Aim: Human leukocyte antigens (HLA) are polymorphic proteins involved in cell surface recognition and complement activation. In transplantation, antibodies against HLA (i.e. donor specific antibodies [DSA]), can be detrimental for allograft survival. However, some DSA-positive graft recipients do not experience organ rejection. Kinetic characterization and affinity measurements of these antibodies is of crucial importance. Here we report a study in which the kinetic profiles of four different monoclonal antibodies (mAb1-mAb4) are very different despite their similar MFI signals on single antigen beads (SAB). Comparisons of antibody kinetics may help stratify DSA by their allele complex lifetime. We seek to understand if this technology can further characterize DSA in clinical settings.
Methods: HLA alleles, antibodies, and treated sera were obtained from One Lambda, Inc. (a part of Thermo Fisher) (West Hills, CA, U.S.A). We used surface plasmon resonance to characterize antibody association/dissociation from each allele.
Results: Multiple HLA alleles were printed to chips to measure their activity. W6/32 antibodies were used to test allele function. Subsequently, studies with mAb1-mAb4 were performed. Significant differences in mAb1-mAb4 dissociation rates from HLA alleles were observed by SPR, while SAB assays indicated similar MFI readings. Affinity measurements showed a range of binding strengths. The mAb1 and mAb2 antibodies, were also spiked into patient’s sera, showing identical kinetic profiles within the sera matrix. Treated sera samples did not show significant background adherence to non-HLA printed chip spots. These observations suggest that HLA chip arrays may allow for measuring the kinetics of many alleles and allow for high throughput.
Conclusion: This study points out the importance of understanding the detrimental characteristics of DSA. It will be important to establish whether there is a clinical connection between antibody off-rates and allograft rejection. Our work supports that SAB assays are crucial for antibody detection, but SPR assays may resolve kinetic characteristics of the corresponding pathogenic antibodies.
