Validated bioanalytical methods such as ELISA assays are vital for the success of drug development studies. ELISA assays can quantify drugs and their metabolites in complex biological matrices, making them critical for nonclinical and clinical studies. The validated method ensures that the data generated is reliable and can support the safety and effectiveness data of drugs and biological products.
The US FDA has clear guidelines for ELISA assay validation. Whether assessing cytokines in study samples or acquiring PK data clinical trials, researchers must always conform to these regulatory guidelines. The primary ELISA assay validation parameters include specificity, sensitivity, precision, accuracy, and linearity. Let’s dive deep into each of these parameters and understand how pushing the limits of these parameters can unlock next-level performance.
Specificity means that a particular assay can differentiate between different components. Researchers can confirm specificity by increasing the concentrations of drugs or related molecules in sample matrices and measuring them within the working assay range.
Specificity is assessed during ELISA optimization and validation because, more data about drug behavior is available during optimization and validation experiments. Moreover, researchers should test specificity using quality control samples. ELISA assays tested with quality control samples delivering satisfactory results are considered valid. Experiments with poor specificity results mean the method is insufficient for assessing samples.
Detection limits and range define assay linearity. Linearity is assay values that can be represented graphically in a straight line. Linearity helps demonstrate direct proportionality among different concentrations of analytes present in the sample. However biological data is not always linear and hence requires mathematical transformation.
On the other hand, the assay range is determined from linearity and the obtained data. The assay range typically includes the upper and lower level of detection. The upper limit of quantification is the mean value of 10 points of maximum absorbance in a linear part. On the other hand, the lower limit of quantification is the lowest analyte concentration that can be quantified reliably with precision and accuracy.
Sensitivity can also be described as the limit of detection. However, it is vital to understand that the limit of detection is different from the lower limit of quantification. The limit of detection is the lowest analyte concentration, distinguishable from the assay background. Especially for ELISA assays working on biological samples, the limit of detection values is lower than the lower limit of quantification values.
Accuracy is the ability of the ELISA assay to describe the closeness of a value compared to the nominal concentration. In practice, quality control samples and standards are critical for defining accuracy. However, a thoroughly developed quality control sample can be used for assay validation. Besides, researchers should assess the accuracy of spiked and quality control samples.
Precision describes the reproducibility of an ELISA assay. Precision is when assay results are closer to each other. However, it is common to confuse accuracy and precision. But both these values are different. Accuracy is achieving the actual value, while precision describes reaching the mean value repeatedly.
Robust ELISA assay development and validation will remain crucial for generating reliable, accurate and reproducible study results.