Multiplex Immunoassay Profiling
Multiplex immunoassays allow for the rapid profiling of biomarker proteins in biological fluids, using less sample and labor than single immunoassays. This chapter details the methods to develop and manufacture multiplex assays for the Luminex® platform.
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Introduction A large majority of immunoassays rely on antibodies to capture and detect the analyte of interest in a biological matrix. Traditional immunoassays detect the presence of a single analyte and most rely on enzyme-driven detections. New technologies have been developed that allow multiple analytes to be measured simultaneously on a single sample, all in a single reaction vessel. The method described here is for the development of a multiplex assay for the Luminex® system, although the same principal will apply to other technologies (Fig. 1). For information on multiplexing technologies, there are several recent publications that review the latest developments [1–4]. As with any new technology, there are unique advantages and disadvantages that the user encounters. For example, the ability to simultaneously measure multiple analytes in a single sample maximizes the amount of information that can be obtained from single sample, reduces laboratory analysis time and sample volume requirement, and provides cost savings. However, multiplexed assays also present unique challenges for the user that would not be encountered if single assays were used for each individual analyte. Examples of these include different detection ranges, the potential
Paul C. Guest (ed.), Multiplex Biomarker Techniques: Methods and Applications, Methods in Molecular Biology, vol. 1546, DOI 10.1007/978-1-4939-6730-8_13, © Springer Science+Business Media LLC 2017
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Laurie Stephen Anti-target 1
Anti-target 2
Anti-target 3
Specific antibodies coupled to distinct beads
Beads filled with red and infrared dyes at different ratios - fluoresce at different wavelengths when excited with a laser
Add bead mixture Quantity
Identity
Target 1
Target 5 Target 4 Add sample
Add biotinylated anti-body
Read in detector
DETECTOR
Fig. 1 Overview of multiplex immunoassay technique. Samples are added to dye-coded microbead-antibody conjugates that capture specific targets. Following incubation with a second antibody containing a biotinylated label to form a “sandwich” configuration, the mixtures are streamed through the Luminex instrument that uses lasers for the identification of the antibody-microbead conjugates and quantitation of the bound molecules. The example shows a triplex assay capable of binding targets 1, 2, and 3. Since the sample only contains target 1, this is the only target bound and quantified
for cross-reactivity, increased matrix interference, and potential for false positives due to antibody interactions. These challenges, if not carefully addressed, can generate misleading results. Several papers have addressed the issue of increased interference in multiplex immunoassays [5–7] and others have described the additional challenges involved in the validation stage [8–10]. This chapter will describe a method to develop a multiplex immunoassay and strategies for minimizing interference and interactions within the assay.
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Materials
2.1 Bead Conjugation
1. Magnetic separator. 2. Sonicating bath. 3. Copolymer tubes a
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