Immunoreactivity of human tissue mast cells: nonspecific binding of primary antibodies against regulatory peptides by ionic linkage.

Abstract

Tissue mast cells (TMC) are known to react with antibodies against various regulatory peptides (RP). The specificity of such reactions was investigated by various methods in this study. When normal immunohistochemical staining procedures were employed. TMC in the vermiform appendix and in a cutaneous mastocytoma reacted with antibodies against ACTH, Leu-enkephalin, Met-enkephalin, and peptide histidine isoleucine (PHI). Antibody specificity was tested by absorption controls, and staining specificity by varying the concentration of the primary antibodies and the pH and sodium chloride concentration of the buffer used for rinsing and diluting. In absorption controls, staining of the TMC by anti-PHI was diminished but staining by anti-ACTH, anti-Leu-enkephalin, and anti-Met-enkephalin remained unchanged. Unlike control reactions, immunostaining of TMC with antibodies against RP exhibited marked dependence on antibody concentration and the pH and sodium chloride concentration of the buffer. Alkalization of the buffer led to an obvious increase in the reaction with antibodies against RP, and lowering the pH to 6.0 usually resulted in abolition of the reaction. These results indicate that the immunostaining of TMC with antibodies against RP, including PHI, was nonspecific. It is postulated that the granules of TMC bind certain antibodies by a cation-exchange mechanism involving ionic interactions with positively charged groups in the F(ab')2 and/or Fc segments.