Development of Reverse Dot Blot Test Strips for HLA DRB1*01 Group Alleles

Knowledge of allele groups and specific alleles present in individuals has important implications in organ and stem cell transplantation and in disease association studies. In organ transplantation application of molecular HLA typing allowed to improve typing quality, leading to a more precise matching assessment with better clinical outcomes. In this study, we have developed the reverse Dot-Blot (RDB) hybridization technique as a rapid and simple method for detection of HLA-DRB1*01 group alleles. For high-resolution typing of HLA-DRB1*01 group alleles, we performed amplification of the alleles of this group using allele-specific primers. Productive DNA amplification occurs if an allele perfectly be complementary to the two primers chosen is present. This technique is often referred to as PCR-SSP. Then for specific discrimination between these alleles (HLA-DRB1*0101, 0102, 0103, 0104), we hybridized PCR product, labeled with biotinylated primers during the amplification, with spots of immobilized probes on a membrane. Variety of immobilization methods could be used. We developed a covalent attachment between 5´-amino probes and carboxylated membrane surfaces. The presence of the PCR product bound to the specific probes at specific locations is detected using streptavidin-AP conjugate and NBT/BCIP as a chromogenic substrate. The presence of each specific allele was detected by the appearance of a dot on the membrane. Obtained results were compatible with those of expected for standard allele samples. Furthermore, results obtained for several other samples which were typed by our technique, were confirmed using sequencing. Our results suggest that HLA typing by RDB method has proved to be a high-resolution, high specificity, rapid and accurate, suitable for clinical application with a greater precision than serological techniques.