Development of a Quantitative Bead Capture Assay for Soluble IL-7 Receptor Alpha in Human Plasma

IL-7 is an essential cytokine in T-cell development and homeostasis. It binds to the IL-7R receptor, a complex of the IL-7Rα (CD127) and common γ (CD132) chains. There is significant interest in evaluating the expression of CD127 on human T-cells as it often decreased in medical conditions leading to lymphopenia. Previous reports showed the usefulness of CD127 as a prognostic marker in viral infections such as HIV, CMV, EBV and HCV. A soluble CD127 (sCD127) is released in plasma and may contribute to disease pathogenesis through its control on IL-7 activities. Measuring sCD127 is important to define its role and may complement existing markers used in lymphopenic disease management. We describe a new quantitative assay for the measurement of sCD127 in plasma and report sCD127 concentrations in healthy adults.We developed a quantitative bead-based sCD127 capture assay. Polyclonal CD127-specific antibodies were chosen for capture and a biotinylated monoclonal anti-CD127 antibody was selected for detection. The assay can detect native sCD127 and recombinant sCD127 which served as the calibrator. The analytical performance of the assay was characterized and the concentration and stability of plasma sCD127 in healthy adults was determined. The assay's range was 3.2–1000 ng/mL. The concentration of plasma sCD127 was 164±104 ng/mL with over a log variation between subjects. Individual sCD127 concentrations remained stable when measured serially during a period of up to one year.This is the first report on the quantification of plasma sCD127 in a population of healthy adults. Soluble CD127 plasma concentrations remained stable over time in a given individual and sCD127 immunoreactivity was resistant to repeated freeze-thaw cycles. This quantitative sCD127 assay is a valuable tool for defining the potential role of sCD127 in lymphopenic diseases

QASI, an international quality management dystem for CD4 T-Cell enumeration focused to make a global difference

Background: A significant worldwide mobilization effort to treat people with HIV disease began in 2003. Most guidelines for initiating antiretroviral therapy require reliable and reproducible CD4 T-cell counting. Therefore, any effort that improves global availability of quality managed assessment schemes for CD4 T-cell enumeration is a positive achievement for the clinical management of AIDS on a worldwide scale. Methods: The Canadian QASI-Quality Management System (QMS) has been in operation for over a decade. More recently, QMS has fine-tuned its strategy to optimize its global impact in the fight against the HIV/AIDS pandemic. Three modifications were implemented: (1) introduction of skills and knowledge transfer workshops pertaining to the initiation of national quality management programs for CD4 counting, (2) introduction of a road map to establish domestic EQAP for countries that are ready, and (3) introduction of a statistical analysis package which permits continuous monitoring of global impact of the QASI-QMS. Results: Based on QASI-QMS distribution of specimens over four consecutive participation cycles, there was decreased interlaboratory variation for both low and medium CD4 T-cell levels. After three cycles of consecutive participation, there is an average of 38 and 26% error reduction reported for the mid and low CD4 levels, respectively. Conclusion: The program improvements mentioned earlier appear to have had a profound effect with regard to enhancing the performance of laboratories participating in the QASI-QMS. Specifically, there is a significant reduction in interlaboratory variability of CD4 T-cell counts resulting from continuous participation in the QASI-QMS. \ua9 2009 Clinical Cytometry SocietyPeer reviewed: YesNRC publication: Ye

Calibration curve of the sCD127 capture bead assay.

<p>Recombinant sCD127-Fc chimera was serially diluted from 0.1 to 1000 ng/mL in assay diluent (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006690#s4" target="_blank">Materials and Methods</a>) and used as the assay's calibrator. The recombinant sCD127 was captured on beads conjugated with sCD127 polyconal antibodies. Bound sCD127 was detected with a biotinylated monoclonal anti-CD127 and streptavidin-PE. The curve was resolved using a 5-parameter logistic equation (MasterPlex QT software). Insert: Standard curve inter-assay variation from 12 assays done in duplicates. The curve's range was 3.2–1000 ng/mL.</p

Soluble CD127 concentration distribution in healthy adults and normal Gaussian curve.

<p>Plasma concentrations of sCD127 (n = 74) were determined using a sCD127 capture bead assay. Each bar represents the number of cases for each interval of sCD127 values and the curve represents the predicted normal distribution. The mean sCD127 was 164.3±104.5 ng/mL.</p

Detection of native sCD127 using the sCD127 capture bead assay.

<p>Detection of native sCD127 in the culture supernatant of unstimulated (Ctrl) and IL-7-stimulated WI-26VA4 cells using the sCD127 capture bead assay. Insert: Specificity of the sCD127 capture bead assay for native sCD127. Inhibition of recombinant sCD127-Fc chimera binding to sCD127 assay's capture antibody by native sCD127. In these experiments, recombinant sCD127-Fc chimera binding to sCD127 capture antibody conjugated to bead was detected using a Fc-specific detection antibody. The residual binding is expressed as the percent of the fluorescence signal (MFI) in presence over the signal in absence of native sCD127 from unstimulated (empty circles) or IL-7-stimulated (filled circles) WI-26VA4 cell culture supernatants.</p

Effect of freeze-thawing on sCD127 immunoreactivity.

a<p>sCD127 concentration in ng/mL (mean±SD).</p>b<p>CV calculation: SD/mean ×100.</p

Serial measurements of sCD127 concentrations in plasma of healthy individuals.

<p>The concentration of sCD127 was measured in the plasma of repeat donors (n = 15) tested 2–5 times over a one-year period. Individual concentrations of sCD127 remained within 14.1±11.3% of their original values (range 0–42%).</p