Fluorescent and Colorimetric Dual-Readout Immunochromatographic Assay for the Detection of Phenamacril Residues in Agricultural Products.

The fungicide phenamacril has been employed to manage Fusarium and mycotoxins in crops, leading to persistent residues in the environment and plants. Detecting phenamacril is pivotal for ensuring environmental and food safety. In this study, haptens and artificial antigens were synthesized to produce antiphenamacril monoclonal antibodies (mAbs). Additionally, gold nanoparticles coated with a polydopamine shell were synthesized and conjugated with mAbs, inducing fluorescence quenching in quantum dots. Moreover, a dual-readout immunochromatographic assay that combines the positive signal from fluorescence with the negative signal from colorimetry was developed to enable sensitive and precise detection of phenamacril within 10 min, achieving detection limits of 5 ng/mL. The method's reliability was affirmed by using spiked wheat flour samples, achieving a limit of quantitation of 0.05 mg/kg. This analytical platform demonstrates high sensitivity, outstanding accuracy, and robust tolerance to matrix effects, making it suitable for the rapid, onsite, quantitative screening of phenamacril residues.

Immunochromatographic POC-CCA Test for the diagnosis of intestinal schistosomiasis in a high endemic region in Brazil: Differences in the interpretation of results.

The POC-CCA test is subject to variations in reading interpretations depending on the intensity of its results, and trace test reading have implications for determining prevalence. The aim of this study was to assess whether the readings obtained from the POC-CCA tests, conducted using a semi-quantitative scale (the G-score classification for test determination), exhibited concurrence with the direct visual interpretation (positive, negative, or trace) performed by two distinct analysts, using photographs from previously performed POC-CCA test carried out in the municipality of Maruim, in the state of Sergipe-Brazil, a region of high endemicity. The devices used to read the photographs were smartphones, so as to simulate field usage, and a desktop, a tool with higher image quality that would help the researchers in the evaluation and establishment of the final result at a later. In direct visual interpretation of the POC-CCA photographs, the most discordant results occurred in the identification of the trace response (T). The Kappa index established for the direct visual interpretation between the two analysts, in which T is considered as positive, in the desktop was κ=0.826 and in the smartphone, κ=0.950. When we use the G-score as a reading standardization technique and classify the results according to the manufacturer, with trace being evaluated as positive, the highest level of agreement was obtained. Some disagreement remains between the direct visual interpretation and the G-score when performed on the desktop, with more individuals being classified as negative in the direct visual interpretation, by both analysts. However, this result was not statistically significant. The use of the G-score scale proved to be an excellent tool for standardizing the readings and classifying the results according to the semi-quantitative scale showed greater concordance of results both among analysts and among the different devices used to view the photographs.

Exploring features in chromatographic profiles as a tool for monitoring column performance.

In the biopharmaceutical industry, chromatography resins have a finite number of uses before they start to age and degrade, typically due to losses of ligand integrity and/or density. The "health" of a column is predicted and validated by running multiple cycles on representative scale-down models and can be followed by real-time on-going validation during commercial production. Principal Component Analysis (PCA), Partial Least Square (PLS), Similarity Scores and Single One Point-MultiParameter Technique (SOP-MPT) along with machine learning principles were applied to explore the hypothesis that there is predictive capability of latent variables in chromatography absorbance profiles for process performance (step yield) and product quality (aggregates, fragments, host cell proteins (HCP) and DNA, and Protein A ligand). The first stage of this study is described in this paper: a MabSelect SuRe™ chromatography column was cycled with a method to establish the "normal" baseline for process performance and product quality, followed by runs using a harsher NaOH Cleaning in Place (CIP) procedure (with a higher NaOH concentration than that recommended by the vendor) to accelerate resin degradation. The different mathematical analytical tools correlated with resin degradation of the column (reflected in decreasing step yield and binding capacity with increasing running cycle), specifically when using the Wash, Elution and Strip phases of the chromatography method. Monomer, HCP and DNA content were not significantly impacted and therefore a correlation with product quality was inconsequential. Importantly, this work shows proof-of-concept that while more traditional methods of measuring resin integrity such as the height equivalent to a theoretical place (HETP) and Asymmetry (As) measurements could not detect changes in the integrity of the resin, PCA, PLS, Similarity Scores and SOP-MPT (to a lesser extent) applied to the absorbance data were capable of anticipating issues in the chromatography bed by identifying atypical outcomes.

