Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 164
Filter
1.
J Agric Food Chem ; 69(45): 13331-13338, 2021 Nov 17.
Article in English | MEDLINE | ID: mdl-34714660

ABSTRACT

The xMAP food allergen detection assay (xMAP FADA) is an advanced multiplex immunoassay with multiple antibodies for each of 15 target food allergens and gluten, allowing for signal confirmation and antigenic profiling to occur in a single analysis. Botanicals used as spices are complex matrices for allergen analysis because they can exhibit inherent cross-reactivity with antibodies employed by the assays. Preliminary examinations of botanicals revealed chili peppers to have notably high levels of cross-reactivity with Brazil nut and hazelnut antibody bead sets in the xMAP FADA. This in-depth investigation of 29 pre-ground and whole chili peppers indicated Brazil nut and hazelnut cross-reactivity to be consistent among most members of genusCapsicum, although cross-reactive signals generated by chili peppers were distinguishable from signals indicative of target allergen detection. Using the requirements that complementary antibodies used in the assay generated positive responses and that the various secondary end points were characteristic of the target analytes, xMAP FADA reactivity to chilis of the genus Capsicum was categorized as cross-reactivity instead of confirmed detection of target allergenic foods.


Subject(s)
Capsicum , Food Hypersensitivity , Allergens , Antibodies , Cross Reactions
2.
J Agric Food Chem ; 69(24): 6860-6869, 2021 Jun 23.
Article in English | MEDLINE | ID: mdl-34125514

ABSTRACT

The xMAP Food Allergen Detection Assay (xMAP FADA) is a powerful analytical method by virtue of its ability to simultaneously detect multiple antigenic elements with a repertoire of antibodies targeting 15 food allergens plus gluten. Further, by incorporating multiple levels of redundancy, it can also be used to distinguish between homologous cross-reactive analytes. The power of its analytical capabilities is especially critical when working with botanicals. In this research, 95 botanicals used in dietary supplements and spices were analyzed for cross-reactivity with common food allergens and gluten using the xMAP FADA. Complementary antibody ratios were calculated, and, with most samples, ratios generated by homologous cross-reactive epitopes were easily distinguished from true reactivity. In very few cases, sample ratios were comparable to the ratios generated by the calibration standards, indicating the probable detection of relatively minor quantities of target food allergen. With the xMAP FADA, distinguishing signal indicating target allergen detection from cross-reactivity in botanicals is possible using redundant antibodies and multiple confirmatory end points.


Subject(s)
Allergens , Food Hypersensitivity , Cross Reactions , Dietary Supplements , Humans , Spices
3.
J Agric Food Chem ; 69(27): 7742-7754, 2021 Jul 14.
Article in English | MEDLINE | ID: mdl-34184885

ABSTRACT

The protein/peptide profiles of gluten during yogurt fermentation were evaluated using an optimized multiplex-competitive ELISA by preparing yogurts incurred with gluten at different concentrations and by varying certain fermentation conditions. Analysis indicated that epitope-specific responses with antibody binding to glutenin epitopes decreased less during longer fermentation times or at higher starter culture concentrations relative to gliadins. Incomplete proteolysis was observed after 24 h of fermentation, which became more efficient as fermentation time was increased. Western blot confirmed the results of ELISA. Cluster analysis indicated that out of the investigated parameters, fermentation time is the only parameter that could affect the overall gluten protein/peptide profiles during yogurt fermentation. This parameter needs consideration in evaluating the suitability of calibrant(s) to be used with the multiplex-competitive ELISA or any other methods to ensure accurate quantitation of gluten in yogurts and potentially in other foods with similar fermentation chemistry. A small-scale multilaboratory evaluation indicated that the multiplex-competitive ELISA has good analytical reproducibility (average interlaboratory % CV of 28-41%).


Subject(s)
Glutens , Yogurt , Enzyme-Linked Immunosorbent Assay , Fermentation , Glutens/metabolism , Hydrolysis , Reproducibility of Results
4.
PLoS One ; 15(7): e0234899, 2020.
Article in English | MEDLINE | ID: mdl-32645020

