Assay Performance Characteristics Research Articles

Evaluation of a Semiautomated System for the Quantitation of Human Adenovirus DNA from Clinical Samples.

Human adenoviruses (HAdVs) cause severe disease in immunocompromised patients. Quantitation of HAdV DNA in peripheral blood is used to assess the risk of disseminated disease and to monitor response to therapy. The lower limit of detection, precision, and linearity of the semiautomated AltoStar adenovirus quantitative PCR (qPCR) was evaluated using reference HAdV-E4 in EDTA plasma and respiratory virus matrix. Qualitative and quantitative agreement was determined using 122 clinical EDTA plasma specimens previously tested using a laboratory-developed HAdV qPCR. The 95% lower limit of detection (LLOD) was 33 IU/mL (95% confidence interval [CI], 10 to 56) for EDTA plasma and 188 IU/mL (95% CI, 145 to 304) for respiratory swab matrix. In both matrices, the AltoStar HAdV qPCR was linear from 7.0 to 2.0 log10 IU/mL. For the clinical specimens, overall agreement was 96.7% (95% CI, 91.8 to 99.1), positive percent agreement was 95.5% (95% CI, 87.6 to 98.5), and negative percent agreement was 98.2% (95% CI, 88.5 to 99.7). Passing-Bablok analysis of specimens quantifiable by both methods revealed a regression line of Y = 1.11 · X + 0.00; there was positive proportional bias (95% CI of the slope, 1.05 to 1.22) but no systematic bias (95% CI of the Y-intercept, -0.43 to 0.23) compared to the reference. The AltoStar platform provides accurate quantitation of HAdV DNA and provides a semiautomated option for the clinical monitoring of HAdV following transplantation. IMPORTANCE Accurate quantification of human adenovirus DNA in the peripheral blood plays a critical role in the management of adenovirus infections in transplant recipients. Many laboratories utilize in-house laboratory-based PCR assays for the quantification of human adenovirus, as there are few commercial options available. Here, we describe the analytical and clinical performance of the semiautomated AltoStar adenovirus quantitative PCR (Altona Diagnostics). This platform provides sensitive, precise, and accurate quantification of adenovirus DNA that is well suited for virological testing following transplantation. Prior to implementing a new quantitative test in the clinical laboratory, a rigorous evaluation is required to determine assay performance characteristics and to correlate results to current in-house methods of quantitation.

Assessment of the clinical and analytical performance of the Aptima SARS-CoV-2 assay using the VALCOR protocol

BackgroundThe COVID-19 pandemic highlighted the importance of diagnostic testing against curbing the spread of SARS-CoV-2. The urgent need and scale for diagnostic tools resulted in manufacturers of SARS-CoV-2 assays receiving emergency authorization that lacked robust analytical or clinical evaluation. As it is highly likely that testing for SARS-CoV-2 will continue to play a central role in public health, the performance characteristics of assays should be evaluated to ensure reliable diagnostic outcomes are achieved.MethodsVALCOR or “VALidation of SARS-CORona Virus-2 assays” is a study protocol designed to set up a framework for test validation of SARS-CoV-2 virus assays. Using clinical samples collated from VALCOR, the performance of Aptima SARS-CoV-2 assay was assessed against a standard comparator assay. Diagnostic test parameters such as sensitivity, specificity and overall per cent agreement were calculated for the clinical performance of Aptima SARS-CoV-2 assay.ResultsA total of 180 clinical samples were tested with an addition of 40 diluted clinical specimens to determine the limit of detection. When compared to the standard comparator assay Aptima had a sensitivity of 100.0% [95% CI 95.9–100.0] and specificity of 96.7% [95% CI 90.8–99.3]. The overall percent agreement was 98.3% with an excellent Cohen’s coefficient of κ = 0.967 [95% CI 0.929–1.000]. For the limit of detection, Aptima was able to detect all of the diluted clinical samples.ConclusionIn conclusion. validation of Aptima SARS-CoV-2 assay using clinical samples collated through the VALCOR protocol showed excellent test performance. Additionally, Aptima demonstrated high analytical sensitivity by detecting all diluted clinical samples corresponding to a low limit of detection.

Performance characteristics of diagnostic assays for schistosomiasis in Ontario, Canada.

