Assay Methodology Research Articles

Measurement and clinical applications of C-reactive protein in gastrointestinal diseases of dogs.

C‐reactive protein (CRP) is a positive acute‐phase protein, serum concentrations of which increase nonspecifically in response to inflammatory processes of the dog. As such, it can aid in the identification of inflammatory disease and, maybe more importantly, the objective monitoring of disease progression. In dogs, CRP is frequently used to evaluate dogs with gastrointestinal diseases, such as chronic inflammatory enteropathies (also termed idiopathic inflammatory bowel disease), acute pancreatitis, canine parvovirus infection, hepatic disease, acute abdomen, and protein‐losing enteropathy. The diversity of the assays available to measure CRP in dogs is nearly as numerous as the diseases in which serum concentrations of this protein are increased. Assay methodologies include laser nephelometric immunoassays, enzyme‐linked immunosorbent assays, immunoturbidimetric assays, and time‐resolved immunofluorometric assays. While many of these assays are acceptable for clinical use in the dog, the same assay and analyzer should be used to measure a patient's CRP concentration longitudinally. By looking at the uses of CRP in human gastroenterology, including reducing the duration of antibiotic therapy, the veterinary profession can gain insight into novel ways in which serum CRP concentration measurements might be applied in veterinary medicine in the future.

CMG helicase activity on G4-containing DNA templates.

G-quadruplexes (G4s) are non-canonical nucleic acid structures that form in G-rich regions of the genome and threaten genome stability by interfering with DNA replication. However, the underlying mechanisms are poorly understood. We have recently found that G4s can stall eukaryotic replication forks by blocking the progression of replicative DNA helicase, CMG. In this paper, we detail the methodology of DNA unwinding assays to investigate the impact of G4s on CMG progression. The method details the purification of recombinantly expressed CMG from the budding yeast, Saccharomyces cerevisiae, purification of synthetic oligonucleotides, and covers various aspects of DNA substrate preparation, reaction setup and result interpretation. The use of synthetic oligonucleotides provides the advantage of allowing to control the formation of G4 structures in DNA substrates. The methods discussed here can be adapted for the study of other DNA helicases and provide a general template for the assembly of DNA substrates with distinct G4 structures.

Studies of GenK and OxsB, two B12-dependent radical SAM enzymes involved in natural product biosynthesis.

The B12-dependent radical SAM enzymes are an emerging subgroup of biological catalysts that bind a cobalamin cofactor in addition to the canonical [Fe4S4] cluster characteristic of radical SAM enzymes. Most of the B12-dependent radical SAM enzymes that have been characterized mediated methyltransfer reactions; however, a small number are known to catalyze more diverse reactions such as ring contractions. Thus, Genk is a methyltransferase from the gentamicin C biosynthetic pathway, whereas OxsB catalyzes the oxidative ring contraction of 2'-deoxyadenosine 5'-phosphates to generate an oxetane aldehyde during the biosynthesis of oxetanocin A. The preparation and in vitro characterization of such enzymes is complicated by the presence of two redox sensitive cofactors in addition to challenges in obtaining soluble protein for study. This chapter describes expression, purification and assay methodologies for GenK and OxsB highlighting the use of denaturation/refolding protocols for solubilizing inclusion bodies as well as the use of cluster assembly and cobalamin uptake machinery during in vivo expression.

A Systematic Study and Potential Limitations of Proton-ELISA Platform for α-Synuclein Antigen Detection

