abstract
- A simple, sensitive ELISA that is performed in 96-well microtiter plates and that requires less than 90 minutes to complete was developed for 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 treatment included exposure to 0.4 N NaOH to remove the nonbiotinylated sense strand, addition of a fluorescein-labeled oligonucleotide probe, one or more 5-minute stringency washes, addition of an alkaline-phosphatase-labeled anti-FL FAB, and then alkaline-phosphatase substrate and amplifier. An intense red-violet color developed within 15 minutes in positive wells and could be quantitated by OD readings at 490-495 nm. To control for stringency and to establish threshold OD values for positive reactions, biotin-labeled antisense oligos that were complementary to the probe or that differed by one or more bases were immobilized in wells in place of PCR products. The assay was sensitive to < 0.05 pmol (approximately 4 ng)/well and required only standard incubators and waterbaths and an optional microplate reader. All reagents were commercially available. The method should facilitate oligotyping of both class I and class II alleles and is adaptable for analysis of other polymorphic gene products.