Due to lower intensity of infection and greater intervals from last exposure, parasitologic detection methods for schistosomiasis are poorly sensitive in non-endemic areas, challenging accurate diagnosis. We evaluated parasitologic versus indirect detection methods for schistosomiasis. We included specimens submitted for Schistosoma serology, and stool for ova and parasite microscopy. Three real-time PCR assays targeting Schistosoma mansoni and S. haematobium were performed. Primary outcome measures were sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), where both microscopy and serology were the composite reference standard against serum PCR. Of 8168 serum specimens submitted for Schistosoma serology, 638 (7.8%) were reactive and 6705 (82.1%) were non-reactive. Of 156,771 stool specimens submitted for ova and parasite testing, 46 (0.03%) were positive for eggs of S. mansoni. Four (0.5%) urine specimens were positive for eggs of S. haematobium. Combined serum PCRs targeting S. mansoni had a sensitivity and specificity of 27.8% (95% CI = 18.3-39.1%) and 100% (95% CI = 83.9-100%), respectively, with PPV of 100% (95% CI = 100%) and NPV of 26.9% (95% CI = 24.3-29.7%). The one serum sample positive for S. haematobium was also detectable by our S. haematobium PCR. No cross-reactivity was observed for all three PCR assays. Although serology is highly sensitive, parasitologic tests signify active infection, but are limited by low population-level sensitivity, particularly in non-endemic settings. Although serum PCR offered no performance advantage over stool microscopy, its role in diagnostic parasitology should be pursued due to its high-throughput and operator-independent nature.

Highly sensitive liquid biopsy Duplex sequencing complements tissue biopsy to enhance detection of clinically relevant genetic variants.

Liquid biopsy (LB) is a promising complement to tissue biopsy for detection of clinically relevant genetic variants in cancer and mosaic diseases. A combined workflow to enable parallel tissue and LB analysis is required to maximize diagnostic yield for patients. We developed and validated a cost-efficient combined next-generation sequencing (NGS) workflow for both tissue and LB samples, and applied Duplex sequencing technology for highly accurate detection of low frequency variants in plasma. Clinically relevant cutoffs for variant reporting and quantification were established. We investigated assay performance characteristics for very low amounts of clinically relevant variants. In plasma, the assay achieved 100% sensitivity and 92.3% positive predictive value (PPV) for single nucleotide variants (SNVs) and 91.7% sensitivity and 100% PPV for insertions and deletions (InDel) in clinically relevant hotspots with 0.5-5% variant allele frequencies (VAFs). We further established a cutoff for reporting variants (i.e. Limit of Blank, LOB) at 0.25% VAF and a cutoff for quantification (i.e. Limit of Quantification, LOQ) at 5% VAF in plasma for accurate clinical interpretation of analysis results. With our LB approach, we were able to identify the molecular cause of a clinically confirmed asymmetric overgrowth syndrome in a 10-year old child that would have remained undetected with tissue analysis as well as other molecular diagnostic approaches. Our flexible and cost-efficient workflow allows analysis of both tissue and LB samples and provides clinically relevant cutoffs for variant reporting and precise quantification. Complementing tissue analysis by LB is likely to increase diagnostic yield for patients with molecular diseases.

Characterization by image analysis of the dose vs response curve for a qualitative serum hCG lateral flow immunoassay

BackgroundAs an adjunct to verification of performance characteristics of a qualitative serum hCG lateral flow immunoassay (LFI), we performed image analysis to characterize the dose vs response curve (visibility of the test line), as a means of understanding the transition from negative to positive as a function of increasing [hCG]. MethodsUsing serum samples of known [hCG], device images were obtained using a scanner at the prescribed reading time (5 min). Image analysis (using Python and R) was used to obtain the integral (S) of the test-line color as a function of [hCG]. ResultsData for S as a function of [hCG] were well characterized by a simple hyperbola: S = Smax [hCG]/([hCG] + K), where K = 202 mIU/ml (r = 0.997). Replicates of S at K had CV of 7.3 %. By eye, uncertainty of test results among users occurred only below the assay's stated sensitivity of 10 mIU/ml, in region of S < 3 % of Smax, and signal:noise ratio < 3. ConclusionsBy image analysis, the dose vs response (Test line integral) for this qualitative serum hCG LFI was a simple hyperbola. Characterization of the dose vs response curve was useful in verification of the assay's performance characteristics.

A robust, low-cost instrument for real-time colorimetric isothermal nucleic acid amplification.

The COVID-19 pandemic has highlighted the need for broader access to molecular diagnostics. Colorimetric isothermal nucleic acid amplification assays enable simplified instrumentation over more conventional PCR diagnostic assays and, as such, represent a promising approach for addressing this need. In particular, colorimetric LAMP (loop-mediated isothermal amplification) has received a great deal of interest recently. However, there do not currently exist robust instruments for performing these kinds of assays in high throughput with real-time readout of amplification signals. To address this need, we developed LARI, the LAMP Assay Reader Instrument. We have deployed over 50 LARIs for routine use in R&D and production environments, with over 12,000 assays run to date. In this paper, we present the design and construction of LARI along with thermal, optical, and assay performance characteristics. LARI can be produced for under $1500 and has broad applications in R&D, point-of-care diagnostics, and global health.