Evaluating multiproduct chromatography Protein A resin reuse for monoclonal antibodies in biopharmaceutical manufacturing.

In good manufacturing practice (GMP) facilities in the biopharmaceutical industry, chromatography resins are largely underutilized during purification of single drug products during clinical production. Chromatography resins are dedicated to a specific product and disposed of, after only a fraction of their lifetime due to concerns of potential product carryover from one program to another. In this study, we follow a resin lifetime methodology typically used for commercial submissions and apply it to determine the feasibility of purifying different products on a Protein A MabSelect PrismA™ resin. Three distinct monoclonal antibodies were used as model molecules. Column performance was monitored through chromatogram profiles, yield, clearance capability of selected media components, pressure and product quality. A protein carryover study was designed to demonstrate that the column cleaning procedures reduced protein carryover to safe cleanliness levels regardless of multiple product contact cycles and the order in which the mAbs are captured. Data show that up to 90 total cycles (30 cycles per antibody), there was negligible protein carryover and impact on process performance. Product quality was consistent, with the only meaningful trends found for the leached Protein A ligand, without affecting the conclusion of the study. While the study was restricted to three antibodies, the proof of concept for resin reuse was demonstrated.

Immunochromatography screening devices for cannabinoids in oral fluid sample

Abstract Cannabis sativa L. is one of the most consumed drugs in the world and recent studies have associated its use with an increase in the number of traffic accidents in different countries. In many countries, like Brazil, simple and reliable methodologies are still needed for the detection of drugs on site, mainly cannabinoids, considering its prevalence of use and oral fluid (OF) has been proved as an appropriate biological matrix for this purpose. Considering that, this work aims to review previous studies on immunochromatographic devices for on-site detection of cannabinoids in OF, discussing their sensitivity, specificity, cut-offs values and confirmatory methods. This data shows the importance of choosing a screening device and it reinforces the need for its implementation in Brazil. The research was conducted on 5 databases and all original articles, published in the last 10 years, were selected. A total of 32 articles were found, providing data for 17 screening devices of distinct brands. Only 2 screening devices showed satisfactory sensitivity and specificity in the evaluated studies (≥80% and ≥90% respectively). However, it should be considered that the screening devices still have some limitations, such as a higher cut-off than those recommended by international guidelines (cut-off > 2 ng/mL), therefore demonstrating the need for more studies in the area and the importance of confirmatory analysis usually fulfilled by LC-MS/MS, GC-MS/MS or GC-MS. Thus, the screening analyzes should not be evaluated by itself, but in association with confirmatory results and observational traits (behavioral changes), for a better understanding of the traffic scenario

An evaluation of the diagnostic performance characteristics of the Yellow Fever IgM immunochromatographic rapid diagnostic test kit from SD Biosensor in Ghana.

Yellow fever is endemic in Ghana and outbreaks occur periodically. The prodromal signs due to Yellow Fever Virus (YFV) infection are non-specific, making clinical signs unreliable as the sole criteria for diagnosis. Accurate laboratory confirmation of suspected yellow fever cases is therefore vital in surveillance programs. Reporting of ELISA IgM testing results by laboratories can delay due to late arrival of samples from the collection sites as well as limited availability of ELISA kits. In this study, the diagnostic performance characteristics of a rapid immunochromatographic Standard Q Yellow Fever IgM test kit (SD Biosensor) was evaluated for the rapid diagnosis of Yellow Fever infection in Ghana. A panel of 275 sera, comprising 81 confirmed YFV positives and 194 negatives were re-tested in this study using the Standard Q Yellow Fever IgM test kit. Using the CDC/WHO Yellow Fever IgM capture ELISA as a benchmark, the sensitivity, specificity and accuracy of the Standard Q Yellow Fever test kit were 96.3%, 97.9% and 97.5%, respectively. The false positivity rate was 5.1% and there was no cross-reactivity when the Standard Q Yellow Fever test kit was tested against dengue, malaria and hepatitis B and C positive samples. In addition, inter-reader variability and invalid rate were both zero. The results indicate that the diagnostic performance of the Standard Q Yellow Fever IgM test kit on serum or plasma is comparable to the serum IgM detection by ELISA and can be used as a point of care rapid diagnostic test kit for YFV infection in endemic areas.

