Multiplex immunoassays enable the detection of multiple different analytes in the same sample. A well-designed multiplex assay could mean that only one vial of blood might need to be drawn from a patient instead of 8 or more vials. Therefore, one multiplex test can answer multiple questions at the same time. Immunoassays capitalize upon the specific affinity between antibody and antigen. Some immunoassays use antigen as the probe in order to detect the binding of antibody target, while other immunoassays use antibody probes in order to detect antigen targets. Most immunoassays are single-plex, meaning that they can only be used to detect one antibody-antigen pair at a time; this is generally the case with most ELISA (Enzyme Linked ImmunoSorbent Assay) tests. The idea of a multiplex ELISA is attractive, and it mostly indicates a type of multiplex immunoassay that relies upon antibody-antigen binding events. The traditional ELISA is single-plex, and requires multiple binding and washing steps as well as an enzymatic system that produces a colorimetric or chemiluminiscent label as a quantitative readout of target concentration in a sample.
Enzymes used in ELISA
When ELISA is used to detect antigen, the most common format is a sandwich ELISA. This format uses an antibody probe attached to a surface, most commonly a 96-well polystyrene plate. If the corresponding antigen is present in the sample, then it will bind to the capture antibody. Now, the complicated detection steps of ELISA are needed to provide a readout of this binding event. Another antibody (detection antibody) is needed to detect the antigen that is already bound to the capture antibody, but this antibody needs to recognize a different epitope of the antigen than the capture antibody did. The detection antibody is also conjugated with an enzyme such as horse radish peroxidase (HRP) or alkaline phosphatase (AP). In the final step of the ELISA, the substrate is added to the wells, and if the antigen was present, then the detection antibody would have bound, and the conjugated enzyme will use the substrate to create a colored product or a chemiluminiscent product. The sandwich ELISA would be very complicated to make into a multiplex ELISA because there would need to be capture antibodies for as many target antigens as desired. This greatly increases the chances of cross-reactivity and decreases the selectivity of the assay. It has been done, however, by microarraying different capture molecules into regular patterns on a glass slide. Then the slide can be incubated in each solution all at the same time. A multiplex ELISA in a 96-well plate would quickly get too large and complicated to manage.
Other multiplex immunoassay formats
The gold standard multiplex immunoassay is performed on luminex technology. This is a flow-based system that works on the same principle as the ELISA, but using fluorescent microspheres and detection antibodies for quantitative readout. Combinations of bead fluorescence wavelength and label wavelength indicate which targets are present in the sample. Each capture antibody is conjugated to a microsphere with a unique emission wavelength. If the antigen is present it will bind to the capture antibody, and the labeled detection antibody will also bind to create a sandwich. The detection antibody is labeled with a fluroescent tag. The luminex system records the fluorescence of every bead that passes through the system and provides a quantitative readout of how many beads bound target.
Newer multiplex immunoassays are coming to the market that don’t require any labels at all. One example is arrayed imaging reflectometry (AIR), which is an optical multiplex immunoassay that is capable of detecting the antibody-antigen binding event directly due to the change in refractive index at the chip surface when the binding event occurs. This change in mass and refractive index is visible as an increase in light intensity, and is quantifiable. The label-free multiplex immunoassay has the potential of being the most sensitive and selective format, and also has the benefit of simplicity and being very easy to use.