György Lustyik

A flow cytometry based competitive fluorescent microsphere immunoassay (CFIA) system for detecting up to six mycotoxins.

BACKGROUND Exposure to multiple mycotoxins through the food chain represents a major potential health hazard to both humans and livestock. They can cause a variety of severe acute as well as chronic diseases. Eliminating mycotoxins from various grain crops is a global health priority. According to the Food and Agriculture Organization (FAO), world food production needs to double by 2050. Innovative solutions will be required to sustain toxin free grain supplies worldwide. METHODS A competitive flow cytometry based multiplexed assay with fluorescent microspheres has been developed. The new multiplexed method can analyze simultaneously any one or all six major mycotoxins. They include: Ochratoxin A (OTA), Aflatoxin B1 (AFB1), Fumonisin B1 (FB1), T-2 toxin (T-2), Deoxynivalenol (DON) and Zearalenone (ZEA), which are all potential human health hazards. The CFIA described here includes a simplified single extraction step for mycotoxins from specimens and a comprehensive post acquisition software module. The new assay system was developed with a FACSArray™ BD Bioanalyzer flow cytometer (BD Biosciences, Belgium). RESULTS The CFIA performs favourably when compared to commercial ELISA. Sensitivity range with CFIA increased between 13% and 100% with an average improvement of 50% for the six mycotoxins. CONCLUSIONS The multiplexed assay presented here has the unique capacity to quantify up to six mycotoxins simultaneously from a single specimen extraction. CFIAs poly-mycotoxin detection sensitivity exceeds standard ELISA. CFIA may be part of a comprehensive assay system that will provide reliable and effective safeguard for agricultural commodities to be free of mycotoxins.

Biological microbeads for flow-cytometric immunoassays, enzyme titrations, and quantitative PCR

Introduction of microbeads into flow‐cytometry has created a new scenario, making quantitative measurement of molecules dispersed in a homogeneous phase, with an extremely wide realm of already realized and potential applications possible. Development of this field has lead to specialized instrumentation and microbead arrays, dedicated to certain applications.

Methionine requirement of sporulation in a Streptomyces fradiae mutant

SUMMARY: When an Arg- mutant of Streptomyces fradiae was grown on sucrose-nitrate agar supplemented with L-citrulline, it produced only non-sporulating aerial mycelia. Sporulation could be induced by adding 0.1 to 10.0 mM-L-methionine or 0.01 mM-cyanocobalamin; several other compounds including homocysteine, other amino acids, vitamins and precursors of nucleic acids were ineffective. Growth of the mutant did not differ significantly with or without methionine. The mutant was more sensitive to L-norleucine (a methionine analogue) than the parent strain. Spontaneous mutants, resistant to this analogue, were obtained. They all sporulated well without exogenously added methionine. In medium-exchange experiments, methionine was required for sporulation only after 48 h cultivation. The phenomenon reported here is most probably based on partially impaired methionine biosynthesis caused by an as yet uncharacterized non-auxotrophic mutation, since Arg+ revertants retained their methionine requirement for sporulation, and were also sensitive to L-norleucine.

Characterization of intracellular calcium oscillations induced by extracellular nucleotides in HEp-2 cells

The effect of extracellular nucleotides on the cytosolic calcium concentration of fluo-3-loaded HEp-2 cells was examined using confocal microscopy. Extracellular ATP and UTP at micromolar concentration induced cytosolic calcium oscillations in 42-66% of the cells. Oscillations were usually sinusoid and their frequency depended only slightly on agonist concentration. Oscillations developed in calcium-free medium but were diminished by depletion of intracellular calcium stores with thapsigargin, indicating periodic calcium release from internal stores. Inhibition of phospholipase C with U73122 prevented the development of oscillations, while ryanodine did not abolish the response to extracellular nucleotides. Activation of protein kinase C with 4beta-phorbol 12-myristate 13-acetate also prevented the development of oscillations. These results indicate that extracellular nucleotides induce periodic calcium release from inositol 1,4,5-trisphosphate-sensitive pools in HEp-2 cells and that the inhibitory effect of protein kinase C on the phosphatidylinositol signaling pathway can contribute to the development of intracellular calcium oscillations.

Comparison and evaluation of seven different bench-top flow cytometers with a modified six-plexed mycotoxin kit.

Many bench‐top flow cytometers (b‐FCs) are compatible with microsphere‐based multiplexed assays. Disciplines implementing b‐FCs–based assays are expanding; they include monitoring and validating food quality. A multiplexed platform protocol was evaluated for poly‐mycotoxin assays, which is compatible with a variety of b‐FC models. The seven instruments included: BD FACSCalibur™, BD FACSArray™ Bioanalyzer, Accuri C6, Partec CyFlow® Space, Beckman Coulter FC 500, Guava EasyCyte Mini, and Luminex 100 ™. Current reports related to the food industry describe fungal co‐infections leading to poly‐mycotoxin contamination in grain (Sulyok M, Berthiller F, Krska R, Schuhmacher R, Rapid Commun Mass Spectrom 2006;20:2649–2659). It is imperative to determine whether b‐FC–based assays can replace traditional single‐mycotoxin enzyme‐linked immunosorbent assay (ELISA). A six‐plexed poly‐mycotoxin kit was tested on seven different b‐FCs. The modified kit was initially developed for the BD FACSArray™ Bioanalyzer (BD Biosciences) (Czeh A, Mandy F, Feher‐Toth S, Torok L, Mike Z, Koszegi B, Lustyik G, J Immunol Methods 2012;384:71–80). With the multiplexed platform, it is possible to identify up to six mycotoxin contaminants simultaneously at regional grain collection/transfer/inspection facilities. In the future, elimination of contaminated food threat may be better achieved with the inclusion of b‐FCs in the food protection arsenal. A universal protocol, matched with postacquisition software, offers an effective alternative platform compared to using a series of ELISA kits. To support side‐by‐side evaluation of seven flow cytometers, an instrument‐independent fluorescence emission calibration was added to the protocol. All instrument performances were evaluated for strength of agreement based on paired sets of evaluation to predicate method. The results suggest that all b‐FCs were acceptable of performing with the multiplexed kit for five of six mycotoxins. For OTA, the detection sensitivity was consistent only for five of the seven instruments.

Photobleaching Measurements of Diffusion in Cell Membranes and Aqueous Cell Compartments

This commentary unit discusses in great detail the theoretical nature of fluorescence recovery after photobleaching (FRAP). This information is crucial to an understanding of how and why FRAP works in a cell system. Further, understanding how to interpret the data sets requires a sound knowledge of the processes involved. Of primary importance are the nature of membrane diffusion and the nature of the multiple compartments into which fluorescent dyes can enter. The unit provides a complete discussion of all aspects of FRAP from the perspective of cellular measurements.