Improve Assay Sensitivity Research Articles

Methods for Enhanced Fluorescence Detection of Proteins by using Entrapped Gold Nanoparticles in Membranes.

Measuring protein levels from biofluids can provide important insight into human health and disease during various physiological and pathological conditions. In many situations, sensitive methods are required for protein quantification because at the early stages of many diseases, proteins in biofluids are present at very low concentrations. Here, a new and simple method is presented in the form of Basic and Alternative Protocols for an immunoassay performed on a nitrocellulose membrane, followed by the addition of gold nanoparticles prior to measuring fluorescence with a microscope. The assay protocol was optimized to achieve 3D metal-enhanced fluorescence (MEF) with increased antibody-binding capacity and enhanced fluorescence signals, improving assay sensitivity. Using different concentrations of spiked fluorescently labeled IgGs in pooled normal human plasma, a lower detection limit of 29 ng/ml was achieved. This limit of detection was found to be a thousand-fold lower than the conventional 2D assay and one order of magnitude lower than when the assay was performed on a 3D membrane without MEF. This method provides an easy way to improve immunoassay sensitivity, and it can be simply transferred to other labs. It also can extend to fluorescence detection of other analytes beyond proteins. © 2022 Wiley Periodicals LLC. Basic Protocol: Assay in nitrocellulose membrane with entrapped AuNPs using commercially available AuNPs Alternative Protocol: Assay in nitrocellulose membrane with entrapped AuNPs using lab-made AuNPs.

Picomolar detection of carbohydrate-lectin interactions on piezoelectrically printed microcantilever array

Recent advances in micro-electromechanical systems (MEMS) has allowed unprecedent perspectives for label-free detection (LFD) of biological and chemical analytes. Additionally, these LFD technologies offer the potential to design high resolution and high throughput sensing platforms, with the promise of further miniaturization. However, the immobilization of biomolecules onto inorganic surfaces without impacting their sensing abilities is crucial for designing these LFD technologies. Currently, covalent functionalization of self-assembled monolayers (SAMs) present promising pathways for improving assay sensitivity, reproducibility, surface stability and proximity of binding sites to the sensor surface. Herein, we investigate the use of chemical vapor deposition of 3-(glycidyloxypropyl)-trimethoxysilane (GOPTS) as a versatile SAM for the covalent functionalization of a SiO2 microcantilever array (MCA) for carbohydrate-lectin interactions with picogram sensitivity. Additionally, we demonstrate glycan immobilization to MCA is feasible using traditional piezoelectric microarray printer technology. Given the complexity of the glycome, the ability to spot samples in a high-throughput manner establishes our MCA as robust, label-free, and scalable means to analyze carbohydrate-protein interactions These findings demonstrate that GOPTS SAMs provide a suitable biofunctionalization route for MEMS and provides the proof of principle that can be extended to various LFD technologies toward a truly high-throughput and high-resolution platform.

A genosensor for detecting single-point mutations in dendron chips after blocked recombinase polymerase amplification.

A biosensing system was developed to accurately detect a single nucleotide change in the target organism genome, integrating a selective isothermal amplification and a sensitive dendron-mediated DNA hybridization assay in the array format. The novelty arises from the coupling reactions of the dendron and its use as a crosslinker. The allele-specific probes were oligonucleotide-dendron conjugates prepared by fast and clean click-chemistry (thiol-yne reaction) and coupled onto the photo-activated surface of polycarbonate substrates (carbodiimide reaction). The output was forest-array chips with multipoint-site crosslinkers and compatible with current microarray fabrication technologies. The products of blocked recombinase polymerase amplification (blocked RPA), formed at 37 °C, were hybridized with attached probes for specific nucleotide genotyping. The developed approach exhibited sensitive recognition of DNA variants compared to chips based on linear crosslinkers (10-100 fold), showing excellent analytical performances for planar chip and fluidic formats. The methodology was successfully applied to detect the H1047R mutation in the PIK3CA gene (c.3140A > G) from clinical samples of human cancer tissues, the results being consistent with sequencing techniques. The colorimetric biosensing method was reliable, versatile, low cost, sensitive (detection limit genomic DNA: 0.02 ng μL-1), and specific (accuracy >95%).

