Phage display selection of human single domain antibodies towards karilysin, a metalloproteinase and secreted virulence factor from Tannerella forsythia.

Metalloproteases derived from microbial pathogens are important virulence factors contributing to evasion of antimicrobial mechanisms of the innate immune system. Karilysin is a metalloprotease recently discovered in the periodonto-pathogen Tanneralla forsythia and currently no monoclonal antibodies exist against karilysin, which is a gap in the molecular toolbox for structure-function studies of karilysin. In this study we have used phage display for fast selection of single domain antibodies (VHs) towards the karilysin catalytic domain (Kly18) using a human domain library based on a VH framework.

Nanobody-based microfluidic human Fc assay for preclinical plasma quantification of IgG1/1.1 and IgG1-Fc-conjugates.

Background: Therapeutic antibodies and Fc-conjugates are becoming increasingly popular for disease management and accurate and sensitive pharmacokinetic measurements are critical in lead candidate selection in pre-clinical drug discovery.

Methods And Study Design: Human Fc-specific intact monoclonal antibodies, polyclonal antibodies, Fab fragments, aptamers, affibodies and nanobodies were screened for potential as biotinylated capture moieties in a microfluidic assay. Test compounds were Bevacizumab, Rituximab, Infliximab as well as an in-house IgG1.

Plasma marker for systemic inflammation is increased in Mexican Tarahumara following ultra-distance running.

Objectives: Previous studies have suggested that acute exercise-induced cardiac and kidney damage following ultra-distance running is low in Mexican Tarahumara even though C-reactive protein (CRP) remained elevated 24 hours post-race. We aimed to study if the plasma biomarker, soluble urokinase-type plasminogen activator receptor (suPAR), could replace or complement CRP as a systemic inflammation biomarker in Tarahumara men and women following ultra-distance running.

Methods: Plasma samples were collected pre-race and at three to six different time points post-race in Mexican Tarahumara competing in three independent ultramarathons; men running 78 km (GroupI, n = 9), women running 52 km (GroupII, n = 3), and men running 63 km (GroupIII, n = 10).

An IgY-based immunoassay to evaluate the biomarker potential of the Tannerella forsythia virulence factor karilysin in human saliva.

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.

Salivary levels of MPO, MMP-8 and TIMP-1 are associated with gingival inflammation response patterns during experimental gingivitis.

Aim: This study aimed to investigate the association between salivary levels of myeloperoxidase (MPO), neutrophil elastase (NE), soluble urokinase-type plasminogen activator receptor (suPAR), matrix metalloproteinase (MMP)-8 and tissue inhibitor of matrix metalloproteinases (TIMP)-1 and gingival inflammation development during an experimental gingivitis study.

Methods: A three-week experimental gingivitis study was conducted. Clinical recordings of dental plaque biofilm (Modified Quigley Hein Plaque Index, TQHPI) and gingival inflammation (Modified Gingival Index, MGI) were made at specific time points for each of the 42 participants.

Soluble urokinase-type plasminogen activator receptor is associated with signs of periodontitis in adolescents.

Owing to its molecular stability in body fluids, soluble urokinase-type plasminogen activator receptor (suPAR) is used as a biomarker for the level of systemic inflammation. This study compares the suPAR levels in serum with those in the saliva of adolescents and evaluates their association with the periodontal conditions. Adolescents identified as screen positive (n = 87) or screen negative (n = 73) for periodontitis had saliva and serum samples taken, along with subgingival plaque samples.

Structural insights into a high affinity nanobody:antigen complex by homology modelling.

Porphyromonas gingivalis is a major periodontitis-causing pathogens. P. gingivalis secrete a cysteine protease termed RgpB, which is specific for Arg-Xaa bonds in substrates.

A parallel panning scheme used for selection of a GluA4-specific Fab targeting the ligand-binding domain.

A method for development of murine Fab fragments towards extracellular domains of a surface receptor is presented. The GluA4 ionotropic glutamate receptor is used as a model system. Recombinant GluA4 ectodomain comprising both the N-terminal domain (NTD) and the ligand-binding domain (LBD) in one molecule was used for immunization.

Selection and characterization of camelid nanobodies towards urokinase-type plasminogen activator.

Urokinase-type plasminogen activator (uPA) is a trypsin-like serine protease that plays a vital role in extracellular conversion of inactive plasminogen into catalytically active plasmin. Activated plasmin facilitates the release of several proteolytic enzymes, which control processes like pericellular proteolysis and remodeling of ECM. uPA and the receptor uPAR, are overexpressed in a number of malignant tumours and uPA/uPAR play major roles in adhesion, migration, invasion and metastasis of cancer cells.

Structure of the catalytic domain of the Tannerella forsythia matrix metallopeptidase karilysin in complex with a tetrapeptidic inhibitor.

Karilysin is the only metallopeptidase identified as a virulence factor in the odontopathogen Tannerella forsythia owing to its deleterious effect on the host immune response during bacterial infection. The very close structural and sequence-based similarity of its catalytic domain (Kly18) to matrix metalloproteinases suggests that karilysin was acquired by horizontal gene transfer from an animal host. Previous studies by phage display identified peptides with the consensus sequence XWFPXXXGGG (single-letter amino-acid codes; X represents any residue) as karilysin inhibitors with low-micromolar binding affinities.

A phage display selected 7-mer peptide inhibitor of the Tannerella forsythia metalloprotease-like enzyme Karilysin can be truncated to Ser-Trp-Phe-Pro.

Tannerella forsythia is a gram-negative bacteria, which is strongly associated with the development of periodontal disease. Karilysin is a newly identified metalloprotease-like enzyme, that is secreted from T. forsythia.

Diagnostic evaluation of a nanobody with picomolar affinity toward the protease RgpB from Porphyromonas gingivalis.

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 used the RgpB gingipain as a biomarker for P.

Superparamagnetic bead interactions with functionalized surfaces characterized by an immunomicroarray.

Magneto-resistive sensors capable of detecting superparamagnetic micro-/nano-sized beads are promising alternatives to standard diagnostic assays based on absorbance or fluorescence and streptavidin-functionalized beads are widely used as an integral part of these sensors. Here we have developed an immunomicroarray for systematic studies of the binding properties of 10 different micro-/nano-sized streptavidin-functionalized beads to a biotin substrate immobilized on SiO(2) with or without surface modification. SiO(2) surface cleaning, immobilized substrate concentration and surface blocking conditions were optimized.

Towards on-site pathogen detection using antibody-based sensors.

In this paper, the recent progress within biosensors for plant pathogen detection will be reviewed. Bio-recognition layers on sensors can be designed in various ways, however the most popular approach is to immobilise antibodies for specific capture of analytes. Focus will be put on antibody surface-immobilisation strategies as well as the use of antibodies in the widely used sensors, quartz crystal microbalance, surface plasmon resonance and cantilevers.