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Organisationseinheit der BAM
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.
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.
A surface-labeled lyophilized lymphocyte (sLL) preparation has been developed using human peripheral blood mononuclear cells prelabeled with a fluorescein isothiocyanate conjugated anti-CD4 monoclonal antibody. The sLL preparation is intended to be used as a reference material for CD4+ cell counting including the development of higher order reference measurement procedures and has been evaluated in the pilot study CCQM-P102. This study was conducted across 16 laboratories from eight countries to assess the ability of participants to quantify the CD4+ cell count of this reference material and to document cross-laboratory variability plus associated measurement uncertainties. Twelve different flow cytometer platforms were evaluated using a standard protocol that included calibration beads used to obtain quantitative measurements of CD4+ T cell counts. There was good overall cross-platform and counting method agreement with a grand mean of the laboratory calculated means of (301.7 ± 4.9) µL-1 CD4+ cells. Excluding outliers, greater than 90% of participant data agreed within ±15%. A major contribution to variation of sLL CD4+ cell counts was tube to tube variation of the calibration beads, amounting to an uncertainty of 3.6%. Variation due to preparative steps equated to an uncertainty of 2.6%. There was no reduction in variability when data files were centrally reanalyzed. Remaining variation was attributed to instrument specific differences. CD4+ cell counts obtained in CCQM-P102 are in excellent agreement and show the robustness of both the measurements and the data analysis and hence the suitability of sLL as a reference material for interlaboratory comparisons and external quality assessment.
This study aimed to assess the measurement capabilities of participating National Metrology Institutes/ Designated Institutes (NMIs/DIs) and expert laboratories in determining of low-polarity organics in surface water. This comparison was organized by Government Laboratory, Hong Kong (GLHK).
At the CCQM Organic Analysis Working Group (OAWG) Meeting held in November 2012 in Hong Kong, GLHK initially proposed a CCQM key comparison and a parallel pilot study programme on pharmaceuticals in surface water. Further discussion at the CCQM Meeting held in April 2014 in Paris, the OAWG approved a CCQM Track C comparison (CCQM-K126) on low polarity pharmaceuticals in surface water. In the meeting, the programme was supported by more than six National Metrology Institutes/ Designated Institutes (NMIs/ DIs).
CCQM-K126 officially commenced in July 2014 and had registration from nine NMIs/DIs. Participants were provided two bottles (40 mL each) of surface water and were requested to determine the mass fraction of spiked carbamazepinein in surface water. The coordinator received nine sets of results from eight NMIs/DIs in February 2015. Apart from one using an immunoassay technique, all participants applied isotope dilution liquid chromatography-tandem mass spectrometry (ID-LCMS/MS) technique as their determination technique.
In this work we report a novel paper-based analytical device read-out via LED-induced fluorescence detection (FPAD) for the quantification of the emerging pollutant ethinylestradiol (EE2) in river water samples. The PAD was used as a reaction platform for a competitive enzyme immunoassay. For the PAD development, microzones of filter paper, printed by a wax printing method, were modified with amino-functionalized SBA-15 and subsequently, anti-EE2 specific antibodies were covalently immobilized. The determination of EE2 in water was
carried out by adding a fixed concentration of EE2 conjugated with the enzyme horseradish peroxidase (HRP) to samples and standards. Then, the FPAD were added and incubated for 10 min. Finally, the detection was performed by the reaction of 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) whose oxidation is catalyzed by HRP in the presence of H2O2, obtaining the highly fluorescent resorufin (R). Resorufin was detected by LED excitation at 550 nm, observing emission at 585 nm. The EE2 concentration in the samples was inversely proportional to the relative fluorescence obtained from the enzymatic reaction products. The FPAD assay showed a detection Limit (LOD) of 0.05 ng L−1 and coefficients of variation (CV) below 4.5% within-assay and below 6.5% between-assay, respectively. The results obtained show the potential suitability of our FPAD for the selective and sensitive quantification of EE2 in river water samples. In addition, it has the PADs advantages of being disposable, easy to
apply and inexpensive.
We report a novel and innovative electrochemical paper-based immunocapture assay (EPIA) to address the need for ultrasensitive detection of emerging pollutants without regulatory status and whose effects on environment and human health are not completely yet understood. In particular, we present the application of this system toward highly sensitive detection of the emerging pollutant ethinyl estradiol (EE2). The EPIA approach is based on the use of paper microzones modified with silica nanoparticles (SNs) and anti-EE2 specific antibodies for capture and preconcentration of EE2 from river water samples. After the preconcentration procedure, the paper microzones are placed onto a screen-printed carbon electrode modified with electrochemically reduced graphene (RG). The bound EE2 is subsequently desorbed adding a diluted solution of sulfuric acid on the paper microzones. Finally, recovered EE2 is electrochemically detected by OSWV. The proposed novel methodology showed an appropriate LOD and linear range for the quantification of EE2 for water samples with different origins. The nonsophisticated equipment required, the adequate recovery values obtained (from 97% to 104%, with a RSD less than 4.9%), and the appropriate LOD and linear range value (0.1 ng L−1 and 0.5−120 ng L−1, respectively) achieved by our immunocapture sensor present significant analytical figures of merit, particularly when the routine quantification of EE2 is considered. In addition, our System was based on electrochemical paper-based technology, which allows obtainment of portable, easy-to-use, inexpensive, and disposable devices. The EPIA can also serve as a general-purpose immunoassay platform applicable to quantitation of other drugs and emerging pollutants in environmental samples.