216.17: Multiplex Digital PCR for the Detection of Donor-derived Cell-free DNA

Introduction: Graft damage and failure are major problems after solid-organ transplantation. Biomarkers for monitoring transplanted patients provide new opportunities to substantially improve patient management, graft lifespan, and patient survival. There is strong evidence that donor-derived cell-free DNA (DD-cfDNA) increases when transplanted organs are damaged. The majority of this information is derived from tests using next-generation sequencing. Here we present an evaluation of new technology, multiplex digital PCR (mdPCR), which has the potential to revolutionize DD-cfDNA testing. Method: This mdPCR method takes advantage of Luminex’s DigiMAP multiplex PCR chemistry and the QuantStudio Absolute Q Digital PCR platform (Thermo Fisher Scientific, previously Combinati). The bench work for mdPCR is completed in less than five hours. Data is analyzed using a custom R package. Assay performance characteristics were determined using artificial mixtures of genomic DNA, and inter-laboratory reproducibility was assessed. Blood was collected from kidney and heart transplant recipients, cf-DNA was isolated using a variety of methods, and mdPCR was used to quantify DD-cfDNA levels. Results: This methodology distinguished donor and recipient DNA and provided absolute quantification of the number of DNA copies. The following performance characteristics were determined: sensitivity, the limit of detection, and linear range, inter- and intra- assay consistency. Several factors affected performance including: DNA characteristics, probe design, primer design, and PCR reaction components. Results showed that high levels of DD-cfDNA are consistent with clinical observations and that rapid testing is feasible (< five hours). Conclusion: The mdPCR approach has high promise to revolutionize diagnostic testing in cancer, infectious disease, and transplant monitoring. Our study demonstrates the advantages of this technology for the detection of DD-cfDNA which includes absolute quantitation, sensitivity, ease of use, short turn-around-time, and a relatively low cost when compared to next-generation sequencing techniques. A test with these features could transform the management of transplant recipients and lead to prolonged graft and patient survival.

Qualification of a Biolayer Interferometry Assay to Support AZD7442 Resistance Monitoring.

AZD7442, a combination of two long-acting monoclonal antibodies (tixagevimab [AZD8895] and cilgavimab [AZD1061]), has been authorized for the prevention and treatment of coronavirus disease 2019 (COVID-19). The rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants requires methods capable of quickly characterizing resistance to AZD7442. To support AZD7442 resistance monitoring, a biolayer interferometry (BLI) assay was developed to screen the binding of tixagevimab and cilgavimab to SARS-CoV-2 spike proteins to reduce the number of viral variants for neutralization susceptibility verification. Six spike variants were chosen to assess the assay's performance: four with decreased affinity for tixagevimab (F486S:D614G and F486W:D614G proteins) or cilgavimab (S494L:D614G and K444R:D614G proteins) and two reference proteins (wild-type HexaPro and D614G protein). Equilibrium dissociation constant (KD) values from each spike protein were used to determine shifts in binding affinity. The assay's precision, range, linearity, and limits of quantitation were established. Qualification acceptance criteria determined whether the assay was fit for purpose. By bypassing protein purification, the BLI assay provided increased screening throughput. Although limited correlation between pseudotype neutralization and BLI data (50% inhibitory concentration versus KD) was observed for full immunoglobulins (IgGs), the correlations for antibody fragments (Fabs) were stronger and reflected a better comparison of antibody binding kinetics with neutralization potency. Therefore, despite strong assay performance characteristics, the use of full IgGs limited the screening utility of the assay; however, the Fab approach warrants further exploration as a rapid, high-throughput variant-screening method for future resistance-monitoring programs. IMPORTANCE SARS-CoV-2 variants harbor multiple substitutions in their spike trimers, potentially leading to breakthrough infections and clinical resistance to immune therapies. For this reason, a BLI assay was developed and qualified to evaluate the reliability of screening SARS-CoV-2 spike trimer variants against anti-SARS-CoV-2 monoclonal antibodies (MAbs) tixagevimab and cilgavimab, the components of AZD7442, prior to in vitro pseudovirus neutralization susceptibility verification testing. The assay bypasses protein purification with rapid assessment of the binding affinity of each MAb for each recombinant protein, potentially providing an efficient preliminary selection step, thus allowing a reduced testing burden in the more technically complex viral neutralization assays. Despite precise and specific measures, an avidity effect associated with MAb binding to the trimer confounded correlation with neutralization potency, negating the assay's utility as a surrogate for neutralizing antibody potency. Improved correlation with Fabs suggests that assay optimization could overcome any avidity limitation, warranting further exploration to support future resistance-monitoring programs.

