A comparative study of qualitative immunochemical screening assays for the combined measurement of T-2/HT-2 in cereals and cereal-based products

Many different immunochemical platforms exist for the screening of naturally occurring contaminants in food from the low cost enzyme linked immunosorbent assays (ELISA) to the expensive instruments such as optical biosensors based on the phenomenon of surface plasmon resonance (SPR). The primary aim of this study was to evaluate and compare a number of these platforms to assess their accuracy and precision when applied to naturally contaminated samples containing HT-2/T-2 mycotoxins. Other important factors considered were the speed of analysis, ease of use (sample preparation techniques and use of the equipment) and ultimately the cost implications. The three screening procedures compared included an SPR biosensor assay, a commercially available ELISA and an enzyme-linked immunomagnetic electrochemical array (ELIME array). The qualitative data for all methods demonstrated very good overall agreements with each other, however on comparison with mass spectrometry confirmatory results, the ELISA and SPR assay performed slightly better than the ELIME array, exhibiting an overall agreement of 95.8% compared to 91.7%. Currently, SPR is more costly than the other two platforms and can only be used in the laboratory whereas in theory both the ELISA and ELIME array are portable and can be used in the field, but ultimately this is dependent on the sample preparation techniques employed. Sample preparative techniques varied for all methods evaluated, the ELISA was the most simple to perform followed by that of the SPR method. The ELIME array involved an additional clean-up step thereby increasing both the time and cost of analysis. Therefore in the current format, field use would not be an option for the ELIME array. In relation to speed of analysis, the ELISA outperformed the other methods.

Development and validation of rapid disequilibrium enzyme-linked immunosorbent assays for the detection of Methyl Yellow and Rhodamine B dyes in foods

Sensitive and specific enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of two illegal synthetic dyes: Methyl Yellow (MY) and Rhodamine B (RB) in food. Polyclonal antibodies were raised against synthesised immunogens and employed in unique direct disequilibrium ELISAs. The time of the assays was only twenty minutes (five minutes for each incubation step with sample and enzyme conjugate and ten minutes with enzyme substrate). The IC50 for MY was in the range 1.4-4.2 ng mL(-1) and for RB 0.1-0.5 ng mL(-1). A simple sample preparation method was developed for the analysis of a range of sauces. In the case of spices a dispersive solid phase extraction was applied to purify the extracts. The testing of twenty samples took approximately one and a half hours (including sample preparation and analysis). Both assays were validated according to the Commission Decision 2002/657/EC criteria for use in sauces and spices. The detection capability for MY in sauces and spices was determined to be less than 15 ng g(-1) and 50 ng g(-1), respectively and for RB, 10 ng g(-1) for both types of food samples. The precision of the developed assays was determined in a repeatability study. The intra-and inter-assay coefficients of variation were less than 25% for both tests and matrix types. The simplicity and performance of both assays indicate that they will be very reliable screening methods for the detection of the illegal dyes MY and RB in a range of food products.

Development and validation of dry reagent time-resolved fluoroimmuno assays for zeranol and alpha-zearalenol to assist in distinguishing zeranol abuse from Fusarium spp. toxin contamination in bovine urine

Zeranol, an oestrogenic growth promoter in food animals, is banned within the European Union (EU). However, commercially available immunoassay kits for zeranol cross-react with toxins formed by naturally occurring Fusarium spp. fungi, leading to false-positive screening results. This paper describes the validation of a specificity enhanced, rapid dry reagent time-resolved fluoroimmunoassay (TR-FIA) for zeranol (recovery 99%, limit of detection 1.3 ng ml(-1)) demonstrating that up to 150 ng ml(-1) of Fusarium spp. toxins in urine do not lead to false-positive results. This assay will assist EU Member States to implement Council Directive 961 23\EC, which requires states to monitor for potential abuses of zeranol. A similar TR-FIA for the Fusarium spp. toxin a-zearalenol, using the same sample extract, is also described (recovery 68%, limit of detection 5.6 ng ml(-1)). Only the addition of diluted sample extract is required to perform these dry-reagent TR-FIAs, the results being available within 1 h of extract application. The EU-funded project 'Natural Zeranol' (FAIR5-CT97-3443) will use these fluoroimmunoassays to screen bovine urine in four Member States to gather data on the seasonality of Fusarium spp. toxin contamination of urine and the incidence of zeranol screening test positives.

Validation and application of reporter gene assays for the determination of estrogenic and androgenic endocrine disruptor activity in sport supplements.