Application of dried blood spot sample pooling strategies for Plasmodium 18S rRNA biomarker testing to facilitate identification of infected persons in large-scale epidemiological studies

BackgroundPlasmodium 18S rRNA is a sensitive biomarker for detecting Plasmodium infection in human blood. Dried blood spots (DBS) are a practical sample type for malaria field studies to collect, store, and transport large quantities of blood samples for diagnostic testing. Pooled testing is a common way to reduce reagent costs and labour. This study examined performance of the Plasmodium 18S rRNA biomarker assay for DBS, improved assay sensitivity for pooled samples, and created graphical user interface (GUI) programmes for facilitating optimal pooling.MethodsDBS samples of varied parasite densities from clinical specimens, Plasmodium falciparum in vitro culture, and P. falciparum Armored RNA® were tested using the Plasmodium 18S rRNA quantitative triplex reverse transcription polymerase chain reaction (qRT-PCR) assay and a simplified duplex assay. DBS sample precision, linearity, limit of detection (LoD) and stability at varied storage temperatures were evaluated. Novel GUIs were created to model two-stage hierarchy, square matrix, and three-stage hierarchy pooling strategies with samples of varying positivity rates and estimated test counts. Seventy-eight DBS samples from persons residing in endemic regions with sub-patent infections were tested in pools and deconvoluted to identify positive cases.ResultsAssay performance showed linearity for DBS from 4 × 107 to 5 × 102 parasites/mL with strong correlation to liquid blood samples (r2 > 0.96). There was a minor quantitative reduction in DBS rRNA copies/mL compared to liquid blood samples. Analytical sensitivity for DBS was estimated 5.3 log copies 18S rRNA/mL blood (28 estimated parasites/mL). Properly preserved DBS demonstrated minimal degradation of 18S rRNA when stored at ambient temperatures for one month. A simplified duplex qRT-PCR assay omitting the human mRNA target showed improved analytical sensitivity, 1 parasite/mL blood, and was optimized for pooling. Optimal pooling sizes varied depending on prevalence. A pilot DBS study of the two-stage hierarchy pooling scheme corroborated results previously determined by testing individual DBS.ConclusionsThe Plasmodium 18S rRNA biomarker assay can be applied to DBS collected in field studies. The simplified Plasmodium qRT-PCR assay and GUIs have been established to provide efficient means to test large quantities of DBS samples.

A novel xenonucleic acid-mediated molecular clamping technology for early colorectal cancer screening.

BackgroundColorectal cancer (CRC) is one of the leading causes of cancer-related death. Early detection is critical to reduce CRC morbidity and mortality. In order to meet this need, we developed a molecular clamping assay called the ColoScape TM assay for early colorectal cancer diagnostics.MethodsNineteen mutations in four genes (APC, KRAS, BRAF and CTNNB1) associated with early events in CRC pathogenesis are targeted in the ColoScapeTM assay. Xenonucleic Acid (XNA)-mediated qPCR clamping technology was applied to minimize the wild-type background amplification in order to improve assay sensitivity of CRC mutation detection. The assay analytical performance was verified and validated, cfDNA and FFPE CRC patient samples were evaluated, and an ROC curve was applied to evaluate its performance.ResultsThe data showed that the assay analytical sensitivity was 0.5% Variant Allele Frequency, corresponding to ~7–8 copies of mutant DNA with 5 ng total DNA input per test. This assay is highly reproducible with intra-assay CV of <3% and inter-assay CV of <5%. We have investigated 380 clinical samples including plasma cfDNA and FFPE samples from patients with precancerous and different stages of CRC. The preliminary assay clinical specificity and sensitivity for CRC cfDNA were: 100% (95% CI, 80.3–97.5%) and 92.2% (95% CI, 94.7–100%), respectively, with AUC of 0.96; 96% specificity (95% CI, 77.6–99.7%) and 92% sensitivity (95% CI, 86.1–95.6%) with AUC of 0.94 for CRC FFPE; 95% specificity (95% CI, 82.5%–99.1%) and 62.5% sensitivity (95% CI, 35.8%–83.7%) with AUC of 0.79 for precancerous lesions cfDNA.ConclusionsThe XNA-mediated molecular clamping assay is a rapid, precise, and sensitive assay for the detection of precancerous lesions cfDNA and CRC cfDNA or FFPE samples.

