Samples were randomized and blinded prior to analysis on the ELISA microarray chips; un-blinding of samples was performed after analysis was complete. sites. By now, investigators are well aware of the need for uniformity in all aspects of sample collection when employing proteomics in biomarker discovery studies, in order to minimize confounding factors [3]. However, archived samples exist from many clinical studies in which sample collection protocols were not Rabbit Polyclonal to Cytochrome P450 8B1 rigidly controlled or properly recorded. Alternatively, some studies employ assays that MT-3014 are not sensitive to differences in sample type or sample collection. For example, the Diabetes Autoantibody Standardization Program (DASP), a collaboration of MT-3014 the Immunology of Diabetes Society and the U.S. Centers for Disease Control and Prevention, has as its goal the improvement of the performance of assays for islet cell autoantibodies [4; 5]. The measurement of islet cell autoantibodies is commonly performed using solution-based radiobinding assays [6], which are insensitive to differences in sample type. Thus, samples collected from patients with recently diagnosed type 1 diabetes and contributed to the DASP may be either serum or plasma. Unfortunately, this represents a confounding factor for potential proteomic analyses of DASP samples for discovery of novel biomarkers of type 1 diabetes, particularly when information regarding sample type is incomplete. Therefore, it is critical to classify samples from this and similar studies as plasma or serum before proteomic analyses are conducted. Traditional methods of distinguishing serum and plasma rely on selectively inducing coagulation in plasma samples [7], and MT-3014 require larger volumes of sample than are typically available from sample archives. The enzyme-linked immunosorbent assays (ELISA) is an attractive option for differentiating plasma and serum samples [8], as it is sensitive and selective, and can efficiently process large numbers of samples [9]. Furthermore, the biochemistry of fibrinogen processing can be exploited when designing an appropriate sandwich ELISA assay. Intact fibrinogen is present only in plasma. In the conversion of plasma to serum, fibrinogen is proteolytically cleaved by thrombin to form fibrin and fibrinopeptide A [10]. In theory, it should be possible to distinguish between the intact and processed fibrinogen using a sandwich ELISA. This is because the sandwich ELISA utilizes two antibodies to detect distal epitopes on a single protein. The first antibody, called the capture antibody, isolates and concentrates the targeted antigen. The second, or detection antibody, binds to a distal epitope. The detection antibody is tagged to allow for signal quantification, which is directly proportional to the antigen concentration under non-saturating conditions. Therefore, it should be possible to select antibodies that bind on reverse ends of fibrinogen, such that only the uncleaved protein will give a transmission with this sandwich ELISA. That is definitely, the use of two antibodies that detect distal portions of the undamaged fibrinogen protein should be useful for distinguishing between plasma, which has high levels of undamaged fibrinogen, and serum, which only has trace amounts of undamaged fibrinogen. Current commercial ELISA packages for measuring fibrinogen are based on polyclonal antibodies that were raised against undamaged fibrinogen. These antibodies typically detect both the whole fibrinogen molecule found in plasma and the proteolytic products in serum, and therfore cannot be used to distinguish between plasma and serum. Herein, we describe the development of a sandwich ELISA microarray assay for whole fibrinogen that can be used specifically as a rapid and high-throughput test to distinguish between plasma and serum. This assay can use either of two capture antibodies for undamaged fibrinogen, as well as a detection antibody to fibrinopeptide A. Fibrinopeptide A was chosen because it is the 1st cleavage event in the multi-step conversion of fibrinogen to fibrin [11]. Materials and Methods Antibody Resource and Preparation The fibrinogen antibodies used as capture antibodies in the sandwich ELISA were mouse monoclonal anti-Fibrinopeptide A (49D2; Product No. ab14801; Abcam, Cambridge, MA), rabbit polyclonal anti-Fibrinogen -chain precursor recombinant protein (HPA001900; Product No. HPA001900; Sigma-Aldrich, St. MT-3014 Louis, MO), and goat polyclonal anti-whole fibrinogen (F8512; Product No. F8512; Sigma-Aldrich). Antibody F8512 was partially.