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Visentin, J.; Leu, D. le; Mulder, A.; Jambon, F.; Badier, L.; Lee, J.H.; ... ; Taupin, J.L. (2019)

Measuring anti-HLA antibody active concentration and affinity by surface plasmon resonance: Comparison with the luminex single antigen flow beads and T-cell flow cytometry crossmatch results

Article / Letter to editor
Background: Although the Luminex single antigen flow beads (SAFB) and the flow cytometry crossmatch (FCXM) are the most sensitive assays used for anti-HLA antibodies characterization in transplant recipients, their semi quantitative fluorescence read-out is not closely linked to graft outcome.Methods: Surface plasmon resonance (SPR) was implemented to determine truly quantitative parameters of five human monoclonal anti-class I HLA antibodies (mAbs): first the active concentration and then the binding constants. The results were compared to those obtained with SAFB and T-cell FCXM (T-FCXM).Results: The five mAbs displayed different rate and equilibrium constants for their cognate antigens. No correlation was evidenced between SAFB MFI or T-FCXM ratio and the binding parameters measured by SPR. Some mAbs amino acid substitutions within the epitope that influenced SAFE MFI resulted in affinity variations evidenced by SPR.Conclusion: The SAFB MFI and T-FCXM ratio, both semi...Show moreBackground: Although the Luminex single antigen flow beads (SAFB) and the flow cytometry crossmatch (FCXM) are the most sensitive assays used for anti-HLA antibodies characterization in transplant recipients, their semi quantitative fluorescence read-out is not closely linked to graft outcome.Methods: Surface plasmon resonance (SPR) was implemented to determine truly quantitative parameters of five human monoclonal anti-class I HLA antibodies (mAbs): first the active concentration and then the binding constants. The results were compared to those obtained with SAFB and T-cell FCXM (T-FCXM).Results: The five mAbs displayed different rate and equilibrium constants for their cognate antigens. No correlation was evidenced between SAFB MFI or T-FCXM ratio and the binding parameters measured by SPR. Some mAbs amino acid substitutions within the epitope that influenced SAFE MFI resulted in affinity variations evidenced by SPR.Conclusion: The SAFB MFI and T-FCXM ratio, both semi-quantitative parameters, only partially reflected the subtlety of the anti-HLA antibody/antigen interaction as it can be analyzed by SPR. Future clinical studies using SPR for anti-HLA antibodies characterization could bring novel insights into the understanding of HLA/anti-HLA interaction and therefore anti-HLA antibodies pathogenicity.Show less
Anti-HLA antibody
Surface plasmon resonance
Single antigen flow beads
Flow cytometry crossmatch
Active concentration
Affinity
All authors
Visentin, J.; Leu, D. le; Mulder, A.; Jambon, F.; Badier, L.; Lee, J.H.; Guidicelli, G.; Bouthemy, C.; Ralazamahaleo, M.; Claas, F.; Primo, C. di; Taupin, J.L.
Date
2019-04-01
Journal
Molecular Immunology
Volume
108
Pages
34 - 44
DOI
doi:10.1016/j.molimm.2019.02.006