Journal: Journal of Oral Microbiology

Article Title: Anti-FimA monoclonal antibodies reveal dual roles in porphyromonas gingivalis pathogenesis

doi: 10.1080/20002297.2026.2729552

Figure Lengend Snippet: Expression of FimA protein and generation of mAbs targeting FimA of P. gingivalis . (A) SDS-PAGE analysis of purified of recombinant FimA-His (rFimA) protein. (B) Schematic of the immunisation protocol. (C) Determination of serum anti-FimA antibody titre by ELISA using rFimA-coated ELISA plates. (D) ELISA-based detection of anti-FimA mAbs in culture supernatants from five hybridoma cell lines. (E) SDS-PAGE analysis of five anti-FimA mAbs purified from mouse ascites via protein A affinity chromatography. (F) Isotype determination of anti-FimA mAbs by ELISA using HRP-conjugated antibodies specific for mouse IgM, IgG1, IgG2a, IgG2b, IgG3, IgA, κ , and λ chains.

Article Snippet: The P. gingivalis FimA-deficient (ΔfimA) mutant in the ATCC 33277 background was constructed as previously described with minor modifications [ ].

Techniques: Expressing, SDS Page, Analysis, Purification, Recombinant, Enzyme-linked Immunosorbent Assay, Affinity Chromatography

Journal: Journal of Oral Microbiology

Article Title: Anti-FimA monoclonal antibodies reveal dual roles in porphyromonas gingivalis pathogenesis

doi: 10.1080/20002297.2026.2729552

Figure Lengend Snippet: Binding analysis of anti-FimA mAbs to FimA. (A) Binding kinetics of anti-FimA mAbs to rFimA protein determined by biolayer interferometry (BLI). Sensorgrams show a dilution series of antibodies (200 to 12.5 nM), with data fitted globally using a 1:1 binding model. (B) Representative ELISA binding curves of the mAbs to immobilised rFimA protein. Bound antibodies were detected with HRP-conjugated anti-mouse IgG secondary antibody and reported as absorbance at 450 nm. (C) Summary of binding affinity (KD), association rate (ka), dissociation rate (kdis), and half-maximal effective concentration (EC50) values for each mAb. (D) Representative immunofluorescence images of mAb binding to P. gingivalis cells FimA fimbriae. Bacterial DNA was stained with Hoechst (blue). Cells were incubated with control IgG or anti-FimA mAbs, followed by Alexa Fluor 546-conjugated anti-mouse IgG antibody (red). Merged images are shown in the bottom panel. Scale bar, 2 μm.

Article Snippet: The P. gingivalis FimA-deficient (ΔfimA) mutant in the ATCC 33277 background was constructed as previously described with minor modifications [ ].

Techniques: Binding Assay, Analysis, Enzyme-linked Immunosorbent Assay, Association Rate Constant, Dissociation Rate Constant, Concentration Assay, Immunofluorescence, Staining, Incubation, Control

Journal: Journal of Oral Microbiology

Article Title: Anti-FimA monoclonal antibodies reveal dual roles in porphyromonas gingivalis pathogenesis

doi: 10.1080/20002297.2026.2729552

Figure Lengend Snippet: Specificity and epitope diversity of anti‑FimA mAbs. (A) Western blot analysis of anti-FimA mAbs using whole-cell lysates of P. gingivalis and other bacteria; rFimA protein was included as a positive control. (B) Cross-reactivity of anti-FimA mAbs assessed by ELISA. (C-D) Flow cytometry analysis of mAbs binding to P. gingivalis ATCC33277 (C) and other bacteria (D) compared with PBS (red) and isotype IgG (black) controls. Median fluorescence intensity (MFI) was determined from three independent experiments (mean ± SD; one‑way ANOVA with Dunnett’s test, ***p < 0.001). (E) Epitope binning by BLI competition assay. (F) Summary of BLI-based competitive binding results among the five anti-FimA mAbs.

Article Snippet: The P. gingivalis FimA-deficient (ΔfimA) mutant in the ATCC 33277 background was constructed as previously described with minor modifications [ ].

