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 (8) + Submit your own case

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
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
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
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
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
Poor Standard Curve Discrimination
Standard curve achieved but shows low or flat curve with poor discrimination between concentration points
💡 6 · ✓ 8
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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