Immunoassay of in vitro activated human tissue transglutaminase
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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2013, Neurobiology of AgingCitation Excerpt :Details of brains, patients, antibodies, measurement of transamidation and tTG, and immunohistochemistry are provided in the Supplementary data. Assay of tTG was performed as (1) sandwich enzyme-linked immunosorbent assay of protein tTG (ptTG, Wolf et al., 2011a); and (2) immunodetection of activated tTG (atTG) after its binding to an acyl donor (Wolf et al., 2011b). Assay of ptTG, atTG, and transamidation in homogenates of frontal cortex revealed no significant differences between controls and AD patients (Fig. 1A–C).
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