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Disease Markers
Volume 14 (1998), Issue 1, Pages 9-19
http://dx.doi.org/10.1155/1998/350145

Time-Resolved Fluorometry Based Sandwich Hybridisation Assay for HLA-DQA1 Typing

Minna Sjöroos,1,2 Jorma Ilonen,2 Helena Reijonen,2 and Timo Lövgren1

1Department of Biotechnology, University of Turku, FIN-20520 Turku, Finland
2Department of Virology, University of Turku, FIN-20520 Turku, Finland

Received 9 December 1999; Accepted 9 December 1999

Copyright © 1998 Hindawi Publishing Corporation. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.