RAPID HLA-DR OLIGOTYPING BY AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY PERFORMED IN MICROTITER TRAYS

A simple, sensitive ELISA that is performed in 96-well microtiter plat es and that requires less than 90 minutes to complete was developed fo r HLA-DRB oligotyping. The second exon of HLA-DRB1 was amplified using an unlabeled forward primer and a biotinylated reverse primer and the PCR product was immobilized in avidin-coated wells. Subsequent treatm ent included exposure to 0.4 N NaOH to remove the nonbiotinylated sens e strand, addition of a fluorescein-labeled oligonucleotide probe, one or more 5-minute stringency washes, addition of an alkaline-phosphata se-labeled anti-FL FAB, and then alkaline-phosphatase substrate and am plifier. An intense red-violet color developed within 15 minutes in po sitive wells and could be quantitated by OD readings at 490-495 nm. To control for stringency and to establish threshold OD values for posit ive reactions, biotin-labeled antisense oligos that were complementary to the probe or that differed by one or more bases were immobilized i n wells in place of PCR products. The assay was sensitive to <0.05 pmo l(similar to 4 ng)/well and required only standard incubators and wate rbaths and an optional microplate reader. All reagents were commercial ly available. The method should facilitate oligotyping of both class I and class II alleles and is adaptable for analysis of other polymorph ic gene products.