RAPID MULTIPLEX HLA DRB TYPING USING FLOWMETRIX TECHNOLOGY.
BL Ray, RT Cunningham, I Balazs, Lifecodes Corporation, Stamford, CT. 

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Flow-PRA methods have been successfully developed to assay HLA antigen/antibody interactions using antigen-coated microspheres. We have employed the Flowmetrix? technology (Luminex Corp, Austin, TX) to develop a microsphere-based, multiplex assay for HLA DRB DNA typing. The method presented can rapidly type an individual DNA sample for HLA DRB in a single assay. The method utilizes microspheres that can be classified based on characteristic internal fluorescence. By attaching a specific probe to a particular class of microsphere, the binding of Cy3-labeled target to a particular class of microsphere, and hence to a specific probe, can be quantified. Quantification is performed on the Luminex-100 instrument which is capable of simultaneously monitoring up to 100 different classes of microspheres.

Employing the Flowmetrix? technology, we have demonstrated that individual blood samples can be typed for HLA DRB using a set of 31 probes. The process involves 1) isolating genomic DNA from samples, 2) PCR amplifying the genomic DNA; 3) hybridizing the PCR product to probes attached to microspheres; 4) quantifying PCR product hybridized to the different microsphere classes; 5) assigning positive or negative attributes for each probe, and 6) computer-aided typing of the sample based on probe assignments. Once the PCR reaction is completed, the remainder of the process can be completed in 30 minutes. Thus, the entire process takes approximately 4.5 hrs with little labor involved. Ninety-six samples can be easily processed by one individual in an 8 hr day. The technology is simple and can be easily extended to other HLA typings.