Summary A combination of TLC‐bioautography, MALDI‐TOF‐MS and LC–MS/MS methods was used to identify flavonoids with anti‐α‐amylase activity in extracts of Lathyrus pratensis L. (herb), L . polyphillus L. (fruits), Thermopsis lanceolata R. Br. (herb) and S . japonica L. (buds). After the TLC‐autobiography assay, substances with anti‐amylase activity were identified by MALDI‐TOF‐MS followed by confirmation of the result by LC–MS/MS. Results of the study revealed that the flavonoids apigenin, luteolin, formononetin, genistein and kaempferol display marked anti‐α‐amylase activity. Formononetin showed the largest activity. Compared with LC–MS/MS, MALDI‐TOF‐MS is a quick and convenient method; results can be obtained within minutes; and only minor sample amounts are required which allows us to analyse mixtures of substances without preliminary separation. However, the inability to distinguish between isomers is the main limitation of the method.

BPA is a chemical commonly used in the production of polymer-based materials that can have detrimental effects on the thyroid gland and impact human reproductive health. Various expensive methods, such as liquid and gas chromatography, have been suggested for detecting BPA. The fluorescence polarization immunoassay (FPIA) is an inexpensive and efficient homogeneous mix-and-read method that allows for high-throughput screening. FPIA offers high specificity and sensitivity and can be carried out in a single phase within a timeframe of 20-30 min. In this study, new tracer molecules were designed that linked the fluorescein fluorophore with and without a spacer to the bisphenol A moiety. To assess the influence of the C6 spacer on the sensitivity of an assay based on the respective antibody, hapten-protein conjugates were synthesized and assessed for performance in an ELISA setup, and this resulted in a highly sensitive assay with a detection limit of 0.05 g/L. The lowest limit of detection was reached by employing the spacer derivate in the FPIA and was 1.0 μg/L, working range from 2 to 155 μg/L. The validation of the methods was conducted using actual samples compared to LC-MS/MS, which served as the reference method. The FPIA and ELISA both demonstrated satisfactory concordance.

Protein immobilization for the functionalization of particles is used in various applications, including biosensors, lateral-flow immunoassays (LFIA), bead-based assays, and others. Common methods for the quantification of bound protein are measuring protein in the supernatant before and after coating and calculating the difference. This popular approach has the potential for a significant overestimation of the amount of immobilized protein since layers not directly bound to the surface (soft protein corona) are usually lost during washing and handling. Only the layer directly bound to the surface (hard corona) can be used in subsequent assays. A simplified amino acid analysis method based on acidic hydrolysis and RP-HPLC-FLD of tyrosine and phenylalanine (aromatic amino acid analysis, AAAA) is proposed to directly quantify protein bound to the surface of gold nano- and latex microparticles. The results are compared with indirect methods such as colorimetric protein assays, such as Bradford, bicinchoninic acid (BCA), as well as AAAA of the supernatant. For both particle types, these indirect quantification techniques show a protein overestimation of up to 1700% compared to the direct AAAA measurements. In addition, protein coating on latex particles was performed both passively through adsorption and covalently through EDC/sulfo-NHS chemistry. Our results showed no difference between the immobilization methodologies. This finding suggests that usual protein determination methods are no unambiguous proof of a covalent conjugation on particles or beads.

Zeolitic imidazolate framework (ZIF) hybrid fluorescent nanoparticles and ZIF antibody conjugates have been synthesized, characterized, and employed in lateral-flow immunoassay (LFIA). The bright fluorescence of the conjugates and the possibility to tailor their mobility gives a huge potential for diagnostic assays. An enzyme-linked immunosorbent assay (ELISA) with horseradish peroxidase (HRP) as label, proved the integrity, stability, and dispersibility of the antibody conjugates, LC-MS/MS provided evidence that a covalent link was established between these metal-organic frameworks and lysine residues in IgG antibodies.

When antibiotics are given to nursing women, clarithromycin (CLA), roxithromycin (ROK), erythromycin (ERI), azithromycin (AZI), dirithromycin (DIR), and oleandomycin (OLE) are excreted in breast milk. A rapid group-specific antibody test for these analytes in breast milk has been developed as a self-monitoring tool for safe feeding in infants.

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.

