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Background: The SARS-CoV-2 virus has mutated and evolved since the inception of the COVID-19 pandemic bringing into question the future effectiveness of current vaccines and antibody therapeutics. With evolution of the virus updated methods for the evaluation of the immune response in infected and vaccinated individuals are required to determine the durability of the immune response to SARS-CoV-2 variants., Methods: We developed a multiplexed surrogate virus neutralization test (plex-sVNT) that simultaneously measures the ability of antibodies in serum to inhibit binding between angiotensin converting enzyme-2 (ACE2) and 7 SARS-CoV-2 trimeric spike protein variants, including wild type, B.1.1.7(α), B.1.351(β), P.1(γ), B.1.617.2(δ), B.1.617.1(κ), and B.1.429(ε). The assay was validated against a plaque reduction neutralization test (PRNT).We evaluated 170 samples from 97 COVID-19 patients and 281 samples from 188 individuals that received the Pfizer-BioNTech or Moderna mRNA vaccines., Results: The plex-sVNT demonstrated >96% concordance with PRNT. Antibody neutralization activity was significantly reduced for all SARS-CoV-2 variants compared to wild type in both the infected and vaccinated cohorts. There was a decline in overall antibody neutralization activity, within both cohorts, out to 5 months post infection or vaccination, with the rate of decline being more significant for the vaccinated., Conclusions: The plex-sVNT provides a correlative measure to PRNT and a convenient approach for evaluating antibody neutralization against SARS-CoV-2 variants. Neutralization of SARS-CoV-2 variants is reduced compared to wild type and declines over the ensuing months after exposure or vaccination within each cohort, however it is still unknown what degree of neutralizing capacity is protective., (© American Association for Clinical Chemistry 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Biomarkers have been widely explored for coronavirus disease 2019 diagnosis. Both viral RNA or antigens (Ag) in the respiratory system and antibodies (Ab) in blood are used to identify active infection, transmission risk, and immune response but have limitations. This study investigated the diagnostic utility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (N-Ag) in serum., Methods: We retrospectively studied 208 randomly selected cases with SARS-CoV-2 infection confirmed by viral RNA test in swabs. N-Ag concentrations were measured in remnant serum samples, compared to viral RNA or Ab results, and correlated to electronic health records for clinical value evaluation., Results: Serum N-Ag was detected during active infection as early as day 2 from symptom onset with a diagnostic sensitivity of 81.5%. Within 1 week of symptom onset, the diagnostic sensitivity and specificity reached 90.9% (95% CI, 85.1%-94.6%) and 98.3% (95% CI, 91.1%-99.9%), respectively. Moreover, serum N-Ag concentration closely correlated to disease severity, reflected by highest level of care, medical interventions, chest imaging, and the length of hospital stays. Longitudinal analysis revealed the simultaneous increase of Abs and decline of N-Ag., Conclusions: Serum N-Ag is a biomarker for SARS-CoV-2 acute infection with high diagnostic sensitivity and specificity compared to viral RNA in the respiratory system. There is a correlation between serum N-Ag concentrations and disease severity and an inverse relationship of N-Ag and Abs. The diagnostic value of serum N-Ag, as well as technical and practical advantages it could offer, may meet unsatisfied diagnostic and prognostic needs during the pandemic., (© American Association for Clinical Chemistry 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Immunoassays based on label-free technologies (label-free immunoassay [LFIA]) offer an innovative approach to clinical diagnostics and demonstrate great promise for therapeutic drug monitoring (TDM) of monoclonal antibody (mAb) drugs. An LFIA measures immunocomplex formation in real time and allows for quantification on initial binding rate, which facilitates fast measurement within a few minutes., Methods: Based on thin-film interferometry (TFI) technology, open-access LFIAs were developed for the quantification of the mAb drugs adalimumab (ADL) and infliximab (IFX) and for the detection of the antidrug antibodies (ADAs) to the mAb drugs (ADL-ADAs and IFX-ADAs)., Results: The LFIAs for active mAb drugs (ADL and IFX) and for ADAs (ADL-ADAs and IFX-ADAs) were validated. The analytical measurement range (AMR) for both ADL and IFX was from 2 to 100 μg/mL. The AMR for ADL-ADAs was from 5 to 100 μg/mL and for IFX-ADAs was 10 to 100 μg/mL. In the comparison of LFIAs and reporter gene assays, the correlation coefficient was 0.972 for the quantification of ADL and 0.940 for the quantification of IFX. The concordance rate was 90% for the detection of ADL-ADAs and 76% for the detection of IFX-ADAs., Conclusions: The LFIAs for active mAb drugs and ADAs were appropriate for the TDM of ADL and IFX. The TFI technology has unique advantages compared with other technologies used for the measurement of mAb drugs. Label-free technologies, especially those allowing for open-access