Confident identification of citrullinated peptides. Abbreviations: (OMV) outer membrane vesicles, (WT) P. gingivalis W83 wild-type, (ΔPPAD) W83 knockout mutant of peptidyl arginine deiminase, (C351A) W83 expressing PPAD with the active site cysteine mutated to alanine, (2D) two-dimensional HFBA-based, (LC-MS) liquid chromatography mass spectrometry, (PAD) humane peptidyl arginine deiminase, (PPAD) bacterial P. gingivalis peptidyl arginine deiminase, (RA) rheumatoid arthritis, (PGE2) prostaglandin E2, (ACPA) anti-citrullinated protein antibodies, (RgpA/B) Argspecific gingipains, (MS) mass spectrometry, (HFBA) heptafluorobutyric acid, (FA) formic acid, (HPLC) high performance liquid chromatography, (AAA) amino acid analysis, (mgf) Mascot Generic File format, (mgx) Mascot Generic eXtended, (RT) retention time, (TIC) total ion count, (T9SS) type IX secretion system,

Porphyromonas gingivalis is one of the major periodontitis-causing pathogens. P. gingivalis secretes a group of proteases, termed gingipains, and in this study we have utilized the RgpB gingipain as a biomarker for P. gingivalis. We constructed a naïve camel nanobody library and used phage display to select one nanobody towards RgpB with picomolar affinity. The nanobody was used in an inhibition assay for detection of RgpB in buffer as well as in saliva. The nanobody was highly specific for RgpB as it did not bind to the homologous gingipain HRgpA. This indicated a presence of a binding epitope within the immunoglobulin-like domain of RgpB. A subtractive inhibition assay was used to demonstrate that the nanobody could bind native RgpB in the context of intact cells. The nanobody bound exclusively to the P. gingivalis membrane-bound RgpB isoform (mt-RgpB) and to secreted, soluble RgpB. Further cross-reactivity studies with P. gingivalis gingipain deletion mutants showed that the nanobody could discriminate between native RgpB and native Kgp and RgpA in complex bacterial samples. This study demonstrates that RgpB can be used as a specific biomarker for P. gingivalis detection and that the presented nanobody-based assay could supplement existing methods for P. gingivalis detection.

Tannerella forsythia is a gram-negative anaerobic bacterium that is associated with the development of destructive periodontal disease. T. forsythia secretes the metalloprotease-like enzyme karilysin. Using in vitro systems karilysin has been shown to modulate the host immune response by

No abstract

Antigen specific humoral immunity can be characterized by the analysis of serum antibodies. While serological assays for the measurement of antibody levels and of neutralization potential against SARS-CoV-2 are available, these are not quantitative in the biochemical sense. Yet, understanding the biology of COVID-19 would need an unambiguous, complete, quantitative, comparable measurement of specific serum antibodies. Here we describe a fluorescent, dual-titration immunoassay, which provides the physico-chemical parameters that are both necessary and sufficient to quantitatively characterize the humoral immune response. We used recombinant Receptor Binding Domain of SARS-CoV-2 as antigen on microspot arrays and varied the concentration of both the antigen and serum antibodies from vaccinated persons to obtain a measurement matrix of binding data. Binding curves were fitted using a novel algorithm to obtain thermodynamic variables of binding. We defined the standard state for a system of serum antibodies and antigen and showed how a normalized generalized logistic function is related to thermodynamic activity, standard concentration and activity coefficient. The utility of the method is demonstrated by defining the composition of tested sera with respect to immunoglobulin classes, affinity, concentration, and thermodynamic activity. The proposed fluorescent dual-titration microspot immunoassay can generate truly quantitative serological data that is suitable for immunological, medical and systems biological analysis.