Stroma-derived extracellular vesicle mRNA signatures inform histological nature of prostate cancer.

Histological assessment of prostate cancer is the key diagnostic test and can predict disease outcome. This is however an invasive procedure that carries associated risks, hence non‐invasive assays to support the diagnostic pathway are much needed. A key feature of disease progression, and subsequent poor prognosis, is the presence of an altered stroma. Here we explored the utility of prostate stromal cell‐derived vesicles as indicators of an altered tumour environment. We compared vesicles from six donor‐matched pairs of adjacent‐normal versus disease‐associated primary stromal cultures. We identified 19 differentially expressed transcripts that discriminate disease from normal stromal extracellular vesicles (EVs). EVs isolated from patient serum were investigated for these putative disease‐discriminating mRNA. A set of transcripts including Caveolin‐1 (CAV1), TMP2, THBS1, and CTGF were found to be successful in discriminating clinically insignificant (Gleason = 6) disease from clinically significant (Gleason > 8) prostate cancer. Furthermore, correlation between transcript expression and progression‐free survival suggests that levels of these mRNA may predict disease outcome. Informed by a machine learning approach, combining measures of the five most informative EV‐associated mRNAs with PSA was shown to significantly improve assay sensitivity and specificity. An in‐silico model was produced, showcasing the superiority of this multi‐modal liquid biopsy compared to needle biopsy for predicting disease progression. This proof of concept highlights the utility of serum EV analytics as a companion diagnostic test with prognostic utility, which may obviate the need for biopsy.

Diverse predictors of treatment response to active medication and placebo in gambling disorder

Gambling disorder creates a significant public health burden. Despite decades of clinical trials, there are no licensed pharmacological treatments for gambling disorder. Contributing factors to this are the high placebo response rates seen in clinical trials, the heterogeneity of the disorder and high rates of psychiatric comorbidities. Indeed, a number of demographic and clinical variables have previously been associated with altered responses to pharmacotherapy, psychotherapy and placebo. Which variables are likely to predict response to one modality over another remains uncertain. We carried out multiple linear regression analyses in a pooled dataset from six treatment studies in gambling disorder with the aim of identifying predictors of treatment response. Potential predictors were identified a priori through hypothesis and entered into models including all patients, and subsequently for those randomized to active medication or placebo separately. We found that baseline severity of gambling symptoms and number of weeks completed in a trial were predictors of active medication response, while decreased baseline symptoms of anxiety, increased baseline symptoms of depression, and non-Caucasian ethnicity were associated with placebo response. Sensitivity analyses showed that these associations were robust to choices made during the analysis. Further research is required to understand whether controlling for these variables, or using enriched samples, improves assay sensitivity in placebo-controlled clinical trials for gambling disorder.

Plasma metagenomic sequencing to detect and quantify bacterial DNA in ICU patients suspected of sepsis: A proof-of-principle study.

Timely recognition of sepsis and identification of pathogens can improve outcomes in critical care patients but microbial cultures have low accuracy and long turnaround times. In this proof-of-principle study, we describe metagenomic sequencing and analysis of nonhuman DNA in plasma. We hypothesized that quantitative analysis of bacterial DNA (bDNA) levels in plasma can enable detection and monitoring of pathogens. We enrolled 30 patients suspected of sepsis in the surgical trauma intensive care unit and collected plasma samples at the time of diagnostic workup for sepsis (baseline), and 7 days and 14 days later. We performed metagenomic sequencing of plasma DNA and used computational classification of sequencing reads to detect and quantify total and pathogen-specific bDNA fraction. To improve assay sensitivity, we developed an enrichment method for bDNA based on size selection for shorter fragment lengths. Differences in bDNA fractions between samples were evaluated using t test and linear mixed-effects model, following log transformation. We analyzed 72 plasma samples from 30 patients. Twenty-seven samples (37.5%) were collected at the time of infection. Median total bDNA fraction was 1.6 times higher in these samples compared with samples with no infection (0.011% and 0.0068%, respectively, p < 0.001). In 17 patients who had active infection at enrollment and at least one follow-up sample collected, total bDNA fractions were higher at baseline compared with the next sample (p < 0.001). Following enrichment, bDNA fractions increased in paired samples by a mean of 16.9-fold. Of 17 samples collected at the time when bacterial pathogens were identified, we detected pathogen-specific DNA in 13 plasma samples (76.5%). Bacterial DNA levels in plasma are elevated in critically ill patients with active infection. Pathogen-specific DNA is detectable in plasma, particularly after enrichment using selection for shorter fragments. Serial changes in bDNA levels may be informative of treatment response. Epidemiologic/Prognostic, Level V.

