Questions around internal standards in ELISA methods are common for drug development and ELISA services. A proper standard curve is the first step in ELISA assays. A standard curve ensures that the operation protocol and kit quality are adequate for generating reliable results. Hence, a robust standard curve will accelerate drug discovery and development projects. Drug discovery, by definition, is a process to screening and identifying potential molecules that can be converted into drug products. Hence, the FDA has several suggestions for the drug development process. Internal standards fall under one such recommendation.
In elisa lab assays, unknown quantities are determined by comparing them with standards of known concentration. The assay standard must be tested at different concentration ranges to generate data for low to high signals. These concentration ranges help researchers produce standard curves for the analyte of interest. Researchers then interpolate the analyte value by seeing where the point lies on the standard curve. Internal standards are widely used to measure the analyte of interest. An internal standard is a structurally similar test compound or is sometimes a stable isotope-labeled compound that is added to samples and calibration standards at constant and known concentrations to quantify the analyte of interest.
Best practices for internal standards
Following is a comprehensive list of best practices ELISA method validation and service providers can follow for internal standards.
- Biological matrices contain potential interfering substances such as metabolites, xenobiotics, and concomitant medications. Therefore, sponsors must thoroughly assess internal standards to avoid interference with the target analyte. Besides, the zero calibrators and blanks should be interference-free at the retention times of the internal standard and the target analyte.
- The blank internal standard response must not exceed 5% of the average responses of internal standard calibrators and quality control samples.
- If an internal standard is suitable for the intended use, sponsors do not have to provide evidence of purity or CoA.
Besides, researchers receive sera in smaller quantities. Therefore, they develop controls by pooling samples. Following are recommendations for controls.
- Sponsors must collect positive and negative samples separately. Once sufficient quantities are received, pool similar study samples and mix them thoroughly.
- Perform serial dilution of positive sera with negative serum in small quantities.
- Each dilution must be assayed following standard test protocol. Identify the most comparable dilution to sample-to-negative or sample-to-positive values that are ideal for monitoring. Generate larger quantities of that dilution.
- Researchers must then filter the prepared controls through a 0.45-micron membrane. Besides, they can further filter the control through a 0.20-micron filter membrane.
- Aliquot the internal control in vials. Label and store them, possibly at -20°C to -80°C.
- Researchers can use this internal control by thawing them and mixing them according to other routine samples. Run these internal controls next to the test controls. However, do not refreeze the in-house controls.
Internal standards closely resemble the sample matrix. Hence, they are crucial in quantifying analytes in study samples. Besides, FDA’s guidelines for ELISA validation recommend using internal standards while quantifying target analytes in complex biological samples.
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