Previously developed estrogen and androgen mammalian reporter gene assays (RGAs) were assessed for their potential use as a quantitative screening method in the detection of estrogenic and androgenic endocrine disruptors (EDs) in sport supplements. The validation of both RGAs coupled with dispersive solid phase extraction (dSPE) was performed in accordance with European Commission Decision EC/2002/6579 for biological screening methods. Decision limits (CCa) and detection capabilities (CCß) were established for both the estrogen and androgen RGAs. All samples were compliant with CCa and CCß in both bioassays. Recovery rates were 96 % for 17ß-estradiol and 115 % for dihydrotestosterone as obtained in their corresponding RGA. Both estrogens and androgens were stable in samples for more than 3 weeks, when stored at -20 °C. Specificity, good repeatability (coefficients of variation (CV), 12–25 %), reproducibility and robustness of both bioassays were also observed. Four different ED modes of action were determined for estrogens and androgens in 53 sport supplements, using the validated RGAs. This study revealed that 89 % of the investigated sport supplements contained estrogenic EDs and 51 % contained androgenic compounds. In conclusion, both bioassays are suitable for sport supplement screening of estrogenic and androgenic EDs.

A tripartite microbial reporter gene system for real-time assays of soil nutrient status

Plant-derived carbon is the substrate which drives the rate of microbial assimilation and turnover of nutrients, in particular N and P, within the rhizosphere. To develop a better understanding of rhizosphere dynamics, a tripartite reporter gene system has been developed. We used three lux-marked Pseudomonas fluorescens strains to report on soil (1) assimilable carbon, (2) N-status, and (3) P-status. In vivo studies using soil water, spiked with C, N and P to simulate rhizosphere conditions, showed that the tripartite reporter system can provide real-time assessment of carbon and nutrient status. Good quantitative agreement for bioluminescence output between reference material and soil water samples was found for the C and P reporters. With regard to soil nitrate, the minimum bioavailable concentration was found to be greater than that analytically detectable in soil water. This is the first time that bioavailable soil C, N and P have been quantified using a tripartite reporter gene system.

Detection of Protein-Protein Interactions Using Protein-Fragment Complementation Assays (PCA)

Protein-protein interactions play a central role in many cellular processes. Their characterisation is necessary in order to analyse these processes and for the functional identification of unknown proteins. Existing detection methods such as the yeast two-hybrid (Y2H) and tandem affinity purification (TAP) method provide a means to answer rapidly questions regarding protein-protein interactions, but have limitations which restrict their use to certain interaction networks; furthermore they provide little information regarding interaction localisation at the subcellular level. The development of protein-fragment complementation assays (PCA) employing a fluorescent reporter such as a member of the green fluorescent protein (GFP) family has led to a new method of interaction detection termed Bimolecular Fluorescent Complementation (BiFC). These assays have become important tools for understanding protein interactions and the development of whole genome interaction maps. BiFC assays have the advantages of very low background signal coupled with rapid detection of protein-protein interactions in vivo while also providing information regarding interaction compartmentalisation. Modified forms of the assay such as the use of combinations of spectral variants of GFP have allowed simultaneous visualisation of multiple competing interactions in vivo. Advantages and disadvantages of the method are discussed in the context of other fluorescence-based interaction monitoring techniques.

Establishing optimal quantitative-polymerase chain reaction assays for routine diagnosis and tracking of minimal residual disease in JAK2-V617F-associated myeloproliferative neoplasms:a joint European LeukemiaNet/MPN&MPNr-EuroNet (COST action BM0902) study

Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21,500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6-85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.

Comparative evaluation of 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8) rapid colorimetric assays for antimicrobial susceptibility testing of staphylococci and ESBL-producing clinical isolates

A new generation of water soluble tetrazolium salts have recently become available and in this study we compared a colorimetric assay developed using one of these salts, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8), with a previously developed 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) colorimetric assay to determine which agent is most suitable for use as a colorimetric indicator in susceptibility testing. The MICs of 6 antibiotics were determined for 33 staphylococci using both colorimetric assays and compared with those obtained using the British Society for Antimicrobial Chemotherapy reference broth microdilution method. Absolute categorical agreement between the reference and test methods ranged from 79% (cefuroxime) to 100% (vancomycin) for both assays. No minor or major errors occurred using either assay with very major errors ranging from zero (vancomycin) to seven (cefuroxime). Analysis of the distribution of differences in the log2 dilution MIC results revealed overall agreement, within the accuracy limits of the standard test (± 1 log2 dilution), using the XTT and WST-8 assays of 98% and 88%, respectively. Further studies on 31 ESBL-producing isolates were performed using the XTT method with absolute categorical agreement ranging from 87% (nitrofurantoin) to 100% (ofloxacin and meropenem). No errors were noted for either ofloxacin or meropenem with overall agreement of 91%. The data suggests that XTT is more reliable and accurate than WST-8 for use in a rapid antimicrobial susceptibility test.

