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The non-steroidal anti-inflammatory drug (NSAID) diclofenac (DCF) is an important environmental contaminant occurring in surface waters all over the world, because, after excretion, it is not adequately removed from wastewater in sewage treatment plants. To be able to monitor this pollutant, highly efficient analytical methods are needed, including immunoassays. In a medical research project, monoclonal antibodies against diclofenac and its metabolites had been produced. Based on this monoclonal anti-DCF antibody, a new indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed and applied for environmental samples. The introduction of a spacer between diclofenac and the carrier protein in the coating conjugate led to higher sensitivity. With a test midpoint of 3 mg L−1 and a measurement range of 1–30 mg L−1, the system is not sensitive enough for direct analysis of surface water. However, this assay is quite robust against matrix influences and can be used for wastewater. Without adjustment of the calibration, organic solvents up to 5%, natural organic matter (NOM) up to 10 mg L−1, humic acids up to 2.5 mg L−1, and salt concentrations up to 6 g L−1 NaCl and 75 mg L−1 CaCl2 are tolerated. The antibody is also stable in a pH range from 3 to 12. Cross-reactivity (CR) of 1% or less was determined for the metabolites 40-hydroxydiclofenac (40-OH-DCF), 5-hydroxydiclofenac (5-OH-DCF), DCF lactam, and other NSAIDs. Relevant cross-reactivity occurred only with an amide derivative of DCF, 6-aminohexanoic acid (DCF-Ahx), aceclofenac (ACF) and DCF methyl ester (DCF-Me) with 150%, 61% and 44%, respectively. These substances, however, have not been found in samples. Only DCF-acyl glucuronide with a cross-reactivity of 57% is of some relevance. For the first time, photodegradation products were tested for cross-reactivity. With the ELISA based on this antibody, water samples were analysed. In sewage treatment plant effluents, concentrations in the range of 1.9–5.2 mg L−1 were determined directly, with recoveries compared to HPLC-MS/MS averaging 136%.
Concentrations in lakes ranged from 3 to 4.4 ng L−1 and were, after pre-concentration, determined with an average recovery of 100%
Heterogeneous sandwich immunoassays are widely used for biomarker detection in bioanalysis and medical diagnostics. The high analyte sensitivity of the current “gold standard” enzyme-linked immunosorbent assay (ELISA) originates from the signal-generating enzymatic amplification step, yielding a high number of optically detectable reporter molecules. For future point-of-care testing (POCT) and point-of-need applications, there is an increasing interest in more simple detection strategies that circumvent time-consuming and temperature-dependent enzymatic reactions. A common concept to aim for detection limits comparable to those of enzymatic amplification reactions is the usage of polymer nanoparticles (NP) stained with a large number of chromophores. We explored different simple NP-based signal amplification strategies for heterogeneous sandwich immunoassays
that rely on an extraction-triggered release step of different types of optically detectable reporters. Therefore, streptavidinfunctionalized polystyrene particles (PSP) are utilized as carriers for (i) the fluorescent dye coumarin 153 (C153) and (ii) hemin (hem) molecules catalyzing the luminol reaction enabling chemiluminescence (CL) detection. Additionally, (iii) NP labeling with hemin-based microperoxidase MP11 was assessed. For each amplification approach, the PSP was first systematically optimized regarding size, loading concentration, and surface chemistry. Then, for an immunoassay for the inflammation marker C- eactive protein (CRP), the analyte sensitivity achievable with optimized PSP
ystems was compared with the established ELISA concept for photometric and CL detection. Careful optimization led to a limit of detection (LOD) of 0.1 ng/mL for MP11-labeled PSP and CL detection, performing similarly well to a photometric ELISA (0.13 ng/mL), which demonstrates the huge potential of our novel assay concept.
Immunoassays, based on the recognition and capture of analytes by highly selective antibodies, are now used extensively in all areas of diagnostics, but the challenge is to further integrate them into online sensors. To improve the transition from laboratory immunoassays to immunosensors, we have developed a complete flow system, based on a microfluidic core flow cell to enable automated detection of one of the most commonly used immunoassay substrates, TMB, by chronoamperometry. The architecture and fluidic optimisation of the system showed that a specially designed 3D flow cell allows higher flow rates (500 μL min−1) than a standard enlarged microfluidic channel (50 μL min−1) resulting in a significantly shorter detection time of 30 seconds per sample and making the system more robust against interferences due to bubble formation in the chip. The electrochemical measurements showed an improved signal-to-noise ratio (SNR) and thus higher sensitivity for a model immunoassay for diclofenac (SNR = 59), compared to the analytical performance of a conventional laboratory microplate-based assay with optical detection (SNR = 19). In general, this system facilitates the conversion of any conventional immunoassay into an immunosensor with automatic and continuous detection.
