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Luminex-Based Methods in High-Resolution HLA Typing

  • Manuela TestiEmail author
  • Marco Andreani
Part of the Methods in Molecular Biology book series (MIMB, volume 1310)

Abstract

Luminex-based technology has been applied to discriminate between the different Human Leukocyte Antigens (HLA) alleles. The typing method consists in a reverse-SSO assay: Target DNA is PCR-amplified using biotinylated group-specific primers. A single PCR reaction is used for each HLA locus. The biotinylated PCR product is chemically denatured using a pH change and allowed to rehybridize to complementary DNA probes conjugated to microspheres. These beads are characterized by two internal fluorescent dyes that create a unique combination of color, make them identifiable. Washes are performed to eliminate any additional PCR product that does not exactly match the sequence detected by the probe. The biotinylated PCR product bound to the microsphere is labelled with streptavidin conjugated with R-phycoerythrin (SAPE). A flow analyzer identifies the fluorescent intensity SAPE on each microsphere. Software is used to assign positive or negative reactions based on the strength of the fluorescent signal. The assignment of the HLA typing is based on positive and negative probe reactions compared with published HLA gene sequences. Recently kits characterized by an extensive number of probes/beads designed to potentially reduce the number of ambiguities or to directly lead to an allele level typing, have been made available.

Key words

HLA typing Luminex PCR-SSO LABType® High resolution 

References

  1. 1.
    Hansen JA, Petersdorf E, Martin PJ, Anasetti C (1997) Hematopoietic stem cell transplants from unrelated donors. Immunol Rev 157:141–51CrossRefPubMedGoogle Scholar
  2. 2.
    Lee SJ, Klein J, Haagenson M et al (1997) High-resolution donor-recipient HLA matching contributes to the success of unrelated donor marrow transplantation. Blood 110:4576–83CrossRefGoogle Scholar
  3. 3.
    The MHC sequencing consortium (1999) Complete sequence and genemap of a human major histocompatibility complex. Nature 401:921–923CrossRefGoogle Scholar
  4. 4.
    Vandiedonck C, Knight JC (2009) The human major histocompatibility complex as a paradigm in genomics research. Brief Funct Genomic Proteomic 8:379–394CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Park M, Seo JJ (2012) Role of HLA in hematopoietic stem cell transplantation. Bone Marrow Res. doi: 10.1155/2012/680841

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Laboratory of Immunogenetics and Transplant Biology – IME FoundationPolyclinic of the University of Roma Tor Vergata UniversityRomeItaly

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