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Human platelet antigen genotyping by using sequence‐specific primers and the particle gel agglutination assay
Author(s) -
Meyer O.,
AbouChaker K.,
Heymann G.,
Bombard S.,
Kiesewetter H.,
Salama A.
Publication year - 2005
Publication title -
vox sanguinis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.68
H-Index - 83
eISSN - 1423-0410
pISSN - 0042-9007
DOI - 10.1111/j.1423-0410.2005.00630.x
Subject(s) - agglutination (biology) , biotinylation , microbiology and biotechnology , streptavidin , fluorescein isothiocyanate , genotyping , polymerase chain reaction , biology , fluorescein , chemistry , antigen , gene , biotin , fluorescence , biochemistry , genetics , physics , quantum mechanics , genotype
Background and Objectives Polymerase chain reaction using sequence‐specific primers (PCR–SSP) is currently the most widely used technique for human platelet antigen (HPA) genotyping. Here, we describe a novel particle gel‐agglutination technique for simplified visualization of the amplified products. Materials and Methods Biotinylated primers were used to amplify HPA‐1, ‐2, ‐3, ‐4, ‐5, ‐6, and ‐15, and the PCR products were incubated with streptavidin particles. Fluorescein isothiocyanate (FITC)‐labelled primers [amplifying a fragment of the human growth hormone ( HGH ) gene] and anti‐FITC‐coated particles were used as internal controls. Agglutination of the particles in or on top of the gel indicated specific amplification. A total of 100 samples from blood donors was tested by using this new technique and a standard PCR–SSP protocol. Results The use of biotinylated sequence‐specific primers resulted in PCR products that agglutinated streptavidin particles, and the FITC‐labelled HGH primers led to agglutination of anti‐FITC‐coated particles. Negative reactions were clearly distinguishable from positive reactions. The results of the particle gel agglutination method were in concordance with those of the electrophoretic visualization in all cases tested. Conclusions The new particle agglutination method is reliable and easy to use.