To evaluate point-of-care testing (POCT) for the potential early detection of biomarkers of Parkinson’s disease, a systematic investigation of portable and low-cost platforms is performed based on the Proton-enzyme-linked immunosorbent assay (Proton-ELISA) methodology. The detection of the α-synuclein antigen was first presented by biotin-relative linkers, and glucose substrate solution was first performed with a systematic experimental design to optimize the sensing results. All materials in this study are commercially available. Three different experiments with the partitional check were performed to investigate the Proton-ELISA platform, including proton catalyzed efficiency, blocking efficiency, and full Proton-ELISA procedure. The response time was selected as 15 min by the time-dependent curves of a full reaction. The limit of detection of conventional ELISA kits is 0.169 ng/mL, which is much lower than the Proton-ELISA results. The final response of the full Proton-ELISA procedure to pH changes was approximately 0.60 and 0.12 for α-synuclein antigen concentrations of 100 ng/mL and 4 ng/mL, respectively. With the partitional check, pH changes of pure glucose substrate and conjugated oxidase and interference of the nonspecific binding are 1.7 and 0.04, respectively. The lower pH changes far from the partitional check results can be concluded for the properties of glucose oxidase conjugation, including the isoelectric point and binding affinity modification by the testing environment. This preliminary guideline can be used as a lesson learnt to speed up following studies of the evaluation and optimization of other antigen detection. Therefore, Proton-ELISA can be suggested for some special applications with the help of custom-designed conjugation in the environment with less degradation or interference and a proper detection concentration range.

Current status of CD34+cell enumeration assay in clinical laboratories and its improvement

Objective: To investigate the current status and problems of CD34+ cell enumeration in clinical laboratories and provide suggestions for the development of quality improvement programs. Methods: A total of 101 laboratories participating in the national external quality assessment program of CD34+cell enumeration were surveyed. Questionnaires and quality assessment materials were distributed to collect information on assay methodology and testing results. Quality control requirements for CD34+cell enumeration were determined according to the international guidelines, and the compliance of the surveyed laboratories was analyzed. Testing results were analyzed in groups and compared with the College of American Pathologists (CAP) quality assessment data. Results: A total of 97 laboratories returned the questionnaires and 99 laboratories returned the results of quality assessment materials. The questionnaire data showed high compliance rates of quality control requirements such as gating protocols, pipetting methods, and the number of cells acquired (92.8%, 83.9%, and 82.5% respectively). However, these laboratories had relatively low compliance rates such as the use of whole blood quality control materials for internal quality control, selection of erythrocyte lysing reagents, sample processing method, whether to report absolute count results, and quality control of equipment (5.2 %, 28.9%, 39.2%, 46.4%, and 55.7%, respectively). Testing results demonstrated that the coefficient of variation (CV) of percent counts was similar to the CAP quality assessment data, but the CV of absolute counts was greater than the CAP quality assessment data. Conclusions: Clinical laboratories have poor compliance with some quality control requirements and the variability of absolute count results between different laboratories is not satisfactory. Therefore, it is recommended that clinical laboratories should strengthen the training related to the quality control of CD34+cell enumeration, especially the absolute counting.

Performance of the T-SPOTⓇ.COVID test for detecting SARS-CoV-2-responsive T cells

ObjectiveTo evaluate the performance of the T-SPOT.COVID test for identifying SARS-CoV-2-responsive T-cells in participants with SARS-CoV-2 infection. MethodsThe T-SPOT.COVID test uses ELISpot interferon-gamma release assay (IGRA) methodology to measure T cell responses to SARS-CoV-2 spike S1 and nucleocapsid peptides. T-SPOT.COVID and anti-N immunoglobulin (Ig) G serology tests were performed on blood from 186 patients with nucleic acid amplification test (NAAT)-confirmed-SARS-CoV-2 infection and 100 control group participants. ResultsIn the 2–8 weeks after NAAT-diagnosed SARS-CoV-2 infection, the T-SPOT.COVID test detected 98.4% (63 of 64) of infected participants, while anti-N IgG serology detected 82.8%. In the first 2 weeks after diagnosis, during adaptive immune response activation, there were less reactive T-SPOT.COVID responses (75.7%, 28 of 37 infected participants) and many less seropositive responses (32.4%). Response numbers tapered after 8 weeks; however, T-SPOT.COVID test continued to detect most participants with confirmed infection (83.6%, 56 of 67) and continued to out-perform serology (52.2%). T-SPOT.COVID response due to cross-reactive T cells was ruled out by demonstrating that, of 44 control group participants with T cells responsive to 4 human common cold coronavirus peptides, only 1 was T-SPOT.COVID reactive. ConclusionThe T-SPOT.COVID test performed well in detecting SARS-CoV-2-sensitized T-cells over many months.

