title: Quantification of human tissue transglutaminase by a luminescence sandwich enzyme-linked immunosorbent assay.
abstract: Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes crosslinking of peptidic glutamine residues with primary amines via isopeptide bonds and hydrolysis of ATP or GTP. The enzyme exerts a variety of functions at the cellular and tissue levels that may be disturbed in disease. Its role in pathoprocesses is poorly understood. For investigation of the involvement of tTG in disease, sensitive and specific assays should be available. We have developed the first sandwich enzyme-linked immunosorbent assay (ELISA) based on two monoclonal antibodies (mabs) against human tTG. tTG is captured by mab 3C10 and detected by biotinylated mab 10F3. After incubation with peroxidase-conjugated streptavidin, bound tTG is visualized by peroxidase reaction applying a luminescence substrate. The detection limit was 40 pg/ml. The assay was highly reproducible. Recovery of spiked tTG in crude samples was greater than 92%. The enzyme could be detected in cellular lysates and tissue homogenates of humans. The effect of typical effectors (retinoic acid and interferon-γ) on tTG expression could be demonstrated. A low signal was also obtained in mice samples, suggesting cross-reactivity of the mabs with murine tTG. The new sandwich ELISA may be successfully applied for investigation of physiological functions of tTG and of disorders associated with inadequate tTG expression.
authors.affiliation: Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, D-04103 Leipzig, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Wolf
authors.forename: Ingolf
authors.initials: I
authors.lastname: Lachmann
authors.forename: Uta
authors.initials: U
authors.lastname: Wagner
authors.forename: Awad A
authors.initials: AA
authors.lastname: Osman
authors.forename: Thomas
authors.initials: T
authors.lastname: Mothes
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D002469:Cell Separation; D003124:Colorimetry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D049449:Luminescence; D051379:Mice; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D012015:Reference Standards; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D007371:Interferon-gamma; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins