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 (24) + 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

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

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

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

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

B/B0 Ratio at Curve Endpoint Outside 5-95% Range

The B/B0 ratio (bound/maximum bound signal) for the highest or lowest standard concentration exceeds 95% or falls below 5%, indicating the standard curve dynamic range does not adequately cover the analyte concentration range.

💡 4 · ✓ 4

Poor Assay-to-Assay Reproducibility

Inconsistent results between different assay runs with same samples

💡 7 · ✓ 9

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

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

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

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

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