Labtype sso hd kits

Study samples were HLA genotyped by Luminex-based sequence-specific oligonucleotide typing assays (Supplemental Table 5). Genomic DNA was extracted from cells using the Qiagen EZ-1 machine. Intermediate resolution of all HLA loci (HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPA1, and HLA-DPB1) were performed using the LABType SSO HD kits (One Lambda). Briefly, the DNA was amplified using primers that were specific for each of the 11 HLA loci. The PCR product was biotinylated and denatured to make single-stranded DNA. This DNA was then hybridized to a series of probes specific for nucleotide sequences that were used to define the HLA genes. Each probe was bound to a uniquely fluorescent coded Luminex bead. The mixture was then washed to remove any excess, unbound PCR product. The bound PCR product was labeled with streptavidin conjugated with a PE fluorescent tag (SAPE). The DNA-bead complexes were run on a Luminex 100 platform and analyzed using HLA-FUSION v3.5.6 software.

HLA typing was performed with genomic high resolution typing (second field) of HLA- A, B, C, DRB1, DRB3, DRB4, DRB5, DQA, DQB, DPA and DPB. All loci were analyzed by PCR-sequence specific oligonucleotide (PCR-SSO) probing using LABType SSO HD kits (One Lambda). A Luminex 200 (Luminex Corporation) flow analyzer was used to identify the fluorescence intensity of PE (phycoerythrin) of PCR products bound to fluorescently coded microspheres. The data were imported into HLA Fusion software version 4.4.0 (One Lambda) for analysis.