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Updated: Nov 4, 2025
HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL
Published on: April 11, 2011
Establishment of a novel cell-based assay using HLA-transfected cells to detect HLA antibodies
Manabu Nakano1, Daisuke Takahashi2, Toru Miyazaki1
1Japanese Red Cross Hokkaido Block Blood Center, Sapporo, Hokkaido, Japan.
Insights
This study introduces a modified immunocomplex capture fluorescence analysis (m-ICFA) using HLA-A transfected cells. This method enhances HLA antibody detection, improving accuracy and accessibility for clinical laboratories.
Area of Science:
- Immunogenetics
- Transplantation immunology
- Clinical diagnostics
Background:
- Detection of Human Leukocyte Antigen (HLA) antibodies is crucial for managing conditions like platelet transfusion refractoriness and transfusion-related lung injury.
- Conventional HLA detection methods, such as immunocomplex capture fluorescence analysis (ICFA), face challenges due to difficulties in preparing intact cell panels.
- Peripheral blood mononuclear cells (PBMC) are often used but present preparation complexities.
Purpose of the Study:
- To develop a modified ICFA (m-ICFA) using engineered HEK293 cells stably expressing the HLA-A locus.
- To evaluate the reactivity, sensitivity, and stability of these HLA-A transfectants for HLA antibody detection.
- To assess the potential of m-ICFA to overcome limitations of existing capture assays.
Main Methods:
- Developed HEK293 cell lines stably transfected with the HLA-A locus.
- Utilized these transfectants as targets in a modified ICFA (m-ICFA) protocol.
- Compared the performance of m-ICFA with conventional methods (WAKFlow® ICFA) and assessed antigenicity and stability of transfectants.
Main Results:
- All 20 tested anti-HLA-A antisera showed reactivity with the transfectants, confirming correct cell surface expression and antigenicity.
- HLA-A transfectants demonstrated excellent stability, with similar expression levels after 6 years of cryopreservation.
- m-ICFA exhibited higher reaction indices compared to WAKFlow® ICFA and showed potential to avoid false-negative reactions common in other capture assays.
Conclusions:
- The developed m-ICFA using HLA-A transfectants is a sensitive, stable, and reliable method for HLA antibody detection.
- This approach can potentially mitigate false-negative results seen in enzyme-linked immunosorbent assays (ELISA) and other capture assays.
- m-ICFA offers a practical alternative for clinical laboratories, as it does not require specialized cell culture facilities.
Abstract:
The detection of HLA antibodies is important in clinical practice, such as platelet transfusion refractoriness and transfusion-related lung injury. However, difficulties are associated with the preparation of panel cells for conventional HLA detection systems using intact cells, such as the immunocomplex capture fluorescence analysis (ICFA). Based on an ICFA analysis, HEK293 cells stably transfected with the HLA-A locus were used instead of peripheral blood mononuclear cells (PBMC). The reactivity, sensitivity, and stability of transfectants were examined. All 20 antisera to HLA-A identified by LABScreen® Single Antigen class I (LS-SA1) were reactive to our modified-ICFA (m-ICFA) and showed the same specificities as those in LS-SA1, indicating the cell surface expression and correct antigenicity of the HLA-A locus in transfectants. The expression of HLA class I antigens was similar between transfectants frozen for 6 years and those prior to freezing. In the reaction of the anti-A24 or anti-A33 antibody vs each transfectant, the index of m-ICFA was higher than that of WAKFlow® ICFA. Our m-ICFA also showed that false negative reactions sometimes observed in capture assays may be avoided. By using HLA-A transfectants as ICFA targets, we herein developed m-ICFA. Our m-ICFA may avoid false negative reactions of capture assay like enzyme-linked immunosorbent assay and can also be carried out in almost any laboratory without cell culture facilities.