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.
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
Electrochemical methods offer great promise in meeting the demand for user-friendly on-site devices for Monitoring important parameters. The food industry often runs own lab procedures, for example, for mycotoxin analysis, but it is a major goal to simplify analysis, linking analytical methods with smart technologies. Enzyme-linked immunosorbent assays, with photometric detection of 3,3’,5,5’-tetramethylbenzidine (TMB),form a good basis for sensitive detection. To provide a straightforward approach for the miniaturization of the detectionstep, we have studied the pitfalls of the electrochemical TMB detection. By cyclic voltammetry it was found that the TMB electrochemistry is strongly dependent on the pH and the electrode material. A stable electrode response to TMB could be achieved at pH 1 on gold electrodes. We created a smartphonebased, electrochemical, immunomagnetic assay for the detection of ochratoxin A in real samples, providing a solid basis forsensing of further analytes.
Der Bedarf nach schnellen und kostengünstigen Analysemethoden in der (Trink-)Wasseranalytik steigt mit dem zunehmenden Eintrag pharmazeutisch aktiver Substanzen in die Umwelt.
Insbesondere die Kontamination mit Antibiotika erscheint neben ökotoxikologischen Effekten vor allem in Hinblick auf die Evolution resistenter Keime im Wasser besorgniserregend.
Wir berichten hier über die Entwicklung eines magnetpartikelbasierten Immunoassays (MBBA) zur Detektion des Breitbandantibiotikums Amoxicillin (AMX) in Wasserproben, der im Vergleich zum entsprechenden Enzyme-linked Immunosorbent Assay (ELISA) eine wesentlich kürzere Analysendauer sowie einen breiteren Messbereich mit niedrigerer Nachweisgrenze aufweist. Das angewendete Assayprinzip ist dabei leicht auf weitere Analyten übertragbar und ermöglicht die Implementierung in einen Immunosensor.
Contamination of waters with pharmaceuticals is an alarming problem as it may support the evolution of antimicrobial resistance. Therefore, fast and cost-effective analytical methods for potential on-site analysis are desired in order to control the water quality and assure the safety of its use as a source of drinking water. Antibody-based methods, such as the enzyme-linked immunosorbent assay (ELISA), can be helpful in this regard but can also have certain pitfalls in store, depending on the analyte. As shown here for the class of β-lactam antibiotics, hydrolysis of the β-lactam ring is a key factor in the immunochemical analysis as it influences antibody recognition. With the antibody used in this study, the limit of detection (LOD) in the immunoassay could be significantly reduced by hydrolysis for the five tested penicillins, with the lowest LOD for carbenicillin (0.2 nmol/L) and the greatest impact on penicillins G and V (reduction by 85%). In addition to enhanced quantification, our strategy also provides access to information about the degree of hydrolysis in water samples as shown for the most abundant penicillin amoxicillin.
The primary screening of hybridoma cells is a time-critical and laborious step during the development of monoclonal antibodies. Often, critical errors occur in this phase, which supports the notion that the generation of monoclonal antibodies with hybridoma technology is difficult to control and hence, a risky venture. We think that it is crucial to improve the screening process to eliminate most of the critical deficits of the conventional approach. With this new microarray-based procedure, several advances could be achieved: Selectivity for excellent binders, high-throughput, reproducible signals, avoidance of misleading avidity (multivalency) effects, and performance of simultaneous competition experiments. The latter can also be used to select clones of desired cross-reactivity properties. In this paper, a model system with two excellent clones against carbamazepine, two weak clones, and blank supernatant containing fetal bovine serum was designed to examine the effectiveness of the new system. The excellent clones could be detected largely independent of the immunoglobulin G (IgG) concentration, which is usually unknown during the clone screening since the determination and subsequent adjustment of the antibody concentration are not feasible in most cases. Furthermore, in this approach, the enrichment, isolation, and purification of IgG for characterization is not necessary. Raw cell culture supernatant can be used directly, even when fetal calf serum (FCS) or other complex media is used. In addition, an improved method for the oriented antibody-immobilization on epoxy-silanized slides is presented. Based on the results of this model system with simulated hybridoma supernatants, we conclude that this approach should be preferable to most other protocols leading to many false positives, causing expensive and lengthy elimination steps to weed out the poor clones.