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10 Key Metrics for Validation of ELISA Results: The Parameters You Need to Know

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10 Key Metrics for Validation of ELISA Results: The Parameters You Need to Know

(Photo : Bermix Studio on Unsplash)

ELISA, otherwise known as enzyme-linked immunosorbent assay, is a biochemical assay used to detect and measure the concentration of complex biological antigens in a sample, such as proteins, peptides, antibodies, or hormones. The ELISA assay has become a popular diagnostic tool in clinical laboratories, primarily because it is a relatively simple procedure and offers high sensitivity and specificity. However, before performing an ELISA assay, it is essential to validate the results by assessing the assay's performance under different conditions.

The significance of validation cannot be overemphasized, as a false positive or negative result can have significant implications for the patient or research subject. The essential validation categories include specificity, linearity, sensitivity, accuracy, precision, robustness, selectivity, parallelism, recovery, and sample stability.

Specificity

The specificity of an assay is the ability to identify a target antigen correctly and differentiate it from other non-target antigens. The specificity of an ELISA assay can be assessed by performing a series of test sera that contain increasing concentrations of the target antigen. The assay should be able to detect and accurately quantitate the target antigen at all concentrations.

Linearity

The linearity of an assay is the ability to measure antigen concentrations in a linear fashion. The linearity of an ELISA assay can be assessed by performing a series of test sera that contain increasing concentrations of the target antigen. The assay should produce a linear curve that accurately reflects the concentration of the target antigen.

Sensitivity

The lower detection limit of a test is the smallest amount of measurable analyte. It is important to know the sensitivity of your assay to ensure you are detecting the analyte of interest. One must consider the background noise of the assay when determining the lower limit of detection to avoid falsely detecting the analyte of interest.

Accuracy

The accuracy of an assay is the percentage of error between the experimental value and the accepted reference value for samples with known amounts of the analyte.

Precision

The precision of an assay is the degree of variability among replicate measurements. How close the measurements are to one another indicates the accuracy of the assay.

Robustness

The ability of an assay to remain unchanged in the presence of minor variability in the test system and environmental conditions. Robustness is determined by evaluating the precision and accuracy of the assay in different laboratories using different lots of reagents.

Selectivity

The selectivity of an assay is the ability to differentiate between analytes and interferences in the presence of other compounds. This is determined by the specificity of the assay reagents and the lack of cross-reactivity between analytes and interferences.

Parallelism

The parallelism of an assay is the ability to produce equivalent results when different amounts of sample are used. The difference in antibody binding (between high and low concentrations) should be minimal.

Recovery

The recovery of an assay is the percentage of the analyte that is present in the sample. This is determined by comparing the concentration of the analyte in the original sample to the concentration of the analyte in the assay sample.

Sample Stability

The stability of a sample is determined by how well the analyte remains in the sample over time. This is usually determined by incubating a sample at different temperatures for a set period and measuring the analyte concentration at different time points.

These are the key metrics that one should assess when validating an ELISA assay. By following these guidelines, laboratories can ensure that their ELISA results are reliable and accurate, and can be used for diagnosis or research.

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