Today there is a growing need to develop immunoassays for assessing therapeutic responses of a drug product. The increased demand for testing needs sustainable options that reduce costs and optimize samples for longer studies. Multiplex immunoassays are the obvious result of such increased pressure on bioanalytical scientists.
Mesoscale Discovery (MSD) and Luminex immunoassays are the leading players in multiplex systems. The costs saved just on the reagents can make a huge difference financially. And when considering the reduced assay times, multiplex immunoassays have massive advantages over conventional single-analyte immunoassays. However, an increase in assay complexities gives rise to several technical challenges. This current article, presents you with three crucial factors while developing high-quality multiplex immunoassays. Following are the three key factors researchers must consider while designing multiplex assays such as MSD and Luminex ELISA assays.
Identifying antibody pairs
The primary choice of an assay method for multiplex immunoassays is often sandwich ELISA.. This type of assay method involves two antibodies, one captures the analyte of interest, and the second antibody measures the captured analyte. Identifying the exact antibody pairs is a challenging and time-consuming task. It also requires extensive knowledge and understanding of antibodies to produce reliable and efficient results. However, assay validation often ensures reliable results to a great extent. A thorough assay validation also helps clear any early red flags associated with poor assay quality.
An ideal assay buffer
Identifying an ideal assay buffer with adequate performance for all analytes of interest is a significant task. Designing studies with buffers for each analyte is not at all possible. Although this may be true, managing matrix effects and optimizing assay buffers for recovery and linearity are crucial for the success of multiplex immunoassays. Hence, scientists use an assay diluent to work with a wide range of analytes for better results. Assay diluents increase the opportunity of multiplexing up to several dozen analytes in a single multiplex assay.
Validation of immunoassays ensures that the assay is robust enough to detect analytes in biological samples. Immunoassay validation comprises characterizing several key parameters around assay performance. Antibody pairs are validated through the Western blotting technique. This technique confirms that the antibodies accurately detect the protein with the correct molecular weight. Validating sample linearity and testing recovery ensures that the generated results are precise and accurate. Moreover, inter and intra assay precision help evaluate assay reliability of generating reproducible results between studies and users and within the same assay.
In addition to these three factors, matrix selection, proper controls, calibration curve, and variability among immunoassays are additional parameters researchers should focus on while designing a quality immunoassay.
Although designing multiplex immunoassays have technical challenges, the ability to analyze multiple analytes and reduce costs and time has been a massive advantage in bioanalysis. From early drug discovery research studies to therapeutic monitoring in clinical trials, multiplex immunoassays are critical in the success of drug development projects. However, regardless of where multiplex assays are conducted, researchers should focus on the technical parameters for developing high-quality MSD and Luminex assays.