Validated sandwich ELISA for the quantification of tissue transglutaminase in tissue homogenates and cell lysates of multiple species.
Tissue transglutaminase (tTG) is a calcium dependent enzyme that displays diverse functions in various physiological processes. In addition to these physiological functions, there is strong evidence for the implication of tTG in a number of pathologies, including celiac disease, cancer and neurodegeneration. To explore the expression and function of tTG during (patho)physiological conditions, it is of utmost importance to have an assay that specifically measures tTG protein levels in various species and matrices. Therefore, we have developed a sensitive sandwich ELISA to measure tTG protein levels in tissue homogenates and cell lysates of human, rat and mouse origin. The ELISA uses commercially available antibodies, and human recombinant tTG as the standard protein. The limit of detection is 100 pg/ml; the coefficients of intra- and inter-assay variation range from 2.4% to 6.6% and from 12.7% to 15.1%, respectively. Clear detectable levels of tTG protein were measured in human and rat liver and cerebral cortex, as well as in brain-derived neuronal and glial cells. tTG levels in mouse tissues were much lower than observed in human and rat tissues. No cross-reactivity against keratinocyte TG (TG1), epidermal TG (TG3) or blood coagulation factor XIII was observed. The tTG specific sandwich ELISA presented in this paper is a sensitive and reliable tool to accurately measure tTG protein levels in different matrices (cell/tissue) of rat, mouse and human origin. It provides a better alternative for the widely used transglutaminase activity assay with respect to sensitivity and specificity, and may serve as a valuable tool to investigate protein expression levels as part of the approach to unravel the contribution of tTG to health and disease.
Brevé
John J P
JJ
VU University Medical Center, Department of Anatomy & Neurosciences, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.
Drukarch
Benjamin
B
van Strien
Miriam
M
van Dam
Anne-Marie
AM
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000937:Antigen-Antibody Reactions; D002457:Cell Extracts / Q000737:chemistry*; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D051379:Mice; D009928:Organ Specificity; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis* / Q000276:immunology; D015203:Reproducibility of Results; D013045:Species Specificity; D013347:Subcellular Fractions / Q000201:enzymology*; D011503:Transglutaminases / Q000032:analysis* / Q000276:immunology
D000906:Antibodies; D002457:Cell Extracts; D011994:Recombinant Proteins; D011503:Transglutaminases