Microfluidic antibody affinity profiling of alloantibody-HLA interactions in human serum

Schneider, Matthias M.; Scheidt, Tom; Priddey, Ashley J.; Xu, Catherine K.; Hu, Mengsha; Meisl, Georg; Devenish, SeanR.A.; Dobson, Christopher M.; Kosmoliaptsis, Vasilis; Knowles, Tuomas P. J.

doi:10.1016/j.bios.2023.115196

Cited by 11 publications

(30 citation statements)

References 59 publications

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“…These results demonstrate that previously reported affinity differences between the two mAbs are reproducible. 2 and their obtained affinity results in Table 3, together with previously reported affinities 26,27,29,50 ). Comparison between solid-phase assay-and in-solution assay-derived results confirmed overall higher K D -values (i.e.…”

Section: Graphical 3d Representation Of Hla Moleculessupporting

confidence: 72%

“…To date, several biophysical methods have been used for determination of HLA-antibody affinity: Surface Plasmon Resonance (SPR), [24][25][26][27] Bio-Layer Interferometry (BLI), 28,29 Microfluidic Diffusional Sizing (MDS)/Microfluidic Antibody Affinity Profiling (MAAP) 29,30 and SAB assays. 26,31 Their main characteristics are summarized in Table 1.…”

Section: Introductionmentioning

confidence: 99%

“…However, the few publications mainly provide data on HLA-specific monoclonal antibodies (mAbs). 26,27,29 Determination of HLA antibody affinity in patient serum is more challenging. While Visentin et al spiked HLA-specific mAbs into negative control serum to mimic the usual matrix for their SPR experiments, 27 Schneider et al applied real patient serum samples for their MAAP measurements but only provided affinity values from four allo-antibodies.…”

Section: Introductionmentioning

confidence: 99%

“…While Visentin et al spiked HLA-specific mAbs into negative control serum to mimic the usual matrix for their SPR experiments, 27 Schneider et al applied real patient serum samples for their MAAP measurements but only provided affinity values from four allo-antibodies. 29 Immobilization-free ligand binding technologies for in-solution analysis of size, quantity and binding properties of pre-labeled fluorescent biomolecules offer several advantages for antibody affinity determination, especially for complex matrices such as patient serum. Some of the benefits of these assays are a better control of antigen concentration and antigen accessibility, a relatively short experimental hands-on time, and comparatively low costs.…”

Section: Introductionmentioning

confidence: 99%

“…Furthermore, serum samples do not have to be pre-treated as the method is less vulnerable to complement-interference known to cause false-negative results in solid-phase binding assays such as SAB. 32,33 Particularly noteworthy are two methodologies/platforms that characterize antibody-antigen binding using the M approach described above: MDS 34 /MAAP) 29,30 and Flowinduced dispersion analysis (FIDA). 35 In this study, we used the latter technology/platform to determine HLA antibody affinity.…”

Section: Introductionmentioning

confidence: 99%

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HLA antibody affinity determination: From HLA‐specific monoclonal antibodies to donor HLA specific antibodies (DSA) in patient serum

Hug,

Keller,

Marty

et al. 2023

HLA

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Organs transplanted across donor‐specific HLA antibodies (DSA) are associated with a variety of clinical outcomes, including a high risk of acute kidney graft rejection. Unfortunately, the currently available assays to determine DSA characteristics are insufficient to clearly discriminate between potentially harmless and harmful DSA. To further explore the hazard potential of DSA, their concentration and binding strength to their natural target, using soluble HLA, may be informative. There are currently a number of biophysical technologies available that allow the assessment of antibody binding strength. However, these methods require prior knowledge of antibody concentrations. Our objective within this study was to develop a novel approach that combines the determination of DSA‐affinity as well as DSA‐concentration for patient sample evaluation within one assay. We initially tested the reproducibility of previously reported affinities of human HLA‐specific monoclonal antibodies and assessed the technology‐specific precision of the obtained results on multiple platforms, including surface plasmon resonance (SPR), bio‐layer interferometry (BLI), Luminex (single antigen beads; SAB), and flow‐induced dispersion analysis (FIDA). While the first three (solid‐phase) technologies revealed comparable high binding‐strengths, suggesting measurement of avidity, the latter (in‐solution) approach revealed slightly lower binding‐strengths, presumably indicating measurement of affinity. We believe that our newly developed in‐solution FIDA‐assay is particularly suitable to provide useful clinical information by not just measuring DSA‐affinities in patient serum samples but simultaneously delivering a particular DSA‐concentration. Here, we investigated DSA from 20 pre‐transplant patients, all of whom showed negative CDC‐crossmatch results with donor cells and SAB signals ranging between 571 and 14899 mean fluorescence intensity (MFI). DSA‐concentrations were found in the range between 11.2 and 1223 nM (median 81.1 nM), and their measured affinities fall between 0.055 and 24.7 nM (median 5.34 nM; 449‐fold difference). In 13 of 20 sera (65%), DSA accounted for more than 0.1% of total serum antibodies, and 4/20 sera (20%) revealed a proportion of DSA even higher than 1%. To conclude, this study strengthens the presumption that pre‐transplant patient DSA consists of various concentrations and different net affinities. Validation of these results in a larger patient cohort with clinical outcomes will be essential in a further step to assess the clinical relevance of DSA‐concentration and DSA‐affinity.

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Section: Graphical 3d Representation Of Hla Moleculessupporting

confidence: 72%