Home › Immunology › How to coat your own plate and run an Invitrogen ELISA kit
Steps
  1. 1 Prepare and coat plate with capture antibody 00:14
  2. 2 Prepare wash buffer and wash plate 00:42
  3. 3 Add blocking solution and wash plate 01:26
  4. 4 Prepare protein standard and serial dilutions 01:47
  5. 5 Add standards, controls, samples and detection antibody 02:33
  6. 6 Incubate plate, wash, and add streptavidin HRP 03:16
  7. 7 Wash plate, add substrate, and develop color 03:56
  8. 8 Stop reaction and read absorbance 04:12
Immunology Thermo Fisher Scientific

How to coat your own plate and run an Invitrogen ELISA kit

Protocol
Difficulty
intermediate

Steps

1
Prepare and coat plate with capture antibody

Dilute the concentrated coating buffer according to protocol, then use it to dilute the capture antibody to the recommended concentration. Add the antibody solution to the plate wells and incubate overnight at 2-8°C to allow antibody absorption.

▶ 00:14
2
Prepare wash buffer and wash plate

Allow wash buffer concentrate to reach room temperature and mix to dissolve any precipitated salts, then dilute in deionized water. Wash the plate by filling wells with wash buffer, soaking, and inverting to decant, repeating according to protocol and tapping dry.

▶ 00:42
3
Add blocking solution and wash plate

Dilute blocking solution with water and add to the plate wells, then incubate as recommended. After incubation, wash the plate using the same method as previously described.

▶ 01:26
4
Prepare protein standard and serial dilutions

Reconstitute protein standard vial by gently swirling and inverting five times or briefly vortexing, then let sit for at least ten minutes. Perform a 1:2 serial dilution by adding assay diluent to tubes, then adding reconstituted standard and mixing thoroughly with fresh pipette tips.

▶ 01:47
5
Add standards, controls, samples and detection antibody

Add standards, controls, and samples to the ELISA plate, pre-diluting samples if needed. Dilute biotin conjugate concentrate in assay buffer and add to the plate to allow the biotin-labeled detector antibody to bind antigen at a different site than the capture antibody.

▶ 02:33
6
Incubate plate, wash, and add streptavidin HRP

Cover the plate with an adhesive seal and incubate as recommended. After incubation, wash the plate to remove unbound detection antibody. Dilute streptavidin HRP concentrate in assay buffer and add to the plate for enzymatic amplification of the signal.

▶ 03:16
7
Wash plate, add substrate, and develop color

Wash the plate to remove excess enzyme. Add chromogenic substrate and incubate at room temperature in the dark for 30 minutes, during which the color intensity develops proportionally to the protein amount present.

▶ 03:56
8
Stop reaction and read absorbance

Add stop solution to halt the enzymatic reaction, turning the solution yellow. Measure the absorbance at 450 nanometers using an appropriate plate reader to obtain your ELISA results.

▶ 04:12

🚨 Failure Case Library (11) + Submit your own case

severe
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
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
High CV from Plate Edge Effects
Outer (edge) wells show systematically different absorbance values compared to inner wells, with CV >15%. Edge wells may show higher or lower signal depending on evaporation or temperature variation. Visual inspection from the side reveals lower buffer levels in edge wells.
💡 5 · ✓ 5
severe
Excessive Signal - Entire Plate Turned Blue
Whole plate turned uniformly blue indicating non-specific signal saturation
💡 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
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
severe
Poor Reproducibility Between Duplicate Wells
Coefficient of variation (CV) between duplicate or triplicate wells exceeds 10-15%. Individual replicates show inconsistent absorbance values that cannot be attributed to biological variation.
💡 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
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
Poor Standard Curve Discrimination
Standard curve achieved but shows low or flat curve with poor discrimination between concentration points
💡 6 · ✓ 8
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