Home › Immunology › How to Run an ELISA Assay – Invitrogen Kit Step-by-Step Tutorial
Steps
  1. 1 Unpack kit and prepare plate 00:02
  2. 2 Prepare wash buffer and standards 00:48
  3. 3 Perform serial dilution of standard 01:24
  4. 4 Add standards, samples, and detection antibody 01:47
  5. 5 Wash plate and add streptavidin-HRP 02:33
  6. 6 Wash plate and add chromogenic substrate 03:36
  7. 7 Stop reaction and read absorbance 03:54
Immunology Thermo Fisher Scientific

How to Run an ELISA Assay – Invitrogen Kit Step-by-Step Tutorial

Protocol
Difficulty
intermediate

Steps

1
Unpack kit and prepare plate

Remove the pre-coated ELISA plate from the foil pouch and separate any unused 8-well strips. Unused strips can be resealed and stored at 2-8°C for future use.

▶ 00:02
2
Prepare wash buffer and standards

Allow wash buffer concentrate to reach room temperature and mix to dissolve any precipitated salts, then dilute with deionized water. Reconstitute protein standard vial by gentle swirling or brief vortexing and allow to sit for at least 10 minutes.

▶ 00:48
3
Perform serial dilution of standard

Create a 1:2 serial dilution by adding assay diluent to each tube, then transferring reconstituted protein standard through the tubes. Mix thoroughly by pipetting up and down, changing tips between tubes.

▶ 01:24
4
Add standards, samples, and detection antibody

Prepare SA buffer and dilute biotin conjugate concentrate. Add diluted biotin-conjugated detector antibody to the plate with standards, controls, and samples, then seal and incubate the plate.

▶ 01:47
5
Wash plate and add streptavidin-HRP

Wash the plate manually or with automated washer by filling with buffer, decanting, and repeating according to protocol. Dilute streptavidin-HRP concentrate in assay buffer, add to plate, and incubate.

▶ 02:33
6
Wash plate and add chromogenic substrate

Wash the plate again to remove excess enzyme. Add chromogenic substrate and incubate at room temperature in the dark for 30 minutes to develop color.

▶ 03:36
7
Stop reaction and read absorbance

Add stop solution to terminate the enzymatic reaction, which turns the wells yellow. Measure absorbance at 450 nanometers using a plate reader.

