Semiautomated HLA-DQB1 typing by fluorescent dye photometry of amplified DNA on microtiter plates.
We report a simple semiautomated HLA class II typing method that suits the demands of a 24-hour-duty transplantation service for preorgan retrieval donor typing. The procedure consists of sequence-specific amplification of HLA alleles by the polymerase chain reaction (nested PCR-SSP) followed by fluorescent dye photometry of the resulting PCR products on microtiter plates. The entire typing procedure is semiautomated and completed in less than 90 minutes after DNA isolation. The test was evaluated for the definition of the specificities DQ1-DQ9 (generic HLA-DQB1 typing). Sensitivity and specificity as judged by DNA reference typing in a different laboratory was 99.5% (n = 202 alleles).
Bein
G
G
Institute of Immunology and Transfusion Medicine, University of Lübeck Medical School, Germany.
Haase
D
D
Schult
J
J
Eiermann
T H
TH
Kirchner
H
H
D000483:Alleles; D001483:Base Sequence; D002460:Cell Line; D002472:Cell Transformation, Viral; D004247:DNA / Q000032:analysis* / Q000302:isolation & purification; D017931:DNA Primers; D005456:Fluorescent Dyes; D005838:Genotype; D006683:HLA-DQ Antigens / Q000032:analysis*; D059866:HLA-DQ beta-Chains; D006801:Humans; D008969:Molecular Sequence Data; D010783:Photometry; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity
D017931:DNA Primers; D005456:Fluorescent Dyes; D006683:HLA-DQ Antigens; D059866:HLA-DQ beta-Chains; C069051:HLA-DQB1 antigen; D004247:DNA