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Under the auspices of the Protein Analysis Working Group (PAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a pilot study, CCQM-P216, was coordinated by the Chinese National Institute of Metrology (NIM), National Research Council of Canada (NRC) and the Bureau International des Poids et Mesures (BIPM). Eleven Metrology Institutes or Designated Institutes and the BIPM participated in the first phase of the pilot study (Part 1). The purpose of this pilot study was to develop measurement capabilities for larger proteins using a recombinant humanized IgG monoclonal antibody against Spike glycoprotein of SARS-CoV-2 (Anti-S IgG mAb) in solution. The first phase of the study was designed to employ established methods that had been previously studies by the CCQM Protein Analysis Working Group, involving the digestion of protein down to the peptide or amino acid level. The global coronavirus pandemic has also led to increased focus on antibody quantitation methods. IgG are among the immunoglobulins produced by the immune system to provide protection against SARS-CoV-2. Anti-SARS-CoV-2 IgG can therefore be detected in samples from affected patients. Antibody tests can show whether a person has been exposed to the SARS-CoV-2, and whether or not they potentially show lasting immunity to the disease. With the constant spread of the virus and the high pressure of re-opening economies, antibody testing plays a critical role in the fight against COVID-19 by helping healthcare professionals to identify individuals who have developed an immune response, either via vaccination or exposure to the virus. Many countries have launched large-scale antibody testing for COVID-19. The development of measurement standards for the antibody detection of SARS-CoV-2 is critically important to deal with the challenges of the COVID-19 pandemic. In this study, the SARS-CoV-2 monoclonal antibody is being used as a model system to build capacity in methods that can be used in antibody quantification. Amino acid reference values with corresponding expanded uncertainty of 36.10 ± 1.55 mg/kg, 38.75 ± 1.45 mg/kg, 18.46 ± 0.78 mg/kg, 16.20 ± 0.67 mg/kg and 30.61 ± 1.30 mg/kg have been established for leucine, valine, phenylalanine, isoleucine and proline, respectively. Agreement between nearly all laboratories was achieved for the amino acid analysis within 2 to 2.5 %, with one participant achieving markedly higher results due to a technical issue found in their procedure; this result was thus excluded from the reference value calculations. The relatively good agreement within a laboratory between different amino acids was not dissimilar to previous results for peptides or small proteins, indicating that factors such as hydrolysis conditions and calibration procedures could be the largest sources of variability. Peptide reference values with corresponding expanded uncertainty of 4.99 ± 0.28 mg/kg and 6.83 ± 0.65 mg/kg have been established for ALPAPIEK and GPSVFPLAPSSK, respectively. Not surprisingly due to prior knowledge from previous studies on peptide quantitation, agreement between laboratories for the peptide-based analysis was slightly poorer at 3 to 5 %, with one laboratory's result excluded for the peptide GPSVFPLAPSSK. Again, this level of agreement was not significantly poorer than that achieved in previous studies with smaller or less complex proteins. To reach the main text of this paper, click on Final Report.
The environmental fate of the frequently used broad-spectrum β-lactam antibiotic amoxicillin (AMX) is of high concern regarding the potential evolution of antimicrobial resistance (AMR). Moreover, it is known that AMX is prone to hydrolysis, yielding a variety of hydrolysis products (HPs) with yet unknown effects. Studies to identify those HPs and investigate their formation mechanisms have been reported but a long-term study on their stability in real water samples was missing. In this regard, we investigated the hydrolysis of AMX at two concentration levels in four distinct water types under three different storage conditions over two months. Concentrations of AMX and four relevant HPs were monitored by an LC-MS/MS method revealing pronounced differences in the hydrolysis rate of AMX in tap water and mineral water on the one hand (fast) and surface water on the other(slow). In this context, the occurrence, relative intensities, and stability of certain HPs are more dependent on the water type than on the storage condition. As clarified by ICP-MS, the main difference between the water types was the content of the metals copper and zinc which are supposed to catalyze AMX hydrolysis demonstrating an effective method to degrade AMX at ambient conditions.
A certain group of mycotoxins, the ergot alkaloids, has caused countless deaths throughout human history. They are found in rye and other cereals and ingesting contaminated foods can cause serious health problems. To identify contaminated food exceeding the legal limits for ergot alkaloids, a portable and cost-effective test system is of great interest to the food industry. Rapid analysis can be achieved by screening for a marker compound, for which we chose ergometrine. We developed a magnetic bead-based immunoassay for ergometrine with amperometric detection in a flow injection system using a handheld potentiostat and a smartphone. With this assay a limit of detection of 3 nM (1 μg/L) was achieved. In spiked rye flour, ergometrine levels from 25 to 250 μg/kg could be quantified. All results could be verified by optical detection. The developed assay offers great promise to meet the demand for on-site ergometrine detection in the food industry.
