Technologies such as LC-MS/MS analysis and Meso Scale Discovery (MSD) are accelerating clinical and drug development initiatives. MSD technologies offer a robust platform for developing immunoassays for a spectrum of clinical and preclinical research. MSD offers ultra-low detection capacities and a broader dynamic range and handles complex study matrices. However, similar to LC-MS/MS method development, MSD tests require adequate development and validation initiatives.
Although MSD protocols and solutions are streamlined to accelerate each step of the drug discovery process, researchers still face challenges while implementing them. Hence, the current article addresses the five common challenges faced with MSD protocols and solutions.
High Backgrounds
Nonspecific binding without sample and capture antibodies may generate higher background signals. One must test alternate blocking solutions and assay diluents to reduce nonspecific binding. Besides, nonspecific binding between detection and capture antibodies can be minimized by lowering antibody concentrations or adding different blocking agents. Moreover, researchers can test MSD’s complete blocker kit to reduce background signals. On the other hand, researchers can reduce the background signal by decreasing the ratio of detecting antibodies when conjugating the Sulfo-Tag labels.
Low Assay Signals
Inefficient conjugation of detection antibodies may generate low assay signals. Besides, there may be different reasons for low assay signals, such as the presence of interfering substances during the labeling reaction, for example, glycine, tris, azide, and histidine. One must ensure that the antibody is in a carrier-free and amine-free buffer. Also, increasing the conjugation ratio may generate elevated signals. Often researchers improve assay signals by using higher or alternate affinity antibodies.
Reagent Storage
Not storing reagents at appropriate conditions is one of the leading causes of assay variability. Researchers must prepare detection antibodies just before use. Besides, all solutions must be covered when not in use. One must thaw and mix diluents before use. The recommended thaw-freeze cycles should not be exceeded. Researchers must also ensure that the Blocker B reagent is kept dry. Also, the Blocker B reagent should completely dissolve before use.
Also Read: Advances in MSD Biomarker Discovery and Validation in 2023
Dissociation Rates
MSD assays generate electrochemical signals when molecules are near the bottom of the well. Before the final washing step, the assay reaction is close to equilibrium. However, prolonged incubation in the read buffer or wash buffer may lead to the dissociation of assay components. As MSD assays are proximity assays, dissociation may lead to a decrease in the assay signal. Hence, the assay must not be kept in the wash buffer for extended periods, and the interval between the read buffer and assay analysis must be consistent.
Excipients Affecting Assay Performance
Both the capture and detection antibodies can affect assay performance. Excipients such as glycerol, gelatin, and other proteins may influence the drying conditions. Therefore, capture antibodies must be free from these excipients. Moreover, large amounts of carrier proteins will compete with the capture antibodies. Hence, adequate care is needed to capture antibodies.
Similarly, detection antibodies must also be free from carrier proteins, glycerol, histidine, tris, and glycine. One may eliminate glycine, histidine, tris, or azide by buffer, exchanging the protein into PBS with pH 7.4 to 7.9.
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