In-cell Western assay

The newly available Odyssey and Aerius infrared imaging systems make it possible to probe whole cells with two-color infrared fluorescently labeled antibodies (anti-phosphopeptide, for example) that are used to detect changes in intracellular kinase signaling (Chen et al., 2005). The advantage of using infrared-labeled probes lies in the increased sensitivity and dynamic range and consequent reduction in the use of reagents. 

These assays resemble a hybrid of an immunohistochemistry assay and ELISA. Whole cells are fixed, for example with 3.7% formaldehyde, to MTPs permeabilized by repetitive washing with 0.1% Triton X-100, blocked with a protein solution, probed with primary antibodies (phospho-spe-cific, and non-phospho-specific), washed, and subsequently the secondary antibodies labeled with infrared fluorescent tags are added. After washing, these assays are read in a reader (such as the Odyssey or Aerius) designed for high sensitivity detection of two colors. The two colors are useful because one color can be used to accommodate a stain assigned as a total protein or cell number control or as an antibody to total protein, which allows for normalization. These assays may only require a single antibody versus the dual antibody sandwich required for ELISA. 

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Egorina EM, Sovershaev MA, Osterud B. In-cell Western assay a new approach to visuahze tissue factor in human monocytes. J. Thromb. Haemost. 2006 4 614-620. 

Dickey et al. (403) reported a list of tau-reducing compounds identified from an initial in-cell Western screening assay. Drugs resulting in more than 25% reductions in tan levels with less than 10% reductions in GAPDH include aggregation inhibitors (diazaquone, methylene blue) antibiotics (alexidine HCl) antiproliferatives (colchicine, albendazole, chelidonine, rotenone) and steroids (norethindrone) (403). 

A modified version of the ECVAM-approved embryonic stem cell test (EST) (52) was recently developed by Hunter and coworkers in NHEERL (31). This assay is capable of quantitatively assessing cytotoxicity and cardiomyocyte differentiation and was used to test the ToxCast Phase I chemical library (32). Briefly, male murine J1 mES cells were seeded onto gelatin-coated 96-well plates at a known density in differentiation media on day 0. On day 1, cells were treated with chemicals ranging from 0.0125 to 12.5 pM, and on day 9 In-Cell Western (Li-Cor Biosciences) assays were assayed for a- and P-cardiac Myosin Heavy Chain (MYH6/MYH7)... 

FIGURE 5.5 (A) In-cell Western target modulation assay. (B) Potency of 17AAG to downregulate client pro-... 

In principle, intensity measurement is no different from the well-based immunoblot or other homogeneous well-based assay format such as an in-cell Western. However, because individual nuclei are identified and used as the mask, precise normalization to cell number can be achieved. In addition, microscopy with high quality cameras offers much higher signal-to-noise ratios, thus making many more assays possible. Finally, if a threshold can be set and cells classified as responders or non-responders, an intensity-based assay can be turned into a cytometry assay. 

Some patients will have repeatedly nonspecific reactivity in an ELISA assay, since the HIV-1 antigen used for the antibody assays is produced in cultured human T cells. It is not unexpected that occasional false positive assays occur in human sera from individuals with autoimmune diseases a history of multiple pregnancies or multiple transfusions or antibodies to certain class II histocompatibility antigens (especially HLA-DR4). Block reagents have been added to specimen diluents to minimize cross-reactions in these sera. This necessitates the use of confirmatory tests, especially the Western blot. With the use of both ELISA and Western blots, false positives decrease to less than 1 per 100,000. 

The first functional cell-based assay for the determination of pan-histone deacetylase activity was achieved by detecting hyperacetylated histones by Western blotting. The histone acetylation level in cells offers a measure of candidate HDACI activity and has been linked to antiproliferative and cytotoxic effects. Beckers et al. used a cellular histone hyperacetylation assay for quantification of the cellular efficacy of HDACIs using the Cellomics Array Scan II platform in 96 wells [14]. The program... 

Fig. 6. Effect of L-PDMP on the content and activity of p42 MAPKin the cortical cells. Cells were treated in the absence (open bar) or presence (closed bar) of 20 fiM. L-PDMP for 2 (1—3 DIV), 5 (1-6 DIV), and 8 days (1-9 DrV). (A) p42 MAPK content of the cell extract was measured by Western blotting as described in methods. (B) Specific activity of p42 MAPK was shown. Immuno-precipitated MAPK activity was measured using the in-gel kinase assay and normalized for MAPK content measured by Western blotting. (C) To compare the total activity of p42 MAPK in the cortical cells, the specific activity was multiplied by MAPK content. Data are expressed as a mean percentage (duplicate) of MAPK content or activity of the cells taking each control value at 1 DIV as 100% (immediately before the addition of L-PDMP).

A western blot assay on treated cancer-derived cell lines (Fig. 8.9a, b) was performed to determine the in-cell specificity of WBZ 4. The immunoblots revealed specificity toward C-Kit consistent with the selective anticancer activity on the GIST882 cell line that expresses C-Kit. Thus, the activating phosphorylation of C-Kit at sites Tyr703 and Tyr721 in ST882 cells is inhibited by WBZ 4 in a dose-sensitive manner similar to imatinib (Fig. 8.9a). By contrast, phosphorylation of Bcr-Abl at Tyr 412 (22) in K562 cells was not significantly inhibited (<15%) by WBZ 4, while densitometry revealed an imatinib-induced inhibition of 85% (Fig. 8.9b). 

A simple way of evaluating the affect of a single nucleotide polymorphism on enzyme function is to perform transient transfection assays in COS-1 cells. Western blot analysis, with anti-UGTl A antibody, is used to determine protein expression levels of the variants, which often correlate with protein stability. Enzyme kinetic analysis is used to investigate the functional impact of amino acid substitutions. 

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