Multiplex assessment of SARS-CoV-2 antibodies improves assay sensitivity and correlation with neutralizing antibodies

ObjectivesDetection of antibodies to multiple SARS-CoV-2 antigens in a single assay could increase diagnostic accuracy, differentiate vaccination from natural disease, and aid in retrospective exposure determination. Correlation of binding antibody assessment in clinical assays with neutralizing antibodies is needed to better understand the humoral response to SARS-CoV-2 infection and establish of correlates of protection. MethodsA cohort of 752 samples was used to assess specificity, sensitivity, and comparison to 6 other Conformitè Europëenne serologic assays for the BioRad SARS-CoV-2 IgG multiplex assay which measures receptor binding domain IgG (RBD), spike-S1 IgG (S1), spike-S2 IgG (S2), and nucleocapsid IgG (N). A subset of serial specimens from 14 patients was also tested for neutralizing antibodies (n = 61). ResultsSpecificity for RBD and S1 IgG was 99.4% (n = 170) and 100% for S2 and N IgG (n = 170) in a cohort selected for probable interference. Overall assay concordance with other assays was >93% for IgG and total antibody assays and reached 100% sensitivity for clinical concordance at >14 days as a multiplex assay. RBD and S1 binding antibody positivity demonstrated 79–95% agreement with the presence of neutralizing antibodies. ConclusionsThe BioRad SARS-CoV-2 IgG assay is comparable to existing assays, and achieved 100% sensitivity when all markers were included. The ability to measure antibodies against spike and nucleocapsid proteins simultaneously may be advantageous for complex clinical presentations, epidemiologic research, and in decisions regarding infection prevention strategies. Additional independent validations are needed to further determine binding antibody and neutralizing antibody correlations.

Use of Liquid Biopsy in the Care of Patients with Non-Small Cell Lung Cancer.

Recent technological advances have enabled the development of liquid biopsy-based approaches, which have revolutionized the diagnostic world. The analysis of circulating tumor DNA (ctDNA) has several clinical applications. First, ctDNA genotyping is becoming widely used for non-invasive biomarker testing. Of note, in lung cancer patients in whom biopsies are difficult to obtain, ctDNA has led to significant improvement in the diagnosis and identification of therapeutic targets. In addition, ctDNA quantification over the course of the disease can be useful for tumor response to treatment monitoring and for early detection of resistance mutations. ctDNA levels per se are also of prognostic significance and could be used to tailor treatments. Finally, improvements in assay sensitivity are facilitating the development of liquid biopsy-based tests for the detection of ctDNA at very low allele frequencies (AFs), which can be used for the measurement of minimal residual disease and ultimately for the development of strategies (by complementing imaging techniques) aimed to improve the efficiency of lung cancer screening programs.

Mapping Epitopes Recognised by Autoantibodies Shows Potential for the Diagnosis of High-Grade Serous Ovarian Cancer and Monitoring Response to Therapy for This Malignancy.

