ROUTINE LOW AND HIGH-RESOLUTION TYPING OF THE HLA-DRB GENE USING THE PCR-MPH (MICROTITRE PLATE HYBRIDIZATION) METHOD

We describe HLA-DRB1 typing using polymerase chain reaction-based micr otitre plate hybridization (PCR-MPH), which can process large numbers of samples. MPH typing is similar to an enzyme-linked immunosorbent as say (ELISA), in which a tandemly ligated sequence-specific oligonucleo tide is immobilized on microtitre wells. The typing procedure consiste d of two steps. Tn the first, PCR-MPH with 16 probes was performed to determine the specificities of the serological levels (DR1, DR2, DR3, DR4, DR11, DR12, DR13, DR14, DR7, DR8, DR9 and DR10) after generic amp lification ('low resolution typing'). In the second step, DR1, DR2, DR 4, DR12/8 and DR3/11/13/14 were group-specifically amplified based on the results of the first PCR-MPH, and microtitre plate hybridization p roceeded in a similar manner to the first step ('high resolution typin g'). Low resolution typing was completed within 2 h after generic ampl ification, and the results of high resolution typing were obtained in another 3.5 h after amplification. The allelic types classified using PCR-MPH were completely concordant with those obtained by PCR-single-s trand conformation poly morphism or PCR-restriction fragment length po lymorphism.