ELISA optimization tips

Optimization tips for your ELISA assay

Below you will find optimization tips for every step of your ELISA assay. The tips below will help you maintain consistency and standardization, essential for assay reproducibility and accuracy.

In addition to optimizing your ELISA as described below it is also essential to ensure that all pipettes used throughout the ELISA are regularly calibrated to prevent variation and a multi-channel pipette used whenever possible.

1. Capture antibody concentration optimization

Prepare different concentrations of capture antibody in coating buffer and proceed to detection. Choose a concentration which gives a strong signal with low background.

2. Blocking buffer optimization

Prepare different blocking buffer solutions. Apply an equal volume of the different blocking buffer solutions to each well. Choose a blocking buffer which gives a strong signal and low background noise. Pre-formulated blocking buffers may be provided with your ELISA kit.

3. Standard Diluent optimization

Its is important to match the standard diluent used to the matrix of the sample. An optimum standard diluent will have a good dynamic range for the standard curve and linearity of dilution for the sample. If the standard curve generated has a poor dynamic range it may be necessary to choose a different diluent. If the sample has poor linearity when diluted in the diluent there may be an imbalance between the sample matrix and standard diluent, in such instances spike in recovery should be performed.

4. Sample concentration optimization

Prepare different sample concentrations using standard diluent. Test a wide range of sample concentrations. Note the detection limit of the substrate being used. Look for a strong signal with low background. Confirm the biological sample matrix is not masking or enhancing the signal by performing spike recovery and linearity experiments.

5. Detection antibody Concentration optimization

Prepare different detection antibody concentrations. Choose the concentration with a strong signal with low background noise.

6. Enzyme conjugate concentration

Prepare different enzyme conjugates concentrations in standard diluent. Ensure that the concentration is within the range described for the substrate. Choose the concentration with a strong signal with low background noise

7. Signal detection optimization

If the antigen can be detected over a dynamic range the substrate used is appropriate for the assay performed.

8. Matrix effects

Matrix effects occur in plasma and serum samples. A series of components can cause interference, these can include cross-reactive and non-specific interactions with substances in the matrix or breakdown products which develop during the handling processes. Matrix effects can be reduced by dilution of the sample.

9. ELISA Wash steps

During your ELISA plate washing steps, be sure to cover wells with appropriate volume of your ELISA wash buffer to remove unwanted complexes.

Recommended starting concentrations for coating and detection antibodies

*(Recommendation only further optimisation may be required)

Source Coating Antibody 5-15 µg/ml

Polyclonal Serum

5-15 µg/ml

1-10 µg/ml

Affinity purified monoclonal

1-12 µg/ml

0.5-5 µg/ml

Affinity purified polyclonal

1-12 µg/ml

0.5-5 µg/ml

Recommended conjugated Secondary antibody concentrations

*(Recommendation only further optimisation may be required)

Enzyme System Concentration



25-50 ng/ml


10-100 ng/ml


20-200 ng/ml

Alkaline Phosphatase


40-200 ng/ml


100-200 ng/ml