Multiplex immunoassay for persistent organic pollutants in tilapia: comparison of imaging- and flow cytometry-based platforms using spectrally encoded paramagnetic microspheres

Recent developments in spectrally encoded microspheres (SEMs)-based technologies provide high multiplexing possibilities. Most SEMs-based assays require a flow cytometer with sophisticated fluidics and optics. A new imaging super-paramagnetic SEMs-based alternative platform transports SEMs with considerably less fluid volume into a measuring chamber. Once there SEMs are held in a monolayer by a magnet. Light-emitting diodes (LEDs) are focused on the chamber to illuminate the SEMs – instead of lasers and they are imaged by a charge-coupled device (CCD) detector, offering a more compact sized, transportable and affordable system. The feasibility of utilising this system to develop a 3-plex SEMs-based imaging immunoassay (IMIA) for the screening of persistent organic pollutants (POPs) was studied. Moreover the performance characteristics of 3-plex IMIA were critically compared with the conventional 3-plex flow cytometric immunoassay (FCIA). Both SEM technologies have potential for the multiplex analysis of polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and polycyclic aromatic hydrocarbons (PAHs) in buffer and fish extract with insignificant differences in assay sensitivities. Furthermore, we developed a faster and simpler, modified QuEChERS-like generic POPs extraction from tilapia fillet using sodium hydrogen carbonate as one of the salt additives and dispersive solid-phase extraction (dSPE) as a clean-up. Finally, a preliminary in-house validation using 40 different blank and spiked tilapia fillet samples was performed in both systems and the results obtained were critically compared. The lower-cost imaging SEMs-based system performed similarly to the original flow cytometer and, in combination with the new quicker QuEChERS-like extraction, it has high potential for future rapid screening of POPs in several other sample matrices such as other fish species, vegetable refined oils and environmental samples

Isolation and characterization of shiga toxin-producing escherichia coli serogroups O26, O45, O103, O111, O113, O121, O145, and O157 shed from range and feedlot cattle from postweaning to slaughter

Cattle are the main reservoirs for Shiga toxin-producing Escherichia coli (STEC) strains. E. coli O26, O45, O103, O111, O121, O145, and O157 are among the STEC serogroups that cause severe foodborne illness and have been declared as adulterants by the U.S. Department of Agriculture, Food Safety and Inspection Service. The objectives of this study were (i) to estimate the prevalence of non-O157 STEC and E. coli O157 in naturally infected beef cows and in steer calves at postweaning, during finishing, and at slaughter and (ii) to test non-O157 STEC isolates for the presence of virulence genes stx1, stx2, eaeA, and ehlyA. Samples were collected from study animals during multiple sampling periods and included fecal grabs, rectal swabs, and midline sponge samples. Laboratory culture, PCR, and multiplex PCR were performed to recover and identify E. coli and the virulence genes. The prevalence of non-O157 STEC (serogroups O26, O45, O103, O111, O121, O113, and O145) fecal shedding ranged from 8% (4 of 48 samples) to 39% (15 of 38 samples) in cows and 2% (1 of 47 samples) to 38% (9 of 24 samples) in steer calves. The prevalence of E. coli O157 fecal shedding ranged from 0% (0 of 38 samples) to 52% (25 of 48 samples) in cows and 2% (1 of 47 samples) to 31% (15 of 48 samples) in steer calves. In steer calves, the prevalence of non-O157 STEC and E. coli O157 was highest at postweaning, at 16% (15 of 96 samples) and 23% (22 of 96 samples), respectively. Among the 208 non- O157 STEC isolates, 79% (164 isolates) had stx1, 79% (165 isolates) had stx2, and 58% (121 isolates) had both stx1 and stx2 genes. The percentage of non-O157 STEC isolates encoding the eaeA gene was low; of the 165 isolates tested, 8 (5%) were positive for eaeA and 135 (82%) were positive for ehlyA. Findings from this study provide further evidence of non-O157 STEC shedding in beef cows and steer calves particularly at the stage of postweaning and before entry into the feedlot

