Surface immunoglobulins of rat immunocytes: Quantitation and fate of cell‐bound peroxidase‐labeled antibody and Fab fragment

Abstract

Abstract By using antibody‐peroxidase conjugates, quantitative data were obtained concerning events following interactions between anti‐Ig antibodies and surface Ig. About 5 × 10 5 molecules of peroxidase‐labeled anti‐rat Ig antibodies were bound per positive lymph node cell, at conjugate excess, at 4 °C. Similar quantities of anti‐Fcγ or anti‐Fab were bound per positive cell, suggesting that cell surface IgG antigenic determinants are largely exposed. Experiments performed on thymocytes showed that the number of peroxidase molecules bound per 10 6 cells was 12 to 16 times higher with lymph node lymphocytes than with thymocytes. After incubation of cells at 4 °C with peroxidase‐labeled rabbit anti‐rat Ig, the cells were washed and post incubated for various times at 37 °C. The fate of the peroxidase conjugates was followed by measuring enzymatic activity on the cell surface, in the cell, and in the incubation medium. During the first 15 min of incubation at 37 °C, peroxidase activity disappeared rapidly from the cell membrane, while enzymatic activity appeared and rapidly increased in the cell supernatant. After this time, membrane‐bound peroxidase activity was found to be constant, while enzymatic activity of the supernatant increased slightly. After 1 h of incubation at 37 °C, about 20 % of the peroxidase activity had been eluted into the culture medium, 20 % remained on the cell surface, and about 35 % had been pinocytozed. Similar results were obtained with peroxidase‐labeled sheep‐Fab anti‐rat Ig, except that more peroxidase activity was found in the supernatant and pinocytosis was less pronounced than with whole antibody. Immunoadsorbents were used to determine if cell‐derived Ig was linked to the peroxidase liberated into the supernatant at 37 °C. Polyacrylamide beads to which were linked anti‐rat Ig antibodies or Fab fragments fixed 40 % of the peroxidase activity. Molecular filtration of the supernatants showed that about 60 % of the peroxidase activity was eluted in the void volume of a Sephadex G‐200 column, the remainder being eluted as free conjugate. Thus, part of the conjugate initially fixed on cells at 4 °C was eluted as cell surface Ig‐anti‐Ig complexes, while another part was apparently released uncomplexed.