Simple SummaryMost women are diagnosed with high-grade serous ovarian cancer (HGSOC) at stage III, when the cancer has already spread, contributing to poor survival outcomes. However, while earlier diagnosis increases survival rates, there is a lack of early diagnosis biomarkers. Previously, autoantibodies specific for phosphorylated heat shock factor 1 (HSF1-PO4) were suggested as a potential diagnostic biomarker for early-stage HGSOC. In the present study, specific regions within HSF1 were identified, tested and confirmed as useful biomarkers, with comparable diagnostic potential to the full protein, across two separate clinical cohorts. Additionally, antibody responses to HSF1-PO4 and the corresponding peptides were found to increase following a round of standard first-line chemotherapy. Together, our data suggest that the identified short peptide sequences could be used as practical alternatives to support early diagnosis or monitor responses to chemotherapy.Autoantibodies recognising phosphorylated heat shock factor 1 (HSF1-PO4) protein are suggested as potential new diagnostic biomarkers for early-stage high-grade serous ovarian cancer (HGSOC). We predicted in silico B-cell epitopes in human and murine HSF1. Three epitope regions were synthesised as peptides. Circulating immunoglobulin A (cIgA) against the predicted peptide epitopes or HSF1-PO4 was measured using ELISA, across two small human clinical trials of HGSOC patients at diagnosis. To determine whether chemotherapy would promote changes in reactivity to either HSF1-PO4 or the HSF-1 peptide epitopes, IgA responses were further assessed in a sample of patients after a full cycle of chemotherapy. Anti-HSF1-PO4 responses correlated with antibody responses to the three selected epitope regions, regardless of phosphorylation, with substantial cross-recognition of the corresponding human and murine peptide epitope variants. Assessing reactivity to individual peptide epitopes, compared to HSF1-PO4, improved assay sensitivity. IgA responses to HSF1-PO4 further increased significantly post treatment, indicating that HSF1-PO4 is a target for immunity in response to chemotherapy. Although performed in a small cohort, these results offer potential insights into the interplay between autoimmunity and ovarian cancer and offer new peptide biomarkers for early-stage HGSOC diagnosis, to monitor responses to chemotherapy, and widely for pre-clinical HGSOC research.

Pushing the detection limits: strategies towards highly sensitive optical-based protein detection.

Proteins are one of the main constituents of living cells. Studying the quantities of proteins under physiological and pathological conditions can give valuable insights into health status, since proteins are the functional molecules of life. To be able to detect and quantify low-abundance proteins in biofluids for applications such as early disease diagnostics, sensitive analytical techniques are desired. An example of this application is using proteins as biomarkers for detecting cancer or neurological diseases, which can provide early, lifesaving diagnoses. However, conventional methods for protein detection such as ELISA, mass spectrometry, and western blotting cannot offer enough sensitivity for certain applications. Recent advances in optical-based micro- and nano-biosensors have demonstrated promising results to detect proteins at low quantities down to the single-molecule level, shining lights on their capacities for ultrasensitive disease diagnosis and rare protein detection. However, to date, there is a lack of review articles synthesizing and comparing various optical micro- and nano-sensing methods of enhancing the limits of detections of the antibody-based protein assays. The purpose of this article is to critically review different strategies of improving assay sensitivity using miniaturized biosensors, such as assay miniaturization, improving antibody binding capacity, sample purification, and signal amplification. The pros and cons of different methods are compared, and the future perspectives of this research field are discussed.

Abstract LB059: Dual methylation and mutation analysis from a single input of plasma cfDNA without sample splitting

Abstract Genomic and epigenetic changes are known to be the underlying mechanism of cancer development. Assessing the genomic (mutation) and epigenetic (methylation) changes has become increasingly important for cancer detection and treatment selection, especially in the field of cfDNA-based liquid biopsy. Targeted Next-Generation Sequencing (NGS) enables in-depth analysis of specific genomic regions that is essential for the clinical application of liquid biopsy. However, current targeted NGS assays are complicated, lengthy, and inefficient. Two major hurdles of existing targeted NGS methods make them impractical for the clinical setting. First, there is no comprehensive, end to end, kit solution available for targeted methylation sequencing (TMS) from sample to sequencing ready library. Second, the low yield of cfDNA from clinical blood samples presents a major challenge for multi-modal analyses on one sample. Thus, an assay that is capable of both methylation and mutation analysis would be optimal for clinical research and future diagnostic assays.Here, we report the performance of Point-n-Seq, a kit solution that can provide in-depth DNA analysis with highly flexible and customizable focused panels to enable both methylation and mutation analysis without sample splitting. Custom panels of 10 to 1000s of markers can be designed with &amp;gt;99% first-pass success rate. We conducted both performance validation and multi-center, multi-operator, reproducibility studies. With spike-in titration of cancer cell-line gDNA with known mutation and methylation profile, we achieved a reliable detection level down to 0.003% of tumor DNA with a linear relationship between the measured and expected fractions.In a pilot study, we designed a colorectal cancer (CRC) TMS panel covering 560 methylation markers and a mutation panel with &amp;gt;350 hotspot mutations in 22 genes. From only 1ml of plasma from late stage CRC patients, we detected cancer-specific methylation signals in all samples tested, and hotspot mutations with clinical implications. Next, a tumor-informed mutation panel covering approximately 100 personalized mutations for each patient was compared to the tumor-independent CRC methylation panel to analyze 23 stage I and II CRC patient plasma samples. The initial results showed that tumor-independent TMS assay achieved a comparable or superior limit of detection compared to the personalized tumor-informed approach. Moreover, to further improve assay sensitivity, metrics for cfDNA size can be integrated into the analysis of the same workflow without sample splitting. The Point-n-Seq kit, with a highly streamlined workflow and dual methylation and mutation analysis, provides a highly sensitive and robust non-invasive method for cancer detection, monitoring and treatment guidance. Citation Format: Grace Q. Zhao, Yun Bao, Heng Wang, Vanita S. Natu, Jayashree Joshi, Yingmin Wang, Elena Arzumanyan, Wanping Wang, Jianmin Wang, John Coller, Shengrong Lin. Dual methylation and mutation analysis from a single input of plasma cfDNA without sample splitting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB059.

