Traditional immunoassays such as the enzyme-linked immunosorbent assay (ELISA) are able to measure the presence or absence of only one analyte per reaction. Multiplex immunoassays measure dozens of different analytes in a single reaction. This is particularly beneficial for precious samples, and when only a small volume is collected for analysis. The multiplex immunoassay also saves working time since multiple assays can be completed simultaneously.
Multiplex immunoassays similar to the ELISA in that they both rely on the specificity of antigen-antibody binding. They also need a detection system. The traditional ELISA relies on an enzymatic reaction while most modern multiplex assays rely on fluorescent probes.
There are two general categories of multiplex immunoassays, and they are divided based on the type of surface on which the antibodies or antigens are bound.
- A solid planar surface on which the targets for each analyte are spatially separated
- antigen- or antibody-bound beads containing an identifying dye
The multiplex immunoassay beads
The bead based multiplex immunoassay requires beads, typically made of polystyrene, that are impregnated with a mixture of red and near-infrared dyes. Manufacturers can create 100 or more uniquely identifiable bead sets by altering the ratio of these dyes. Each analyte to be measured is assigned to a particular bead set, and antibodies are immobilized on the surface. The beads are incubated with the sample for an appropriate time to enable binding between the antibodies and antigens. A secondary probe is also introduced during this incubation period.
The secondary identification probe for multiplex immunoassays
The secondary probe consists of a fluroescent probe conjugated to an antigen or antibody complementary to the analyte. This antigen or antibody is the same type that is conjugated to a bead. As an example, lets say that we are doing an assay for the presence of specific antibodies in a clinical sample. One bead set will be bound with antibodies specific to antigen A. Another bead set will be bound with antibodies specific to antigen B. A third bead set will be bound with antibodies specific to antigen C, and so on. Additionally, free antigens specific to type A, B, and C are bound to green fluorescent probes. All of these components are mixed with the sample. If antigen A is in the sample it will bind to the bead set bound with antibody A, and the free-floating probe-conjugated antibody will also bind to the same antigen and form a sandwich. This joins the red fluorescent bead and the secondary green fluorescent probe together into one conjugated system. If antigen B is present in the sample it will form its own conjugated system. If antigen C is not present in the sample, then the red fluorescent bead will not be conjugated to a green fluorescent probe because there is no antigen to link the two together.
Quantification by flow cytometry
A flow cytometer is used for data acquisition. The flow cytometer sends a stream of beads through laser beams one bead at a time. One laser is red and the other is green. The red laser identifies the specific analyte being measured by identifying the unique color of the bead set. The green laser identifies whether or not the secondary probe was conjugated to the bead via the analyte of interest. In the example above, beads conjugated to antigens A and B will fluoresce green, while beads from set C will not.
Superparamagnetic beads for multiplex immunoassays
Superparamagnetic beads are being introduced to multiplex immunoassays. The magnetic beads are coated with a dye-impregnated polymer in order to have both magnetic and fluorescent properties. The magnetic property allows for automated washing steps to be added to the protocol to remove contaminants or unbound probes. This can help the flow cytometry process proceed smoothly. Alternatively, the magnetic beads allow other quantification methods that don't require an expensive flow cytometer and lasers. The magnetic beads can be drawn to a surface and immobilized while an LED-based quantification system measures the fluorescent signals.
Other applications for muliplex assays
The fluroescent bead-based multiplex system is also useful for the detection of nucleic acids. In this case, a single strand sequence is covalently attached to the bead surface. This sequence is complementary to the target sequence. The bead is included in a polymerase chain reaction of the sample, and will be extended by DNA polymerase. Some of the bases used by the DNA polymerase are modified to contain a binding site for a fluroescent probe. The presence of the target sequence is therefore indirectly measured by the multiplex system.
The developers of this bead-based-fluroescent multiplex assay also sell premade kits containing beads conjugated with an array of common targets. If a premade kit doesn’t contain all of the markers to meet your unique experimental goals, it is easy to order a custom-made kit. There is no need for the researcher to conjugate each bead set his or herself. This service makes it quick and easy to perform multiplex assays on a variety of targets.
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