Non-radioisotopic typing of human leukocyte antigen class II genes on microplates.
We describe a new nonradioisotopic method for HLA class II molecular typing performed in 96-well plates of the same size as those used in enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA). Biotinylated sequence-specific oligonucleotide (SSO) probes are bound to avidin-coated plates. Digoxigenin-labeled PCR-amplified DNA samples are hybridized, washed and detected with a peroxidase conjugated antibody assay. The method was tested by performing a partial HLA DQA1 and DQB1 typing on 69 randomly selected blood samples. The results are completely concordant with a traditional SSO-PCR typing performed on the same samples. This procedure is simple, fast and could be adapted for performance in semi-automated or automated fashion using equipment already available for ELISA and RIA assays.
Giorda
R
R
Department of Pediatrics, University of Pittsburgh, School of Medicine, Rangos Research Center Children's Hospital of Pittsburgh, PA 15213.
Lampasona
V
V
Kocova
M
M
Trucco
M
M
D001360:Avidin; D001710:Biotin; D004247:DNA / Q000032:analysis; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology; D004076:Digoxigenin; D005802:Genes, MHC Class II; D006683:HLA-DQ Antigens / Q000032:analysis / Q000235:genetics; D059848:HLA-DQ alpha-Chains; D059866:HLA-DQ beta-Chains; D000949:Histocompatibility Antigens Class II / Q000032:analysis* / Q000235:genetics; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D007124:Immunoenzyme Techniques; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction
D006683:HLA-DQ Antigens; D059848:HLA-DQ alpha-Chains; D059866:HLA-DQ beta-Chains; C069050:HLA-DQA1 antigen; C069051:HLA-DQB1 antigen; D000949:Histocompatibility Antigens Class II; D015345:Oligonucleotide Probes; D001360:Avidin; D001710:Biotin; D004247:DNA; D004076:Digoxigenin