Microtiter Immunocyto- chemical ELISA Assay

Quantitative assessment of immunoreactivity and protein expression in cultured cells can be time-consuming and subjective. The problem is magnified when rapid and efficient screening is required, and the supply of cells is limited. Immunoblot analysis requires protein extraction, quantification of protein content and gel electrophoresis, which make the method impractical for evaluating multiple treatment effects or screening for inducible gene expression in a large number of stably transfected clones. Although the use of bioluminescent reporter genes has substantially reduced some of the problems related to clone selection, not all systems can take advantage of such reagents. In this report, we describe the microtiter immunocytochemical enzyme-linked immunosorbent assay (ELISA), called the MICE assay, which is a rapid method for objectively quantifying levels of immunoreactivity in cultured cells without the need for protein extraction, gel electrophoresis or cell counting. The MICE assay differs from the cellular ELISA in that a correction for cell density is incorporated into the procedure, thereby permitting comparisons of protein expression following different treatments, even if the effect of the treatment includes cell death or proliferation. In addition, the MICE assay measures levels of protein expression and intracellular immunoreactivity, whereas cellular ELISAs are designed to detect surface immunoreactivity (2,3,10). The Midwestern assay applies ELISA technology to tissue sections (13), but it provides no means to compare levels of protein expression in relation to cell density. Also, the assay is performed on microscope slides, rendering it difficult to conduct multiple simultaneous analyses. This report describes two examples in which the MICE assay was used to measure changes in protein expression. One study examined the inhibitory effects of ethanol on insulin-stimulated protein expression, and the other measured nitric-oxide synthase 3 (NOS3) levels in cells infected with recombinant adenovirus vectors (Adv) that express full-length NOS3 or green fluorescent protein (GFP) cDNA under the control of a cytomegalovirus (CMV) promoter. The objective was to demonstrate that modulation of endogenous genes and genes over-expressed by infection or transfection of foreign cDNAs is readily detected and quantified using the MICE assay. Insulin stimulation studies. Insulin stimulation modulates cell proliferation and energy metabolism by activating complex intracellular signal transduction pathways (11,15). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an insulin-responsive gene (1), and insulin-stimulated DNA synthesis is also associated with increased levels of proliferating cell nuclear antigen (PCNA) (4). Ethanol has profound inhibitory effects on insulin-stimulated DNA synthesis and gene expression (4,16). These adverse effects of ethanol are partly mediated by inhibition of the insulin-stimulated phosphorylation of molecules that are required for intracellular signaling (12,14). NIH3T3 cells were used to examine the inhibitory effects of ethanol on insulin-stimulated GAPDH and PCNA expression. The cells were grown for 4 days in either the presence or absence of 100 mM ethanol (6), then re-seeded (2 × 105 cells per 10-cm2 petri dish or 2 × 104 cells per well of 96-well, flat-bottom plates) and maintained in sealed chambers equilibrated with 5% CO2: 95% air mixture ± 8 mg/mL ethanol in the reservoir tray (9). After 6 h, the freshly seeded cells were serum-starved for 24 h, then stimulated with 100 nM insulin for 24 h. GAPDH and PCNA expression were assessed by western blot analysis and the MICE assay. NOS3 expression in neuronal cells infected with recombinant Adv. SHSy5y neuroblastoma cells express very low endogenous levels of NOS3 (5). Therefore, these cells were ideally suited to measure increased levels of NOS3 after infection with recombinant Adv that express a full-length cDNA encoding NOS3 or GFP under control of a CMV promoter (8). This model system was used to demonstrate that increased levels of a gene product expressed after