1. Raysyan A, Galvidis IA, Schneider RJ, Eremin SA, Burkin MA. Development of a latex particles-based lateral flow immunoassay for group determination of macrolide antibiotics in breast milk. J Pharm Biomed Analysis. 2020;189:113450. doi:10.1016/j.jpba.2020.113450 CrossRef Google Scholar

A lateral flow immunoassay (LFIA) using latex particles labeled with antibody to BSA-clarithromycin (CLA) was developed for the rapid simultaneous group determination of six macrolide antibiotics. Optimization of antigen spotting on the membrane and latex probe loading allowed improving visual detectability (vLOD) 100 times, which was 1, 1, 10, 10, 50, and 1000 ng/mL for CLA, roxithromycin, erythromycin, dirithromycin, azithromycin, and oleandomycin in buffer, respectively. The calculated limits of instrumental detection (cLOD) were respectively 0.12, 0.15, 1.4, 2.1, 2.4, and 3.3 ng/mL. To avoid a strong influence of breast milk of a very diverse and variable composition, a sample pretreatment is proposed. The six macrolides mentioned can be visually detected in breast milk after 20 min pretreatment at concentrations of 10-1000 ng/mL or instrumentally with cLOD of 4.0, 2.5, 30, 42, 42 and 180 ng/mL. The recovery rate from the spiked samples carried out using a strip scanner device ranged from 71 % to 110 %, and precision expressed as relative standard deviation was between 3-14 %. The described rapid on site diagnostic assay format can be useful for monitoring the content of antibiotics in breast milk during macrolide treatment to ensure safe breastfeeding of infants. (C) 2020 Elsevier B.V. All rights reserved.

INTRODUCTION:Aflatoxin B1 (AFB1) is a toxic low-molecular-weight secondary metabolite of Aspergillus flavus and A. parasiticus. AFB1 was classified as a Group I carcinogen by the World Health Organisation for Research on Cancer in 1993. AFB1 is an unavoidable natural contaminant of some herbal medicine, able to cause serious health issues for humans consuming the related medicine.OBJECTIVE:Therefore, this study aimed to develop an efficient fluorescence polarisation immunoassay (FPIA) and a rapid, low-cost, and easy-to-use membrane-based flow-through immunoassay (MBA) for determination of AFB1 in herbal medicine Origanum vulgare L., Rubus idaeus L., Urtica dioica L. and Sorbus aucuparia L.RESULTS:A cut-off level of the developed MBA was 0.8 ppb. Validation of the developed test was performed with blank and spiked samples. Using three naturally contaminated or three artificially spiked samples. The FPIA showed a linear working range of 8.6 to 64 ppb, and a half maximal inhibitory concentration (IC50 ) of 24 ppb.CONCLUSION:The results were in good correlation with the enzymelinked immunosorbent assay (ELISA) results (the IC50 0.1 ppb). Both the sample preparation and analysis are simple, cost-effective and easy to perform on-site in non-laboratory environments. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used as a confirmatory technique.

Improvements in bioanalytical technologies are driven by evolving threats and challenges to the global community. These challenges include emerging diseases, the need for substance screening and increasing environmental pollution. Here, we present a new bioanalytical concept that employs nanosized Metal-Organic Framework (MOF) particles as labels for antibody-based analytical methods, including enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay (LFIA). A strategy for covalently linking antibodies to form stable, colloidal Zeolitic Imidazolate Frameworks (ZIFs) conjugates is described. The proof of principle for their use in LFIA is presented. In particular, ZIF-antibody conjugates displayed excellent behaviour in dispersion and good mobility via lateral flow on a solid substrate while retaining full selectivity of the antibody. Moreover, protein aggregation of the antibodies was prevented, suggesting protection against degradation in an exoskeleton-like manner. Our study could inspire future work to address global bioanalytical and diagnostic challenges.

Bisphenol A (BPA) is a common industrial chemical that is widely used as a monomer or additive in the production of polycarbonate plastics and other polymeric materials. The primary way of exposure to BPA for most people is assumed to be a direct con-tamination of food and water through use of containers, bottles, and other plastic stor-age. BPA has been identified as an important endocrine disrupting compound (EDC). It can interfere with hormonal activities like biosynthesis, metabolism and elimination of natural blood-borne hormones. Moreover, BPA can originate environmental problems. A spectrum of methods have been developed with the aim of determining BPA in water. Immunoassays appear to comply with sensitivity, rapidity, and low cost requirements for effective monitoring. In this work a comparative analysis was conducted of the effect of temperature and the choice of buffers, as well as the substrate at various stages of an ELISA (enzyme-linked immunosorbent assay). The method has been optimized and has shown good repeatability and sensitivity. The results obtained from ELISA will be used as the basis for the development of a Lateral Flow ImmunoAssay (LFIA).