629 Performance monitoring of a streamlined and scalable non-invasive gene expression assay for pigmented lesions

The DermTech Pigmented Lesion Assay (PLA) is a qPCR-based assay that aids in the detection of melanoma by assessing up-regulation of two melanoma-associated genes, LINC00518 and PRAME, in skin samples collected by non-invasive adhesive patches. This study is to establish assay performance characteristics of the PLA post optimization, and to investigate the prognostic potential of the assay by comparison of qPCR amplification cycle in PLA positive samples with the histopathologic diagnoses of cutaneous pigmented lesions. RNA isolated from non-invasively collected skin cells was analyzed from seventy-five pigmented lesions. The PLA was optimized to simplify the laboratory workflow and increase scalability of the assay through a series of process improvements including RNA quantification by 1-step qRT-PCR (vs. reverse transcription followed by qPCR) and assay miniaturization. PLA result was determined by evaluating qPCR amplification of LINC00518 and PRAME multiplexed with housekeeping genes β-Actin and PPIA respectively. Samples that demonstrated over-expression of LINC00518 and PRAME relative to the housekeeping genes were considered PLA positive. Blinded to the PLA results, biopsy specimens from the pigmented lesions were examined and classified by a dermatopathologist as invasive melanoma (n=22); melanoma in situ (MIS) (n=26); benign nevus (24); or other (n=3). All twenty-two lesions classified as invasive melanoma by histopathology were PLA-positive, with an average LINC00518 Ct of 32.2 and PRAME Ct of 30.9. Seventeen of the twenty-six samples classified as MIS were PLA-positive, with an average LINC00518 Ct of 33.8 and PRAME Ct of 33.2. Of the twenty-seven samples not classified as melanoma, five were PLA-positive, with an average LINC00518 Ct of 34.4 and PRAME Ct of 35.8. The results indicate that PLA Ct values are highly correlated with histopathologic diagnoses. The optimized PLA assay demonstrated a sensitivity of 100% for invasive melanoma and 65% for MIS, with an overall specificity of 81%.

Validation of a Commercial Indirect ELISA Kit for the Detection of Bovine alphaherpesvirus1 (BoHV-1)-Specific Glycoprotein E Antibodies in Bulk Milk Samples of Dairy Cows.

Simple SummaryRecent studies have assessed the feasibility of using commercial indirect enzyme-linked immunosorbent assays (ELISA) on bulk milk (BM) samples. Today is a need to validate a diagnostic method for granting and maintaining a correct surveillance strategy for infectious bovine rhinotracheitis (IBR). However, the poor availability of reference materials for diagnostic tests on milk samples and, the low sensitivity of blocking glycoprotein E (gE) ELISAs using milk as a matrix, represent limitations for developing new assays to control infectious diseases in livestock. This study aimed to validate a commercial indirect ELISA kit to detect antibodies to gE of Bovine alphaherpesvirus 1 (BoHV-1) from BM samples, according to the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. The samples were collected from an IBR outbreak. The findings demonstrated high diagnostic performances. Furthermore, the validated kit is an easy-to-use and economical method to discriminate between BoHV-1 infected or immunised animals with gE-deleted markers vaccines using BM samples. Moreover, milk sampling represents a fast and non-invasive procedure for animals because it avoids negative effects, such as stress, and reduces the total cost of surveillance. In this study, we validated a commercial indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies to glycoprotein E (gE) of Bovine alphaherpesvirus 1 (BoHV-1) in bulk milk (BM) samples using the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. The assay performance characteristics were evaluated using a panel of positive (n = 36) and negative (n = 80) samples with known infectious bovine rhinotracheitis (IBR) status. The assay showed adequate repeatability (within-run and between-run), with a coefficient of variability (CV%) of replicates below 30%; only two 1:40 diluted samples had a CV% above 20%. Additionally, an agreement analysis of the qualitative results of replicates led to a Gwet’s agreement coefficient of 0.99 (95% confidence interval (CI): 0.96–1.00, p < 0.001). The estimated diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were 100% (95% CI: 90.3–100%) and 97.5% (95% CI: 91.3–99.7%), respectively. Overall, a good level of agreement was observed between the assay results and the true IBR status of samples (weighted Cohen’s κ: 0.96, 95% CI: 0.78–1.00). The findings demonstrate that the indirect ELISA kit validated here is an easy-to-use and economical method to differentiate infected and gE-deleted marker vaccine-immunised animals using BM samples.

Comparison of six regression-based lot-to-lot verification approaches.

Detection of between-lot reagent bias is clinically important and can be assessed by application of regression-based statistics on several paired measurements obtained from the existing and new candidate lot. Here, the bias detection capability of six regression-based lot-to-lot reagent verification assessments, including an extension of the Bland-Altman with regression approach are compared. Least squares and Deming regression (in both weighted and unweighted forms), confidence ellipses and Bland-Altman with regression (BA-R) approaches were investigated. The numerical simulation included permutations of the following parameters: differing result range ratios (upper:lower measurement limits), levels of significance (alpha), constant and proportional biases, analytical coefficients of variation (CV), and numbers of replicates and sample sizes. The sample concentrations simulated were drawn from a uniformly distributed concentration range. At a low range ratio (1:10, CV 3%), the BA-R performed the best, albeit with a higher false rejection rate and closely followed by weighted regression approaches. At larger range ratios (1:1,000, CV 3%), the BA-R performed poorly and weighted regression approaches performed the best. At higher assay imprecision (CV 10%), all six approaches performed poorly with bias detection rates <50%. A lower alpha reduced the false rejection rate, while greater sample numbers and replicates improved bias detection. When performing reagent lot verification, laboratories need to finely balance the false rejection rate (selecting an appropriate alpha) with the power of bias detection (appropriate statistical approach to match assay performance characteristics) and operational considerations (number of clinical samples and replicates, not having alternate reagent lot).

Liquid chromatography-tandem mass spectrometry based quantification of arginine metabolites including polyamines in different sample matrices

The conditionally essential amino acid arginine and its metabolic products play an important role in different biological processes, such as metabolic regulation of the immune response, including macrophage activation and polarization and regulation of T cell function. Furthermore, the polyamine spermidine has a role in aging and age-related diseases. Additionally, altered polyamine metabolism may be associated with neurodegenerative diseases, while polyamine levels may present useful biomarkers associated with severity of Parkinson's disease or with progression of non-alcoholic fatty liver disease. In the present study, a simple, derivatization-free hydrophilic interaction liquid chromatography based tandem mass spectrometry (LC-MS/MS) method is described, that allows the accurate quantification of arginine and related amine, polyamine and acetylated polyamine metabolites in different experimental sample matrices, such as cell lysates, cell culture supernatants and tissues. Ten arginine metabolites, including citrulline, agmatine, ornithine, putrescine, spermidine, spermine, N1-acetylspermidine, N1-acetylspermine, N1,N12-diacetylspermine and arginine in conjunction with the metabolic cofactors S-adenosylhomocysteine and S-adenosylmethionine are simultaneously analyzed within a total LC-MS/MS run time of 9.5 min. The assay is suitable to quantify concentration ranges over multiple orders of magnitude for all metabolites with averaged accuracies observed at 103.2% ± 6.8%, 99.0% ± 4.2% and 100.4% ± 4.3% in cell lysates, cell culture supernatant and tissue extracts, respectively. Inter-day coefficients of variation ranged from 5.9 to 14.8% in cell lysates, 6.7 to 14.6% in cell culture supernatants and 5.3 to 12.0% in tissue extracts. The method was successfully applied to cell culture systems of different origin as well as different murine tissues and organs. The herein described LC-MS/MS method provides a simple tool for a fast and simultaneous analysis of arginine metabolites, including polyamines and their respective metabolic cofactors. Assay performance characteristics demonstrate suitability for applications in different experimental and preclinical settings.

Dynamic Monitoring of Seroconversion using a Multianalyte Immunobead Assay for Covid-19

Multiplex technologies for interrogating multiple biomarkers in concert have existed for several decades; however, methods to evaluate multiple epitopes on the same analyte remain limited. This report describes the development and optimization of a multiplexed immunobead assay for serological testing of common immunoglobulin isotypes (e.g., IgA, IgM, and IgG) associated with an immune response to SARS-CoV-2 infection or vaccination. Assays were accomplished using a flow-based, multiplex fluorescent reader with dual-channel capability. Optimizations focused on analyte capture time, detection antibody concentration, and detection antibody incubation time. Analytical assay performance characteristics (e.g., assay range (including lower and upper limits of quantitation); and intra- and inter-assay precision) were established for either IgG/IgM or IgA/IgM serotype combination in tandem using the 'dual channel' mode. Analyte capture times of 30 min for IgG, 60 min for IgM, and 120 min for IgA were suitable for most applications, providing a balance of assay performance and throughput. Optimal detection antibody incubations at 4 µg/mL for 30 min was observed and are recommended for general applications, given the overall excellent precision (percent coefficient of variance (%CV) ≤ 20%) and sensitivity values observed. The dynamic range for the IgG isotype spanned several orders of magnitude for each assay (Spike S1, Nucleocapsid, and Membrane glycoproteins), which supports robust titer evaluations at a 1:500 dilution factor for clinical applications. Finally, the optimized protocol was applied to monitoring Spike S1 seroconversion for subjects (n = 4) that completed a SARS-CoV-2 vaccine regimen. Within this cohort, Spike S1 IgG levels were observed to reach maximum titers at 14 days following second dose administration, at a much higher (~40-fold) signal intensity than either IgM or IgA isotypes. Interestingly, we observed highly variable Spike S1 IgG titer decay rates that were largely subject-dependent were observed, which will be the topic of future studies.

Dynamic Monitoring of Seroconversion using a Multianalyte Immunobead Assay for Covid-19.

Multiplex technologies for interrogating multiple biomarkers in concert have existed for several decades; however, methods to evaluate multiple epitopes on the same analyte remain limited. This report describes the development and optimization of a multiplexed immunobead assay for serological testing of common immunoglobulin isotypes (e.g., IgA, IgM, and IgG) associated with an immune response to SARS-CoV-2 infection or vaccination. Assays were accomplished using a flow-based, multiplex fluorescent reader with dual-channel capability. Optimizations focused on analyte capture time, detection antibody concentration, and detection antibody incubation time. Analytical assay performance characteristics (e.g., assay range (including lower and upper limits of quantitation); and intra- and inter-assay precision) were established for either IgG/IgM or IgA/IgM serotype combination in tandem using the 'dual channel' mode. Analyte capture times of 30 min for IgG, 60 min for IgM, and 120 min for IgA were suitable for most applications, providing a balance of assay performance and throughput. Optimal detection antibody incubations at 4 µg/mL for 30 min was observed and are recommended for general applications, given the overall excellent precision (percent coefficient of variance (%CV) ≤ 20%) and sensitivity values observed. The dynamic range for the IgG isotype spanned several orders of magnitude for each assay (Spike S1, Nucleocapsid, and Membrane glycoproteins), which supports robust titer evaluations at a 1:500 dilution factor for clinical applications. Finally, the optimized protocol was applied to monitoring Spike S1 seroconversion for subjects (n = 4) that completed a SARS-CoV-2 vaccine regimen. Within this cohort, Spike S1 IgG levels were observed to reach maximum titers at 14 days following second dose administration, at a much higher (~40-fold) signal intensity than either IgM or IgA isotypes. Interestingly, we observed highly variable Spike S1 IgG titer decay rates that were largely subject-dependent were observed, which will be the topic of future studies.

Methods paper: Performance characteristics of novel assays for circulating levels of proglucagon-derived peptides and validation in a placebo controlled cross-over randomized clinical trial

BackgroundThe measurement of proglucagon-derived peptides (PGDPs) is a challenging task mainly due to major overlaps in their molecular sequence in addition to their low circulating levels. Here, we present the technical characteristics of novel ELISA assays measuring C-peptide and all six PGDPs including, for the first time, major proglucagon fragment (MPGF), and we validate them by performing a pilot in vivo cross-over randomized clinical trial on whether coffee consumption may affect levels of circulating PGDPs. MethodsThe performance and technical characteristics of novel ELISA assays from Ansh measuring GLP-1, GLP-2, oxyntomodulin, glicentin, glucagon, MPGF and C-peptide were first evaluated in vitro in procured samples from a commercial vendor as well as in deidentified human samples from three previously performed clinical studies. Their performance was further evaluated in vivo in the context of a cross-over randomized controlled trial, in which 33 subjects consumed in random order and together with a standardized meal, 200 ml of either (a) instant coffee with 3 mg/kg caffeine, or (b) instant coffee with 6 mg/kg caffeine, (c) or water. ResultsAll assays demonstrated high accuracy (spike and recovery and average linearity recovery ±15%), precision (inter-assay CV ≤ 6.4%), specificity (no significant cross-reactivities) and they were sensitive in low concentrations. Measurements of glicentin in archived random human samples using the Ansh assay correlated strongly with the glicentin measurements of Mercodia assay (r = 0.968) and of GLP-1 modestly with Millipore GLP-1 assay (r = 0.440). Oxyntomodulin, glicentin and glucagon concentrations were 2–5 fold higher in plasma compared to serum and serum concentrations correlated modestly (for oxyntomodulin and glicentin) or poorly (for glucagon) with the plasma concentrations. The evaluated assays detected a postprandial increase of gut-secreted PGDPs (GLP-1, GLP-2, oxyntomodulin and glicentin) and a postprandial decrease of pancreas-secreted PGDPs (glucagon, MPGF) in response to consuming coffee in comparison to consuming water with breakfast (enter here composition of breakfast). Only coffee consumption at the high dose alter levels of gut-secreted PGDPs and both at low and high dose to lower levels of pancreas-secreted PGDPs compared to water consumption during breakfast. ConclusionAccurate, precise and specific measurement of six PGDPs is possible with novel assays. A randomized controlled trial demonstrated in vivo utility of those assays and supports the notion that coffee may exert part of its beneficial effects on glucose homeostasis in the short term through the regulation of PGDPs.

Development of a loop-mediated isothermal amplification (LAMP) assay based on the C962R gene for african swine fever virus detection

Aim. The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) assay for African swine fever virus (ASFV) detection. Methods. Primer design was performed using publicly available full genome sequences of ASFV. A panel of heterologous DNA samples and reference ASFV DNA samples were used for the assay specificity testing. The limit of detection (LOD) was assessed using purified and quantified serial dilution of the amplified target sequence. LAMP product detection was performed via gel-electrophoresis and via ethidium bromide fluorescence under UV after adding the ethidium bromide directly to the tube with the LAMP product. Results. Three primer sets amplifying different regions of ASFV gene C962R were developed, of which the set № 2 providing the most intense product synthesis with the most vivid and clear pattern was selected for further studies. The optimal concentration of reaction mix components for the most effective primer set was established. In the final protocol the LAMP reaction was carried out at 60 °C for 40 min. The limit of detection (LOD) of the assay was 50 copies of the target sequence per reaction. In a preliminary testing the assay proved specific, using 10 reference and 4 heterologous viral and two bacterial DNA samples. Our LAMP assay detected ASFV genotypes I and II that are currently spread in Europe, Asia, and the Pacific and IX, occurring in Africa. Conclusion. A LAMP assay was developed based on the C962R gene that proved in preliminary validation to be specific and sensitive and was able to detect down to 50 copies per reaction of purified target gene within 40 minutes. Classical gel electrophoresis and direct staining using ethidium bromide were used for product visualisation in this study. Colorimetric approaches or the use of lateral flow devices in the visuali- sation step could make the assay less equipment dependent. Further validation of the assay, determining analytical specificity, selectivity and reproducibility performance characteristics also using clinical samples under field condi- tions and inclusion of an internal control would possibly enable its use as a test of choice at point-of-care and at low resource laboratories.

Minimal Residual Disease (MRD) Data in Hematologic Malignancy Drug Applications and Prescribing Information: FDA Analysis

Introduction: In the past decade, multiple studies have reported the prognostic and predictive value of MRD status in specific hematologic malignancies (HM). Because clinical trials are increasingly incorporating MRD status as a biomarker and efficacy endpoint, the adequacy of the MRD data to inform the prescribing information (PI) is relevant for the design and conduct of pivotal clinical trials. We present an analysis of the trends in inclusion of MRD data in pivotal trials in HMs and regulatory decisions made by the U.S. Food and Drug Administration (FDA).Methods: We reviewed FDA internal databases for original and supplemental new drug applications (NDAs) and biologics licensing applications (BLAs) submitted 1/2014-12/2020 to support approval of therapies (drugs, biologics, and cellular therapies) for HM. MRD data were evaluated for two time periods to inform potential trends: 1/2014-6/2017 (period 1) and 7/2017-12/2020 (period 2). Clinical study reports, selected datasets, FDA clinical reviews, and the proposed and approved PIs were examined for inclusion of MRD data, and FDA assessments of the adequacy of the MRD data for inclusion in the PI were reviewed.Results: Of 196 NDAs or BLAs involving HM submitted between 2014-2021, 53 (27%) had MRD data, including 53 pivotal trials. The trials included patients with chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, acute lymphocytic leukemia, and multiple myeloma. Twenty-one applications and pivotal trials with MRD data were submitted in period 1, and 32 applications and 35 trials were submitted in period 2. Three trials were resubmitted in period 2. MRD evaluation was specified as a secondary and exploratory endpoint in 35 (66%) and 19 (36%) of the trials, respectively. Of the 53 trials, MRD data was proposed by the Applicant for inclusion in the PI in 41 (77%) but was ultimately included in 25 (47%). Of the trials for which MRD data was proposed in labeling, MRD data were deemed adequate by FDA in 81% of studies in period 1 (13/16) and 48% of studies in period 2 (12/25). MRD assays in the PI included polymerase chain reaction, flow cytometry, and next-generation sequencing in 18 (72%), 5 (20%) and 4 (16%) of the trials, respectively, with the clinical threshold for test positivity ranging from 10 -3 to 10 -5. For 11 trials with MRD data in the PI (44%), the MRD was evaluated regardless of clinical response, and in 14 trials (56%) MRD was evaluated in patients achieving a specific clinical response. The leading reasons for excluding MRD data from the PI were analytical and test validation deficiencies (e.g., incomplete test characteristics data, lack of test validation overall or in that disease) followed by performance issues (e.g., high amount of test failure, inability to identify a clone) and issues with trial conduct or design (e.g., inadequate data collection, statistical issues).Conclusion: A quarter of HM drug applications, including 53 pivotal trials, submitted to the FDA between 2014-2020 included MRD data. Characterization of regulatory actions showed that despite the increasing number of submissions proposing MRD data for inclusion in the PI, rates of inclusion of MRD data in the PI did not reflect this increase. Improvements in assay validation and performance characteristics, robust collection of MRD data, and appropriate statistical planning can enable greater representation of MRD data in prescription drug labeling. DisclosuresNo relevant conflicts of interest to declare.

Liquid Biopsy Hotspot Variant Assays: Analytical Validation for Application in Residual Disease Detection and Treatment Monitoring.

Analysis of circulating tumor DNA (ctDNA) in plasma is a powerful approach to guide decisions in personalized cancer treatment. Given the low concentration of ctDNA in plasma, highly sensitive methods are required to reliably identify clinically relevant variants. We evaluated the suitability of 5 droplet digital PCR (ddPCR) assays targeting KRAS, BRAF, and EGFR variants for ctDNA analysis in clinical use. We investigated assay performance characteristics for very low amounts of variants, showing that the assays had very low limits of blank (0% to 0.11% variant allele frequency, VAF) and limits of quantification (0.41% to 0.7% VAF). Nevertheless, striking differences in detection and quantification of low mutant VAFs between the 5 tested assays were observed, highlighting the need for assay-specific analytical validation. Besides in-depth evaluation, a guide for clinical interpretation of obtained VAFs in plasma was developed, depending on the limits of blank and limits of quantification values. It is possible to provide comprehensive clinical reports on actionable variants, allowing minimal residual disease detection and treatment monitoring in liquid biopsy.

Development of a novel reporter gene assay to evaluate antibody-dependent cellular phagocytosis for anti-CD20 therapeutic antibodies

More than 100 monoclonal antibodies (mAbs) have been approved by FDA. The mechanism of action (MoA) involves in neutralization of a specific target via the Fab region and Fc effector functions through Fc region, while the latter include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). ADCP has been recognized one of the most important MoAs, especially for anti-cancer mAbs in recent years. However, traditional bioassays measuring ADCP always introduced primary macrophages and flow cytometry, which are difficult to handle and highly variable. In this study, we engineered a monoclonal Jurkat/NFAT/CD32a-FcεRIγ effector cell line that stably expresses CD32a-FcεRIγ chimeric receptor and NFAT-controlled luciferase. The corresponding mAb could bind with the membrane antigens on the target cells with its Fab fragment and CD32a-FcεRIγ on the effector cells with its Fc fragment, leading to the crosslinking of CD32a-FcεRIγ and the resultant expression of subsequent NFAT-controlled luciferase, which represents the bioactivity of ADCP based on the MoA of the mAb. With rituximab as the model mAb, Raji cells as the target cells, and Jurkat/NFAT/CD32a-FcεRIγ cells as the effector cells, we adopted the strategy of Design of Experiment (DoE) to optimize the bioassay. Then we fully validated the established bioassay according to ICH-Q2(R1), which proved the good assay performance characteristics of the bioassay, including specificity, accuracy, precision, linearity, stability and robustness. This RGA can be applied to evaluate the -ADCP bioactivity for anti-CD20 mAbs in lot release, stability testing as well as biosimilar comparability. The engineered cells may also potentially be used to evaluate the ADCP bioactivity of mAbs with other targets.

Feasibility of singlicate-based analysis in bridging ADA assay on Meso-Scale Discovery platform: comparison with duplicate analysis.

Aim: To investigate the feasibility of singlicate analysis in anti-drug antibody (ADA) assay by comparing performance characteristics for assays qualified in duplicate and singlicate formats. Materials & methods: We employed modeling to assess and quantify the impact of singlicate to cut point factor (CPF) in scenarios with the duplicate precision from 1-20% and the proportion of well-to-well variance to overall assay variance from 0.01-0.90. The impact to CPF by singlicate is marginal if the well-to-well coefficient of variation is <10% and represents <25% of the overall variability. Results & conclusion: The assay parameters including sensitivity, precision, selectivity, drug and target tolerance were comparable between singlicate and duplicate based assays. Our results suggested the minimal impact of singlicate analysis on ADA assay with good duplicate precision. The study provided additional supportive evidence that the singlicate-based analysis is feasible in ADA ligand bindingassays.