HLA CLASS-II TYPING IN A MICROTITRE PLATE FORMATE USING DIGOXIGENIN-LABELED AMPLIFIED DNA AND BIOTIN-LABELED OLIGONUCLEOTIDE PROBES

We describe a new, non-radioactive microtitre plate assay for the analysis of genetic variations at the DNA level. The new method combines hybridization of oligonucleotides with PCR amplified DNA in liquid phase with detection in solid phase using an ELISA-reader. Genomic DNA is labelled with digoxigenin during PCR using a nucleotide mix containing DIG-11-2'-deoxy-uridine-5'-triphosphate (DIG-11-dUTP). The DIG labelled, amplified genomic DNA is hybridized in solution with an oligonucleotide which is labelled with one biotin at its 3'-end, using biotin-16,2',3'-dideoxy-uridine-5'-triphosphate (BIO-16-ddUTP) and DNA deoxynucleotidylexo-transferase (TdT). The hybridized complex is immobilized in a streptavidin (SA) coated microtitre plate via the biotin and detection of digoxigenin is performed using anti-digoxigenin horseradish peroxidase, fab fragments (anti-DIG-POD), and the colorimetric substrate 2,2'-Azino-di-(3-ethylbenzthiazolinsulfonat[6]) (ABTS(R)). The resulting absorbtion of the assay is analysed in a microtitre plate reader. This method results in highly specific and sensitive hybridization signals and with the 15 oligonucleotides chosen, allows the typing of DR1-DR10.