Revisiting the placental clock: Early corticotrophin-releasing hormone rise in recurrent preterm birth.

ObjectiveTo determine if maternal plasma CRH and preterm birth history were associated with recurrent preterm birth risk in a high-risk cohort.Study designSecondary analysis of pregnant women with a prior preterm birth ≤35 weeks receiving 17-alpha hydroxyprogesterone caproate for the prevention of recurrent spontaneous preterm birth. All women with a 24-week blood sample were included. Maternal plasma CRH level at 24- and 32-weeks’ gestation was measured using both enzyme-linked immunosorbent assay (ELISA) and extracted radioimmunoassay (RIA) technologies. The primary outcome was spontaneous preterm birth <37 weeks. The association of CRH, prior preterm birth history, and the two combined was assessed in relation to recurrent preterm birth risk.ResultsRecurrent preterm birth in this cohort of 169 women was 24.9%. Comparing women who subsequently delivered <37 versus ≥37 weeks, mean levels of CRH measured by RIA were significantly different at 24 weeks (111.1±87.5 vs. 66.1±45.4 pg/mL, P = .002) and 32 weeks (440.9±275.6 vs. 280.2±214.5 pg/mL, P = .003). The area under the receiver operating curve (AUC) at 24 and 32 weeks for (1) CRH level was 0.68 (95% CI 0.59–0.78) and 0.70 (95% CI 0.59–0.81), (2) prior preterm birth history was 0.75 (95% CI 0.67–0.83) and 0.78 (95% CI 0.69–0.87), and (3) combined was 0.81 (95% CI 0.73–0.88, P = .001) and 0.81 (95% CI 0.72–0.90, P = .01) respectively for delivery <37 weeks. CRH measured by ELISA failed to correlate with gestational age or other clinical parameters.ConclusionIn women with a prior preterm birth, CRH levels were higher and had an earlier rise in women who experienced recurrent preterm birth. Second trimester CRH may be useful in identifying a sub-group of women with preterm birth due to early activation of the placenta-fetal adrenal axis. Assay methodology is a variable that contributes to difficulties in reproducibility of CRH levels in the obstetric literature.

Myeloma patients with COVID‐19 have superior antibody responses compared to patients fully vaccinated with the BNT162b2 vaccine

SummaryPatients with multiple myeloma (MM) have a suboptimal antibody response following vaccination against severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and lower seroconversion rates following coronavirus disease 2019 (COVID‐19) compared with healthy individuals. In this context, we evaluated the development of neutralising antibodies (NAbs) against SARS‐CoV‐2 in non‐vaccinated patients with MM and COVID‐19 compared with patients after vaccination with two doses of the BNT162b2 vaccine. Serum was collected either four weeks post confirmed diagnosis or four weeks post a second dose of BNT162b2. NAbs were measured with a Food and Drug Administration‐approved enzyme‐linked immunosorbent assay methodology. Thirty‐five patients with COVID‐19 and MM along with 35 matched patients were included. The two groups did not differ in age, sex, body mass index, prior lines of therapy, disease status, lymphocyte count, immunoglobulin levels and comorbidities. Patients with MM and COVID‐19 showed a superior humoral response compared with vaccinated patients with MM. The median (interquartile range) NAb titre was 87·6% (71·6–94%) and 58·7% (21·4–91·8%) for COVID‐19‐positive and vaccinated patients, respectively (P = 0·01).Importantly, there was no difference in NAb production between COVID‐19‐positive and vaccinated patients who did not receive any treatment (median NAb 85·1% vs 91·7%, P = 0·14). In conclusion, our data indicate that vaccinated patients with MM on treatment without prior COVID‐19 should be considered for booster vaccine doses.