An immunochromatography strip with peroxidase-mimicking ferric oxyhydroxide nanorods-mediated signal amplification and readout.

Immunochromatography testing strips (ICTs) promise to become the point-of-care test format for early diagnosis due to their convenience, low cost, and simplification. However, the insufficient signal intensity and limited sensitivity of this format hamper their application. Herein, we overcame these limitations by integrating rod-like ferric oxyhydroxide (ß-FeOOH) nanoparticles with ICTs. By varying the concentration of PEI, a one-pot, mild-temperature hydrolysis method was adapted for the synthesis and morphology regulation of ß-FeOOH nanorod. Due to the excellent enzyme-like catalytic activity toward peroxidase substrates (TMB) in the presence of hydrogen peroxide (H2O2), the ß-FeOOH nanorod in ICTs served as a signal generator and the nanozyme for signal amplification. The proof-of-concept work was performed for the detection of human chorionic gonadotropin (hCG). A two fold improvement of detection sensitivity was achieved compared to the sensitivity of conventional Au NPs-based ICTs. These results show that ß-FeOOH-based ICT has a potential application in POCT detection in clinical diagnostics.

Rapid Detection of Carbapenemase-producing Enterobacteriaceae From Blood Culture Bottles of Known CPE Carriers: Real-world Experience.

We used a rapid antigen test for the detection of carbapenemases directly from positive blood culture bottles of pediatric hemato-oncologic patients, known carriers of carbapenemase-producing enterobacteriaceae. Resistance mechanism was detected within 15 minutes of observing Gram-negative bacilli from a positive bottle, leading to treatment modification. This simple-to-use, inexpensive assay shortens the interval between empiric to tailored antimicrobial therapy.

Lateral flow devices for samples collected by straw sampling method for postmortem canine rabies diagnosis.

The direct fluorescent antibody test (dFAT) using brain sample after opening the skull is the standard rabies diagnostic test in animal rabies. However, it is not feasible in many resource-limited settings. Lateral flow devices (LFD) combined with a simple sampling methodology is quicker, simpler, and less hazardous than the standard test and can be a useful tool. We conducted a prospective on-site study to evaluate the diagnostic accuracy of the LFD with the straw sampling method compared with that of the dFAT with the skull opening procedure for post-mortem canine rabies diagnosis. We collected 97 rabies-suspected animals between December 1, 2020 and March 31, 2021. Among the 97 samples, 53 and 50 cases were positive tests for dFAT and LFD, respectively. The sensitivity and specificity of LFD with straw sampling method were 94.3% (95% confidence interval [CI], 84.3-98.8%) and 100% (95% CI, 92.0-100%), respectively. The performance of LFD by the straw sampling method showed relatively high sensitivity and 100% specificity compared with that of dFAT performed on samples collected after opening the skull. This methodology can be beneficial and is a strong tool to overcome limited animal surveillance in remote areas. However, because of our limited sample size, more data using fresh samples on-site and the optimizations are urgently needed for the further implementation in endemic areas.

Evaluation of microcolumn stability in ultrafast affinity extraction for binding and rate studies.