Critical Comparison between Large and Mini Vertical Flow Immunoassay Platforms for Yersinia Pestis Detection.

Yersinia pestis is a Gram-negative bacterium that is the causative agent of plague and is widely recognized as a potential biological weapon. Due to the high fatality rate of plague when diagnosis is delayed, the development of rapid, sensitive, specific, and cost-effective methods is needed for its diagnosis. The Y. pestis low calcium response V (LcrV) protein has been identified as a potential microbial biomarker for the diagnosis of plague. In this paper, we present a highly sensitive, paper-based, vertical flow immunoassay (VFI) prototype for the detection of LcrV and the diagnosis of plague. An antigen-capture assay using monoclonal antibodies is employed to capture and detect the LcrV protein, using a colorimetric approach. In addition, the effect of miniaturizing the VFI device is explored based on two different sizes of VFI platforms, denoted as "large VFI" and "mini VFI." Also, a comparative analysis is performed between the VFI platform and a lateral flow immunoassay (LFI) platform to exhibit the improved assay sensitivity suitable for point-of-care (POC) diagnostics. The analytical sensitivity or limit of detection (LOD) in the mini VFI is approximately 0.025 ng/mL, that is, 10 times better than that of the large VFI platform or 80 times over a standard lateral flow configuration. The low LOD of the LcrV VFI appears to be highly suitable for testing clinical samples and potentially diagnosing plague at earlier time points. In addition, optimization of the gold nanoparticle (AuNP) concentration, nanomaterial plasmonic properties, and flow velocity analysis could improve the performance of the VFI. Furthermore, we developed automated image analysis software that shows potential for integrating the diagnostic system into a smartphone. These methods and findings demonstrate that the VFI platform is a highly sensitive device for detecting the LcrV and potentially many other biomarkers.

Fast and Sensitive Determination of the Fungicide Carbendazim in Fruit Juices with an Immunosensor Based on White Light Reflectance Spectroscopy.

Carbendazim is a systemic benzimidazole-type fungicide with broad-spectrum activity against fungi that undermine food products safety and quality. Despite its effectiveness, carbendazim constitutes a major environmental pollutant, being hazardous to both humans and animals. Therefore, fast and reliable determination of carbendazim levels in water, soil, and food samples is of high importance for both food industry and public health. Herein, an optical biosensor based on white light reflectance spectroscopy (WLRS) for fast and sensitive determination of carbendazim in fruit juices is presented. The transducer is a Si/SiO2 chip functionalized with a benzimidazole conjugate, and determination is based on a competitive immunoassay format. Thus, for the assay, a mixture of an in-house developed rabbit polyclonal anti-carbendazim antibody with the standards or samples is pumped over the chip, followed by biotinylated secondary antibody and streptavidin. The WLRS platform allows for real-time monitoring of biomolecular interactions carried out onto the Si/SiO2 chip by transforming the shift in the reflected interference spectrum caused by the immunoreaction to effective biomolecular adlayer thickness. The sensor is able to detect 20 ng/mL of carbendazim in fruit juices with high accuracy and precision (intra- and inter-assay CVs ≤ 6.9% and ≤9.4%, respectively) in less than 30 min, applying a simple sample treatment that alleviates any “matrix-effect” on the assay results and a 60 min preincubation step for improving assay sensitivity. Excellent analytical characteristics and short analysis time along with its small size render the proposed WLRS immunosensor ideal for future on-the-spot determination of carbendazim in food and environmental samples.

Future Perspectives in Detecting EGFR and ALK Gene Alterations in Liquid Biopsies of Patients with NSCLC.

Non-small-cell lung cancer (NSCLC) is a major cause of death worldwide. Alterations in such genes as EGFR and ALK are considered important biomarkers in NSCLC due to the existence of targeted therapies with specific tyrosine kinase inhibitors (TKIs). However, specific resistance-related mutations can occur during TKI treatment, which often result in therapy inefficacy. Liquid biopsies arise as a reliable tool for the early detection of these types of alterations, allowing a non-invasive follow-up of the patients. Furthermore, they can be essential for cancer screening, initial diagnosis and to check surgery success. Despite the great advantages of liquid biopsies in NSCLC and the high input that next-generation sequencing (NGS) approaches can provide in this field, its use in oncology is still limited. With improvement of assay sensitivity and the establishment of clinical guidelines for liquid biopsy analysis, it is expected that they will be used in routine procedures. This review focuses on the usefulness of liquid biopsies of NSCLC patients as a means to detect alterations in EGFR and ALK genes and in disease management, highlighting the impact of NGS methods.

Quantification of Surrogate Monoclonal Antibodies in Mouse Serum Using LC–MS/MS

Background: Surrogate monoclonal antibodies (mAbs) used in preclinical in vivo studies can be challenging to quantify due to lack of suitable immunoaffinity reagents or unavailability of the mAb protein sequence. Generic immunoaffinity reagents were evaluated to develop sensitive LC-MS/MS assays. Peptides of unknown sequence can be used for selective LC-MS quantification. Results: anti-mouse IgG1 was found to be an effective immunoaffinity reagent, enabling quantification of mouse IgG1mAbs in mouse serum. Selective peptides of unknown sequence were applied for multiplex LC-MS quantification of two rat mAbs co-dosed in mouse. Conclusion: Generic anti-mouse IgG subtype-specific antibodies can be used to improve assay sensitivity and peptides of unknown sequence can be used to quantify surrogate mAbs when the mAb protein sequence in unavailable.

A facile one-step gold nanoparticles enhancement based on sequential patterned lateral flow immunoassay device for C-reactive protein detection

A gold nanoparticles (AuNPs) enhancement strategy for lateral flow immunoassays (LFIAs) was previously reported to improve assay sensitivity. The multi-steps of detection resulted in a tedious procedure. In this report, to reduce this limitation, a sequential flow LFIA was introduced to perform the AuNPs enhancement in only single step operation using C-reactive protein (CRP) as a model analyte. The delay and non-delay channels created on nitrocellulose membrane (NCM) using wax-printing method were used to deliver immunoassay and AuNPs enhancement reagents to the detection zone sequentially. For the enhancing step, AuNPs were enlarged by the catalytic reaction of KAuCl4 and NH2OH⋅HCl. Under optimal conditions, a calibration curve was found in a range of 0.1−5 μg⋅mL−1 with a good linear correlation (R2 = 0.9953). The limits of detection (LOD) were found to be 0.1 and 0.001 μg⋅mL-1 for naked eye observation and calculation (3SD/slope), respectively. Furthermore, the device shows no cross-reactivity with common interferences for CRP. The platform was successfully applied to detect CRP in certified human serum samples within 15 min, demonstrating the simplicity and speed of the AuNPs enhancement strategy platform. These promising results suggest that the new device may be used as an alternative tool for CRP detection and other biomarkers for POCT analysis.

Performance of an automated chemiluminescence SARS-CoV-2 IG-G assay

IntroductionWe describe our evaluation of the Abbott SARS-CoV-2 IgG assay on the Architect immunoassay analyser. MethodsWe assessed assay precision, sensitivity, specificity, positive/negative predictive values (PPV/NPV), cross-reactivity (influenza/dengue/hepatitis B and C/rheumatoid factor/anti-nuclear/double-stranded DNA/syphilis) and sample throughput in samples from real-time polymerase chain reaction (RT-PCR) positive patients/healthcare workers (HCWs)/pre-pandemic samples. We compared the cut-off indexes (COIs) between all control samples (HCWs and pre-pandemic) to generate an optimised COI limit for reactivity. ResultsThe assay specificity was 99.8% (n = 980) and sensitivity was 45.9–96.7% (n = 279). When tested ≥ 14 days post-positive RT-PCR (POS), the PPV/NPV was 96.4%/99.8%. The difference between the COIs of HCWs/pre-pandemic samples was small (0.01, p < 0.0001). There was minimal cross-reactivity with other antibodies. A lower COI limit for reactivity (≥0.55, using the 99th percentile COI of our controls and ROC analysis) improved diagnostic sensitivity, especially at 0–6 days POS (45.9–55.8%), with a small decrease in specificity (98.9%). The assay throughput was 100 samples in 70 min. ConclusionThe Abbott SARS-CoV-2 IgG assay shows excellent performance in patients ≥ 14 days POS. The difference between the COIs of HCWs and pre-pandemic samples was numerically small. A lower COI limit improves assay sensitivity with a slight decrease in specificity.

Abstract LB-284: A novel xenonucleic acid-mediated fusion gene detection method for cancer diagnostic

Abstract Introduction: Fusion transcript are mRNA containing sequences from more than one gene and can be produced through chromosomal rearrangement or special splicing events. Some fusions have strong connection to certain cancers. For example, EML4-ALK fusion was identified in 6.7% of all non-small cell lung carcinoma patients. Patients with this fusion can be effectively treated with ALK inhibitors, making this fusion an ideal biomarker. Our protocol aims to target these actionable biomarkers. Previously fusions have been detected using FISH, RT-PCR, RNA-seq and targeted sequencing. Targeted sequencing provides better sensitivity and accuracy than the other methods mentioned above. In addition, our protocol incorporates Xenonucleic Acid (XNA), a molecule that can bind to and block the amplification of targeted wildtype sequences. It has been shown to improve assay sensitivity in detecting mutant sequences. In this case, XNA can block the amplification of wildtype transcripts and improve the sensitivity of fusion transcript detection. The addition of XNA can provide higher sensitivity than any currently commercially available kits. Methods: Total RNA was converted to cDNA using 5' RACE (Rapid amplification of cDNA ends) technology, adding a common tail to one end of all cDNA transcripts for PCR steps. Targeted multiplex PCR with XNA spike in was then performed, followed by another nesting multiplex PCR to further improve sensitivity. The libraries were indexed with an additional PCR step. Libraries were then pooled and sequenced using Illumina MiSeq. Data was analyzed with Qiagen CLC Genomic Workbench using a custom workflow. Results: TACC3 XNA can successfully block the amplification of wild type TACC3 gene while allow amplification of FGFR3-TACC3 fusion transcripts. FGFR3-TACC3 fusion read percentage (fusion reads/[fusion read+ WT reads]) increased noticeably from 2.68% to 60.12%. No false positive was detected in the negative control. Negative control also exhibit decrease in wild type TACC3 reads following the introduction of TACC3 XNA, from 10.45% of total reads (fusion/total reads) to 0.88%. This indicated that wildtype was blocked effetely by XNA. Preliminary tests with NTRK1 and ROS1 XNA showed no interference in detecting TPM3-NTRK1, CD74-ROS1, and SLC34A-ROS1 fusions, but increase its detection. Conclusion: XNA can effectively suppress the amplification from WT RNA transcript and enhances fusion transcripts detection during target sequencing. TACC3 XNA successfully increased fusion read percentage from 2.68% to 60.12%. More studies are being conducted to evaluate the effectiveness of XNA on other fusions. Citation Format: Simni Tang, Andrew Y. Fu, Michael J. Powell, Aiguo Zhang, Michael Y. Sha. A novel xenonucleic acid-mediated fusion gene detection method for cancer diagnostic [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-284.