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HLA antigens are cell membrane glycoproteins with key roles in the initiation of the immune response. Current methods for HLA typing define HLA alleles and allele groups using DNA-based methods. Different DNA-based molecular techniques are used depending on the clinical application. Solid organ transplantation requires a low- to intermediate-level typing resolution to determine an individual’s HLA antigens. Bone marrow transplantation requires a high-resolution typing to determine the HLA alleles. Determination of HLA phenotypes is also applied to vaccine development, studies of disease associations and as companion diagnostics for the safe and effective use of therapeutic products.
The Johns Hopkins University Immunogenetics Laboratory uses a combination of reverse Sequence Specific Oligonucleotide (rSSO) and Sequence Based Typing (SBT) molecular technologies for HLA typing.
Reverse SSO is used for low to intermediate HLA typing of HLA-A, -B, -C, -DRB1, -DRB3-5, -DQA, -DQB and -DP loci. This technology uses the polymerase chain reaction (PCR) to amplify the locus of interest. DNA probes of known sequence are allowed to bind with complementary DNA sequences on the PCR product. The combination of bound and unbound probes allows for a snapshot of DNA sequences throughout the PCR product. Testing is performed with a multiplex bead assay on the Luminex® platform.
SBT (Sequenced Based Typing)
SBT is used for high-resolution identification of alleles of HLA-A, -B, -C, - DRB1, -DQB1 and -DPB1. This technology uses PCR to amplify the locus of interest. Sanger sequencing is then used to determine the nucleotide sequence of the PCR product.