MAST-CELL IMMUNOHISTOCHEMISTRY - NONIMMUNOLOGICAL IMMUNOSTAINING MEDIATED BY NONSPECIFIC F(AB')(2)-MAST CELL SECRETORY GRANULE INTERACTION

During investigations of murine and human mast cell immunoreactivity w ith potential anti-interleukin-4 antibodies, non-specific, non-immunol ogical labelling of mouse and human mast cells became apparent. Non-sp ecific, non-immunological labelling was identified by (i) immunolabell ing of mast cells when using control isotype primary antibodies, (ii) ability of conjugated secondary antibodies to label mast cells without prior mast cell exposure to a primary antibody, (iii) extinction of t he non-specific labelling and retention of specific labelling when the pH of the diluting and washing buffers is shifted from pH 7.2 to pH 6 .0, and (iv) reduction/extinction of the labelling when the antibodies are pre-incubated with soluble heparin prior to immunostaining. The s ite of the reactivity on the electron microscope level was shown to be confined to the mast cell secretory granules. The results of this stu dy support the hypothesis that non-specific labelling of mast cells re sults from an ionic interaction between the F(ab')2 segments of antibo dies and the heparin constituent of the mast cell secretory granules. This study points out the necessity of stringent controls when using i mmunohistochemistry to determine mast cell reactivity to various antib odies.