▶ 03:54

🚨 Failure Case Library (45) + Submit your own case

critical
Absent Standard Curve Signal with Normal Zero Standard
No detectable signal is observed for standard curve points containing analyte, while the zero standard (maximum binding control) shows normal OD values, indicating complete loss of competitive inhibition.
💡 4 · ✓ 4
critical
No Signal or Weak Signal in ELISA
After developing the ELISA plate, wells show no color change or very faint signal that is barely above background, even for positive controls or high-concentration standards.
💡 6 · ✓ 6
critical
No Signal or Weak Signal in ELISA
The ELISA plate reader detects no colorimetric signal or signal intensity is significantly below expected values after substrate development. Wells appear clear or only faintly colored even after standard incubation times of 10-30 minutes.
💡 6 · ✓ 6
critical
No Signal for Standard Curve with Normal Zero Standard
All standard concentrations show no signal (high OD in competitive format similar to zero standard), while the zero standard (no competitor) shows normal maximum binding signal.
💡 4 · ✓ 4
critical
Complete Signal Absence
No detectable signal across the entire plate when signal is expected
💡 7 · ✓ 9
severe
B/B0 Ratio Outside Acceptable Range at Curve Endpoints
The B/B0 ratio (bound/maximum bound) for the terminal point of the standard curve is either >95% or <5%, indicating insufficient competitive displacement range.
💡 3 · ✓ 3
severe
Standard Curve Plateau at High Concentration Range
The standard curve reaches a plateau (flat response) at high standard concentrations with correspondingly low OD values, failing to show expected dose-response relationship in competitive ELISA format.
💡 4 · ✓ 4
severe
Non-specific Background Signal Too High in Competitive ELISA
High background optical density is observed across wells, reducing signal-to-noise ratio and making it difficult to distinguish specific binding from non-specific interactions.
💡 4 · ✓ 4
severe
Standard Curve Plateau at High Concentration End
The standard curve reaches a plateau at high analyte concentrations (low OD values in competitive format), showing no further decrease in signal despite increasing standard concentration.
💡 4 · ✓ 4
severe
OD Value of Zero Standard Too Low
The zero standard (maximum binding, no competitor) shows insufficient optical density signal, providing inadequate dynamic range for the competitive assay and poor B/B0 calculations.
💡 4 · ✓ 4
severe
No or Very Low Signal Due to Poor Plate Binding
No detectable signal or extremely weak signal in ELISA even when target protein is expected to be present. Signal may be inconsistent across wells.
💡 4 · ✓ 4
severe
Detection System Lacks Sensitivity for Target Concentration
Low or no signal despite proper assay setup. High concentration samples may show weak signal while low concentration samples are undetectable.
💡 4 · ✓ 4
severe
Buffer Components Inhibit Detection Enzyme
No or very weak signal despite correct antibody binding. Signal loss occurs after addition of detection reagent. Other wells using different buffers show normal signal.
💡 4 · ✓ 4
severe
Non-specific Secondary Antibody Binding Causing High Background
Elevated background signal across wells, including negative controls. Signal appears uniformly high rather than specific to target-containing wells.
💡 4 · ✓ 5
severe
Standard Curve R² Below 0.98 Threshold
The regression coefficient (R²) of the standard curve falls below 0.98, indicating poor curve fit and unreliable quantification of sample concentrations.
💡 5 · ✓ 6
severe
Excessive Signal - Entire Plate Turned Blue
Whole plate turned uniformly blue indicating non-specific signal saturation
💡 5 · ✓ 5
severe
Poor Dynamic Range Between Signal and Background
The difference between maximum signal (high standards) and background (blank wells) is compressed, typically less than 5-fold, making it difficult to distinguish between samples of different concentrations.
💡 5 · ✓ 5
severe
High Non-Specific Background Signal in Competitive ELISA
Elevated background signal is detected across wells, interfering with specific signal measurement. Background noise reduces assay sensitivity and may mask low-concentration analyte detection.
💡 4 · ✓ 4
severe
Inadequate Washing Between Assay Steps
High background with retained signal in negative controls. Edge wells may show higher background than center wells. Residual reagents visible in wells.
💡 4 · ✓ 5
severe
High Inter-Assay CV from Inconsistent Sample Storage
High inter-assay coefficient of variation (>15%) when the same samples are tested across different runs. Sample readings decrease over time or vary unpredictably between runs.
💡 5 · ✓ 5
severe
Incomplete Standard Reconstitution with Visible Particulates
After adding reconstitution buffer to lyophilized standard, visible undissolved material or particulates remain in the vial, leading to inaccurate standard concentrations.
💡 4 · ✓ 6
severe
High CV from Inconsistent Well Washing
