Influence of target cell preparation on binding of monoclonal islet cell reactive antibodies (mc-ICRA) in cellular enzyme-linked immunosorbent assay (CELISA).
A rapid, effective and sensitive CELISA for the detection of monoclonal islet cell reactive antibodies (mc-ICRA) using the insulin-producing rat insulinoma cell line (RIN) is described. RIN cells are a suitable target for this monoclonal antibody assay as shown by a comparative study with normal rat islet cells. We tested the influence of the target cell preparation and obtained the best sensitivity and reliability with the CELISA using desiccated cells or desiccated cell homogenate with a cell number of 5 x 10(4) cells per well rather than an adsorbed cell homogenate. Furthermore, ethanol fixation of RIN cells resulted in a loss of antigenicity as shown particularly by the detection of islet cell surface antibodies. We also compared the binding of mc-ICRA in RIN-CELISA with data obtained by indirect immunofluorescence using viable RIN cells as targets. By permeabilization of the cell membrane by desiccation or sonication, more antibodies are detected in CELISA (surface and cytoplasmic antibodies), whereas in immunofluorescence on viable RIN cells, only surface reactive antibodies are detected.
Schlosser
M
M
Institute of Diabetes, Gerhardt Katsch, Karlsburg, F.R.G.
Witt
S
S
Ziegler
B
B
Ziegler
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions; D001323:Autoantibodies / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D066298:In Vitro Techniques; D007340:Insulinoma / Q000276:immunology; D007515:Islets of Langerhans / Q000276:immunology*; D010190:Pancreatic Neoplasms / Q000276:immunology; D051381:Rats; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; C024211:islet cell antibody