Mannose-binding Lectin Levels and Susceptibility to Tuberculosis

Abstracts standard curves, the sensitivities and ranges of detection for each kit were consistent with manufacturers' technical datasheets but were different among the kits. These data indicate differences in operational issues and sensitivities among kits from various vendors that should be considered when implementing or comparing clinical trials. doi:10.1016/j.clim.2010.03.236

F.15. Establishment of an Efficient Validation Strategy for Reliable High-throughput Protein Quantification with Multiplex Bead-based Luminex Immunoassays Chahrazed Belabani, Gregoire Morisse, Nadia Ouamara, Ada Villalobos, Sathy Rajasekharan, Viviane Poupon, Amit Bar-Or. Montreal Neurological Institute, Montreal, QC, Canada

S79 recognition system. Previous studies have reported conflicting results correlating MBL genotype with susceptibility to tuberculosis. We evaluated the plasma level of MBL in samples with and without tuberculosis, as judged by the QuantiFERON assay in discard, de-identified samples. MBL protein levels were measured in a sandwhich ELISA. Previous studies have shown that serum samples from patients deficient in MBL showed a MBL protein level of b 100 ng/mL. In a small initial study of 10 samples the rate of MBL deficiency was the same regardless of TB status. Interestingly, the rate of MBL deficiency in both groups was higher (40%) than would be predicted for the normal population. A larger study analyzing an additional 40 samples is currently underway. MBL deficiency may predispose patients to bacterial infections including TB. Other researchers have suggested that MBL deficiency may protect patients from infection with TB by limiting entry into host cells. Larger studies are needed to evaluate these correlations. doi:10.1016/j.clim.2010.03.238

Multiplex bead-based Luminex immunoassays, which allow simultaneous high throughput detection and quantification of a large number of analytes (either protein, RNA or DNA) within the same sample, have been developed and optimized for a broad range of applications over the past 10 years. Targeted markets were initially in vitro diagnostic and high-throughput research within the pharmaceutical industry, two sectors where validation studies and GLP practices are mandatory. In such settings, the validation procedure can be highly timeconsuming and cost-intensive. As this technology represents a valuable tool for researchers in an academic setting, we have developed and implemented within our program a validation protocol that addresses the key steps to accurately measure concentrations of proteins using any given bead-based Luminex assay. We present this method, applied to determination of protein concentrations in serum obtained from adult healthy volunteers, using three commercially available Luminex detection kits that measure disease-relevant proteins as metalloproteases (MMPs) (MMP panel, R&D Systems), cytokines (11-plex human cytokines panel, BioRad), and proteins associated with cardiovascular diseases (CVD panel 3, EMD4biosciences). In summary, with a maximum of five 96-plates and within three to five days we can i) accurately determine the Lower and Upper Limits of Detection (LLOQ and ULOQ), ii) validate sample acceptance criteria by measuring lot number specific intraplate and interplate variability, and iii) establish in-house quality controls for a given Luminex kit. Together, this practical validation protocol enables us to efficiently ascertain the accuracy and reliability of sample measurements using the Luminex platform. doi:10.1016/j.clim.2010.03.237

F.16. Mannose-binding Lectin Levels and Susceptibility to Tuberculosis Michelle Altrich. ViraCor-IBT Laboratories, Lenexa, KS The innate immune system provides the first line of defense against pathogens. The mannose-binding lectin (MBL) pathway is an important part of the innate immune system's pathogen

F.17. Serum IFN-α and TNF-α Levels in Sarcoidosis Patients are Associated with Disease Manifestations and Differ Significantly by Ancestry Nadera Sweiss, Beverly Franek, Silvia Kariuki, Linda Hushaw, Joseph Garcia, Timothy Niewold. University of Chicago, Chicago, IL Objective: Interferon alpha (IFN-α) and tumor necrosis factor alpha (TNF-α) have been implicated in the pathogenesis of sarcoidosis, although the exact role of these cytokines in human disease is not well understood. Methods: Serum samples from 78 sarcoidosis patients (56 African-American, 22 European-American ancestry) were assessed. Serum IFN-α levels were measured using a sensitive reporter cell assay, and serum TNF-α was measured using a commercial ELISA. Clinical data, including potential neurologic, cardiac, skin, and other extra-pulmonary manifestations of sarcoidosis, were abstracted from medical records. Results: Serum TNF-α levels were significantly higher in African-American subjects (mean = 37.6 pg/mL, SD = 74.9 pg/mL) than in European-Americans (mean = 6.1 pg/mL, SD= 16.4) (p= 0.0015). In contrast, there was a non-significant trend toward higher serum IFN-α levels in European-American subjects (p = 0.1), although levels of serum IFN-α in our sarcoidosis cohort were lower than those we observed in either lupus or Sjogren's syndrome (pb 0.0005 for each). In African-American subjects, high TNF-α was correlated with neurologic involvement (p= 0.043), while no clinical associations were observed with IFN-α. In European-American subjects, serum IFN-α was associated with extra-pulmonary manifestations (p = 0.024), and no clinical associations were observed with TNF-α. Conclusions: These results suggest distinct associations between serum levels of IFN-α and TNFα and clinical manifestations in our sarcoidosis cohort which differ significantly by self-reported ancestry. These differences may relate to ancestral differences in the molecular pathogenesis of this heterogeneous disease. doi:10.1016/j.clim.2010.03.239