Functional characteristics and binding pattern toward rADAMTS13 of the 13 human anti-ADAMTS13 whole IgGs generated by EBV transformation of switched memory B cells
| mAbs . | Inhibitory potential assessed by residual ADAMTS13 activity (%) . | Binding pattern toward rADAMTS13 as assesed by . | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Clone . | IgG subclass . | CDR3 motif . | 10 nM . | 100 nM . | KD Biacore (M) . | ELISA 405 nm* . | CLIPS conformational epitope . | Dot-blot reactivity with† . | |
| Full-length . | MDTCS . | ||||||||
| A-1 | IgG4 | 2 | 6.6 | 4.7 | 9.0E-09 | 2.186 | Group 1 | +++ | + |
| A-2 | IgG4 | 2 | 1 | 8.1 | 2.7E-10 | 1.485 | Group 1 | +++ | + |
| A-3 | IgG1 | 1 | 11.0 | 8.5 | 4.0E-09 | 1.304 | Group 1 | +++ | ++ |
| A-5 | IgG4 | 4 | <1 | <1 | 2.8E-09 | 2.817 | Group 1 | +++ | + |
| A-6 | IgG1 | 1 | 100 | 100 | 3.8E-08 | 2.278 | ‡ | − | − |
| B-1 | IgG4 | 2 | 100 | 16.0 | 2.1E-09 | 0.757 | Group 1 | +++ | + |
| B-2 | IgG4 | 5 | 27.4 | 13.7 | 2.8E-08 | 1.090 | Group 3 | ++ | ++ |
| B-3 | IgG1 | 2 | 100 | 20.4 | 2.0E-08 | 1.118 | Group 2 | ++ | + |
| B-4 | IgG1 | 2 | 1 | 4.2 | 4.6E-09 | 1.903 | Group 1 | +++ | ++ |
| B-5 | IgG1 | 6 | 2.4 | 4.4 | 2.9E-09 | 2.255 | ‡ | +++ | + |
| B-6 | IgG1 | 5 | 100 | 5.5 | 4.8E-10 | 0.802 | ‡ | +++ | ++ |
| B-7 | IgG1 | 1 | 22.2 | 5.4 | 4.3E-10 | 1.472 | Group 2 | +++ | +++ |
| B-8 | IgG1 | 4 | 21.5 | 3.2 | 9.5-09 | 1.488 | Group 1 | +++ | + |
| mAbs . | Inhibitory potential assessed by residual ADAMTS13 activity (%) . | Binding pattern toward rADAMTS13 as assesed by . | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Clone . | IgG subclass . | CDR3 motif . | 10 nM . | 100 nM . | KD Biacore (M) . | ELISA 405 nm* . | CLIPS conformational epitope . | Dot-blot reactivity with† . | |
| Full-length . | MDTCS . | ||||||||
| A-1 | IgG4 | 2 | 6.6 | 4.7 | 9.0E-09 | 2.186 | Group 1 | +++ | + |
| A-2 | IgG4 | 2 | 1 | 8.1 | 2.7E-10 | 1.485 | Group 1 | +++ | + |
| A-3 | IgG1 | 1 | 11.0 | 8.5 | 4.0E-09 | 1.304 | Group 1 | +++ | ++ |
| A-5 | IgG4 | 4 | <1 | <1 | 2.8E-09 | 2.817 | Group 1 | +++ | + |
| A-6 | IgG1 | 1 | 100 | 100 | 3.8E-08 | 2.278 | ‡ | − | − |
| B-1 | IgG4 | 2 | 100 | 16.0 | 2.1E-09 | 0.757 | Group 1 | +++ | + |
| B-2 | IgG4 | 5 | 27.4 | 13.7 | 2.8E-08 | 1.090 | Group 3 | ++ | ++ |
| B-3 | IgG1 | 2 | 100 | 20.4 | 2.0E-08 | 1.118 | Group 2 | ++ | + |
| B-4 | IgG1 | 2 | 1 | 4.2 | 4.6E-09 | 1.903 | Group 1 | +++ | ++ |
| B-5 | IgG1 | 6 | 2.4 | 4.4 | 2.9E-09 | 2.255 | ‡ | +++ | + |
| B-6 | IgG1 | 5 | 100 | 5.5 | 4.8E-10 | 0.802 | ‡ | +++ | ++ |
| B-7 | IgG1 | 1 | 22.2 | 5.4 | 4.3E-10 | 1.472 | Group 2 | +++ | +++ |
| B-8 | IgG1 | 4 | 21.5 | 3.2 | 9.5-09 | 1.488 | Group 1 | +++ | + |
Functional tests: inhibitory capacity against plasma-derived ADAMTS13 in a Bethesda-like assay determining the residual ADAMTS13 activity assessed by FRETS-VWF73 (in %). Binding pattern assays included affinity analysis by SPR (Biacore), specificity tests by ELISA (Figure 3), and epitope mapping performed by either conformational CLIPS technology and dot-blot technique on either recombinant full-length or on ADAMTS13 stop mutant (MDTCS-13, consisting of ADAMTS13–metalloprotease–disintegrin–thrombospondin type I repeat number 1–cys-rich and spacer domain MDTCS) (supplemental Materials).
ELISA data in Figure 3.
Dot-blot technique described in supplemental Data.
No binding pattern found by CLIPS technology.