Journal: mBio

Article Title: Revisiting an IgG Fc Loss-of-Function Experiment: the Role of Complement in HIV Broadly Neutralizing Antibody b12 Activity

doi: 10.1128/mBio.01743-21

Figure Lengend Snippet: b12 variant panel. (A) Antibody (Ab) IgG1 Fc variants illustrated on the crystal structure of the broadly neutralizing antibody b12 (PDB accession number 1HZH ) by coloration of the component point mutations. The phenotypically diminished variants to the left of b12 are illustrated in red (e.g., LALA and KA), while phenotypically enhanced variants to the right are in blue (e.g., EG and EFTAE). Accompanying the illustrations is a table of expected qualitative C1q and FcγR binding phenotypes and observed affinity of antigen binding (equilibrium [kinetic] dissociation constant [ K D ] values) of each variant to antigen (HIV-1 YU-2 gp140 trimer). (B) Antigen binding profiles determined by biolayer interferometry (BLI) across a range of concentrations. (C) FcγR binding profiles of each variant as determined by staining antigen-conjugated beads with tetramerized receptor. Error bars represent the ranges from two technical replicates.

Article Snippet: For the detection of C1q binding, cells were incubated with mouse anti-C1q (clone JL-1, catalog number HM2382; Hycult Biotech) at 5 μg/ml and washed, and secondary staining was performed with 1:1,000-diluted anti-mouse IgG-Alexa Fluor 647 (catalog number A-21235; Invitrogen).

Techniques: Variant Assay, Binding Assay, Staining

Journal: mBio

Article Title: Revisiting an IgG Fc Loss-of-Function Experiment: the Role of Complement in HIV Broadly Neutralizing Antibody b12 Activity

doi: 10.1128/mBio.01743-21

Figure Lengend Snippet: The ability of b12 to activate complement is influenced by assay setup and antigen context. (A) The antigen-independent ability of the antibody panel to bind C1q (left) and fix complement (right) was determined by ELISAs via antibody-coated wells. (B and C) Beads conjugated with SHIV SF162P3 gp120 (B) or HIV-1 YU-2 gp140 trimer (C) were used to assay antigen binding (left), C1q binding (center), and complement fragment C3d deposition (right). Antigen beads treated with PNGase F were used to assess the impact of N -linked antigen glycosylation antibody-independent activation and to isolate antibody-dependent C3d deposition (shaded). For C3d deposition on non-PNGase F-treated antigen beads, background activity is reported as the average MFI (dotted line) ± standard deviation (shaded region on the y axis) of anti-C3d detected on beads in replicate wells of pooled NHS ( n = 6) in the absence of antibody. Data are representative of results from two independent experiments. AU, arbitrary units.

Article Snippet: For the detection of C1q binding, cells were incubated with mouse anti-C1q (clone JL-1, catalog number HM2382; Hycult Biotech) at 5 μg/ml and washed, and secondary staining was performed with 1:1,000-diluted anti-mouse IgG-Alexa Fluor 647 (catalog number A-21235; Invitrogen).

Techniques: Binding Assay, Activation Assay, Activity Assay, Standard Deviation

Journal: mBio

Article Title: Revisiting an IgG Fc Loss-of-Function Experiment: the Role of Complement in HIV Broadly Neutralizing Antibody b12 Activity

doi: 10.1128/mBio.01743-21

Figure Lengend Snippet: Complement-enhanced Fc variants of IgG1 b12 do not mediate viral lysis despite detectable deposition activity on viral particles. Antibodies were assayed for their ability to bind to the surface of HIV-1 BaL particles bound to lectin-conjugated magnetic beads (A), recruit C1q to the viral surface (B), and affect terminal complement activities, including C3 deposition (C), C5b-9 complex formation (D), and MAC-mediated lysis (E), determined by the detection of released capsid protein p24. Dotted lines represent average baseline complement deposition on beads in wells containing a nonspecific isotype control (A to D) or antibody-independent baseline complement deposition and heat-inactivated NHS (E). Points and error bars represent means ± standard deviations from technical triplicates, respectively. Data are reported as mean or median fluorescence intensities (mFI or MFI, respectively) and are representative of results from two independent experiments. huIgG, human IgG.

Article Snippet: For the detection of C1q binding, cells were incubated with mouse anti-C1q (clone JL-1, catalog number HM2382; Hycult Biotech) at 5 μg/ml and washed, and secondary staining was performed with 1:1,000-diluted anti-mouse IgG-Alexa Fluor 647 (catalog number A-21235; Invitrogen).

Techniques: Lysis, Activity Assay, Magnetic Beads, Fluorescence

Journal: mBio

Article Title: Revisiting an IgG Fc Loss-of-Function Experiment: the Role of Complement in HIV Broadly Neutralizing Antibody b12 Activity

doi: 10.1128/mBio.01743-21

Figure Lengend Snippet: IgG1 b12 does not measurably direct complement activity against Env-expressing cells, while the CDC activity of enhanced Fc variants depends on the target cell. Antibodies were assayed for their abilities to bind to the surface of HIV-1 JR-FL gp140 transiently expressed on HEK293F cells (A), recruit C1q to HEK JR-FL cells (B), and affect terminal complement activities targeting the antibody-opsonized HEK JR-FL cell surface, including C3 deposition (C), C5b-9 complex formation (D), and MAC-mediated lysis of two distinct Env-expressing cell models (E). Dotted lines represent average baseline complement deposition on cells in wells lacking antibody (A to D). Points and error bars represent means from technical triplicates (A to D and E, top) or from three serum donors (E, bottom) and standard deviations, respectively. Data are representative of results from at least two independent experiments.

Article Snippet: For the detection of C1q binding, cells were incubated with mouse anti-C1q (clone JL-1, catalog number HM2382; Hycult Biotech) at 5 μg/ml and washed, and secondary staining was performed with 1:1,000-diluted anti-mouse IgG-Alexa Fluor 647 (catalog number A-21235; Invitrogen).

Techniques: Activity Assay, Expressing, Lysis