Immunoassay of in vitro activated human tissue transglutaminase

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Abstract

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that exerts a variety of physiological functions and is involved in various pathoprocesses. To characterize the role of tTG in disease, simple assays are necessary for detection. We developed a highly sensitive enzyme-linked immunosorbent assay (ELISA) that combines a transamidation step with immunological detection to determine tTG. This assay is based on covalent binding of in vitro activated tTG to N,N′-dimethylated casein and subsequent detection of coupled tTG by specific immunoglobulin G (IgG) antibodies directed against tTG followed by binding of a secondary biotin-labeled antibody. The assay reaches a detection limit of 0.05 ng of tTG/ml. Type 1 and 3 transglutaminases and factor XIII are not detected. By use of this assay, tTG in several cell lines and the stimulatory effect of retinoic acid on tTG expression could be demonstrated. The new assay represents a promising tool for the study of tTG in normal cell physiology and under pathological conditions.

Section snippets

Chemicals

Recombinant human tTG was obtained from two sources: tTGZ was purchased from Zedira (Darmstadt, Germany) and tTGR was purchased from AJ Roboscreen (Leipzig, Germany). Recombinant human keratinocyte transglutaminase (TG1); recombinant human epidermal transglutaminase (TG3); recombinant human factor XIII, A subunit (FXIIIA); and the specific tTG inhibitor benzylcarbonyl-(6-diazo-5-oxonorleucinyl)-l-valinyl-l-prolinyl-l-leucine methylester (Z-DON) were obtained from Zedira. Monoclonal mouse

Results

With TG100, 10F3, and 3C10, we observed a linear relationship between absorbance and the concentration of tTGZ in the new immunoassay (Fig. 1). For TG100 the standard curve spanned from 0.025 to 2 ng/ml, and for the other mAbs it spanned from 0.05 to 4 ng/ml. The detection limit of the assay, defined as tTG concentration at the twofold optical density of the background signal, was 0.05 for TG100 and 0.1 ng/ml for 10F3 and 3C10. The absorbance of the blank (background), containing all reagents

Discussion

We have described a novel highly sensitive method for specific detection of active tTG. The new assay principally differs from other hitherto described tests. The essential new feature of our assay is that it is based on binding of tTG to N,N′-dimethylated casein, which coupled to the surface of wells in a microtiter plate. Therefore, an important prerequisite for the enzyme to be measured is its transamidation activity. In fact, such catalytic activity of the recombinant human tTG could be

Acknowledgments

The authors thank Ina Heinrich and André Reinhardt for technical assistance. This work was supported by Grant 13534/2312 from the Development Bank of Saxony (Sächsische Aufbaubank–Förderbank [SAB]).

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