Comparison of two HLA-DRB high resolution microtiter plate reverse hybridization typing methods: advantage of a codon-86 valine or glycine PCR segregation

: Two rapid, nonisotopic, high-resolution HLA-DRB typing methods have been developed for DRB1, DRB3, DRB4 and DRB5 alleles. These methods are based on a single procedure consisting of the reverse hybridization of biotinylated amplicons to oligonucleotide probes that are covalently attached to a microtiter plate. Detection is by an enzymatic reaction with a fluorescent substrate. The 1 Generic amplification (1GA) method amplifies all HLA-DRB alleles in the same reaction mix. The 2 Allelic Subset amplification (2SA) method uses two distinct amplification reactions that distributes all DRB alleles into two equal-size subsets, according to the codon 86 Gly or Val polymorphism; this adds an extra discrimination level to the typing. 108 samples were typed using the 1GA and the 2SA methods and no discrepancies were found. Typing indeterminations due to overlapping probe combinations were compared; it was found that the 2SA method, with the extra discrimination level at the PCR step, greatly improved resolution.