Materials And Methods

2.1 Establishment of monoclonal antibodies

As antigen, recombinant human TGase 3 was expressed and purified from bacteria expression system as described previously. Splenocytes from the immunized mice were prepared and hybridomas were established using mouse myeloma Sp2 by standard methods. Among the various hybridomas, the line secreting most specific monoclonal antibody (C2D) against human TGase 3 were selected by ELISA.

2. 2 Immunochemical analysis of TGase 3

For the preparation of total cellular lysates from human skin, frozen epidermis was homogenized in liquid nitrogen. The powder was solubilized with Urea/Tris extraction buffer. Immunohistochemical analysis was performed on formalin-fixed human skin embedded in paraffin using the avidin-biotin peroxidase method. The immunoreaction was detected using diaminobenzidine.

2. 3 Digestion and activation of recombinant TGase 3

Recombinant human cathepsins S and L were used for digestion of recombinant TGase 3. In each case, the molar ratio (cathepsins: TGase) is around 1:100 in an appropriate buffer. In the case of dispase, reaction condition was similar to the method as described previously4. The reaction was stopped by the addition of E-64, an inhibitor for cystein protease upon measurment of enzyme actity. The enzymatic activity was determined by the measurement of incorporated biotinylated cadaverine into dimethylcasein.

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