Ultrafast affinity extraction (UAE) has recently been developed and employed for measuring non-bound (or free) fractions and binding or rate constants for drugs and other targets with soluble binding agents such as serum proteins. This study examined the long-term stability of 10 mm × 2.1 mm i.d. affinity microcolumns when used in UAE at both low and high flow rates (e.g., 0.5 and 3.5 mL/min) over an extended series of injections. This stability was investigated by using immobilized human serum albumin (HSA) and samples containing the drug warfarin with or without soluble HSA as a model system. The free warfarin fractions measured at 0.5 mL/min in the presence of soluble HSA were stable up to 150 injections and changed by <10% at 3.5 mL/min. The association equilibrium constant for warfarin with HSA that was estimated by UAE at 3.5 mL/min had no significant change over 50 injections and a change of only ∼18-22% over 100-150 injections. The dissociation rate constant for warfarin from HSA was found by combining UAE results at 0.5 and 3.5 mL/min and employing a new two-point approach, with no significant changes in this value being seen even after 200 injections. The effects of extended microcolumn use on the retention time, peak width, and peak asymmetry for warfarin, and on the backpressure of the microcolumn, were also considered. These results indicated that UAE and HSA microcolumns could be used to provide consistent values for free solute fractions, binding constants, and rate constants over a large series of injections. These results should be useful in future work by providing guidelines for the assessment, further development, and use of UAE in characterizing interactions involving other drugs and binding agents in solution-based samples.

Characterization and utility of immobilized metal affinity-functionalized cellulose membranes for point-of-care malaria diagnostics.

Immobilized metal affinity chromatography (IMAC) is a well-established technique for protein separation and purification. IMAC has been previously utilized to capture the malaria biomarker histidine-rich protein 2 (HRP2) from blood, enhancing the sensitivity of field-appropriate diagnostic tools such as lateral flow assays. However, little work has been done to translate this technique to a truly field-usable design. In this study, IMAC-functionalized cellulose membranes are created and characterized fully for future use in applied malaria diagnostics. IMAC-functionalized cellulose membranes were investigated across a range of cellulose substrates, IMAC ligands, and divalent transition metals before use in a capture and elution flowthrough workflow. Following characterization and optimization, it was found that iminodiacetic acid bound to Zn(II) was the most promising ligand-metal pair, with three available coordination sites and a molar loading capacity of 57.7 µmol of metal/cm3 of cellulose. Using these parameters, more than 99% of HRP2 was captured from a large-volume lysed blood sample in a simple flow-through assay and 89% of the captured protein was eluted from the membrane using the chelating compound ethylenediaminetetraacetic acid. Use of this enhancement protocol on an in-house HRP2 lateral flow assay (LFA) yielded a limit of detection of 7 parasites/µL, a 15.8x enhancement factor compared to traditional LFA methods.

Comparison of Protein A affinity resins for twin-column continuous capture processes: Process performance and resin characteristics.

Continuous chromatography is a promising technology for downstream processing of biopharmaceuticals. The operation of continuous processes is significantly different to batch-mode chromatography and needs comprehensive evaluation. In this work, the performances of four Protein A affinity resins were studied systematically for twin-column continuous capture processes. A model-based approach was used to evaluate the process performance (productivity and capacity utilization) under varying operation conditions, and the objective was to reveal the crucial resin properties for continuous capture. The trade-off between productivity and capacity utilization was found, and it is necessary to select appropriate resins for different feedstock and operation conditions. The capacity utilization heavily depends on mass transfer, and steep breakthrough curves are favorable for high capacity utilization. The productivity is determined by both equilibrium binding capacity and mass transfer, and the balance of feed amount and feed time is critical. Moreover, the influence of binding capacity and mass transfer on process productivity and parameter sensitivity with two important resin properties (equilibrium binding capacity qmax and effective pore diffusion coefficient De) were assessed by the model, and suitable resin parameter ranges for twin-column continuous capture were determined. The model-based approach is an effective and useful tool to evaluate the complex performance of different resins and guide the design of next-generation resins for continuous processes.

Evaluating a new mixed-mode resin Diamond MMC Mustang using Capto MMC ImpRes as a benchmark.

Diamond MMC Mustang is a relatively new mixed-mode resin, which mediates both cation exchange and hydrophobic interactions. In this work, we evaluated this resin using Cytiva's Capto MMC ImpRes, a well-established mixed-mode resin with similar properties, as a benchmark. The data suggest that in comparison with Capto MMC ImpRes, Diamond MMC Mustang exhibits comparable binding capacity and resolution. In addition, the resin under evaluation shows good lot-to-lot consistency. The information provided in this study allows users to have additional options when selecting mixed-mode resin for intermediate purification or final polishing, which is favourable especially at the present time when the supply chains of many manufacturers are negatively impacted by the coronavirus pandemic.

A custom ÄKTA avant configuration enabling automated parallel protein purification over a range of process scales.

Biologics are making up an increasing proportion of the global drug discovery pipeline. Supporting the expansion of biologics drug discovery requires higher throughput techniques for the expression, purification and characterization of both therapeutic candidates and reagents. Here we describe the programming and development of a novel ÄKTA™ instrument configuration that enables automated parallel and multistep chromatography over a range of scales. The programming strategy is offered as open source and the custom plumbing configuration was developed with off the shelf components available from Cytiva. Combined with high flow resin technology we show how this strategy can reduce the duration of a standard antibody purification process by 4.5X, from 4.5 h down to 1 h per run. An automated loading strategy was also developed to enable true walk away application of up to 24 samples and around the clock processing capability. The techniques used here to accomplish parallel multistep chromatography can be duplicated or modified for specific applications and represent a straightforward and cost-effective means to eliminate protein purification bottlenecks.

Development and evaluation of colloidal gold immunochromatography test strip for rapid diagnosis of nervous necrosis virus in golden grey mullet (Chelon aurata).

A lateral flow immunochromatography strip test, based on antibody-gold nanoparticles specific for nervous necrosis virus (NNV), was developed for rapid, on-site detection of the virus in fish stocks. A monoclonal antibody against NNV was conjugated with colloidal gold as the detector antibody. A rabbit anti-NNV polyclonal antibody and goat anti-mouse IgG antibody were blotted onto the nitrocellulose membrane as the capture antibodies on the test line and control line, respectively. The reaction could be seen by the eye within 15 min and did not cross-react with the other viruses tested. The detection limit of the strip was approximately 103 TCID50 /ml and had good stability after storage at 4°C for 8 months. When brains of 70 naturally infected golden grey mullet, Chelon aurata, were tested with the strip test, the diagnostic specificity and sensitivity of the test compared to real-time RT-PCR were 100% and 74%, respectively. Therefore, the one-step test strip developed here had high specificity, reproducibility, and stability. This, together with its simplicity to use and rapid detection, without the requirement of sophisticated equipment or specialized skills, makes the strip suitable for pond-side detection of NNV in farmed fish.

Bronchoalveolar lavage fluid IMMY Sona Aspergillus lateral-flow assay for the diagnosis of invasive pulmonary aspergillosis: a prospective, real life evaluation.

Prompt and reliable diagnosis of invasive pulmonary aspergillosis (IPA) is essential for early initiation of antifungal therapy. We evaluated bronchoalveolar lavage (BAL) fluid IMMY Sona Aspergillus lateral-flow assay (IMMY LFA) in 92 individuals with suspected pulmonary infection. Sensitivity and specificity (vs. host factor but no IPA) of BAL IMMY LFA for diagnosis of IPA in individuals with any European Organisation for Research and Treatment of Cancer-defied "host factor" were 67% and 85%, respectively. Performance appeared better in individuals with renal transplantation (100%, 100%), compared to those with hematological malignancy and/or allogenic stem cell transplantation (70%, 78%). We found BAL IMMY LFA to be a convenient and useful addition to our diagnostic armory for IPA. LAY ABSTRACT: We evaluated a new test for diagnosing invasive pulmonary aspergillosis from bronchoscopy samples. We tested 92 people and found that it was 67% sensitive and 85% specific (compared to diagnosis according to a set of internationally recognised criteria). We found this test convenient and useful.

Frontal affinity chromatography: An excellent method of analyzing weak biomolecular interactions based on a unique principle.

BACKGROUND: Not only strong biomolecular interactions but also weak interactions play important roles in ensuring appropriate operations of many biological systems. Although a thorough investigation of the latter is essential in understanding life science, few suitable research tools are available because of inherent difficulties. SCOPE OF REVIEW: Frontal affinity chromatography (FAC) is a versatile method that overcomes the inherent difficulties to provide accurate information on weak interactions. Since its concept and merit are not widely recognized, a comprehensive interpretation of FAC is provided in this review to encourage its application among researchers. MAJOR CONCLUSION: FAC is based on a unique principle of measuring the binding strength by the delayed migration of an analyte through an affinity column. Its utility was elucidated via the lectin-glycan interactions. GENERAL SIGNIFICANCE: FAC has a great potential as a research tool to solve many difficult problems in general bioscience that are relevant to almost all researchers.

Semi-quantitative and quantitative detection of ochratoxin A in agricultural by-products using a self-assembling immunochromatographic strip.

BACKGROUND: The toxicity and health risks of mycotoxins have encouraged increased awareness and strict monitoring of these contaminants in agricultural by-products. In this paper, we developed and tested a sensitive, selective, and self-assembling immunochromatographic (IC) strip for on-site detection of ochratoxin A (OTA). We were able to demonstrate semi-quantitative and quantitative detection of OTA in agricultural by-product samples. RESULTS: The optimized IC strip had a limit of detection (LOD) of 0.5 ng mL-1 OTA using the naked eye for semi-quantitative detection. When a digitized strip reader was used to achieve quantitative results, the LOD for OTA was reduced to 0.1 ng mL-1 with a linear detection range of 0.1-10 ng mL-1 . We also evaluated the specificity, stability, accuracy, and precision of the IC strip and demonstrated high performance in all of these areas. We then confirmed the ability of the IC strip to visually detect OTA contamination in authentic agricultural by-product samples from the markets in China. Furthermore, quantitative detection of OTA using the IC strip showed that the concentration of OTA ranged from 1.7 to 101.3 ng g-1 in the positive agricultural by-product samples, correlating well with the measurements obtained via liquid chromatography with tandem mass spectrometry. CONCLUSION: The results indicated that this proposed IC strip was capable of sensitive, semi-quantitative, quantitative, and on-site detection of OTA contamination in agricultural by-product samples. © 2020 Society of Chemical Industry.

Gold nanoparticles-functionalized three-dimensional flower-like manganese dioxide: A high-sensitivity thermal analysis immunochromatographic sensor.

A sensitive photothermal immunochromatographic test strip (PITS) for the detection of deoxynivalenol (DON) was developed using flower-like gold nanoparticle-deposited manganese dioxide nanocarrier (FMD-G NC) labeled antibodies (Abs) as the photothermal-sensing probe. FMD was used as a template to deposit small gold nanoparticles (GNPs) to synthesize FMD-G NC with large specific surface area and significant photothermal conversion property. The FMD-G-Ab probe was competitively captured by DON target and antigen coated on test line (T-line), forming colorimetric signals under naked eyes and photothermal signals under an 808 nm laser. Under optimal conditions, the PITS exhibited sensitive and specific detection of DON from 0.19 ng mL-1 to 12 ng mL-1 with detection limits of 0.013 ng mL-1, which were over 15-fold and 58-fold more sensitive than visual FMD-G-ITS and traditional GNPs-ITS. In addition, the novel FMD-G-PITS possessed a universal applicability, which could be well applied in green bean, corn, and millet.

Fluorescence based immunochromatographic sensor for rapid and sensitive detection of tadalafil and comparison with a gold lateral flow immunoassay.

Tadalafil (TDL) is an illegal additive drug found in drinks and functional foods that could threaten public health. There was a great concern whether the adulteration occurred in coffee added with similar type of herbs. Here we have developed a rapid, simple, sensitive, and semi-quantitative lateral flow immunoassay (LFIA) based on gold and fluorescence labelled monoclonal antibody (mAb) for detection of TDL in coffee sample. Under optimal conditions, the cut off limits using gold nanoparticles labelled mAb (GLM) was found to be 250 ng/mL and 100 ng mL using fluorescent labelled mAb (FLM) in coffee samples. The coffee samples were spiked with TDL, and the LFIA with GLM gave average recoveries of 92-105.3% (intra-assay) and 96.6-105.9% (inter-assay), meanwhile with FLM gave recoveries 97.9-107.3% (intra-assay) and 98.3-108.9% (inter-assay). Results gave LFIA with FLM more sensitive than with GLM and all the test can be completed within 10 min, which would be an option for convenient and rapid assay of TDL detection.