Immunological assays of hemocytes in the Northern Quahog Mercenaria mercenaria

The northern quahog Mercenaria mercenaria (commonly named hard clam) is an important aquaculture and fishery species along the Atlantic west coast. Environmental stresses, such as heat shock, fluctuating salinity, and harmful algal blooms are major challenges for clam aquaculture. In response to environmental stresses, hemocytes would change dynamically for defense and immunity. The goal of this study was to characterize basic immunological assays of hemocytes in the northern quahog by use of flow cytometry. The objectives were to: 1) develop a non-lethal method for hemolymph collection and dilution; 2) verify the capability of flow cytometry for hemocyte count and type identification through comparison with microscopic observation; 3) validate hemocyte viability assay based on plasma membrane integrity, and 4) develop hemocyte phagocytosis assay by use of fluorescein labeled microbeads. A non-lethal hemocyte collection method was developed using needle insertion through the ligament. Osmolality measurement of serum was the same as that of culture seawater. The pH measurement of serum (7.2) was significantly different from that of culture seawater (8.4). By microscopic observation, three types of hemocytes were identified with granulocytes, the dominant cell type (70 ± 16%), agranulocyte (14 ± 4%), and blast-like cell (16 ± 4%), and no differences were found from the measurements by flow cytometer on FSC/SSC plot (cell size/granularity). The viability of hemocytes based on plasma membrane integrity was 88 ± 6% ranging from 70 to 97% (n = 60, three populations), and viability protocol was further validated with the pre-set expected viability (p ≥ 0.424). Phagocytosis assay of hemocytes with fluorescence beads showed a mean capacity of 10 ± 5% (n = 60, three populations). Incubation time (up to 6 h) or bead concentrations (2:1 or 5:1 to hemocytes) did not affect the phagocytosis measurement. Overall, this study reported the basic characteristics of hemolymph (serum and hemocytes) of northern quahogs. It is expected that the assay methodologies will be applied to evaluation of hemocyte responses to environmental stresses for clam aquaculture.

Validation of Single Nucleotide Variant Assays for Human Leukocyte Antigen Haplotypes HLA-B*15:02 and HLA-A*31:01 Across Diverse Ancestral Backgrounds.

The human leukocyte antigen haplotypes HLA-B*15:02 and HLA-A*31:01 have been linked to life-threatening adverse drug reactions to the anticonvulsants carbamazepine and oxcarbazepine. Identification of these haplotypes via pharmacogenetic techniques facilitates implementation of precision medicine to prevent such reactions. Using reference samples from diverse ancestral origins, we investigated the test analytical validity (i.e., ability to detect whether or not the haplotypes were present or absent) of TaqMan assays for single nucleotide variants previously identified as potentially being able to “tag” these haplotypes. A TaqMan custom assay for rs10484555 and an inventoried assay for rs17179220 and were able to identify with 100% sensitivity and 100% specificity HLA-B*15:02 and HLA-A*31:01 respectively. A custom assay for rs144012689 that takes into account a neighboring single nucleotide variant with manual calling was also able to identify HLA-B*15:02 with 100% sensitivity and 100% specificity. A custom assay for rs1061235 identified HLA-A*31:01 with 100% sensitivity and 95% specificity. The slight reduction in specificity for the latter was owing to another haplotype (HLA-A*33:03) also being detected. While any positive call using the rs1061235 assay could therefore be further investigated, as the presence of the HLA-A*31:01 haplotype confers adverse drug reaction risk, the absence of false negatives (indexed by sensitivity) is more important than false positives. In summary, we present validated TaqMan assay methodology for efficient detection of HLA haplotypes HLA-B*15:02 and HLA-A*31:01. Our data are relevant for other genotyping technologies that identify, or have the potential to identify, these haplotypes using single nucleotide variants.

