Time-resolved fluorometry based sandwich hybridisation assay for HLA-DQA1 typing

Dis Markers. 1998 Jun;14(1):9-19. doi: 10.1155/1998/350145.

Abstract

A microtitration plate based time-resolved fluorescence (TRF) hybridisation assay was developed for HLA typing utilising biotinylated sequence-specific catching probes and europium (Eu) labelled gene locus-specific detection probe to allow time-resolved fluorometer reading of the reaction. In an application for HLA-DQA typing a 228 base pair long region of the polymorphic exon 2 of DQA1 gene was amplified and the denatured PCR product distributed into streptavidin-coated microtitration wells together with the detection probe and one of the catching probes. After incubation and washes, the enhancement solution was added and specific hybridisation signal detected by measuring the emitted light. A series of 100 isolated genomic DNA samples were studied using biotinylated probes specific for DQA1*01, *0101/0104, *0103/0201/0601, *0201, *03, *0401/0601, *05 and *0502 alleles with results demonstrating the capacity of the test to detect aimed alleles. A series of whole blood spot samples were also studied and the results confirmed the applicability of this modification of the test.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Primers
  • Fluorometry
  • Genotype
  • HLA-DQ Antigens / genetics*
  • HLA-DQ alpha-Chains
  • Humans
  • Nucleic Acid Hybridization
  • Polymerase Chain Reaction

Substances

  • DNA Primers
  • HLA-DQ Antigens
  • HLA-DQ alpha-Chains
  • HLA-DQA1 antigen