Quantification of glomerular epithelial cell adhesion by using anti-DNA antibodies in ELISA.
A sensitive and reproducible microassay is described for quantification of adhesion of cells to matrix-coated 96-wells plates under different experimental conditions. For this purpose glomerular visceral epithelial cells (GVEC) were used. Attached GVEC were fixed with methanol and incubated with a monoclonal anti-DNA antibody. Following standard procedures, the amount of bound antibody was quantified by ELISA. A positive linear relationship in the range of 800-5000 cells per well was found between OD values and cell numbers obtained by hand-counting (r = 0.94, p less than 0.001). The assay is 10 to 100 times more sensitive than most other adhesion assays. The applicability of the ELISA assay was demonstrated by manipulation of the temperature during adhesion and by using different concentrations of the matrix-molecules fibronectin, EHS-laminin and collagen type I. The ELISA assay was found to be unaffected by non-specific interaction of anti-DNA antibodies with the matrix molecules used for coating. The assay was neither affected by potential release of DNA from the GVEC under these different experimental conditions. In conclusion, this cell adhesion microassay is simple, reliable, sensitive, and cost-effective, since it requires small amounts of GVEC and reagents.
Coers
W
W
Department of Pathology, University of Groningen, The Netherlands.
Huitema
S
S
Smeenk
R J
RJ
Salant
D J
DJ
Grond
J
J
Weening
J J
JJ
D000818:Animals; D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D002448:Cell Adhesion / Q000276:immunology; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells; D004848:Epithelium / Q000276:immunology; D006825:Hybridomas / Q000276:immunology; D007678:Kidney Glomerulus / Q000166:cytology* / Q000276:immunology*; D051381:Rats
D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal