ABSTRACT
Aim: Serotype-specific assays detecting pneumococcal polysaccharides in bodily fluids are needed to understand the pneumococcal serotype distribution in non-bacteremic pneumonia. Methods: We developed a urine antigen detection assay and using urine samples from adult outpatients without pneumonia developed positivity cutoffs for both a previously published 15-valent and the new 21-valent assay. Clinical sensitivity was confirmed with samples from patients with invasive pneumococcal disease. Results: Total assay precision ranged from 7.6 to 17.8% coefficient of variation while accuracy ranged between 80 and 150% recovery, except for three serotypes where recoveries ranged from 32 to 60%. Clinical sensitivity was 86.4% and specificity was 96.5% across all 30 serotypes. Conclusion: The assay could potentially assess serotype-distribution in non-infected and infected participants with pneumococcal disease.
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ABSTRACT
Dilutions are a common source of analytical error, both in terms of accuracy and precision, and a common source of analyst mistakes. When serial dilutions are used, errors compound, even when employing laboratory automation. Direct point dilutions instead of serial dilutions can reduce error but is often impractical as they require either large diluent volumes or very small sample volumes when performed with traditional liquid handling equipment. We evaluated preparation of dilution curves using a picoliter digital dispenser, the HP, Inc. / TECAN D300 which is capable of accurately delivering picoliter volumes directly into sample wells filled with assay diluent. Dilution linearity and variability of the direct dilutions were similar to or less than those generated with a traditional liquid handler as measured using a fluorophore assay and an ELISA used to measure vaccine potency. Minimum concentrations for detergent in the dispensed sample were identified but no correlation with detergent characteristics was observed. The tolerance to protein in the sample was evaluated as well with up to 5% BSA having no impact on dispense linearity and precision. We found the digital dispenser to reduce automation complexity while maintaining or improving assay performance in addition to facilitating complex plate lay-outs.
Subject(s)
Enzyme-Linked Immunosorbent Assay/instrumentation , High-Throughput Screening Assays/instrumentation , Vaccine Potency , Automation, Laboratory , Calibration , Detergents/chemistry , Enzyme-Linked Immunosorbent Assay/standards , Equipment Design , Fluorescent Dyes/chemistry , High-Throughput Screening Assays/standards , Miniaturization , Reference Standards , Reproducibility of Results , Spectrometry, FluorescenceABSTRACT
Pharmacokinetic data derived from assays that accurately and precisely quantitate a therapeutic drug in circulation are critical to appropriately designing suitable dosing schedules. This manuscript describes the validation and implementation of methods to quantitate a therapeutic anti-human PCSK9 monoclonal antibody in rat and monkey sera as well as immunogenicity methods to screen the possible presence of rat and monkey antibodies directed against the antibody. As soluble, endogenous PCSK9 can interfere with a PCSK9-mediated capture step in ELISA, an indirect target-capture assay was used that potentially could capture free and target-engaged therapeutic mAb. Immunogenicity assays were based on a standard bridge ELISA using the therapeutic antibody for capture and detection. Both pharmacokinetic and immunogenicity assays were implemented in preclinical studies of the therapeutic antibody. The methods presented here may enable further pharmacokinetic studies.