High coefficient of variation (>15%) between replicates with uneven background signal across the plate. Some wells show higher background than others after washing steps.
💡 5 · ✓ 5
severe
High Variability Between Replicates (CV >15%)
Replicate wells for the same sample or standard show high coefficient of variation (>15%), with inconsistent absorbance values that suggest uneven treatment or technical errors.
💡 5 · ✓ 5
severe
Poor Assay-to-Assay Reproducibility
Inconsistent results between different assay runs with same samples
💡 7 · ✓ 9
severe
Uneven Color Development Across Plate
Color intensity varies significantly between wells that should be identical. Pattern may show edge effects, gradients across the plate, or random spotty appearance indicating incomplete or inconsistent reagent contact.
💡 4 · ✓ 4
moderate
High Coefficient of Variation at Low Standard Concentrations
Replicate wells at the bottom of the standard curve (low analyte, high OD) show high CVs >15-20%, while mid-curve and high-concentration points demonstrate acceptable reproducibility.
💡 4 · ✓ 4
moderate
Mixed Components from Different ELISA Kits
Unpredictable or absent signal despite following protocol. Results are inconsistent and do not match expected standard curves. Signal may vary dramatically between experiments.
💡 4 · ✓ 4
moderate
Incubation Temperature Below Optimal Range
Consistently weak signal across all samples including positive controls. Signal improves when experiment is repeated with attention to temperature. Longer incubations partially compensate.
💡 4 · ✓ 4
moderate
Over-Washing Removes Bound Detection Reagents
Signal progressively decreases with increased wash steps or aggressive washing. Reducing wash stringency restores signal. Edge wells show lower signal than center wells.
💡 4 · ✓ 5
moderate
Excessive Primary Antibody Concentration Saturating Wells
High background signal with poor signal-to-noise ratio. Standard curve may show compressed dynamic range or plateau at lower concentrations than expected.
💡 3 · ✓ 3
moderate
Precipitate Formation in Wells Upon Substrate Addition
Visible precipitate or cloudiness appears in wells immediately or shortly after substrate addition. Absorbance readings may be abnormally high or variable.
💡 4 · ✓ 4
moderate
High CV from Bubbles in Wells
Inconsistent absorbance readings between replicate wells with coefficient of variation >15%. Visual inspection may reveal bubbles present in wells, particularly when using detergent-containing solutions.
💡 4 · ✓ 4
moderate
High Background Signal
Elevated background noise across the ELISA plate making it difficult to distinguish specific signals from non-specific binding
💡 3 · ✓ 3
moderate
Unexpected or Inconsistent Assay Results
Sample quantification values are outside expected biological range, controls fail to meet acceptance criteria, or results are not reproducible between runs despite similar experimental conditions.
💡 5 · ✓ 5
moderate
High Variability Between Experimental Runs
Standard curves or sample values differ significantly between experiments run on different days, despite using the same reagents and protocol, showing poor reproducibility.
💡 5 · ✓ 5
moderate
Poor Duplicate Reproducibility
Duplicate wells showing inconsistent results with high variation between replicates
💡 6 · ✓ 8
moderate
Very Low Readings Across Plate
Consistently low optical density readings across entire plate including standards
💡 5 · ✓ 6
moderate
Signal Drift Across Plate
Gradual change in signal intensity across the plate suggesting time-dependent variation during assay setup
💡 2 · ✓ 2
moderate
High Coefficient of Variation at Lower Standard Curve
Replicate measurements at the bottom of the standard curve (highest OD values, lowest analyte concentrations) show excessive variability with high coefficient of variation (CV) between replicates.
💡 4 · ✓ 4
moderate
Weak Signal from Inadequate Antibody-Antigen Interaction
Signal is present but consistently lower than expected. Positive controls show weak but detectable signal while samples are near background.
💡 4 · ✓ 4
moderate
B/B0 Ratio at Standard Curve Endpoint Out of Range
The B/B0 ratio (bound/maximum bound) at the lowest or highest standard concentration is either >95% or <5%, indicating the standard curve dynamic range is not properly positioned.
💡 3 · ✓ 3
moderate
High Coefficient of Variation Between Standard Replicates
Duplicate or triplicate wells for the same standard concentration show high variability (CV >10-15%), compromising curve fit and confidence in quantification.
💡 5 · ✓ 6
moderate
Poor Standard Curve Discrimination
Standard curve achieved but shows low or flat curve with poor discrimination between concentration points
💡 6 · ✓ 8
minor
Standard Curve OD Values Differ From Datasheet
The optical density (OD) measurement values for the standard curve vary considerably from the examples shown on the kit datasheet or protocol booklet, causing user concern about assay validity.
💡 4 · ✓ 4
minor
Edge Effects on Plate
Wells at the edge of the plate show different readings compared to central wells
💡 1 · ✓ 2
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