Search Results (5)

A regenerable immunoaffinity layer comprising covalently immobilized orientation-controlled antibodies was developed for use in a surface plasmon resonance (SPR) biosensor. For antibody orientation control, antibody-binding Z-domain-autodisplaying Escherichia coli (E. coli) cells and their outer membrane (OM) were utilized, and a disuccinimidyl crosslinker was employed for covalent antibody binding. To fabricate the regenerable immunoaffinity layer, capture antibodies were bound to autodisplayed Z-domains, and then treated with the crosslinker for chemical fixation to the Z-domains. Various crosslinkers, namely disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS) and poly (ethylene glycol)-ylated bis (sulfosuccinimidyl)suberate (BS(PEG)₅), were evaluated, and DSS at a concentration of 500 μM was confirmed to be optimal. The E. coli-cell-based regenerable HRP immunoassay was evaluated employing three sequential HRP treatment and regeneration steps. Then, the Oms of E. coli cells were isolated and layered on a microplate and regenerable OM-based HRP immunoassaying was evaluated. Five HRP immunoassays with four regeneration steps were found to be feasible. This regenerable, covalently immobilized, orientation-controlled OM-based immunoaffinity layer was applied to an SPR biosensor, which was capable of quantifying C-reactive protein (CRP). Five regeneration cycles were repeated using the demonstrated immunoaffinity layer with a signal difference of <10%. Full article

Contamination of foods by mycotoxins is linked to various health and economic implications. This study evaluated the incidence of mycotoxins in commercial and small-scale maize and evaluated potential health risks for consumers based on South African and international regulations. The sensitivity/specificity of HPLC over other analytical methods used was also ascertained. In total, 100 maize samples were analyzed using immuno-affinity column for extraction and clean-up, thin layer chromatography (TLC), HPLC, and enzyme linked immunosorbent assay (ELISA) for quantification. Results revealed that fumonisinB₁ was the most contaminant mycotoxin in both small-scale and commercial samples with incidence rates of 100% and 98.6%, respectively. Aflatoxins contamination occurred at incidences of 26.7% in small-scale and 25.0% in commercial samples. Furthermore, ochratoxin A had high incidence rates of 97.8% and 93.0% and ranged from 3.60–19.44 µg/kg and 1.60–9.89 µg/kg, respectively, in small-scale and commercial samples, while ZEA occurred in 50% and 55% of small-scale and commercial samples, respectively. Results demonstrate that maize, especially from small-scale farmers, may contribute to dietary exposure to mycotoxins. Farmers and consumers should be alerted to the dangers of mycotoxins contamination in maize with resultant health risks. Additionally, HPLC method was also found to be more specific for mycotoxin detection than ELISA. Full article

As a class of mycotoxins with regulatory and public health significance, aflatoxins (e.g., aflatoxin B₁, B₂, G₁ and G₂) have attracted unparalleled attention from government, academia and industry due to their chronic and acute toxicity. Aflatoxins are secondary metabolites of various Aspergillus species, which are ubiquitous in the environment and can grow on a variety of crops whereby accumulation is impacted by climate influences. Consumption of foods and feeds contaminated by aflatoxins are hazardous to human and animal health, hence the detection and quantification of aflatoxins in foods and feeds is a priority from the viewpoint of food safety. Since the first purification and identification of aflatoxins from feeds in the 1960s, there have been continuous efforts to develop sensitive and rapid methods for the determination of aflatoxins. This review aims to provide a comprehensive overview on advances in aflatoxins analysis and highlights the importance of sample pretreatments, homogenization and various cleanup strategies used in the determination of aflatoxins. The use of liquid-liquid extraction (LLE), supercritical fluid extraction (SFE), solid phase extraction (SPE) and immunoaffinity column clean-up (IAC) and dilute and shoot for enhancing extraction efficiency and clean-up are discussed. Furthermore, the analytical techniques such as gas chromatography (GC), liquid chromatography (LC), mass spectrometry (MS), capillary electrophoresis (CE) and thin-layer chromatography (TLC) are compared in terms of identification, quantitation and throughput. Lastly, with the emergence of new techniques, the review culminates with prospects of promising technologies for aflatoxin analysis in the foreseeable future. Full article

Through orientation control of antibodies, Z-domains autodisplaying Escherichia coli outer cell membrane (OM) may be utilized to improve the sensitivity and limit of detection (LOD) of immunoassays and immunosensors. A regenerative immunoaffinity layer based on Z-domains autodisplaying E. coli OM was developed for the surface plasmon resonance (SPR) biosensor. Regeneration conditions for the Z-domains autodisplaying E. coli OM-based immunoassays and immunosensors were optimized by varying pH and detergent concentration. An E. coli cell-based HRP immunoassay was tested and validated in three sequential regenerative immunoassays under optimal conditions. The OM of Z-domains autodisplaying E. coli was isolated and coated on the two-dimensional substrate (microplate). The OM-based HRP immunoassay was tested and validated in four regenerative immunoassays. This regenerative OM layer was applied to the SPR biosensor. Z-domains autodisplaying OM layered onto the gold surface of SPR biosensors was developed, and the OM-based regenerative immunoaffinity layer with orientation control was tested using CRP analyte. The SPR biosensor regenerative immunoaffinity layer demonstrated that CRP biosensing was repeated for five regeneration cycles with less than 2% signal difference. Therefore, the newly developed regenerative immunoaffinity layer with antibody orientation control may improve biosensing sensitivity and reduce the cost of medical diagnosis. Full article

To determine the hapten number in hapten-carrier protein conjugate matrix-assisted laser desorption/ionization (MALDI) tof mass spectrometry was applied. Highly specific anti-ginsenoside Rb₁ and Rg₁ monoclonal antibodies (MAbs) were prepared. Ginsenosides were developed on thin layer chromatography (TLC) plates which were covered by a polyvinylidene difluoride (PVDF) membrane resulting in blotting. The membrane was treated with NaIO₄ solution to release the aldehyde group on the sugar moiety of the ginsenosides. By treatment of the membrane with a protein solution the ginsenoside-protein conjugation as a Schiff-base occurred, which can function to fix it to the PVDF membrane. A part of the ginsenoside aglycone was reacted with anti-ginsenoside Rb₁ MAb, secondary MAb conjugated with enzyme and finally a substrate was added, resulting in a specific and highly sensitive staining that we named Eastern blotting. Furthermore, it makes one-step isolation of ginsenoside Rb₁ possible using an immuno-affinity column conjugated with anti-ginsenoside Rb₁ MAb. Furthermore, immunoaffinity concentration was carried out allowing high sensitivity analysis of lower concentrations of ginsenoside Rb₁ so that several unknown bands could be structurally determined. Full article