Antimicrobial activity of polyhexamethylene biguanide nanoparticles against mastitis-causing Staphylococcus aureus

Postmilking teat disinfection is one of the main measures used to prevent mastitis caused by contagious pathogens, such as Staphylococcus aureus. The present study evaluated the antimicrobial activity of polyhexamethylene biguanide (PHMB) and PHMB nanoparticles (NP) against mastitis-causing Staph. aureus using a microdilution assay methodology. A total of 20 mastitis-causing Staph. aureus isolates were used to determine the minimum inhibitory concentrations (MIC) of PHMB and PHMB NP compared with 3 disinfectants commonly used for teat disinfection (chlorhexidine digluconate, povidone-iodine, and sodium dichloroisocyanurate). The MIC90 was defined at the concentrations required to inhibit the growth of 90% of Staph. aureus. Our results indicated that PHMB NP presented the lowest MIC value (<0.03 µg/mL) to inhibit 90% of Staph. aureus, followed by chlorhexidine digluconate (≥0.25 µg/mL) and PHMB (≥0.5 µg/mL). On the other hand, sodium dichloroisocyanurate (≥500 µg/mL) and povidone-iodine (≥8,000 µg/mL) presented the highest concentrations to inhibit the growth of most Staph. aureus. Our preliminary results suggested that both PHMB and PHMB NP have antimicrobial activity against mastitis-causing Staph. aureus, which indicates the potential for both to be used as a teat-dip disinfectant to prevent bovine mastitis.

Observations on the Relationships between Endophytic Metarhizium robertsii, Spodoptera frugiperda (Lepidoptera: Noctuidae), and Maize.

Fungi in the genus Metarhizium are entomopathogens that can establish endophytically inside plants and benefit them through growth promotion and pest suppression. Lab- and greenhouse-based experiments were conducted to examine the effects of endophytic M. robertsii colonization in maize (Zea mays) on fall armyworm (FAW) (Spodoptera frugiperda). Maize seeds were inoculated with M. robertsii conidia, plants were evaluated for endophytic colonization, and then relative growth rate (RGR) and feeding behavior of larval FAW fed leaves from inoculated and uninoculated maize were measured. Endophytic M. robertsii was recovered from 60.5% of inoculated maize. In feeding bioassays, the RGR of larval FAW fed leaves of inoculated maize was no different than the RGR of larvae fed leaves from uninoculated maize. The RGR of larval FAW was positively correlated with the proportion of endophytic colonization of maize leaf and root tissues; however, in feeding assays, FAW larvae demonstrated no preference for consuming leaf tissue from inoculated or uninoculated maize. The proportion of leaf tissue consumed was unrelated to the proportion of M. robertsii-colonization of leaf or root tissue from source plants. We discuss possible reasons why FAW were not affected by endophytic M. robertsii in the context of assay methodology, FAW physiology, and induced maize defenses.

Detection of Interleukin-2 and Interleukin-31 among Patients with Uremic Pruritus

Uremic pruritus is a form of chronic itching that affects patients with advanced end-stage renal disease. Itis one of the most common symptoms in patients with end-stage renal disease, with approximately 60–90percent of patients on hemodialysis (HD) experiencing it. The study aims to assess the role of interleukin-31and Interleukin-2 in the pathogenesis of uremic pruritus among hemodialysis patients attending the EbnSina Dialysis Center in Diyala, and Correlation between Interleukin 31 and Interleukin-2 with severityof uremic pruritus. A study was performed from 1 Sep 2020 to 20 Jan 2021 in Ebn Sina Dialysis center,Diyala Governorate. the study was conducted on 150 patients with CKD divided to two groups, 75 patientswith uremic pruritus and 75 patients without uremic pruritus, the control groups was 26 healthy persons.Itch intensity was scored by VAS as mild, moderate and severe. Serum IL-31 levels and serum IL-2 levelswere determined by the enzyme-linked immunosorbent assay methodology. The various characteristics pfpruritus was assessed using an interview questionnaire. MS excel package and SPSS 20 software was usedfor analysis. Among 226 HD patient excluded under 20 age and hepatitis C and Hepatitis B HD patients,33.8% had uremic pruritus. The patients with pruritus had higher serum IL-31 levels and IL-2 than thosewithout uremic pruritus and control groups, it was statically significant. As well as the IL-31 and IL-2showed no significant differences among patients with mild, moderate, and severe itch. IL-31 and IL-2 playimportant role in the pathogenesis of uremic pruritus. Further studies to confirm the role of cytokines in thepathogenesis of UP.