Increasing contamination of environmental waters with pharmaceuticals represents an emerging threat for the drinking water quality and safety. In this regard, fast and reliable analytical methods are required to allow quick countermeasures in case of contamination. Here, we report the development of a magnetic bead-based immunoassay (MBBA) for the fast and cost-effective determination of the analgesic diclofenac (DCF) in water samples, based on diclofenac-coupled magnetic beads and a robust monoclonal anti-DCF antibody. A novel synthetic strategy for preparation of the beads resulted in an assay that enabled for the determination of diclofenac with a significantly lower limit of detection (400 ng/L) than the respective enzyme-linked immunosorbent assay (ELISA). With shorter incubation times and only one manual washing step required, the assay demands for remarkably shorter time to result (< 45 min) and less equipment than ELISA. Evaluation of assay precision and accuracy with a series of spiked water samples yielded results with low to moderate intra- and inter-assay variations and in good agreement with LC–MS/MS reference analysis. The assay principle can be transferred to other, e.g., microfluidic, formats, as well as applied to other analytes and may replace ELISA as the standard immunochemical method.
Mit dem Testsystem zur Probe
(2021)
In this work, we report an electrochemical immunosensor to detect ethinylestradiol in water samples, using electrochemical impedance spectroscopy (EIS) as a detection technique. For the development of this immunosensor, the direct modification of the working electrode of a screen-printed carbon electrode was carried out. First, to reduce the resistance of the electrode, electroreduced graphene was incorporated on the surface.
Second, a porous gold structure was electrodeposited on reduced graphene by electrodeposition and the dynamic hydrogen bubble template assisted method. Thus, a marked increase in surface area was obtained for anti-EE2 antibodies immobilization. Subsequently, the specific anti-EE2 antibodies were covalently immobilized using α-lipoic acid for attaching them to the gold surface.
The electrode modified with the antibodies was incubated for 30 min in the samples containing EE2, producing the specific Antigen antibody binding. As the charge transfer resistance of a redox probe in the electrode surface is governed by the surface blocking effects, the charge transfer resistance was related to the amount of EE2 captured to realize a quantitative determination. For this, the EIS measurements were performed in a 4 mM [Fe(CN)6]4−/3− solution in 0.1 M KCl. The obtained Nyquist diagrams were adjusted using the Randles circuit as an equivalent circuit to obtain the corresponding resistances. The developed methodology showed good selectivity, precision, and sensitivity; although the LOD obtained was higher than those presented in other published articles, it turned out to be an alternative that allows the determination of ethinylestradiol using a simple disposable electrode.
One of the most important chemicals used in the production of polymer plastics and coatings is bisphenol A. However, despite the large number of studies on the toxicity and hormonal activity of BPA, there are still open questions and thus considerable media attention regarding BPA toxicity. Hence, it is necessary to develop a sensitive, simple, cost-efficient, specific, portable, and rapid method for monitoring bisphenol A and for high sample throughput and on-site screening analysis. Lateral flow immunoassays have potential as rapid tests for on-site screening. To meet sensitivity criteria, they must be carefully optimized. A latex microparticle-based LFIA for detection of BPA was developed. The sensitivity of the assay was improved by non-contact printing of spot grids as the control and test lines with careful parameter optimization. Results of the test could be visually evaluated within 10 min with a visual cut-off of 10 µg/L (vLOD). Alternatively, photographs were taken, and image analysis performed to set up a calibration, which allowed for a calculated limit of detection (cLOD) of 0.14 µg/L. The method was validated for thermal paper samples against ELISA and LC–MS/MS as reference methods, showing good agreement with both methods
Portable, antikörperbasierte Analysenverfahren für die Schadstofferfassung im Wasserkreislauf
(2021)
Die Wasseranalytik wird bestimmt von zwei Hauptbedarfen, einerseits der Erfassung der Gewässergüte und Grundwassergüte als Basis für die Bereitstellung einwandfreien Trinkwassers und zum anderen des Monitorings der Wasserqualität in den Prozessen der Wasseraufbereitung und der Abwasserreinigung. Für beide Anwendungsbereiche ist eine laborbasierte Analytik eher hinderlich, werden die Analysenergebnisse doch eigentlich vor Ort benötigt, um etwa Beprobungskampagnen rasch anpassen zu können. Des Weiteren werden die Daten auch schnell benötigt, um zeitnah in die technischen Prozesse einzugreifen. Organische Kontaminanten werden meist über chromatographische Verfahren, häufig gekoppelt mit einer massenspektrometrischen Detektion, in instrumentellen Labormethoden erfasst, und die hochauflösende Massenspektrometrie und die Non-target-Analytik haben viel zum Wissen über das Vorkommen, insbesondere von sogenannten „Emerging Contaminants“, beigetragen, sowie zum Verständnis der beteiligten Eintragspfade und Abbauwege. Moderne, portable Analysenmethoden und insbesondere Sensoren werden zukünftig aber ihren Platz in der Schadstofferfassung im Wasserkreislauf einnehmen. Dies gilt insbesondere für antikörperbasierte Analysenverfahren, da erst diese oft die nötige Selektivität und Sensitivität in die Detektionsmethoden einbringen.
