It has been well established that harboring the allele enhances dementia associated with Alzheimers disease (AD), and several studies have supported a role of proteolysis as an important factor that may contribute to this risk [2,3C10]

It has been well established that harboring the allele enhances dementia associated with Alzheimers disease (AD), and several studies have supported a role of proteolysis as an important factor that may contribute to this risk [2,3C10]. genotype in the plasma or CSF. allele is believed to neither increase nor decrease ones risk of AD, while having the form may decrease ones risk. In contrast, inheritance of one copy of the allele increases disease risk fourfold, while two copies raises the risk tenfold [2]. It has been well established that harboring the allele enhances dementia associated with Alzheimers disease (AD), and several studies have supported a role of proteolysis as an important factor that may contribute to this risk [2,3C10]. Thus, the high susceptibility of to proteolytic cleavage may be the driving force behind the enhanced risk for AD in individuals who CCT251236 are either heterozygous or homozygous for the allele. Recently, we reported the presence of a 17 kDa fragment (p17) of in the AD brain (corresponding to amino acids 1-151) that localized intracellularly, predominantly within the nucleus of microglia and neurons and the levels of this fragment was significantly higher as compared to neuropathological normals (NPNs) [11]. These findings were accomplished using a site-directed cleavage antibody to that is highly specific for a 17 kDa amino-terminal fragment of or but does not react with the mature, full-length form of [11]. In the present study, we sought to examine the utility of this antibody (termed nApoECFp17 antibody) to detect the p17 fragment in either cerebrospinal fluid or plasma samples. The results indicated the levels of the p17 fragment were very low CCT251236 in both fluids and no significant differences between the NPN and AD cohorts were observed. Therefore, the 17 kDa amino-terminal fragment of does not appear to be a viable biomarker for predicting AD. Materials and Methods Materials The in house, rabbit polyclonal nApoECFp17 antibody was synthesized and extensively characterized as previously described and detects the amino-terminal fragment of corresponding to amino acids 1-151 [11]. The nApoECFp17 antibody was synthesized using the 7-mer peptide C-RKRLLRD, which represents the N-terminal upstream neoepitope fragment of and that would be generated following cleavage after the terminal aspartic acid residue at position D151. The anti-N-terminal rabbit polyclonal antibody was purchased from Aviva Systems Biology Corp. (San Diego, CA). Peroxidase-conjugated AffiniPure goat anti-rabbit (IgG) was purchased from Jackson ImmunoResearch Labs (West Grove, PA). This antibody is known to recognize the extreme N-terminal region of human Genotypeantibody as representing the total amount of both full-length and any associated amino-terminal fragments of and specifically detects a 17 kDa fragment in AD cases that localizes predominantly within the nucleus of microglia [11]. Immunohistochemical analysis of AD cases indicated staining of nApoECFp17 was exclusively intracellular with no evidence of immunoreactivity in extracellular, plaque-rich regions. However, we did observe staining along blood vessels and therefore hypothesized that the p17 fragment of may serve as a biomarker in AD. To test this hypothesis, we CCT251236 first examined whether this amino-terminal fragment of could be detected in CSF in both AD and neuropathological normal (NPN) cases and compared these results to those obtained using an CCT251236 antibody that detects full-length and will readily detect CCT251236 amino-terminal fragments of and [11]. As shown in Figure 1, ELISA analysis of CSF using the nApoECFp17 antibody showed no significant differences between NPNs and AD subjects (Figure 1A). Indeed, there was a trend for lower levels of the p17 fragment in AD cases as compared to NPNs. It is noteworthy that all data were expressed in relative units due to lack of any control antigen that could possibly provide for a standard curve. Open in a separate window Figure 1 Examination of CSF for fragmentation by ELISA reveals no significant difference between NPNs and AD subjects. (A): CSF samples were assayed by ELISA utilizing the antibody, nApoECFp17, which is specific for an amino-terminal fragment of fragmentation according to genotype following ELISA utilizing the nApoECFp17 antibody. Due to the rarity of the genotype, heterozygous SERPINB2 cases consisting of at least 1 allele were combined together and are depicted as 2(all) in the bar graph. No significant differences were found across any of the for genotyping groups (p 0.05) Previous studies have.