COMPARISON OF 2 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 be en 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 ail HLA-DRB alleles in the same reaction mix. The 2 Allelic Subset Amplification (2SA) method uses two distinct amplification rea ctions that distributes all DRB alleles into two equal-size subsets, a ccording to the codon 86 Gly or Val polymorphism; this adds an extra d iscrimination level to the typing. 108 samples were typed using the 1G A and the 2SA methods and no discrepancies were found. Typing indeterm inations due to overlapping probe combinations were compared; it was f ound that the 2SA method, with the extra discrimination level at the P CR step, greatly improved resolution.