Portable, antikörperbasierte Analysenverfahren für die Schadstofferfassung im Wasserkreislauf
(2021)
Die Wasseranalytik wird bestimmt von zwei Hauptbedarfen, einerseits der Erfassung der Gewässergüte und Grundwassergüte als Basis für die Bereitstellung einwandfrei-en Trinkwassers und zum anderen des Monitorings der Wasserqualität in den Prozes-sen der Wasseraufbereitung und der Abwasserreinigung. Für beide Anwendungsbe-reiche ist eine laborbasierte Analytik eher hinderlich, werden die Analysenergebnisse doch eigentlich vor Ort benötigt, um etwa Beprobungskampagnen rasch anpassen zu können. Desweiteren werden die Daten auch schnell benötigt, um zeitnah in die technischen Prozesse einzugreifen. Organische Kontaminanten werden meist über chromatographische Verfahren, häufig gekoppelt mit einer massenspektrometrischen Detektion, in instrumentelle Labormethoden erfasst und die hochauflösende Massen-spektrometrie und die Non-target-Analytik haben viel zum Wissen über das Vorkom-men, insbesondere von sog. „emerging contaminants“, beigetragen, sowie zum Ver-ständnis der beteiligten Eintragspfade und Abbauwege. Moderne, portable Analy-senmethoden und insbesondere Sensoren werden zukünftig aber ihren Platz in der Schadstofferfassung im Wasserkreislauf einnehmen. Dies gilt insbesondere für anti-körperbasierte Analysenverfahren, da erst diese oft die nötige Selektivität und Sensitivität in die Detektionsmethoden einbringen.
A vast number of emerging pollutants has been detected in the environment over the last decades. Analytical methods suitable for trace analysis are needed that are desirably also fast, inexpensive and, if possible, robust and portable. Immunoanalytical, i.e. antibody-based, methods which are available in a broad range of formats, can be profitably used here to analyse for the distribution and the trends of concentration levels of contaminants in the environment. Some of these formats are single-analyte but high-throughput methods. In order to use them wisely, indicator substances, sometimes called anthropogenic markers, should be selected and used in screening approaches. Other methods are suitable to be performed on portable instrumentation in the field (on-site) or in facilities such as wastewater treatment plants for on-line monitoring. Furthermore, there are array technologies that allow for parallel (multiplex) analysis of several analytes of interest.
The microtiter-plate based ELISA (Enzyme-linked Immunosorbent Assay) is the method of choice for the analysis of a large number of samples [1]. ELISAs are available to monitor for anthropogenic markers such as the antiepileptic carbamazepine, the analgesic diclofenac, the antihistaminic cetirizine, the steroid hormone estrone, the antimicrobial sulfamethoxazole, the stimulants caffeine and cocaine, the priority pollutant bisphenol A, and the bile acid isolithocholic acid. For on-site screening and monitoring, simpler formats, like mix-and-read assays, e.g. the Fluorescence Polarization Immunoassay (FPIA) [2] or Lateral-flow Immunoassays (LFIA) [3] are more suitable tools, the latter based on dipsticks or cassettes, that is why they are also called pregnancy test-like assays. The suitability of multi-analyte formats such as immunomicroarrays depends on the choice of a signal-producing system that provides small uncertainties and good reproducibility of the measurements. Bead-based (“suspension”) arrays read out in flow cytometers are a powerful platform for multiplex assays [4]. Electrochemical formats run on portable devices provide additional advantages as no light source is required. They are most promising for stand-alone analysers and biosensors