POS0129 ASSOCIATION BETWEEN OSTEOARTHRITIS-RELATED SERUM BIOCHEMICAL MARKERS OVER 11 YEARS AND KNEE MRI-BASED IMAGING BIOMARKERS IN MIDDLE-AGED ADULTS

Background:Serum levels of osteoarthritis (OA)-related cartilage and joint-specific biochemical markers – cartilage oligomeric matrix protein (COMP), matrix metalloproteinase (MMP)-3, and hyaluronan (HA) –are shown to be associated with cartilage degradation, joint tissue degradation, and synovitis in patients with OA. Although these OA-related biochemical markers may initially precede the MRI biomarkers of joint structural changes, such changes detected in MRI can lead to biochemical marker changes later as the condition progresses. However, there is a lack of data on OA-related biochemical markers’ association with MRI-based biomarkers in the middle-aged general population.Objectives:We aimed to describe the associations between OA-related biochemical markers and MRI-based imaging biomarkers in middle-aged adults followed up over 10-13 years.Methods:Blood samples were collected during the Childhood Determinants of Adult Health (CDAH)-1 study at baseline (year: 2004-06, age: 26–36 years) and 10-13 year follow-up (CDAH-3; year: 2014–2019, age: 36–49 years). Serum samples from baseline (n=156) and follow-up (n= 167) were analyzed for three OA-related biomarkers – namely COMP, MMP-3, and HA– using non isotopic ELISA assay methodology. Knee MRI scans were obtained during the CDAH-knee study (year: 2008-10, age: 30-40 years, n=313), and MRIs were assessed for cartilage volume, cartilage thickness, subchondral bone area, cartilage defects, and bone marrow lesions (BML). Univariable and multivariable (adjusted for age, sex, and body mass index (BMI)) linear regression and logistic regression were used to describe the association of biochemical marker at CDAH-1 and MRI-based imaging biomarkers at CDAH-knee, and Tobit regression was used to describe the association of MRI-based imaging biomarkers at CDAH-knee and biochemical markers at CDAH-3.Results:In the multivariable model for the association of biochemical marker with MRI-based imaging biomarkers (assessed after 4 years), we found a significant negative association of COMP with medial femorotibial compartment cartilage thickness (-0.010 (-0.019, -0.000) p=0.045), and MMP-3 with patellar cartilage thickness (-9.075 (-16.344, -1.807) p=0.015) and total bone area (-0.047 (-0.086, -0.007) p=0.020). No significant association was observed between HA and MRI markers.In the multivariable model for association of MRI-based imaging biomarkers with biochemical markers (assessed after 6-9 years), a significant negative association of total cartilage volume (-0.0005 (-0.0008, -0.0002) p=0.001) and total cartilage thickness (-0.628 (-1.143, -0.114) p=0.017) with MMP-3, and total bone area with COMP (0.270 (-0.474, -0.006) p=0.010) was observed. No significant association was observed between MRI-based imaging biomarkers and HA.Conclusion:COMP and MMP-3 levels were negatively associated with knee cartilage thickness assessed 4-years later. Similarly, knee cartilage thickness and volume were negatively associated with COMP and MMP-3 levels assessed 6-9 years later in population-based middle-aged adults, indicating an interdependent negative association of OA-related biochemical markers and MRI-based imaging biomarkers. These results suggest that OA-related biochemical markers may predict future MRI-based imaging biomarkers in middle-aged adults and thus represent possible at-risk populations to target for structure modification interventions.Acknowledgements:This project was funded by the National Health and Medical Research Council of Australia Project Grant and Royal Hobart Hospital Research Foundation Project Grant.Disclosure of Interests:Benny Antony Grant/research support from: Investigator-Initiated Clinical Trial support from Nat Rem Ltd., Ambrish Singh: None declared, Leigh Blizzard: None declared, Alison Venn: None declared, Graeme Jones Speakers bureau: Speaker for various pharma, Grant/research support from: Investigator-Initiated Clinical Trial support, John Burgess: None declared, Venkat Parameswaran: None declared, Flavia Cicuttini: None declared, Lyn March: None declared, Changhai Ding: None declared

IFCC interim guidelines on rapid point-of-care antigen testing for SARS-CoV-2 detection in asymptomatic and symptomatic individuals.

With an almost unremittent progression of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections all around the world, there is a compelling need to introduce rapid, reliable, and high-throughput testing to allow appropriate clinical management and/or timely isolation of infected individuals. Although nucleic acid amplification testing (NAAT) remains the gold standard for detecting and theoretically quantifying SARS-CoV-2 mRNA in various specimen types, antigen assays may be considered a suitable alternative, under specific circumstances. Rapid antigen tests are meant to detect viral antigen proteins in biological specimens (e.g. nasal, nasopharyngeal, saliva), to indicate current SARS-CoV-2 infection. The available assay methodology includes rapid chromatographic immunoassays, used at the point-of-care, which carries some advantages and drawbacks compared to more conventional, instrumentation-based, laboratory immunoassays. Therefore, this document by the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC) Taskforce on COVID-19 aims to summarize available data on the performance of currently available SARS-CoV-2 antigen rapid detection tests (Ag-RDTs), providing interim guidance on clinical indications and target populations, assay selection, and evaluation, test interpretation and limitations, as well as on pre-analytical considerations. This document is hence mainly aimed to assist laboratory and regulated health professionals in selecting, validating, and implementing regulatory approved Ag-RDTs.

Paraneoplastic disorders of the nervous system.

This article on paraneoplastic neurologic disorders provides an update on the diagnostic approach, utility and pitfalls of autoantibody testing and emerging settings in which these disorders are encountered. Recognition of the clinical and neuroimaging features accompanying paraneoplastic neurologic disorders is crucial to select those at highest risk who need neural antibody testing and screening for cancer. Cursory knowledge of the antibody assay methodology being ordered is important as the false positive rate varies by the technique utilized for detection. Antibodies can generally be stratified by the location of the target antigen (intraceullar versus cell-surface/synaptic) which informs frequency of cancer association, treatment response and prognosis. The therapeutic approach generally involves detection of the underlying cancer and combinations of oncologic treatments and immunosuppressant medications. The occurrence of paraneoplastic autoimmune neurologic disorders in novel settings, such as with immune checkpoint inhibitor use, has improved understanding of their pathogenesis and increased the likelihood neurologists will encounter such patients in their practice.

Profiling Colorectal Cancer in the Landscape Personalized Testing-Advantages of Liquid Biopsy.

Drug-specific therapeutic approaches for colorectal cancer (CRC) have contributed to significant improvements in patient health. Nevertheless, there is still a great need to improve the personalization of treatments based on genetic and epigenetic tumor profiles to maximize the quality and efficacy while limiting cytotoxicity. Currently, CEA and CA 19-9 are the only validated blood biomarkers in clinical practice. For this reason, laboratories are trying to identify new specific prognostics and, more importantly, predictive biomarkers for CRC patient profiling. Thus, the unique landscape of personalized biomarker data should have a clinical impact on CRC treatment strategies and molecular genetic screening tests should become the standard method for diagnosing CRC. This review concentrates on recent molecular testing in CRC and discusses the potential modifications in CRC assay methodology with the upcoming clinical application of novel genomic approaches. While mechanisms for analyzing circulating tumor DNA have been proven too inaccurate, detecting and analyzing circulating tumor cells and protein analysis of exosomes represent more promising options. Blood liquid biopsy offers good prospects for the future if the results align with pathologists’ tissue analyses. Overall, early detection, accurate diagnosis and treatment monitoring for CRC with specific markers and targeted molecular testing may benefit many patients.

Label-Free Screening of SARS-CoV-2 NSP14 Exonuclease Activity Using SAMDI Mass Spectrometry.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the global COVID-19 pandemic. Nonstructural protein 14 (NSP14), which features exonuclease (ExoN) and guanine N7 methyltransferase activity, is a critical player in SARS-CoV-2 replication and fidelity and represents an attractive antiviral target. Initiating drug discovery efforts for nucleases such as NSP14 remains a challenge due to a lack of suitable high-throughput assay methodologies. This report describes the combination of self-assembled monolayers and matrix-assisted laser desorption ionization mass spectrometry to enable the first label-free and high-throughput assay for NSP14 ExoN activity. The assay was used to measure NSP14 activity and gain insight into substrate specificity and the reaction mechanism. Next, the assay was optimized for kinetically balanced conditions and miniaturized, while achieving a robust assay (Z factor > 0.8) and a significant assay window (signal-to-background ratio > 200). Screening 10,240 small molecules from a diverse library revealed candidate inhibitors, which were counterscreened for NSP14 selectivity and RNA intercalation. The assay methodology described here will enable, for the first time, a label-free and high-throughput assay for NSP14 ExoN activity to accelerate drug discovery efforts and, due to the assay flexibility, can be more broadly applicable for measuring other enzyme activities from other viruses or implicated in various pathologies.

A Fluorogenic Assay: Analysis of Chemical Modification of Lysine and Arginine to Control Proteolytic Activity of Trypsin

The chemical modification of amino acids plays an important role in the modulation of proteins or peptides and has useful applications in the activation and stabilization of enzymes, chemical biology, shotgun proteomics, and the production of peptide-based drugs. Although chemoselective modification of amino acids such as lysine and arginine via the insertion of respective chemical moieties as citraconic anhydride and phenyl glyoxal is important for achieving desired application objectives and has been extensively reported, the extent and chemoselectivity of the chemical modification of specific amino acids using specific chemical agents (blocking or modifying agents) has yet to be sufficiently clarified owing to a lack of suitable assay methodologies. In this study, we examined the utility of a fluorogenic assay method, based on a fluorogenic tripeptide substrate (FP-AA1-AA2-AA3) and the proteolytic enzyme trypsin, in determinations of the extent and chemoselectivity of the chemical modification of lysine or arginine. As substrates, we used two fluorogenic tripeptide probes, MeRho-Lys-Gly-Leu(Ac) (lysine-specific substrate) and MeRho-Arg-Gly-Leu(Ac) (arginine-specific substrate), which were designed, synthesized, and evaluated for chemoselective modification of specific amino acids (lysine and arginine) using the fluorogenic assay. The results are summarized in terms of half-maximal inhibitory concentrations (IC50) for the extent of modification and ratios of IC50 values (IC50arginine/IC50lysine and IC50lysine/IC50arginine) as a measure of the chemoselectivity of chemical modification for amino acids lysine and arginine. This novel fluorogenic assay was found to be rapid, precise, and reproducible for determinations of the extent and chemoselectivity of chemical modification.

Development of a Novel Label-Free and High-Throughput Arginase-1 Assay Using Self-Assembled Monolayer Desorption Ionization Mass Spectrometry

Arginase-1, an enzyme that catalyzes the reaction of L-arginine to L-ornithine, is implicated in the tumor immune response and represents an interesting therapeutic target in immuno-oncology. Initiating arginase drug discovery efforts remains a challenge due to a lack of suitable high-throughput assay methodologies. This report describes the combination of self-assembled monolayers and matrix-assisted laser desorption ionization mass spectrometry to enable the first label-free and high-throughput assay for arginase activity. The assay was optimized for kinetically balanced conditions and miniaturized, while achieving a robust assay (Z-factor > 0.8) and a significant assay window [signal-to-background ratio > 20] relative to fluorescent approaches. To validate the assay, the inhibition of the reference compound nor-NOHA (Nω-hydroxy-nor-L-arginine) was evaluated, and the IC50 measured to be in line with reported results (IC50 = 180 nM). The assay was then used to complete a screen of 175,000 compounds, demonstrating the high-throughput capacity of the approach. The label-free format also eliminates opportunities for false-positive results due to interference from library compounds and optical readouts. The assay methodology described here enables new opportunities for drug discovery for arginase and, due to the assay flexibility, can be more broadly applicable for measuring other amino acid-metabolizing enzymes.