LFIAs, have great potential for clinical diagnostics., (© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Cannabis use results in impaired driving and an increased risk of motor vehicle crashes. Cannabinoid concentrations in blood and other matrices can remain high long after use, prohibiting the differentiation between acute and chronic exposure. Exhaled breath has been proposed as an alternative matrix in which concentrations may more closely correspond to the window of impairment; however, efficient capture and analytically sensitive detection methods are required for measurement., Methods: Timed blood and breath samples were collected from 20 volunteers before and after controlled administration of smoked cannabis. Cannabinoid concentrations were measured using LC-MS/MS to determine release kinetics and correlation between the 2 matrices., Results: Δ9-Tetrahydrocannabinol (THC) was detected in exhaled breath for all individuals at baseline through 3 h after cannabis use. THC concentrations in breath were highest at the 15-min timepoint (median = 17.8 pg/L) and declined to <5% of this concentration in all participants 3 h after smoking. The decay curve kinetics observed for blood and breath were highly correlated within individuals and across the population., Conclusions: THC can be reliably detected throughout the presumed 3-h impairment window following controlled administration of smoked cannabis. The findings support breath THC concentrations as representing a physiological process and are correlated to blood concentrations, albeit with a shorter window of detection., (© 2019 American Association for Clinical Chemistry.)

Background: Untargeted data acquisition on high-resolution mass spectrometers (HRMSs) has been used in clinical toxicology for screening and identifying unknown compounds in patient samples. A common modality for untargeted HRMS data acquisition is information-dependent acquisition (IDA), which analyzes the most abundant small molecules within an acquisition cycle. This process can potentially lead to false negatives of clinically relevant compounds at low concentrations. Sequential window acquisition of all theoretical fragment ion spectra (SWATH) has emerged as a method of unbiased, untargeted HRMS data acquisition in which no spectral data are lost. SWATH has yet to be optimized and assessed for use in clinical toxicology., Method: We developed a variable-window SWATH method (vSWATH) and compared it to IDA by limit of detection studies in drug-supplemented urine (81 compounds) and against a retrospective cohort of 50 clinical urine samples characterized by LC-MS/MS., Results: vSWATH had a lower limit of detection than IDA for 33 (41%) drugs and metabolites added into urine samples. Both IDA and vSWATH were equivalent in discovering compounds from clinical urine samples and confirmed 26 additional compounds not previously discovered by targeted LC-MS/MS. Lastly, the unbiased acquisition of spectra in vSWATH allowed for identification of 5 low-abundance compounds missed by IDA., Conclusions: This vSWATH method for clinical toxicology demonstrated equivalent analytical sensitivity and specificity for untargeted drug screening and identification in urine samples. vSWATH provided the additional benefit of collecting all tandem mass spectrometry spectra in a sample, which could be useful in discovering low-abundance compounds not discovered by IDA., (© 2019 American Association for Clinical Chemistry.)

Background: Liquid chromatography high-resolution mass spectrometry (LC-HRMS) with untargeted data collection is especially attractive for general unknown drug screening owing to its ability to identify unexpected compounds. LC-HRMS offers several advantages over traditional selected reaction monitoring (SRM) techniques and could be an ideal screening platform as long as its analytical performance is comparable to that of SRM-based methods., Methods: We developed a broad-spectrum drug screen on a high-resolution mass spectrometer [tandem quadrupole time-of-flight (QqTOF)] that collected data in an untargeted manner and compared its performance to a nominal mass instrument [triple quadrupole linear ion trap (QqLIT)] that collected data in a targeted manner. Both methods used information-dependent acquisition of product ion spectra. We evaluated the lower limits of detection and matrix effects for each method and compared their ability to identify drugs in 100 routine clinical urine samples. Additional information (patient prescription history, drug screening results, etc.) was used to confirm discordant results., Results: QqLIT was slightly more analytically sensitive than QqTOF; however, this difference did not significantly affect compound identification in patient samples. QqLIT identified 596 drugs in the urine samples, of which 531 (89%) were confirmed. QqTOF identified 515 drugs, of which 500 (97%) were confirmed. There were 562 instances of a confirmed drug (68 unique drugs) in the 100 urine samples; the methods were concordant in 469 of these instances., Conclusions: Overall, QqTOF performed similarly to QqLIT and could serve as an alternative method for general unknown screening., (© 2015 American Association for Clinical Chemistry.)