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

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Abstract
SUMMARY: We describe HLA‐DRB1 typing using polymerase chain reaction‐based microtitre plate hybridization (PCR‐MPH), which can process large numbers of samples. MPH typing is similar to an enzyme‐linked immunosorbent assay (ELISA), in which a tandemly ligated sequence‐specific oligonucleotide is immobilized on microtitre wells. The typing procedure consisted of two steps. In 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 amplification (‘low resolution typing’). In the second step, DR1, DR2, DR4, DR 12/8 and DR3/11/13/14 were group‐specifically amplified based on the results of the first PCR‐MPH, and microtitre plate hybridization proceeded in a similar manner to the first step (‘high resolution typing’). Low resolution typing was completed within 2 h after generic amplification, 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‐strand conformation polymorphism or PCR‐restriction fragment length polymorphism.