The monoclonal antibody (MAb) AR20.5 is a murine MAb, generated against the tandem repeat protein backbone of the tumor-associated antigen MUC1. MAb AR20.5 reacts strongly with either the soluble form or the cell surface epitope of MUC1 on many human cancer cell lines. It also reacts with a 23-amino acid MUC1 peptide, E23, which includes the core tandem repeat sequence. Epitope mapping confirmed that MAb AR20.5 recognizes a minimum of six residues with the sequence DTRPAP. Inhibition of glycosylation of MUC1 resulted in decreased binding of MAb AR20.5 to cell surface MUC1, suggesting that MAb AR20.5 binding is carbohydrate dependent. The antibody was studied in a human PBL-SCID/beige mouse model to evaluate its effect on progression of NIH:OVARCAR-3 tumors. Tumor reduction was observed in mice injected with MAb AR20.5, but not in mice treated with control murine antibody or PBS (p < 0.001 and p < 0.05, respectively). An anti-tumor effect could also be demonstrated in a CB6F1 mouse model with the MUC1 transfectoma 413BCR.
BackgroundIL-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.Methodology/Principal FindingsWe 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/SignificanceThis 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.
The potential of Immunoglobulin M (IgM) in an Multi Analyte Profiling (xMAP) multiplex suspension assay was determined using Alt a1, a major allergen of Alternaria alternata, as the model target protein. LumAvidin (LAM) and carboxylated (CAM) microspheres, from LUMINEX™ Corporation, were compared. The reproducible standard curves for concentrations between 1 pg/mL and 10 ng/mL and 3 pg/mL and 300 ng/mL of the allergen were generated by the LAM assay, allowing for the quantitation of an unknown test sample. The success of the LAM assay is dependent on avidin molecules, initially bound on the microsphere, along with the use of a phycoerythrin-coupled secondary IgM. The standard curves generated by the CAM assay for the two allergen concentrations were erratic, falling short of quantifying the test sample. This is the first time a monoclonal IgM is successfully applied as a primary and secondary antibody in the xMAP format. The routine application of this assay will be consolidated as new IgM-based xMAP diagnostics become available. 2 M. Abebe et al.
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