Extended Data Figure 3. Lack of pairing between HCAb and co-expressed light chain.
(a) Assay schematic. 293T cells were transfected with expression plasmids for the J3 VHH HCAb, a full-length (FL) conventional human IgG1 derived from Ofatumumab, or its Igκ L chain only (LC), or combinations as indicated. Supernatants, including mixed supernatants as controls, were evaluated by ELISAs based on HIV gp120 binding by the J3 VHH and detection with an anti-VHH antibody (control), or anti-Igκ L chain antibodies to detect pairings between the J3 HCAb and either the FL or LC components. FabALACTICA digestion is expected to cleave FL antibodies into Fab and Fc fragments and may also cleave HCAbs. (b-c) Results of gp120-VHH (b) and gp120-Igκ (c) ELISAs, with or without FabALACTICA digestion (n = 2–4). Cross-pairing was only observed after co-transfection of J3 HCAb and FL antibody, consistent with H chain interactions that were released by FabALACTICA digestion. In contrast, the L chain alone did not pair with the J3 HCAb. Error bars show mean ± SEM. Statistics were performed using 2-way ANOVA. **** p < 0.0001.