Abstract
Antibody (AB) testing or serotesting for reactive ABs against antigenic proteins is broadly used. Parallel examination of many antigens is of high interest to identify autoantibodies (AAB) or differential antigenic reactivities in many biological settings like allergy and infectious autoimmune, cancerous, or systemic disease. The resulting AAB profiles can be used for diagnosis, prognosis, and monitoring of such conditions. Protein microarrays have been used for AB profiling over the past decade but show some significant limitations which make them unsuitable for clinical applications. Alternative multiplexing platforms such as bead arrays were shown to provide a versatile tool for the confirmation and efficient analysis of high numbers of biological samples. Luminex’ bead-based xMAP technology combines advantages such as multiplexing and lower demand for sample volume and at the same time overcomes the challenges of microarrays. It works faster, shows better antigen stability, is more reproducible, and allows the analysis of up to 500 analytes in one sample well. In this chapter we introduce our established workflow for the use of the xMAP technology for AB profiling including an overview of the method principle and protocols for the covalent immobilization of proteins to the MagPlex beads, confirmation of protein coupling, the execution of a multiplexed bead-based protein immunoassay, and subsequent data handling.
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Abbreviations
- AA:
-
Amino acid
- AAB:
-
Autoantibodies
- AB:
-
Antibody
- BSA:
-
Bovine serum albumin
- ddH2O:
-
Distilled water
- EDC:
-
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
- ELISA:
-
Enzyme-linked immunosorbent assay
- h:
-
Hour
- HSA:
-
Human serum albumin
- MES:
-
2-(N-Morpholino)ethanesulfonic acid
- MFI:
-
Median fluorescent intensity
- min:
-
Minute
- PBS:
-
Phosphate-buffered saline
- PE:
-
Phycoerythrin
- rpm:
-
Rounds per minute
- s:
-
Second
- SA-PE:
-
Streptavidin phycoerythrin conjugate
- S-NHS:
-
Sulfo-N-hydroxysuccinimide
- Ttds:
-
Trioxatridecan-succinamic acid
- xMAP:
-
Multi-analyte profiling
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Huber, J. et al. (2023). Accessing Antibody Reactivities in Serum or Plasma to (Auto-)antigens Using Multiplexed Bead-Based Protein Immunoassays. In: Greening, D.W., Simpson, R.J. (eds) Serum/Plasma Proteomics. Methods in Molecular Biology, vol 2628. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2978-9_26
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DOI: https://doi.org/10.1007/978-1-0716-2978-9_26
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