A microcytotoxicity assay for thyroid-specific cytotoxic antibody, antibody-dependent cell-mediated cytotoxicity and direct lymphocyte cytotoxicity using human thyroid cells

Abstract

A microcytotoxicity assay for detection of thyroid-specific complement-dependent cytotoxic antibody (cytotoxic antibody), antibody-dependent cell-mediated cytotoxicity (ADCC) and direct lymphocyte cytotoxicity was developed using human thyroid epithelial cells as targets. Thyroid tissue was obtained from patient with Grave's disease and was treated with collagenase and then trypsin. The red blood cells, interfollicular fibroblasts and infiltrating lymphoid cells in thyroid tissue from patients with Graves' disease could be removed by this procedure. To obtain entirely single cells as target cells, the suspension of dispersed thyroid epithelial cells was allowed to stand for 1 h at 4°C in culture medium to allow cell clumps to settle. For assay of cytotoxic antibody, a mixture of the single target cells, patient's serum and human complement was incubated in microwells for 18 h. After removing the detached cells, remaining target cells in the wells were fixed, stained and counted to assess cytotoxicity. For assay of ADCC and of direct lymphocyte cytotoxicity, target thyroid cells were precultured in the microwells for 18 h. Then effector cells with or without patient's serum were added and cultured further for 24–72 h. Cytotoxicity was assessed as described above. Adherence of effector cells to target thyroid cells sometimes disturbed the enumeration of target cells when the effector/target cell ration was high. With this microassay system 3 different cytotoxic immune reactions against human thyroid cells could be measured quantitatively at the same time on 2–3 ml blood samples.