Flow cytometry based rapid duplexed immunoassay for fusarium mycotoxins.

At small food processing facilities, the most frequently used test to determine if grain-derived mycotoxin concentrations are compliant with legal limits is the enzyme-linked immunosorbent assay (ELISA). Each kit is designed to detect one of the six dangerous mycotoxins. With the increasing occurrence of coinfection of grain with multiple-mycotoxins in the field and/or during storage, ELISA is no longer a cost effective best assay option. With ELISA, each species of mycotoxin requires different sample preparation/extraction and a 45 min incubation. The alternative multiplexed assay presented here, the competitive fluorescent microsphere immunoassay (CFIA), follows current food safety standards. It handles several toxins simultaneously with a single universal extraction protocol. The authors' objective was to modify an existing commercial CFIA kit developed for bench top flow cytometry and extend its utility for point-of-need (PON) applications. The accelerated protocol offers over 60% reduction in total processing time and it detects dual mycotoxin contamination simultaneously. The observed enhanced binding kinetics equations reported here utilizing suspended solid phase particles in liquid phase, are also supported by published theoretical calculations. In the near future portable cytometry may bring rapid multiplexed PON testing to assure the safety of small food processing installations. © 2016 International Society for Advancement of Cytometry.

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.

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. CFIA's 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.

Sandwich type ELISA and a fluorescent cytometric microbead assay for quantitative determination of hepatitis B virus X antigen level in human sera.

The hepatitis B virus X protein (HBxAg) is responsible for severe complications of HBV infections including primary hepatocellular carcinoma. A sandwich type ELISA and a flow cytometric microbead assay for quantitative determination of serum levels of Hbx-Ag are introduced. We have previously developed monoclonal antibody families against well-conserved epitopes on HbxAg, characterized by different immunohistochemical and immunoserological techniques. Special selection of the antibody pairs provided highly sensitive and highly specific tools for quantitative immunoassay development. The resulting assays were tested on human sera (208 samples) collected from patients suffering from different clinical forms of HBV infection. The sensitivity range of the sandwich type ELISA was between 4 and 2000 ng/ml as measured on both the recombinant antigen and the sera of chronic hepatitis patients. A further flow cytometric microbead assay was established and tested in parallel with the ELISA. The quantitative results of these two immunoserological techniques were in strong correlation and they were found to be highly specific and sensitive on clinical samples. The HBxAg ELISA technique is applicable for routine clinical laboratory measurements, and our HBxAg microbead technique is recommended for complex multiparametric measurements combined with other markers.

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

BACKGROUND: 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. METHODS: Formaldehyde-fixed yeast and bacterial cells were conjugated with avidin and applied as microbeads, to establish a simple, convenient, flexible, and inexpensive flow-cytometric platform for various immunological and biochemical assays. RESULTS: We have tested these "biological microbeads" for the simultaneous titration of human alpha-fetoprotein (AFP) and human Chorionic Gonadotropin (betahCG) hormone levels, for the titration of proteolytic and nucleolytic (restriction) enzymes, and for quantitative PCR, using biotinylated and fluorescent primers. CONCLUSIONS: The use of biological microbeads for various immunological and biochemical assays has been demonstrated. The flow-cytometric methods proved to be at least as sensitive as the standard biochemical or immunological tests. For proteinase K activity measurements, a single enzyme molecule in the sample could be detected. The sensitivity, versatility, and low cost of the assays may advance flow-cytometry to become a central methodological platform in most laboratories. The biological microbeads offer virtually unlimited possibilities for fluorescent labeling (addressing), conjugation of ligand binding molecules, and they are easy to handle and perform well in a multiplex format.

Distinct groups of multidrug resistance modulating agents are distinguished by competition of P-glycoprotein-specific antibodies.

P-glycoprotein (Pgp) is one of the ABC transporters responsible for the multidrug resistance of cancer cells. The conformational changes of Pgp that occur in the presence of substrates/modulators or ATP depletion are accompanied by the up-shift of UIC2 monoclonal antibody (mAb) binding. In the case of cyclosporin A, vinblastine or valinomycin, this up-shift was found to be concomitant with the near-complete suppression of labeling with other mAbs specific for Pgp epitopes overlapping with UIC2, while pre-treatment with verapamil or Tween 80 brings about a modest suppression. Here we have extended these observations to 44 Pgp interacting agents, and found that only 8 fall into the cyclosporin-like category, inducing a conformational state characterized by the complete UIC2 dominance. The rest of the drugs either did not affect antibody competition or had a modest effect. Thus, Pgp substrates/modulators can be classified into distinct modalities based on the conformational change they elicit.