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 (30) + 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

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

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

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

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

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

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

Excessive Signal - Entire Plate Turned Blue

Whole plate turned uniformly blue indicating non-specific signal saturation

💡 5 · ✓ 5

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

Low OD Value for Zero Standard in Competitive ELISA

The zero standard (B0, representing maximum antibody binding without competing analyte) produces lower than expected optical density values, reducing overall assay sensitivity and dynamic range.

💡 4 · ✓ 4

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

Poor Assay-to-Assay Reproducibility

Inconsistent results between different assay runs with same samples

💡 7 · ✓ 9

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

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

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

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

Poor Standard Curve Discrimination

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

💡 6 · ✓ 8

Edge Effects on Plate

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

💡 1 · ✓ 2

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