Abstract
Porphyromonas gingivalis (P. gingivalis) is a major etiological agent associated with periodontitis. This study aims to develop antibodies to P. gingivalis in vitro for real‐time detection of bacteria in clinical samples. Lymphocytes were isolated from whole blood of patient treated for periodontitis and were stimulated with P. gingivalis ATCC 33277. B‐cell maturation to long‐living antibody secreting‐plasma cells was studied using flow cytometry and immunofluorescence staining. The antibodies developed in vitro were immobilized onto a CM‐5 sensor chip of a biosensor to detect the presence of P. gingivalis in the gingival crevicular fluid of patients with periodontitis compared to periodontally healthy controls (n = 30). Surface plasmon resonance (SPR) analysis was performed to evaluate specific interactions of bacteria in samples with the immobilized antibodies. The results of SPR analysis were compared to the detection of P. gingivalis in the samples using DNA–DNA checkerboard hybridization technique. A clear and distinct change in lymphocyte morphology upon stimulation with P. gingivalis was observed. Anti‐P. gingivalis antibodies secreted by CD38+ plasma cells showed the presence of all the four IgG subclasses. The results of DNA–DNA checkerboard analysis were in agreement with that of SPR analysis for the detection of P. gingivalis in patient samples. Furthermore, incubation with anti‐P. gingivalis attenuated the bacterial response in SPR. The in vitro method for antibody production developed during this study could be used for an efficient real‐time detection of periodontitis, and the attenuating effects of in vitro antibodies suggest their role in passive immunization to prevent periodontitis and their associated risk factors.
Highlights
Porphyromonas gingivalis is a gram-negative, black-pigmented anaerobic bacterium and a major etiological agent of periodontitis
Lymphocytes stimulated with P. gingivalis exhibited morphological changes, such as increased size and granulation when compared to unstimulated cells (Fig. 1)
Significant increase in CD19+ (B-cells), CD3+ (T-cells), and CD16/56+ (NK-cells) cells was observed in lymphocyte samples stimulated with P. gingivalis, compared to unstimulated lymphocytes (Fig. 2)
Summary
Porphyromonas gingivalis is a gram-negative, black-pigmented anaerobic bacterium and a major etiological agent of periodontitis. Periodontitis is a chronic inflammatory polymicrobial condition leading to tissue destruction and tooth loss (Kadowaki et al, 2007). P. gingivalis and other periodontopathic bacteria evade the host immune responses by a variety of mechanisms such as interfering with host mechanism to detoxify toxins, inhibit adherence and colonization, increase protease activity degrading host enzymes/proteins, block anti-inflammatory cytokines, and prevent leukocyte migration to the infected site (Gupta and Gupta, 2014; Jain and Darveau, 2010). Virulence factors of P. gingivalis include their fimbriae, capsular polysaccharides, hemagglutinin, lipopolysaccharide, gingipains, outer membrane protein, antigen, and heat shock proteins (Holt et al, 1999). Antigenic specificities are important to elicit antibody responses
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