Characterization of a reference material for BCR-ABL (M-BCR) mRNA quantitation by real-time amplification assays:towards new standards for gene expression measurements

Monitoring of BCR-ABL transcripts has become established practice in the management of chronic myeloid leukemia. However, nucleic acid amplification techniques are prone to variations which limit the reliability of real-time quantitative PCR (RQ-PCR) for clinical decision making, highlighting the need for standardization of assays and reporting of minimal residual disease (MRD) data. We evaluated a lyophilized preparation of a leukemic cell line (K562) as a potential quality control reagent. This was found to be relatively stable, yielding comparable respective levels of ABL, GUS and BCR-ABL transcripts as determined by RQ-PCR before and after accelerated degradation experiments as well as following 5 years storage at -20 degrees C. Vials of freeze-dried cells were sent at ambient temperature to 22 laboratories on four continents, with RQ-PCR analyses detecting BCR-ABL transcripts at levels comparable to those observed in primary patient samples. Our results suggest that freeze-dried cells can be used as quality control reagents with a range of analytical instrumentations and could enable the development of urgently needed international standards simulating clinically relevant levels of MRD.

Macroautophagic cargo sequestration assays

Macroautophagy, the process responsible for bulk sequestration and lysosomal degradation of cytoplasm, is often monitored by means of the autophagy-related marker protein LC3. This protein is linked to the phagophoric membrane by lipidation during the final steps of phagophore assembly, and it remains associated with autophagic organelles until it is degraded in the lysosomes. The transfer of LC3 from cytosol to membranes and organelles can be measured by immunoblotting or immunofluorescence microscopy, but these assays provide no information about functional macroautophagic activity, i.e., whether the phagophores are actually engaged in the sequestration of cytoplasmic cargo and enclosing this cargo into sealed autophagosomes. Moreover, accumulating evidence suggest that macroautophagy can proceed independently of LC3. There is therefore a need for alternative methods, preferably effective cargo sequestration assays, which can monitor actual macroautophagic activity. Here, we provide an overview of various approaches that have been used over the last four decades to measure macroautophagic sequestration activity in mammalian cells. Particular emphasis is given to the so-called "LDH sequestration assay", which measures the transfer of the autophagic cargo marker enzyme LDH (lactate dehydrogenase) from the cytosol to autophagic vacuoles. The LDH sequestration assay was originally developed to measure macroautophagic activity in primary rat hepatocytes. Subsequently, it has found use in several other cell types, and in this article we demonstrate a further validation and simplification of the method, and show that it is applicable to several cell lines that are commonly used to study autophagy.

Considerations for optimization of microRNA PCR assays for molecular diagnosis

The remarkable stability of microRNAs in biofluids underlies their potential as biomarkers, but their small size presents challenges for detection by RT-qPCR. The heterogeneity of microRNAs, with each one comprising a series of variants or 'isomiRs', adds additional complexity. Presented here are the key considerations for use of RT-qPCR to measure microRNAs and their isomiRs, with a focus on plasma. Modified nucleotides can be incorporated into primer sequences to enhance affinity and provide increased specificity and sensitivity for RT-qPCR assays. Approaches based upon polyA tailing and use of a common oligo(dT)-based reverse transcription oligonucleotide will detect most isomiRs. Conversely, stem-loop RT oligonucleotides and sequence specific probes can enable detection of specific isomiRs of interest. Next generation sequencing of all the products of a microRNA RT-PCR reaction is a promising new approach for both microRNA quantification and characterization.

A comparison of immunohistochemical assays and FISH in detecting the ALK translocation in diagnostic histological and cytological lung tumor material.

