RAPID HLA CLASS I DNA TYPING USING MICROTITER PLATE-REVERSE HYBRIDIZATION ASSAY (MRHA) BY SIMPLE THERMOREGULATION.
Toyoki Moribe1, Hiromi Hirai1, Toshihiko Kaneshige1 and Hidetoshi Inoko2, 1Diagnostic Science Division, Shionogi & Co., Ltd., Osaka, and 2Department of Molecular Life Science, Tokai University School of Medicine, Kanagawa, Japan
We have established a precise, rapid, simple and economical method for HLA class I (A, B and C) DNA typing using microtiter plate-reverse hybridization assay (MRHA), which is based on the general principle of HLA oligotyping by reverse dot blot hybridization. Amino-modified sequence-specific oligonucleotide (SSO) probes were immobilized covalently onto a carboxylate-modified microtiter plate. In order to perform medium/high-resolution genotyping of the HLA-A, -B and -C loci, the regions of exons 2 and 3 were separately amplified using a pair of locus-specific biotinylated primers. PCR-amplified products were hybridized with SSO probes in hybridization buffer containing formamide for 1 hour at 37 . After post-washing at room temperature, the bound PCR products were detected by horseradish peroxidase-conjugated streptavidine followed by color development. The HLA-A, -B and -C typing was performed using 23 respective SSO probes in which resolution was higher than that obtained by the serological typing using the lymphocytotoxicity assay. Further, HLA-DNA typing by MRHA was easily and exactly performed regardless of sample number. The greatest advantages of this technique are strong positive signals obtained, reproducibility and the ease of thermoregulation for hybridization and washing as compared to previously reported microtiter plate hybridization methods. In conclusion, the HLA class I DNA typing by MRHA enables us to process a large number of samples precisely, rapidly and simply, and allows automated colorimetric reading. Using MRHA for HLA class I DNA typing, the serological typing may ultimately be replaced.