A novel simple assay system for the detection of human platelet antigen 15 (HPA‐15) alloantibodies based on three techniques: an HPA‐15 expressing cell line, a monoclonal antibody‐specific antigen‐capture method and mixed‐passive haemagglutination

摘要

Background and objectives To detect HPA‐15 alloantibodies, we previously developed a human platelet antigen 15 (HPA‐15)‐expressing cell line‐based modified rapid monoclonal antibody immobilization of platelet antigen (CL‐MR‐MAIPA) assay. In this study, the protocol was modified for easier performance by introducing the mixed‐passive haemagglutination (MPHA) principle. Material and methods In total, 20 samples that tested negative for HPA alloantibodies and eight that tested positive for HPA‐15 alloantibodies (two and six positive for HPA‐15a and HPA‐15b antibodies, respectively) by CL‐MR‐MAIPA assay were used in this study. HPA‐15 cell lines were incubated with serum/plasma and then solubilized. The lysate was transferred to a round‐bottom well, which was coated with anti‐human CD109 monoclonal antibodies. After incubation and repeated washings, sheep red blood cells, coated with anti‐human IgG, were added to the wells. Haemagglutination was assessed the next day. Results The proposed cell line‐based immune complex capture‐dependent mixed‐passive haemagglutination (CL‐IC‐MPHA) assay consisted of four steps, but required only 2?h to perform, except for overnight incubation for haemagglutination. Two HPA‐15a alloantibody samples were reactive only for HPA‐15a cells, and six HPA‐15b alloantibody samples were reactive only for HPA‐15b cells with the CL‐IC‐MPHA assay. The 20 samples that tested negative for HPA alloantibodies did not react with HPA‐15a or HPA‐15b cells. These data indicated that the CL‐IC‐MPHA assay was highly specific and sensitive. Unfortunately, the CL‐IC‐MPHA assay's analytic sensitivity was twofold to eightfold lower than that of the CL‐MR‐MAIPA assay. Conclusion A novel, easy‐to‐perform protocol was successfully developed to detect HPA‐15 alloantibodies with high specificity and sensitivity.