SBIR-STTR Award

The use of PCR- based assays in clinical laboratories performing human leukocyte antigen (HLA) typing is growing rapidly. This has created a need for increasingly stringent standards for test validation and laboratory accreditation. However, there are currently no commercially available specific control HLA DNA standards for use with PCR or DNA probe-based test systems. Presently the most widely used HLA DNA typing methods utilize either sequence-specific primers (SSP) to PCR amplify specific HLA alleles or sequence-specific oligonucleotide probes (SSOP) to detect HLA alleles amplified using primers to conserved HLA gene sequences. Although these approaches have proved to be effective HLA typing techniques, the applicant argues that quality assurance remains a concern. The only approach currently available for monitoring the quality of individual primer pairs is to concurrently test control genomic DNA isolated from a panel of reference cell lines. However, maintenance of reference cell lines is labor intensive and costly. The overall goal of this Phase I proposal is to examine the feasibility of producing one set of DNA standards for use with SSP and SSOP HLA typing assays and to compare their performance to currently used reference cell genomic DNA standards. The specific aims are: 1) To construct a set of DNA standards for HLA-DRB typing; 2) To optimize the standards for use with SSP typing; and 3) To optimize the standards for use with SSOP typing

HLA typing methods used in clinical laboratories utilize either sequence-specific primers (SSP) to amplify or sequence certain HLA alleles, or sequence-specific oligonucleotide probes (SSOP) to detect them. However, control DNA standards for primer- and probe-based HLA typing systems are not available commercially, and market research among HLA typing laboratories indicates a desire for kits to monitor primer- and probe- based HLA typing reagents. Phase I studies indicate the feasibility of producing HLA DNA standards that consist of modified gene sequences cloned in a plasmid vector. The authors predict that the constructs will be useful as controls for both SSP-PCR and SSOP assays. The specific aims of this Phase II proposal are: 1) to develop and test additional HLA DNA standards, including those for low resolution HLA-A, -B, and DQB primers and probes, 2) to optimize this set of low-resolution HLA DNA standards, and 3) to evaluate the feasibility of streamlining the standards for future kit development