ELISA Assay Validation: Minimum Required Dilution, Dilution Linearity, Spike Recovery, etc.

popular analytical methods

ELISA is one of the most popular analytical methods used to quantify analytes in complex biological matrices. However, accurate analyte measurements are of great concern for all ELISA services. The method employed should be able to accurately quantifyanalytes in any biological sample. Hence to ensure such accuracy, ELISA method validation becomes crucial in demonstrating the accuracy and effectiveness of ELISA assays.

Dilution linearity and spike recovery are two of the most common series of developmental and calibration parameters achieved for demonstrating adequate ELISA assay validation. These indispensable experiments validate the accuracy of ELISA assays in measuring the actual amount of analyte in biological samples. Moreover, these studies help identify any unwanted issues such as sample matrix and interference issues that may arise from false-negative or false-positive reports.

Dilution linearity assays

ELISA testing services use dilution linearity assays to validate ELISA accuracy and its sample compatibility. Dilution linearity assays involve diluting a sample serially using an analyte with a high endogenous concentration. If a high endogenous concentration is not available, an analyte with a known concentration can be added to dilute the sample. A standard curve is prepared, and the concentration of the antigen is measured against it.

Dilution linearity is demonstrated by diluting the analyte concentration to such an extent that each dilution is 100% of the concentration estimated at the previous dilution. Minimum required dilution is a dilution factor where the prior dilution begins to stay constant or linear and is in the range of expected sample recovery of 80-120%. 

Spike recovery assays

Spike recovery assays are another set of studies crucial for validating assay accuracy. Exogenous analyte with known concentration is added into the diluted sample and then tested against the blank with the same exogenous analyte in spike recovery assays. The unspiked sample should also be tested to measure endogenous analytes. All three; spiked sample, sample without the spike, and spiked blank should be tested against a standard curve to calculate each concentration.

The primary goal is to achieve 100% recovery, which means that both spiked samples and blank samples have identical responses. A recovery range between 80-120% indicates all matrix effects are negated and considered acceptable. A recovery rate outside the designated scale suggests sample interference is likely because of an incorrect selection of sample diluent.

What are the potential solutions for incompatibility?

Dilution linearity and spike recovery are mandatory if ELISA testing services want to demonstrate assay accuracy. Some potential solutions include using a higher sample dilution for avoiding interferences from sample matrices, provided the sample contains high concentrations of the endogenous analyte. Sample diluent can be adjusted to reflect sample matrix complexity. Or, in the case of interferences from heterophilic antibodies, ELISA service can include blockers to sample diluent.

Poor results of spike and recovery and linearity of dilution studies indicate sample matrix and sample diluent incompatibility. Generally, incompatibility is either due to heterophilic antibody interference, incorrect buffer choice, or a result of both. Hence, poor dilution linearity and spike recovery assay results stipulate further ELISA assay optimization.

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