Techniques: Western Blot, Analysis, Bacteria, Positive Control, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Binding Assay, Fluorescence, Epitope Binning, Competitive Binding Assay

Journal: Journal of Oral Microbiology

Article Title: Anti-FimA monoclonal antibodies reveal dual roles in porphyromonas gingivalis pathogenesis

doi: 10.1080/20002297.2026.2729552

Figure Lengend Snippet: Anti-FimA mAbs inhibit P. gingivalis adhesion. (A) SEM images of P. gingivalis adhesion to sHA beads following treatment with control IgG or anti-FimA mAbs. Scale bar, 5 µm. (B) qPCR quantification of bound P. gingivalis 16S rRNA gene copies on sHA beads. (C) Fluorescence microscopy images of DilC18(3)-labelled P. gingivalis (red) binding to SYTO9 stained S. gordonii biofilms (green) following pre-treatment with control IgG or anti-FimA mAbs. Scale bar, 200 µm. (D) Quantification of adhesion was performed in ImageJ based on the area of red fluorescence (Dil) from P. gingivalis colocalized with the green biofilm. Data are presented as mean ± SD and were analysed by one‑way ANOVA with Dunnett’s test, ***p < 0.001.

Article Snippet: The P. gingivalis FimA-deficient (ΔfimA) mutant in the ATCC 33277 background was constructed as previously described with minor modifications [ ].

Techniques: Control, Fluorescence, Microscopy, Binding Assay, Staining

Journal: Journal of Oral Microbiology

Article Title: Anti-FimA monoclonal antibodies reveal dual roles in porphyromonas gingivalis pathogenesis

doi: 10.1080/20002297.2026.2729552

Figure Lengend Snippet: Functional assessment of anti‑FimA mAbs in complement‑mediated killing and phagocytosis of P. gingivalis . (A−B) Complement‑mediated killing assay. P. gingivalis was incubated with guinea pig complement serum in the presence of anti‑FimA mAbs, isotype control IgG (100 µg/mL), or without antibody (Mock). (A) Bacterial suspensions were stained with a LIVE/DEAD viability kit; the percentage of live versus dead cells was quantified based on fluorescent signal using ImageJ software. (B) Bacterial survival was determined by colony-forming unit (CFU) counts and expressed as a percentage of the IgG control. (C-G) Macrophage phagocytosis assay. FITC‑labelled P. gingivalis was pre‑treated with anti‑FimA mAbs or IgG control and then co‑cultured with macrophages at an MOI of 100. (C) Representative fluorescence microscopy images showing macrophages (F-actin, red; nuclei, blue) with ingested bacteria (green). Scale bar, 200 µm. (D) Quantitative analysis based on fluorescent signal area using ImageJ software. (E) Flow cytometry analysis of FITC fluorescence in macrophages, showing the percentage of FITC-positive cells. (F) Percentage of macrophages with internalised bacteria. (G) Phagocytic Index defined as (percentage of macrophages with internalised bacteria) × (mean fluorescence intensity, MFI)/100. All data are presented as mean ± SD from at least three independent experiments and were analysed by one‑way ANOVA with Dunnett’s test ( *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant).

Article Snippet: The P. gingivalis FimA-deficient (ΔfimA) mutant in the ATCC 33277 background was constructed as previously described with minor modifications [ ].

Techniques: Functional Assay, Incubation, Control, Staining, Software, Phagocytosis Assay, Fluorescence, Microscopy, Bacteria, Analysis, Flow Cytometry

Journal: Journal of Oral Microbiology

Article Title: Anti-FimA monoclonal antibodies reveal dual roles in porphyromonas gingivalis pathogenesis

doi: 10.1080/20002297.2026.2729552

Figure Lengend Snippet: Anti‑FimA mAbs induce auto-aggregation and modulate biofilm formation in P. gingivalis . (A) Auto‑aggregation of P. gingivalis induced by anti‑FimA mAbs. Bacteria were treated with the indicated mAbs or isotype control IgG (100 µg/mL) for 2 h at room temperature. Representative images show Gram‑stained bacterial aggregates visualised by light microscopy (Top) and macroscopic appearance (Bottom). (B) Aggregation kinetics monitored by the decrease in OD600 of the culture supernatant over 120 min. Data are normalised to the initial OD600 value (set as 100%). (C) Representative images of crystal violet‑stained biofilms formed in the presence of control IgG or anti‑FimA mAbs. (D) Biofilm biomass was quantified by absorbance at 600 nm. (F) SEM micrographs showing P. gingivalis biofilms formed after 48 h of exposure to control IgG or different anti‑FimA mAbs. All data are mean ± SD from three independent experiments and were analysed by one-way ANOVA analysis with Dunnett’s test ( * * * p < 0.001).

Article Snippet: The P. gingivalis FimA-deficient (ΔfimA) mutant in the ATCC 33277 background was constructed as previously described with minor modifications [ ].

Techniques: Bacteria, Control, Light Microscopy, Analysis