Our assay using two monoclonal antibodies appears to be specific because it accurately detects MLH1 and MSH2 in control cell lines that contain one or the other or both of these proteins (click here Figure 1A) and the assay also detects MLH1 and MSH2 proteins in mixing experiments where these proteins are present in varying proportions

(Figure 1C). Our immunoassay also appears to be sensitive since it will detect MLH1 and MSH2 proteins in a sample from SW480 cells that contains as little as 10 ug of cellular protein (Figure 1B). Moreover, our assay appears to have an acceptable level of precision in that it is highly reproducible (Table 2). The fact that MLH1 and MSH2 are not readily detected in untreated fresh lymphocytes or monocytes is likely due to the fact that they are not rapidly proliferating. HCS assay This is supported by the fact that MLH1 and MSH2 are detectable in immortalized lymphocytes [7], which are proliferative cells by virtue of the fact that they have been transfected with an attenuated

Epstein Barr Virus (EBV) and PHA treatment has little affect on MLH1 and MSH2 levels in these already proliferative cells. It Daporinad price should be noted that colon cancer cell lines (e.g., SW480) are also proliferating cells and have readily detectable levels of MMR proteins. The importance of our finding that PHA stimulation makes MLH1 and MSH2 detectable in fresh lymphocytes has relevance to the development of a practical immunoassay for the identification of carriers of an LS trait in a population-based Flucloronide setting. A second finding is that the distribution of MMR ratios among individuals in a genetic counseling program, which includes carriers of an LS trait, was bimodal (Figure 3) with

one peak close to 1.0 (less likely to be affected) and another lower than 1.0 (more likely to be affected). A bimodal distribution was not seen for healthy controls. This suggests that a subpopulation within the cohort of individuals at high risk for LS has substantially reduced levels of one of the two MMR proteins, which is what we predicted. This finding is consistent with our previous retrospective study [7] that also found a bimodal distribution. That earlier study was done using immortalized lymphocytes and involved individuals with a known MMR genotype, those who carried the LS trait and those who did not. Our findings are consistent with other studies [10, 11] that report microsatellite instability (MSI) in lymphocytes from LS patients – including ones with germline MSH2 or MLH1 mutations. If lymphocytes from LS patients have MSI, it can be assumed that they have reduced levels of the wild type DNA mismatch repair protein caused by the corresponding germline mutation. Another study by Marra et al [12] reported that MSH2 protein levels are decreased in immortalized lymphocytes from LS patients carrying known MSH2 germline mutations.