Immunohistochemical staining of human teeth, and production of monoclonal antibodies against cementum.

Abstract

Influence of various forms of fixation and decalcification on immunohistochemical staining of paraffin-embedded human teeth and surrounding tissues was initially examined using commercially available antibodies against vimentin (V), type I collagen (C) and cytokeratin (K). Secondly, monoclonal antibody (MoAb) was produced against both bovine and human cementum and immunohistochemical screening was subsequently undertaken to test reactivity with different scheme of fixation and decalcification. The combination of neutral buffered paraformaldehyde fixation, Morse solution and unmasking procedure yielded both optimal morphology and immunoreactivity. The application of this method into production of MoAb against human or bovine cementum generated a variety of MoAbs reactive with both human teeth and surrounding tissues. The percentage of hybridoma supernatant reactive with sections of human teeth was 14.0-22.4% and was both higher and much improved compared to results of previous soft tissue studies. Finally, the MoAbs, BC 1 and 2 (isolated following the use of bovine cementum as immunogen) and HC 1 (human cementum immunogen) recognized specific bands of various molecular weight. The three MoAbs showed strong resistance against periodate oxidation and borohydrate reduction and were stable following treatment with proteolytic enzymes. All were immunoreactive with components of cementum.