Introduction: Detection of the ALK rearrangement in a solid tumor gives these patients the option of crizotinib as an oral form of anticancer treatment. The current test of choice is fluorescence in situ hybridization (FISH), but various cheaper and more convenient immunohistochemical (IHC) assays have been proposed as alternatives. 
Methods: Fifteen FISH-positive cases from patients, seven with data on crizotinib therapy and clinical response, were evaluated for the presence of ALK protein using three different commercially available antibodies: D5F3, using the proprietary automated system (Ventana), ALK1 (Dako), and 5A4 (Abcam). A further 14 FISH-negative and three uncertain (<15% rearrangement detected) cases were also retrieved. Of the total 32 specimens, 17 were excisions and 15 were computed tomography-guided biopsies or cytological specimens. All three antibodies were applied to all cases. Antibodies were semiquantitatively scored on intensity, and the proportion of malignant cells stained was documented. Cutoffs were set by receiver operating curve analysis for positivity to optimize correct classification. 
Results: All three IHC assays were 100% specific but sensitivity did vary: D5F3 86%, ALK 79%, 5A4 71%. Intensity was the most discriminating measure overall, with a combination of proportion and intensity not improving the test. No FISH-negative IHC-positive cases were seen. Two FISH-positive cases were negative with all three IHC assays. One of these had been treated with crizotinib and had failed to show clinical response. The other harbored a second driving mutation in the EGFR gene.
Conclusions: IHC with all three antibodies is especially highly specific (100%) although variably sensitive (71%-86%), specifically in cases with scanty material. D5F3 assay was most sensitive in these latter cases. Occasional cases are IHC-positive but FISH-negative, suggesting either inaccuracy of one assay or occasional tumors with ALK rearrangement that do not express high levels of ALK protein.

The clinical significance of cathepsin S expression in human astrocytomas

Early local invasion by astrocytoma. cells results in tumor recurrence even after apparent total surgical resection, leading to the poor prognosis associated with malignant astrocytomas. Proteolytic enzymes have been implicated in facilitating tumor cell invasion and the current study was designed to characterize the expression of the cysteine proteinase cathepsin S (CatS) in astrocytomas and examine its potential role in invasion. Immunohistochemical analysis of biopsies demonstrated that CatS was expressed in astrocytoma cells but absent from normal astrocytes, oligodendrocytes, neurones and endothelial cells. Microglial cells and macrophages were also positive. Assays of specific activity in 59 astrocytoma biopsies confirmed CatS expression and in addition demonstrated that the highest levels of activity were expressed in grade IV tumors. CatS activity was also present in astrocytoma cells in vitro and the extracellular levels of activity were highest in cultures derived from grade IV tumors. In vitro invasion assays were carried out using the U251MG cell line and the invasion rate was reduced by up to 61% in the presence of the selective CatS inhibitor 4-Morpholineurea-LeuHomoPhe-vinylsulphone. We conclude that CatS expression is up-regulated in astrocytoma. cells and provide evidence for a potential role for CatS in invasion.

The conserved Helix C region in the superfamily of interferon-a/interleukin-10-related cytokines corresponds to a high-affinity binding site for the HSP70 chaperone DnaK.

HSP70 chaperones mediate protein folding by ATP-dependent interaction with short linear peptide segments that are exposed on unfolded proteins. The mode of action of the Escherichia coli homolog DnaK is representative of all HSP70 chaperones, including the endoplasmic reticulum variant BiP/GRP78. DnaK has been shown to be effective in assisting refolding of a wide variety of prokaryotic and eukaryotic proteins, including the -helical homodimeric secretory cytokine interferon- (IFN-). We screened solid-phase peptide libraries from human and mouse IFN- to identify DnaK-binding sites. Conserved DnaK-binding sites were identified in the N-terminal half of helix B and in the C-terminal half of helix C, both of which are located at the IFN- dimer interface. Soluble peptides derived from helices B and C bound DnaK with high affinity in competition assays. No DnaK-binding sites were found in the loops connecting the -helices. The helix C DnaK-binding site appears to be conserved in most members of the superfamily of interleukin (IL)-10-related cytokines that comprises, apart from IL-10 and IFN-, a series of recently discovered small secretory proteins, including IL-19, IL-20, IL-22/IL-TIF, IL-24/MDA-7 (melanoma differentiation-associated gene), IL-26/AK155, and a number of viral IL-10 homologs. These cytokines belong to a relatively small group of homodimeric proteins with highly interdigitated interfaces that exhibit the strongly hydrophobic character of the interior core of a single-chain folded domain. We propose that binding of DnaK to helix C in the superfamily of IL-10-related cytokines may constitute the hallmark of a novel conserved regulatory mechanism in which HSP70-like chaperones assist in the formation of a hydrophobic dimeric "folding" interface.