Establishment of K562 cell lines expressing platelet antigen HPA-15 using lentiviral infection method

This study aimed to establish stable K562 cell lines individually overexpressing the HPA-15a and HPA-15b alloantigens for subsequent antigenic functional verification.

A three-plasmid lentiviral system, including CD109 (HPA-15)-GFP, psPAX2, and pMD2.G, was transfected into HEK293T cells to produce lentivirus. K562 cells were infected with the harvested virus, followed by puromycin screening and single-cell cloning. Flow cytometry and Western blot were then performed to identify CD109 expression in cell clones. After blocking IgG Fc receptors on the cell surface, samples with confirmed HPA-15 positive and negative reactivity were applied to characterize the HPA-15 antigen reactivity of established cell clones using a cell-lysis-free MAIPA assay.

Lentiviral packaging in HEK293T cells was verified by GFP fluorescence observation and RT-PCR detection. K562 cell lines stably overexpressing HPA-15a and HPA-15b were successfully generated. Repeated lentiviral infection significantly upregulated HPA-15 expression, with the final expression level approximately 12-fold higher than that in pooled platelets from 10 donors. A strong linear correlation was observed between positive sample volume and MAIPA assay signal intensity. Besides, the assay signals induced by positive samples were markedly higher than those of negative controls, confirming the HPA-15 antigen reactivity of recombinant cell lines.

Stable K562 cell lines individually overexpressing HPA-15a and HPA-15b were successfully constructed. Meanwhile, an optimized cell-lysis-free MAIPA detection system based on IgG Fc receptor pre-blocking, which enabled semi-quantitative detection of HPA-15 alloantibodies, was developed.