Proper sample prep is critical to the success of any immunoassay. Not only must material be collected efficiently to prevent analyte degradation, but it may also be necessary to concentrate certain sample components or include steps to remove unwanted debris. Sample prep can especially be challenging for multiplex immunoassays, where researchers must ensure conditions are suitable for all analytes being measured. Here, we share some general sample-prep recommendations and suggest best practices for handling some commonly used sample types.
Since sample collection and storage represent the first step of any immunoassay protocol, it is important to get them right. Poor sample-prep technique can lead to analytes being broken down by endogenous enzymes, target proteins precipitating out of solution, or undesirable reactions taking place. Moreover, where sample prep is inconsistent, it can cause significant inter- and intra-assay variability that may require experiments to be repeated. Fortunately, by following best practices, many common pitfalls can be avoided.
Irrespective of the sample type or the immunoassay that will be used for analysis, several best practices are key to producing reliable data. First, samples should be either assayed immediately or snap-frozen and stored at or below -20oC upon collection. Aliquoting samples helps to avoid freeze-thawing, while storing sample material in polypropylene tubes (rather than glass containers) is essential to prevent analytes from being depleted by binding to the storage vessel surface.
Other general recommendations include vortexing samples to produce a homogeneous mixture, especially in situations where material has been centrifuged and the supernatant separated off, and using reverse-pipetting for more accurate sample dispensing. It is also important to use a consistent sample type throughout a given study; for example, analyte concentrations may differ between serum and plasma, meaning that switching between the two can yield variable results.
Proper and consistent pipetting technique is key to accurate data.
Plasma is the liquid component of blood, which functions to carry red and white blood cells and platelets. It is often analyzed for the presence of specific plasma proteins, including those associated with conditions such as cancer, autoimmune disorders, or viral infections. When separating plasma from whole blood, it is recommended that samples are centrifuged for 10 minutes at 1000 x g within 30 minutes of collection. It is also suggested that EDTA rather than heparin be used as an anti-coagulant since an excess of heparin will artificially skew results by providing falsely high values.
Serum comprises plasma from which the clotting proteins (e.g., Fibrinogen, Prothrombin, Tissue Factor) have been removed. It is essentially a “cleaner” form of plasma and should be chosen preferentially over plasma for immunoassays where either sample type may be used. It is advised that serum separator tubes are used for separating serum from whole blood, and that the blood be allowed to clot for at least 30 minutes before being centrifuged for 10 minutes at 1000 x g to remove any cells. To address the issue of hemolysis (the release of enzymes from red blood cells that can be a major cause of analyte degradation), it is recommended that protease inhibitors are added to sample collection buffers.
Analyte concentrations may differ between serum and plasma. Be consistent with the use of sample type within a study for best results.
Cell culture supernatants are often tested for the presence of secreted proteins like cytokines, growth factors, or other signaling molecules. These samples should be centrifuged upon collection to remove any suspension cells or debris that might interfere with assay performance. When working with cell culture supernatants, fresh culture medium should be used as the matrix solution for any blanks, standard curves, and controls; however, it is important to confirm that this is free of components such as active proteases, phosphatases, or other supplements that could be a source of unwanted background signal.
Immunoassay kits may have specific requirements, always refer to the kit protocol before planning your experiment.
While plasma, serum, and cell culture supernatants are among the most widely used sample materials, other sample types include tissue lysates, urine, cerebrospinal fluid (CSF), saliva, and many more. Each of these requires careful handling to preserve sample integrity and avoid compromising results, yet researchers can save significant time and money—particularly when multiplexing—by using an off-the-shelf immunoassay for analysis.
MILLIPLEX® multiplex assay kits contain all the components and reagents necessary to detect multiple analytes in parallel and are supplied with detailed, easy-to-follow instructions for sample collection, processing, and analysis. To learn more, visit SigmaAldrich.com/milliplex