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Development of a quantitative bead capture assay for soluble IL-7 receptor alpha in human plasma.

Faucher S, Crawley AM, Decker W, Sherring A, Bogdanovic D, Ding T, Bergeron M, Angel JB, Sandstrom P - PLoS ONE (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: National HIV and Retrovirology Laboratories, Public Health Agency of Canada, Ottawa, Canada. sylvie_faucher@phac-aspc.gc.ca

ABSTRACT

Background: IL-7 is an essential cytokine in T-cell development and homeostasis. It binds to the IL-7R receptor, a complex of the IL-7Ralpha (CD127) and common gamma (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.

Methodology/principal findings: 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.

Conclusions/significance: 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.

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Related in: MedlinePlus

Calibration curve of the sCD127 capture bead assay.Recombinant sCD127-Fc chimera was serially diluted from 0.1 to 1000 ng/mL in assay diluent (Materials and Methods) 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.
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pone-0006690-g002: Calibration curve of the sCD127 capture bead assay.Recombinant sCD127-Fc chimera was serially diluted from 0.1 to 1000 ng/mL in assay diluent (Materials and Methods) 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.

Mentions: The sCD127 capture assay was calibrated with purified recombinant CD127-Fc chimera from 0.1 to 1000 ng/mL (Fig. 2). Curves generated from 12 separate assays from 2 different calibrator's batches and spread over a 9 month-period showed a mean variation in fluorescence signal of 17% (range 10.6–22.1%) over the entire calibrator curve (Fig. 2, insert). Inter-assay precision was established by replicated measures of two samples of known concentration in 7 assays and showed a mean of 322.1±36.5 ng/mL (CV of 11.3%, n = 14) and 1006.9±162.8 ng/mL (CV of 16.2%, n = 13). One sample was tested in replicates of 14 in 3 assays to assess intra-assay precision and showed a mean concentration of 256±16.4 ng/mL with a variation of 6.3% (range CV 5.2–8.1%). The minimum detectable dose, determined as the mean of 30 replicates of the zero calibrator plus 2 SD was 2.6 ng/mL. The lowest measurable concentration of sCD127 was 3.2 ng/mL (CV = 18%).


Development of a quantitative bead capture assay for soluble IL-7 receptor alpha in human plasma.

Faucher S, Crawley AM, Decker W, Sherring A, Bogdanovic D, Ding T, Bergeron M, Angel JB, Sandstrom P - PLoS ONE (2009)

Calibration curve of the sCD127 capture bead assay.Recombinant sCD127-Fc chimera was serially diluted from 0.1 to 1000 ng/mL in assay diluent (Materials and Methods) 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2723935&req=5

pone-0006690-g002: Calibration curve of the sCD127 capture bead assay.Recombinant sCD127-Fc chimera was serially diluted from 0.1 to 1000 ng/mL in assay diluent (Materials and Methods) 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.
Mentions: The sCD127 capture assay was calibrated with purified recombinant CD127-Fc chimera from 0.1 to 1000 ng/mL (Fig. 2). Curves generated from 12 separate assays from 2 different calibrator's batches and spread over a 9 month-period showed a mean variation in fluorescence signal of 17% (range 10.6–22.1%) over the entire calibrator curve (Fig. 2, insert). Inter-assay precision was established by replicated measures of two samples of known concentration in 7 assays and showed a mean of 322.1±36.5 ng/mL (CV of 11.3%, n = 14) and 1006.9±162.8 ng/mL (CV of 16.2%, n = 13). One sample was tested in replicates of 14 in 3 assays to assess intra-assay precision and showed a mean concentration of 256±16.4 ng/mL with a variation of 6.3% (range CV 5.2–8.1%). The minimum detectable dose, determined as the mean of 30 replicates of the zero calibrator plus 2 SD was 2.6 ng/mL. The lowest measurable concentration of sCD127 was 3.2 ng/mL (CV = 18%).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: National HIV and Retrovirology Laboratories, Public Health Agency of Canada, Ottawa, Canada. sylvie_faucher@phac-aspc.gc.ca

ABSTRACT

Background: IL-7 is an essential cytokine in T-cell development and homeostasis. It binds to the IL-7R receptor, a complex of the IL-7Ralpha (CD127) and common gamma (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.

Methodology/principal findings: 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.

Conclusions/significance: 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.

Show MeSH
Related in: MedlinePlus