sample with known high signal) can make it difficult to distinguish between true negatives and a failed assay run (e.g., due to reagent degradation).
Inconsistent placement or omission of spiked matrix or quality control (QC) samples leads to variability between runs and across plates, making it difficult to compare data or validate results.
Omitting a spiked matrix control (where the standard is added to the sample matrix) can mask matrix effects, leading to over- or underestimation of the analyte due to interference from serum, plasma, or tissue lysates.
