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

Steps

1 Unpack kit and prepare plate 00:02
2 Prepare wash buffer and standards 00:48
3 Perform serial dilution of standard 01:24
4 Add standards, samples, and detection antibody 01:47
5 Wash plate and add streptavidin-HRP 02:33
6 Wash plate and add chromogenic substrate 03:36
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 (51) + Submit your own case

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

No Signal or Weak Signal in ELISA

No detectable signal or very weak signal across all wells, including standard wells. The plate reader shows absorbance values near baseline or significantly lower than expected.

💡 6 · ✓ 6

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

Poor or Non-Linear Standard Curve

Standard curve shows irregular shape, poor linearity, or inconsistent serial dilution response. R-squared value below 0.95 or curve does not follow expected sigmoidal or linear pattern across dynamic range.

💡 5 · ✓ 5

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

Complete Absence of Standard Curve Signal with Normal Zero

All standard concentrations show no detectable signal (very low OD), while the zero standard (B0, maximum binding) produces normal expected signal, indicating complete competition failure.

💡 4 · ✓ 4

Complete Signal Absence

No detectable signal across the entire plate when signal is expected

💡 7 · ✓ 9

Excessive Non-Specific Background Signal in Competitive ELISA

High background optical density readings are observed across wells, obscuring the difference between standards and samples. Signal persists even in wells without primary antibody or sample.

💡 4 · ✓ 4

Insufficient Signal from Zero Standard (B0)

The zero standard (no competing antigen, maximum antibody binding) produces OD values that are too low, resulting in compressed standard curve with poor sensitivity.

💡 4 · ✓ 4

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

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

Primary and Secondary Antibody Incompatibility

No signal or minimal signal in indirect ELISA despite proper coating and blocking. Positive controls with matched antibodies work correctly.

💡 4 · ✓ 4

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

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

High CV from Inconsistent Pipetting Technique

High intra-assay coefficient of variation (>15%) between technical replicates. Standard curve shows poor reproducibility between duplicate points.

💡 5 · ✓ 5

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

Excessive Signal - Entire Plate Turned Blue

Whole plate turned uniformly blue indicating non-specific signal saturation

💡 5 · ✓ 5

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

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

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

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

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

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

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

Poor Assay-to-Assay Reproducibility

Inconsistent results between different assay runs with same samples

💡 7 · ✓ 9

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

High Background Signal in ELISA

All wells including negative controls show elevated absorbance readings. Background signal obscures specific signal, reducing signal-to-noise ratio and making data interpretation difficult.

💡 6 · ✓ 6

Standard Curve Plateau at High Concentration End

The top of the standard curve (high analyte concentration, low OD) shows a plateau with minimal signal change across multiple high-concentration standards, reducing dynamic range.

💡 4 · ✓ 4

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

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

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

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

Excessive Signal Amplification in Detection System

Very high signals across all wells including low-concentration standards. Loss of discrimination between different antigen concentrations. Background approaches signal levels.

💡 4 · ✓ 4

High Background Signal

Elevated background noise across the ELISA plate making it difficult to distinguish specific signals from non-specific binding

💡 3 · ✓ 3

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

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

Edge and Drift Effects on ELISA Plate

Wells at the plate edges show systematically higher or lower absorbance than center wells, or a gradient pattern appears across the plate, affecting data quality especially for samples in edge positions.

💡 5 · ✓ 5

Poor Duplicate Reproducibility

Duplicate wells showing inconsistent results with high variation between replicates

💡 6 · ✓ 8

Very Low Readings Across Plate

Consistently low optical density readings across entire plate including standards

💡 5 · ✓ 6

Signal Drift Across Plate

Gradual change in signal intensity across the plate suggesting time-dependent variation during assay setup

💡 2 · ✓ 2

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

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

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

Bacterial Contamination in Wash or Incubation Buffers

Sporadic signal loss or high background that worsens over time during multi-day experiments. Cloudy or turbid buffer appearance. Inconsistent results when using same buffer batch.

💡 4 · ✓ 5

High CVs at Bottom of Standard Curve

Coefficient of variation (CV) among replicates is excessively high at the lowest standard concentrations (highest OD in competitive format), reducing precision at the sensitive end of the curve.

💡 4 · ✓ 4

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

Poor Standard Curve Discrimination

Standard curve achieved but shows low or flat curve with poor discrimination between concentration points

💡 6 · ✓ 8

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

Edge Effects on Plate

Wells at the edge of the plate show different readings compared to central wells

💡 1 · ✓ 2

Plate Contamination or Optical Interference

Random high background in specific wells or patterns. Fingerprints, dust, or residue visible on plate bottom. Erratic readings not following expected pattern.

💡 4 · ✓ 5

Green Color Upon Adding Stop Solution (Streptavidin-HRP)

When sulfuric acid stop solution is added to wells after TMB substrate incubation, a green color appears instead of the expected yellow, indicating incomplete mixing and pH gradient in the well.

💡 2 · ✓ 3

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