TRIPLE-LABEL HYBRIDIZATION ASSAY FOR TYPE-1 DIABETES-RELATED HLA ALLELES

We describe a method for the detection of two type 1 (insulin-dependen t) diabetes susceptibility (0201, *0302) alleles and two protective ( 0301, *0602/0603) alleles of the HLA-DQB1 gene on the human major his tocompatibility complex (MHC). The rest is based on DNA amplification with PCR followed by simultaneous, allele-specific triple-label hybrid ization performed in microtitration wells. In the hybridization, very short allele-specific oligonucleotides labeled with europium (Eu), ter bium (Tb) or samarium (Sm) are used. The labeled probes could be detec ted using time-resolved fluorometry with sensitivities of I x 10(7), 3 x 10(8) and 3 x 10(8) molecules, respectively. Cross-reactions were n ot found among samples containing 14 common DQB1 alleles. To test the utility of the developed assay, 100 DNA and 14 dried blood spot sample s with known DQB1 alleles were analyzed. A 100% agreement with the ref erence method was reached. Thus, this triple-label hybridization assay proved to be suitable evert for detection of a large number of sample s.