Development, Validation, and Clinical Implementation of an Assay to Measure Total Antibody Response to Naglazyme® (Galsulfase)

Naglazyme® (galsulfase, rhASB) was developed as enzyme replacement therapy for mucopolysaccharidosis type VI. Naglazyme generated an IgG antibody response in most patients. To better characterize Naglazyme immunogenicity, a solution phase bridged immunoassay was developed to measure total antibody response regardless of isotype. Overnight incubation of serum dilutions with rhASB labeled with biotin and ruthenium-based tags allowed antibody–antigen complexes to form prior to capture on a streptavidin plate. Neat serum was tolerated in the assay, with a 1:10 screening dilution implemented for testing. At this dilution, the assay was sensitive to 75 ng/ml anti-rhASB. Titers were reported as the highest dilution factor with signal above a 95% confidence interval from naïve individual sera. Precise measurement of titers, within two consecutive dilution factors, was observed across analysts and days. Clinical samples showed similar positive/negative results between the IgG ELISA and the total antibody ECLA, although with an imperfect correlation. Improvements in assay performance and implementation strategy altered some positive clinical samples to negative and vice versa. Comparison of the titer readout for clinical samples with the screening signal illustrates a range of relationships for signal versus sample dilution factor, confirming that signal from a screening dilution cannot directly predict the reported titer

Comparison of Neutralizing Antibody Assays for Receptor Binding and Enzyme Activity of the Enzyme Replacement Therapeutic Naglazyme® (Galsulfase)

Most patients receiving Naglazyme® (galsulfase, rhASB) enzyme replacement therapy for mucopolysaccharidosis type VI develop an antibody response. To evaluate the impact of this response, two in vitro neutralizing antibody (NAb) assays were developed based on the two steps of the mechanism of action. Neutralization of enzyme activity was detected by inhibition of rhASB cleavage of a fluorogenic substrate. Neutralization of receptor binding was detected by decreased binding of labeled rhASB to immobilized soluble receptor. For the enzyme activity NAb assay, serum pretreatment was required to isolate antibodies from interfering phosphate ions, with sensitivity of ≤5 μg/mL. The receptor binding NAb assay used a five-fold dilution, with sensitivity of ≤40 μg/mL. Cutpoints for percent inhibition were based on 95% confidence intervals from naïve sera. Clinical samples were similarly likely to be positive in both assays than positive for neutralization of only one step in the mechanism of action. The two NAb assays yielded complementary information about potential neutralization of rhASB. Relative estimated sensitivity between neutralization assays did not correlate with the number of positive clinical samples or patients. In vitro NAb assays based on a well-understood mechanism of action provide specific information about the NAb mechanism

Monitoring SARS-CoV-2 incidence and seroconversion among university students and employees: a longitudinal cohort study in California, June–August 2020

ObjectivesTo identify incident SARS-CoV-2 infections and inform effective mitigation strategies in university settings, we piloted an integrated symptom and exposure monitoring and testing system among a cohort of university students and employees.DesignProspective cohort study.SettingA public university in California from June to August 2020.Participants2180 university students and 738 university employees.Primary outcome measuresAt baseline and endline, we tested participants for active SARS-CoV-2 infection via quantitative PCR (qPCR) test and collected blood samples for antibody testing. Participants received notifications to complete additional qPCR tests throughout the study if they reported symptoms or exposures in daily surveys or were selected for surveillance testing. Viral whole genome sequencing was performed on positive qPCR samples, and phylogenetic trees were constructed with these genomes and external genomes.ResultsOver the study period, 57 students (2.6%) and 3 employees (0.4%) were diagnosed with SARS-CoV-2 infection via qPCR test. Phylogenetic analyses revealed that a super-spreader event among undergraduates in congregate housing accounted for at least 48% of cases among study participants but did not spread beyond campus. Test positivity was higher among participants who self-reported symptoms (incidence rate ratio (IRR) 12.7; 95% CI 7.4 to 21.8) or had household exposures (IRR 10.3; 95% CI 4.8 to 22.0) that triggered notifications to test. Most (91%) participants with newly identified antibodies at endline had been diagnosed with incident infection via qPCR test during the study.ConclusionsOur findings suggest that integrated monitoring systems can successfully identify and link at-risk students to SARS-CoV-2 testing. As the study took place before the evolution of highly transmissible variants and widespread availability of vaccines and rapid antigen tests, further research is necessary to adapt and evaluate similar systems in the present context