CELISA for rapid screening of monoclonal islet cell surface antibodies using living rat insulinoma cells as target.
An improved rapid cell enzyme-linked immunosorbent assay (CELISA) is described which is suitable for the large scale screening of monoclonal antibodies to islet cell surface antigens. 5 x 10(4) insulin-producing rat insulinoma (RIN) cells were seeded per well in a 96-well flat-bottomed polystyrene plate coated one day before a 0.01% poly-D-lysine solution in PBS. After culture for 4 days in 200 microliters/well RPMI 1640 supplemented with 7.5% heat-inactivated fetal calf serum, the cell number per well was up to 2.1 x 10(5). These monolayer RIN cell cultures were used as a target for the detection of islet cell surface antibodies (ICSA) in the supernatants of hybridomas. The cells were used without fixation to avoid modification of sensitive surface antigens. Poly-D-lysine did not cause non-specific binding of immunoglobulins to the plastic wells as tested with irrelevant monoclonals. The specificity and sensitivity of the method is comparable to indirect immunofluorescence. All mc-ICSA primary screened by indirect immunofluorescence using viable RIN cell suspensions were positive in this CELISA. There was a correlation (r = 0.7; n = 44) between the antibody binding measured by CELISA and the indirect immunofluorescence technique. The advantage of this CELISA is that cell surface structures are well preserved in a viable cell monolayer used as target without chemical fixation. This assay procedure should be generally suitable for the initial screening of monoclonal antibodies to cell surface antigens of cells growing under culture conditions.
Ziegler
B
B
Institute of Diabetes Gerhardt Katsch Karlsburg, Germany.
Schlosser
M
M
Fürll
B
B
Witt
S
S
Ziegler
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000032:analysis*; D001323:Autoantibodies / Q000032:analysis*; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005455:Fluorescent Antibody Technique; D007340:Insulinoma; D007515:Islets of Langerhans / Q000276:immunology*; D010190:Pancreatic Neoplasms; D051381:Rats
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D001323:Autoantibodies; C024211:islet cell antibody