Multiplex Screening of Persistent Organic Pollutants in Fish Using Spectrally Encoded Microspheres

Persistent organic pollutants (POPs) are environmental and food-related contaminants of global public health concern and known to be carcinogenic and endocrine disruptors. Their monitoring is essential, and an easy-to-use, rapid, and affordable multianalyte screening method with simplified sample preparation can be a valuable tool prior to instrumental analysis. For this purpose, a flow cytometric immunoassay (FCIA), based on a spectrally encoded microbeads technology, was developed for the multiplex detection of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (BDEs) in buffer and fish extracts. The sensitivities of the assays in the three-plex FCIA format were similar to the individual FCIAs for the marker compounds benzo[a]pyrene (BaP), 3,3',4,4'-tetrachlorobiphenyl (PCB77), and 2,2',4,4'-tetrabromodiphenyl ether (BDE47) in buffer with IC50 values of 0.4, 20, and 2 µg L–1, respectively. Apart from the three markers, we could detect at least 14 other POPs. Extracts of fish with different fat content, prepared with a simplified extraction and cleanup procedure, had an insignificant influence on the overall three-plex FCIA performance, with the exception of some impact on the PAHs detection. The performance of the three-plex FCIA, in combination with the simple extraction procedure, is adequate for regulatory control in accordance with the required limits

Cloning, expression, and purification of an anti‐desipramine single chain antibody in NS/0 myeloma cells

Drug‐specific monoclonal antibodies and their antigen‐binding Fab fragments reverse acute desipramine toxicity in a rat experimental model by inducing a redistribution of drug from cardiac tissue into serum and extracellular fluid. In order to investigate the use of smaller recombinant antibody fragments such as single chain Fv (sFv) as an antidote, an efficient murine NS/0 myeloma expression system was developed. The variable light (V) and variable heavy (V) domains of a murine anti‐desipramine monoclonal antibody were cloned and sequenced. A 270 amino acid V‐(GlySer)‐V sFv was prepared by overlapping polymerase chain reaction (PCR) amplification of V with heavy chain leader peptide, V, and the linker. This construct was subcloned into a mammalian expression vector which utilizes the SRα promoter, a hybrid promoter consisting of the SV40 early promoter with portions of the human T‐cell leukemia virus type I long terminal repeat and also containing the Escherichia coli xanthine–guanine phosphoribo‐syltransferase gene for selection. NS/0 myeloma cells were transfected by electroporation. Stable recombinant NS/0 clones were screened for expression of sFv using reverse transcriptase‐PCR to detect mRNA and an enzyme‐linked immunosorbent assay (ELISA) to detect sFv. Secreted sFv from clones capable of growth to a cell density of 2–4 × 10 viable cells/mL was purified in a single step using a desipramine affinity column resulting in 12–39 mg/L of purified sFv. Affinity‐purified sFv had comparable desipramine binding activity to Fab when evaluated by competitive ELISA. Copyrigh

Electrocatalytic response of poly(cobalt tetraaminophthalocyanine)/multi-walled carbon nanotubes-Nafion modified electrode toward sulfadiazine in urine*

A highly sensitive amperometric sulfadiazine sensor fabricated by electrochemical deposition of poly(cobalt tetraaminophthalocyanine) (poly(CoIITAPc)) on the surface of a multi-walled carbon nanotubes-Nafion (MWCNTs-Nafion) modified electrode is described. This electrode showed a very attractive performance by combining the advantages of CoIITAPc, MWCNTs, and Nafion. Compared with the bare glassy carbon electrode (GCE) and the MWCNTs-Nafion modified electrode, the electrocatalytic activity of poly(CoIITAPc)-coated MWCNTs-Nafion GCE generated greatly improved electrochemical detections toward sulfadiazine including low oxidation potential, high current responses, and good anti-fouling performance. The oxidation peak currents of sulfadiazine obtained on the new modified electrode increased linearly while increasing the concentration of sulfadiazine from 0.5 to 43.5 μmol/L with the detection limit of 0.17 μmol/L