ELISA Optimization and Validation

Each component of the assay including primary and secondary antibodies, blocking agent, buffers enzyme conjugate and substrate can be optimized for improved performance of a newly developed ELISA working protocol. Different concentrations and time of coating, blocking, sample incubation and detection steps are tested to find the optimum conditions for the assay. Usually, one component is tested at a time, nevertheless in some cases two components can be optimized concurrently by applying a checkerboard titration grid method as shown below:


Figure 2: Checkerboard titration grid method to optimize two ELISA components simultaneously. This example shows component A vs. component B with all other components remaining constant.

Optimizing the Coating process

Different concentrations of coating agent (target antigen or capture antibody) can be prepared in coating buffer and equal volume of each concentration can be applied to execute the ELISA protocol to find the trend for strong signal vs. low background. In some cases, selection of coating buffer and plate coating time may also need to be optimized depending on the nature of the coating agent.

Optimizing the Blocking process

Equal volume of different prepared or commercially available blocking solutions can be tested with the ELISA protocol to ensure least nonspecific binding without compromising the assay signal intensity. If blocking solution is prepared in-house, different concentration of the blocking agent can be adjusted.

Optimizing Sample concentration

Standard diluents for sample preparation to generate the calibration curve should imitate the native sample matrix as closely as possible and test different compositions. Same volume of serially diluted sample concentrations is tested with the ELISA protocol. A good dynamic range of concentrations for the standard curve and dilution linearity can be determined for the sample. Sample diluent may need to be adjusted depending on the spectrum of the dynamic range.

Optimizing the Detection process

Different concentrations of the detection antibody prepared in standard diluent can be tested in equal volumes with the ELISA protocol to find the concentration best suited for the strong signal and low background noise.

Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the two most common reporter enzymes used in ELISA. Different concentrations of the enzyme conjugate or enzyme conjugated secondary antibody prepared in standard diluent are optimized by testing equal volume of each concentration to the plate and proceeding with the ELISA protocol. The concentration resulting the strong signal with the least background noise can be determined. The substrate needs to be sensitive for the target antigen to be detected clearly over the dynamic range and be able to be detected with appropriate instruments.

The following table refers to the recommended concentration ranges for the coating and detection antibodies and enzyme conjugate that can be useful for determining the most suitable experimental conditions for colorimetric ELISA optimization. For the enzyme conjugates, review the manufacturer’s instructions for more specific concentration range. Unpurified antibodies can be used but may require higher concentration than purified antibody and can cause higher background noise. It is recommended to use affinity-purified antibodies for optimal signal to noise ration. The suggested concentration ranges are for guidance only; individual optimization of each component would provide better results.

Table 2: Recommended concentration ranges for the coating and detection antibodies and enzyme conjugate


Coating Antibody

Detection Antibody

Enzyme conjugate


5–15 µg/mL

1–10 µg/mL


Crude ascites

5–15 µg/mL

1–10 µg/mL


Affinity-purified polyclonal

1–12 µg/mL

0.5–5 µg/mL


Affinity-purified monoclonal

1–12 µg/mL

0.5–5 µg/mL





0.02–0.2 µg/mL




0.1–0.2 µg/mL


ELISA validation

Validation of the developed ELISA ensures that the protocol performs as expected and consistently produces the expected result. The validation process can include testing the detection range, precision, sensitivity and specificity, recovery, dilution linearity of the assay.

Quantification range

Quantification range of the assay can be determined by identifying the upper limit and lower limit of quantifications where the highest and lowest standard curve points can reliably and reproducibly detect and quantify the target analyte with accuracy and precision.


Accuracy of the developed protocol can be tested by analyzing a sample with known concentration and compare the result with a reference “gold-standard” or alternative method which has been proven to work before or pre-validated.


Repeatability and reproducibility of the same result for the same target are the main elements of precision of an immunoassay. Coefficient of variation (%CV) within the same assay (intra-assay) and in between assays (inter-assay) needs to be determined for the entire ELISA protocol, where the acceptable inter-assay %CV should be less than 15% and intra-assay %CV should be less than 10%.


Recovery test can be carried out by spiking samples with known amount of standard analyte and analyze the observed results by comparing with the expected values based on non-spiked and undiluted samples. In ideal scenarios, having no diluent or matrix interference will show results as close to 100% recovery. However, the interference from the diluent or matrix inhibiting the capture and binding of the target of interest can cause recovery significantly lower than 100% and require diluent optimization.

 Sensitivity and Specificity

The sensitivity of the developed assay protocol can be identified by testing various dilutions of known sample in a relevant matrix and analyze the ability of the assay to accurately detect the presence of the target of interest in a sample. Specificity of the assay will exhibit the ability of the test to give negative result when the target of interest is not present in a sample. Specificity also refers to the cross-reactivity of the assay that will accurately distinguish the target analyte from other similar substances in a sample.

Dilution linearity

Dilution linearity validation of the developed ELISA protocol can be performed by testing a sample spiked with a concentration more than the upper limit of quantification and diluting to a concentration within the quantification range. A good dilution linearity of the assay will still give accurate and reliable result with the dilution of high concentration samples.