Rapid and simultaneous HLA class I (-A, -B and -C loci) DNA typing using the microtitre plate-reverse hybridization assay (MRHA).

Abstract

We have established a precise, rapid, simple and practical HLA class I DNA typing method using the microtitre plate-reverse hybridization assay (MRHA), which enables us to perform simultaneous DNA typing of the HLA-A, -B and -C loci using the same PCR parameters and hybridization conditions. PCR-amplified products for the HLA-A, -B and -C loci were hybridized, respectively, with sequence-specific oligonucleotide (SSO) probes, which were immobilized covalently onto a microtitre plate, in hybridization buffer containing formamide at 37 degrees C. After washing at room temperature, the bound PCR products were detected by peroxidase-conjugate streptavidine followed by colour development such as enzyme immunoassay (EIA). In addition to the simple thermoregulation for hybridization and postwashing, strong positive signals, low background and high reproducibility, this DNA typing method enabled simultaneous typing of the HLA-A, -B and -C loci using a single microtitre plate as in HLA serotyping. The assignment of the HLA genotype was easily achieved by automated colorimetric reading and computer software, based on the cut-off value (threshold) established for each probe. For routine HLA class I typing, it may be possible to replace serological typing with the HLA class I DNA typing system using our MRHA method.