Rapid HLA class I DNA typing using microtiter plate-reverse hybridization assay (MRHA) by simple thermoregulation: High-resolution subtyping of the HLA-A2 and-B40 antigen groups

We have established a precise, rapid, simple and economical subtyping metho d for alleles encoding the HLA-A2 and -B40 antigens using microtiter plate- reverse hybridization assay (MRHA), which is based on the general principle of HLA oligotyping by reverse doc blot hybridization. Amino-modified seque nce-specific oligonucleotide (SSO) probes were immobilized covalently onto a carboxylate-modified microtiter plate. In order to perform high-resolutio n subtyping of thr HLA-A2 and -B40 antigen groups, thr alpha 1 and alpha 2 domain regions were amplified using a pair of group-specific primers compos ed of an unlabeled sense primer and a biotinylated antisense primer. PCR-am plified products were hybridized mit-h SSO probes in hybridization buffer c ontaining formamide for 1 hour at 37 degrees C. After washing with 2 X SSC at room temperature, the bound PCR products were detected by alkaline phosp hatase-conjugated streptavidine followed by color development. All of 8 HLA -BIO suballeles, all of 2 HLA-B47 suballeles (B40 group-specific primers us ed in this study allowed also B47 amplification) and 17 out of 21 HLA-A2 su balleles were discriminated. The remaining four HLA-AZ suballeles were dete rmined by analysis after exon 4 amplification. HLA-DNA typing by this metho d was easily and exactly performed regardless of sample number. The greates t advantages of this technique are strong positive signals obtained, reprod ucibility and thr ease of thermoregulation for hybridization and washing as compared ro previously reported microtiter plate hybridization methods. (C ) American Society for Histocompatibility: and Immunogenetics, 1999. Publis hed by Elsevier Science Inc.