We have developed a simple cell immunoassay to determine SMN protein levels that can be performed in 96-well plates and potentially higher density formats. To assess the influence of various parameters, we used a lymphoblastoid cell line derived from a type I SMA patient (GM10684) and a similarly established cell line from an age- and gender-matched individual as a control (GM12497) that we have previously characterized [21
]. For each experiment, the common exon-junction complex component protein Y14 was also determined to confirm equal loading of wells. As expected, SMN levels in patient cells were significantly reduced compared to control (Figure ). When expressed as a ratio of SMN to Y14 expression, control SMN levels were 5.31 ± 0.04 and SMA I levels were 2.21 ± 0.05(N = 4). The optimal number of patient lymphoblastoid cells to load into a 96-well plate well, determined for this assay, was 1.5 × 105
cells (Figure ). Immunoblotting performed at the same time confirmed that SMN levels in the patient cells were reduced (Figure and ). The degree of reduction in SMN protein measured by the 96-well plate-based assay (58.3 ± 7.0%, N = 4) was comparable to that measured by Western blot (66.8 ± 10.7%, N = 9).
Figure 1 A) SMN levels in control and SMA I lymphoblastoid cell lines from four separate experiments. B) SMN and Y14 chemiluminescent intensity levels as a function of number of cells loaded per 96-well plate well. C) SMN levels from the same cell lines determined (more ...)
Blood samples are the most practical source of patient cells for SMN protein level determination and can be measured repeatedly over time. We chose to study the peripheral blood mononuclear cells (PBMC) because they are readily isolated and typically represent the physiological cellular environment. One potential source of variability of using the PBMC is cell type heterogeneity, which can vary between different individuals and within the same individual at different times. We therefore compared SMN levels in pure populations of human monocytes and lymphocytes and found no significant difference when the same number of cells was analyzed (Figure ). Moreover, mixing the cell types had no effect on the SMN or Y14 levels. SMN levels from peripheral blood of control individuals are shown in Figure . The intensity values for SMN and Y14 in PBMCs are similar in the pure and mixed monocyte and lymphocyte populations.
Figure 2 SMN (dark grey bars), Y14 (light grey bars) and background (white bars) chemiluminescent intensity levels in (A) sorted monocytes and lymphocytes, including mixed ratios of the two cell types, and in (B) peripheral blood mononuclear cell isolated from (more ...)
The SMN protein cell immunoassay compares SMN expression in an equivalent number of PBMC cells and does not depend on a stable endogenous control. Nonetheless, the use of Y14 as a stable endogenous control in these experiments, as would be done for Western blot analysis, lends further confidence in the assay to detect specific changes in SMN expression between two well-characterized cell lines. The SMN protein cell immunoassay method does not require cell lysis as do western blotting and sandwich ELISA methods. Moreover, there is no transfer of protein to a membrane and a relatively small amount of biological sample is required. We find that SMN levels can be measured in triplicate from a 5 to 8 ml sample of peripheral blood.
Nucleated cells from blood were chosen for protein determinations because the SMN protein is found both in the cytoplasm and the nucleus of cells. The measurement of SMN in the PBMC is valid despite the fact that the cell type composition of the PBMC can vary between individuals and even within the same individual at different times. We addressed the issue of cell type heterogeneity by measuring SMN in pure populations of PBMC components and found no effect of cell type composition. Recently, we also described a reliable assay to measure a well established function of the SMN protein, small nuclear ribonucleoprotein (snRNP) assembly [21
]. In combination, the SMN protein cell immunoassay, described here, with the SMN activity assay can be used to gain insight into the regulation of SMN protein expression and activity. These new assays may significantly add to the present methods available to study SMA.