This report focuses on the characterization of CD4 expression level in terms of equivalent number of reference fluorophores (ERF). Twelve different flow cytometer platforms across sixteen laboratories were utilized in this study. As a first step the participants were asked to calibrate the fluorescein isothiocyanate (FITC) channel of each flow cytometer using commercially available calibration standard consisting of five populations of microspheres. Each population had an assigned value of equivalent fluorescein fluorophores (EFF denotes a special case of the generic term ERF with FITC as the reference fluorophore). The EFF values were assigned at the National Institute of Standards and Technology (NIST). A surface-labelled lyophilized cell preparation was provided by the National Institute of Biological Standards and Control (NIBSC), using human peripheral blood mononuclear cells (PBMC) pre-labeled with a FITC conjugated anti-CD4 monoclonal antibody. Three PBMC sample vials, provided to each participant, were used for the CD4 expression analysis. The PBMC are purported to have a fixed number of surface CD4 receptors. On the basis of the microsphere calibration, the EFF value of the PBMC samples was measured to characterize the population average CD4 expression level of the PBMC preparations. Both the results of data analysis performed by each participant and the results of centralized analysis of all participants' raw data are reported. Centralized analysis gave a mean EFF value of 22,300 and an uncertainty of 750, corresponding to 3.3% (level of confidence 68%) of the mean EFF value. The next step will entail the measurement of the ERF values of the lyophilized PBMC stained with labels for other fluorescence channels. The ultimate goal is to show that lyophilized PBMC is a suitable biological reference cell material for multicolor flow cytometry and that it can be used to present multicolor flow cytometry measurements in terms of ABC (antibodies bound per cell) units.
Gastric cancer (GC) is the 3rd deadliest cancer worldwide, due to limited treatment options and late diagnosis. Human epidermal growth factor receptor-2 (HER2) is overexpressed in similar to 20% of GC cases and anti-HER2 antibody trastuzumab in combination with conventional chemotherapy, is recognized as standard therapy for HER2-positive metastatic GC. This strategy improves GC patients' survival by 2-3 months, however its optimal results in breast cancer indicate that GC survival may be improved. A new photoimmunoconjugate was developed by conjugating a porphyrin with trastuzumab (Trast: Porph) for targeted photodynamic therapy in HER2-positive GC. Using mass spectrometry analysis, the lysine residues in the trastuzumab structure most prone for porphyrin conjugation were mapped. The in vitro data demonstrates that Trast: Porph specifically binds to HER2-positive cells, accumulates intracellularly, co-localizes with lysosomal marker LAMP1, and induces massive HER2-positive cell death upon cellular irradiation. The high selectivity and cytotoxicity of Trast: Porph based photoimmunotherapy is confirmed in vivo in comparison with trastuzumab alone, using nude mice xenografted with a HER2-positive GC cell line. In the setting of human disease, these data suggest that repetitive cycles of Trast: Porph photoimmunotherapy may be used as an improved treatment strategy in HER2-positive GC patients.
The synthetic estrogen ethinylestradiol (EE2) is an active component of oral contraceptives (OCs), considered as an endocrine disrupting compound (EDC). It is excreted from humans and released via sewage treatment plant effluents into aquatic environments. EDCs are any environmental pollutant chemical that, once incorporated into an organism, affects the hormonal balance of various species including humans. Its presence in the environment is becoming of great importance in water quality. This paper describes the development of an accurate, sensitive and selective method for capture, preconcentration and determination of EE2 present in water samples using: magnetic particles (MPs) as bioaffinity support for the capture and preconcentration of EE2 and a glassy carbon electrode modified with multi-walled carbon nanotubes (MWCNTs/GCE) as detection system. The capture procedure was based on the principle of immunoaffinity, the EE2 being extracted from the sample using the anti-EE2 antibodies (anti-EE2 Ab) which were previously immobilized on MPs. Subsequently the analyte desorption was done employing a sulfuric acid solution and the determination of the EE2 in the pre-concentrated solution was carried out by square wave voltammetry (SWV).
This method can be used to determine EE2 in the range of 0.03570 ng L-1 with a detection limit (LOD) of 0.01 ng L-1 and R.S.D. < 4.20%. The proposed method has been successfully applied to the determination of EE2 in water samples and it has promising analytical applications for the direct determination of EE2 at trace levels.