Time-resolved fluorometry based sandwich hybridisation assay for HLA-DQA1 typing

Area of Science:

• Immunogenetics
• Molecular Biology
• Biochemistry

Background:

• Accurate human leukocyte antigen (HLA) typing is crucial for organ transplantation and disease association studies.
• Existing HLA typing methods can be complex and time-consuming.
• Development of rapid and reliable HLA typing assays is an ongoing need.

Purpose of the Study:

• To develop a novel time-resolved fluorescence (TRF) hybridisation assay for HLA typing.
• To evaluate the assay's performance for specific HLA-DQA1 allele detection.
• To assess the applicability of the assay using both isolated genomic DNA and whole blood samples.

Main Methods:

• Development of a microtitration plate-based TRF hybridisation assay.
• Utilisation of biotinylated sequence-specific catching probes and europium (Eu)-labelled detection probes.
• Amplification of polymorphic exon 2 of the DQA1 gene followed by hybridisation assay.
• Detection of specific hybridisation signals using a time-resolved fluorometer.

Main Results:

• The assay successfully detected targeted DQA1 alleles in 100 isolated genomic DNA samples.
• Specific probes were designed for various DQA1 alleles including *01, *0101/0104, *0103/0201/0601, *0201, *03, *0401/0601, *05, and *0502.
• The assay demonstrated applicability and confirmed results when tested with whole blood spot samples.

Conclusions:

• The developed TRF hybridisation assay provides a sensitive and specific method for HLA typing.
• The assay is capable of detecting specific HLA-DQA1 alleles with high accuracy.
• This method offers a viable alternative for HLA typing using both DNA and blood samples.