An improved cell-ELISA for the differential screening of antibodies against cell surface molecules of viable adherent Schwann cells.
In this report we describe a highly sensitive large-scale screening assay that uses viable adherent cells. The newly developed test design combines the advantages of live cell immunocytochemistry with the versatility of a conventional ELISA technique. Culturing of target cells as well as incubation with antibodies is done in the same microtiter plate. No processing of cells prior to incubation with the antibodies, such as fixation or enzymatic detachment of the cells, is performed. This eliminates possible factors that might interfere with antibody-antigen recognition and results in a dramatically increased sensitivity that is comparable to cell-free ELISA procedures with an assay detection limit reproducibly between 0.004 and 0.002 micrograms/ml. Furthermore, this test design is convenient for the rapid identification of antibodies against differentiation-dependent antigens. Incubation in parallel with the same antibody of different cell types (forskolin-stimulated Schwann cells or fibroblasts) or cells grown under different conditions (forskolin-stimulated and non-stimulated Schwann cells) facilitates the selection of antibodies displaying genotypic or phenotypic specificity. Since culture of target cells and the detection of antibodies is done on one microtiter plate, it is not only possible to detect cell surface molecules but also secreted molecules that remain bound to the cell surface or the culture substrate. Compared to other previously introduced cell-ELISAs, our protocol offers the following advantages: (i) increased sensitivity, (ii) convenience for large-scale screening, (iii) optimal identification of antibodies reacting with native molecules, and (iv) identification of differentiation-dependent antigens. Since this assay could also be used in studies examining the regulation of cell surface molecules at a semiquantitative level it may be of general relevance.
Wewetzer
K
K
F. Hoffmann-La Roche Ltd., Preclinical Research, PRPN, Basel, Switzerland.
Heiniger
C
C
Seilheimer
B
B
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000937:Antigen-Antibody Reactions; D015703:Antigens, CD / Q000032:analysis; D002448:Cell Adhesion / Q000276:immunology; D002450:Cell Communication / Q000276:immunology; D002470:Cell Survival / Q000276:immunology; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D008565:Membrane Proteins / Q000276:immunology*; D051381:Rats; D017208:Rats, Wistar; D012583:Schwann Cells / Q000276:immunology*
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D008565:Membrane Proteins