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Application of bead array technology to simultaneous detection of human leucocyte antigen and human platelet antigen antibodies

2008; Wiley; Volume: 96; Issue: 3 Linguagem: Inglês

10.1111/j.1423-0410.2008.01140.x

1423-0410

Koki Fujiwara, K. Shimano, Hidenori Tanaka, Michiko Sekine, Koichi Kashiwase, Makoto Uchikawa, Masahiro Satake, Kazunori Nakajima,

Monoclonal and Polyclonal Antibodies Research

Background Detection of antibodies against human leucocyte antigens (HLA) and human platelet antigens (HPA) is crucial for patients refractory to platelet transfusion therapy. However, a reliable and high‐throughput method for HLA cross‐matching and detecting HPA antibodies has not yet been described. Study Design and Methods Immunocomplex capture fluorescence analysis (ICFA) was developed for high‐throughput, simultaneous detection of HLA and HPA antibodies. Microarray beads were separately coupled with monoclonal antibodies specific for CD36, CD41, CD42b, CD49b, CD61 and HLA class I antigens. Platelets reacting with patient serum were lysed and the lysates were incubated with the bead mixture to specifically capture antigen–antibody complexes via the epitopes on platelet glycoproteins or HLA antigens. The beads capturing immunocomplexes were then subjected to flow cytometric analysis. Results Immunocomplex capture fluorescence analysis was validated using 50 serum samples containing HLA antibodies and 20 serum samples containing HPA antibodies. The method enabled the detection of all the HLA antibodies with a sensitivity comparable to that of the purified HLA antigen‐coated pooled‐bead assay (FlowPRA, One Lambda, Canoga Park, CA, USA). The method also enabled the detection of all the HPA antibodies with a sensitivity higher than that of the mixed passive haemagglutination. Conclusion In this study, we developed a rapid, simple and reliable method for the simultaneous analysis of HLA and HPA antibodies. ICFA can also be used as an alternative to the lymphocyte cytotoxicity test for HLA cross‐matching.