Journal: bioRxiv

Article Title: Human Fcγ-receptors selectively respond to C-reactive protein (CRP) isoforms

doi: 10.1101/2025.03.23.644781

Figure Lengend Snippet: ( A ) Upper: mIL-2 levels produced by BW5147 cells on immobilized IgG1 (titrated from 0.1 to 0.025 µg in 50 µl medium). Each cell line was stably transduced with one FcγR only. Cell medium without BW cells (‘blank’) served as a negative control. Center/lower: mIL-2-levels produced by BW5147 reporter cells on immobilized pCRP (center) and mCRP (lower) (titrated from 2.5 to 0.63 µg in 50 µl medium, with concentrations matched using Qubit Fluorometric Quantitation). A total of 100,000 BW 5147 reporter cells were added to each well in 200 µl RPMI BW medium and incubated overnight at 37°C 5% CO 2 . Data are shown in technical replicates (N=3) with standard deviation for one representative of at least three individual experiments for each cell line. Activation shown as OD in a sandwich mIL-2-ELISA. ( B ) Left: AUCs for activation of BW cells by immobilized IgG1, pCRP and mCRP after normalization of ODs to mean OD of individual experiment. AUCs were calculated and jointly analyzed for three independent experiments normalized to mean OD of individual experiment with three technical replicates each. Two-way ANOVA and Dunnett’s multiple comparisons calculated using GraphPad Prism software. Right: Spider web plot of AUCs normalized to mean OD of individual experiment, created using Microsoft Excel Graph Software. ( C ) Upper : Schematic indicating recognition by conformation-specific monoclonal and polyclonal anti-CRP antibodies. Middle /lower : pCRP/mCRP was titrated from 1 µg to 0.1 µg/well and coated to MaxiSorp wells. Concentrations of pCRP and mCRP preparations were matched using Qubit Fluorometric Quantitation. CRP was detected using conformation specific 8D8 (anti-pCRP), 9C9 (anti-mCRP/pCRP*) and polyclonal goat anti-hCRP antibody.

Article Snippet: Following the same blocking and washing steps as described above, IgG1, pCRP, or mCRP were added in 50 µl ELISA blocking buffer, and binding was detected using goat anti-hCRP antibody (1:3,000; 1 mg/ml; Bethyl: A80-125A) and donkey anti-goat (DAG) POD-conjugated antibody (DAG-POD; 1:5,000; 2,5mg/ml; Invitrogen, Waltham, Massachusetts, USA: A16005) for CRP (pCRP and mCRP) and goat anti-human-IgG-POD (1:3,000, 1 mg/ml; Rockland Immunochemical, Philadelphia, Pennsylvania, USA,, #109-035-003) for IgG1.

Techniques: Produced, Stable Transfection, Transduction, Negative Control, Quantitation Assay, Incubation, Standard Deviation, Activation Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: bioRxiv

Article Title: Human Fcγ-receptors selectively respond to C-reactive protein (CRP) isoforms

doi: 10.1101/2025.03.23.644781

Figure Lengend Snippet: ( A ) MaxiSorp ELISA plates were saturated with 10% FCS. sICs as well as soluble CRP-streptococci complexes (with S. pneumoniae serotype 27) were allowed to incubate for two hours at RT prior to adding them to the experiment. Upper : sICs were added in 100 µl/well medium and consisted of 25 nM Infliximab (149.1 kDa) and 50 nM TNFα monomer (17.5 kDa) to ensure 1:1 stoichiometry (per ml stock of 25 nM ICs: 0.875 µg TNFα + 2.66 µg Infliximab). Selected values of log2 titration depicted in this graph. Central: pCRP in solution assay without pre-incubation with streptococci. CRP was added in 100 µl medium. Lower: 10 µl of streptococci were added to 20/10/5 µg of CRP. Complexes were added to wells in 100 µl medium. 100,000 BW5147 reporter cells were added to each well in another 100 µl of medium. Activation shown as OD in sandwich mIL-2-ELISA. Data are shown with standard deviation (N=2; N=1 for IC controls). ( B ) BWCD64 activation assay comparing coated pCRP and soluble pCRP/ soluble pCRP-streptococci complexes (N=2). Ordinary one-way ANOVA and Tukeýs multiple comparisons test carried out using GraphPad Prism software and selected significances are indicated on the graph. ( C ) Titration of His-tagged hFcγRs from 0.25 µg to 0 µg and coating to ELISA wells. Addition of 0.1 µg IgG1 ( upper ), pCRP ( central ) or mCRP ( lower ) and detection via goat-anti-hCRP antibody and DAG-POD for CRP and anti-human-IgG-POD for IgG1. ODs for 450-620 nm. Data shown with standard deviation for two individual experiments with three technical replicates each. ( D ) Coating of goat F(ab) 2 anti-human IgG (Fab-specific) (0.1 µg in 50 µl/well) was followed by blocking and addition of hIgG1 (0.25 µg in 50 µl/well) before addition of soluble human FcyR-His-proteins titrated as stated in the graph. Detection with rabbit anti-His antibody and GAR-POD was performed. Data shown in technical triplicates for two individual experiments.

Article Snippet: Following the same blocking and washing steps as described above, IgG1, pCRP, or mCRP were added in 50 µl ELISA blocking buffer, and binding was detected using goat anti-hCRP antibody (1:3,000; 1 mg/ml; Bethyl: A80-125A) and donkey anti-goat (DAG) POD-conjugated antibody (DAG-POD; 1:5,000; 2,5mg/ml; Invitrogen, Waltham, Massachusetts, USA: A16005) for CRP (pCRP and mCRP) and goat anti-human-IgG-POD (1:3,000, 1 mg/ml; Rockland Immunochemical, Philadelphia, Pennsylvania, USA,, #109-035-003) for IgG1.

Techniques: Enzyme-linked Immunosorbent Assay, Titration, Incubation, Activation Assay, Standard Deviation, Software, Blocking Assay