Laboratory testing.
Tralesinidase alfa quantification in CSF and K2EDTA plasma was based on an electrochemiluminescence immunoassay (ECLA) technique using standard-bind, 96-well Meso Scale Discovery (MSD) streptavidin-coated plates. Samples were collected before dosing and 0.5, 4, 10, 24, 48, 72, 96, and 168 hours after drug administration at weeks 1 (baseline), 5, 12, and 36 of Study 250-201 Part 2. CSF samples were collected from the lateral ventricle through the i.c.v. port. Methods were validated, and sample analysis was conducted by Charles River laboratories.ADAs were measured in the CSF and serum using an ECLA method with MSD technology. Samples were diluted in a Master Mix with an equal concentration of ruthenylated (sulfo-tagged) tralesinidase alfa and biotinylated tralesinidase in the wells of polypropylene plates. Detection of ADAs is based on the bivalent characteristics of the antibodies; ADAs bind to both sulfo-tagged and biotinylated molecules to form an antibody complex bridge. Samples are dispensed onto streptavidin-coated MSD assay plates to allow binding of biotinylated tralesinidase alfa to the streptavidin in the wells. Only the samples that contain ADAs bound to both the biotinylated and sulfo-tagged tralesinidase alfa generate an ECL signal. In the presence of tripropylamine, ruthenium produces a chemiluminescent signal that is triggered when voltage is applied. The signal produced is proportional to the amount of ADAs present. Endpoint titer determination estimates the relative levels of ADAs for those positive samples. ADA assays were validated and samples were run by ICON Laboratory Services.Neutralizing antibodies were quantified in CSF using a cell-based assay that measured the ability of ADAs to interfere on CI-MPR–mediated uptake of tralesinidase alfa by the human Jurkat cell line. Tralesinidase alfa conjugated to Alexa 647 was monitored and quantified by flow cytometry using median fluorescence intensity (MFI) as the readout. The method was validated and samples were analyzed by Eurofins Pharma Bioanalytics Services US.Levels of tralesinidase alfa–specific IgE were quantified using tralesinidase alfa covalently coupled to ImmunoCAP. After washing away nonspecific IgE, a β-gal–labeled mouse monoclonal antibody against human IgE was added to form a complex. After incubation, unbound enzyme anti-IgE was washed away, and the bound complex was incubated with the ImmunoCAP Development Solution containing 4-methylumbelliferyl-β-Total HS and HS-NRE measurements in CSF and plasma were performed by ARUP Laboratories using a previously described method (