ABSTRACT

The increasing prevalence of individuals with multiple food allergies and the need to distinguish between foods containing homologous, cross-reactive proteins have made the use of single-analyte antibody-based methods (e.g., ELISAs) sometimes insufficient. These issues have resulted in the need to conduct multiple analyses and sometimes employ orthogonal methods like mass spectrometry or DNA-based methods for confirmatory purposes. The xMAP Food Allergen Detection Assay (xMAP FADA) was developed to solve this problem while also providing increased throughput and a modular design suitable for adapting to changes in analytical needs. The use of built-in redundancy provides the xMAP FADA with built-in confirmatory analytical capability by including complementary antibody bead sets and secondary analytical end points (e.g., ratio analysis and multi-antibody profiling). A measure of a method's utility is its performance when employed by analysts of varying expertise in multiple laboratory environments. To gauge this aspect, a multi-laboratory validation (MLV) was conducted with 11 participants of different levels of proficiency. The MLV entailed the analysis of incurred food samples in four problematic food matrices, meat sausage, orange juice, baked muffins, and dark chocolate. Except for a couple of instances, involving two confirmatory components in the analysis of baked muffins, the allergenic foods were detected by all participants at concentrations in the analytical samples comparable to ≤ 10 µg/g in the original food sample. In addition, despite high levels of inter-lab variance in the absolute intensities of the responses, the intra-laboratory reproducibility was sufficient to support analyses based on the calibration standards and direct comparison controls (DCCs) analyzed alongside the samples. In contrast, ratio analyses displayed inter-laboratory %CV (RSDR) values < 20%; presumably because the ratios are based on inherent properties of the antigenic elements. The excellent performance of the xMAP FADA when performed by analysts of varying proficiency indicates a reliability sufficient to meet analytical needs.


Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Food Hypersensitivity/diagnosis , Food Hypersensitivity/immunology , Allergens/immunology , Antibodies/immunology , Biological Assay , Cross Reactions , Food Analysis/methods , Humans , Laboratories , Mass Spectrometry , Reproducibility of Results
5.
J Food Prot ; 83(6): 1050-1056, 2020 Jun 01.
Article in English | MEDLINE | ID: mdl-32050018

ABSTRACT

ABSTRACT: The xMAP food allergen detection assay (xMAP FADA) can simultaneously detect 15 analytes (14 food allergens plus gluten) in one analysis. The xMAP FADA typically employs two antibody bead sets per analyte, providing built-in confirmation that is not available with other antibody-based assays. Before an analytical method can be used, its reliability must be assessed when conditions of the assay procedure are altered. This study was conducted to determine the effects on assay performance associated with changes in incubation temperature, amounts of the antibody bead cocktail, and concentrations of detection antibody and ß-mercaptoethanol in the reduced-denatured extraction buffer. The analysis of buffered-detergent extracts revealed lower responses at 22°C than at 37°C, but temperature had no effect on the analysis of reduced-denatured extracts. Changes in ß-mercaptoethanol and detection antibody concentrations had an effect on the detection of only milk in the reduced-denatured extracts. A slight change in the measured bead count was observed when one-fourth of the bead cocktail was used, and a large decrease in the bead count was noted when one-eighth of the recommended amount was used, but this number (≥25) was still sufficient to provide reliable results. Overall, the xMAP FADA was very robust to changes in the assay procedure, which may inadvertently occur.


Subject(s)
Food Hypersensitivity , Allergens , Animals , Antibodies , Biological Assay , Enzyme-Linked Immunosorbent Assay , Food Hypersensitivity/diagnosis , Reproducibility of Results
6.
J Food Prot ; 83(1): 129-135, 2020 Jan.
Article in English | MEDLINE | ID: mdl-31855617

ABSTRACT

An estimated 0.1 to 0.2% of the North American population is allergic to sesame, and deaths due to anaphylactic shock have been reported. Detecting and quantifying sesame in various food samples is critical to safeguard the allergic population by ensuring accurate ingredient labeling. Because of the modular nature of the xMAP Food Allergen Detection Assay (FADA), it was possible through method extension to add sesame as a validated additional analyte. Because raw and toasted sesame are both commonly used and the two display significantly different antigenicity, three antibodies, one monoclonal and two polyclonal, were conjugated to bead sets to ensure reliable detection. The modified xMAP FADA successfully detected sesame incurred or spiked in baked muffins, spice mix, canola oil, and in both raw and toasted sesame oils with limit of quantitation values ≤ 1.3 ppm of sesame. Canola oil, sesame oil, toasted sesame oil, and olive oil inhibited sesame detection, as did the detection of sesame incurred in foods containing oil (e.g., hummus). Despite this inhibition, the xMAP FADA was still able to reliably detect sesame at levels throughout the dynamic range of the assay (22 to 750 ng of protein per mL) in all the foods examined. Further, the high signal-to-noise ratio of the lowest calibration standard and preliminary studies conjugating the antibodies at higher concentrations indicate an ability to increase the sensitivity of the assay should the need arise.


Subject(s)
Allergens/analysis , Food Hypersensitivity , Immunoassay/methods , Sesamum/chemistry , Antibodies , Humans
7.
J Agric Food Chem ; 67(1): 484-498, 2019 Jan 09.
Article in English | MEDLINE | ID: mdl-30484638

ABSTRACT

An xMAP Food Allergen Detection Assay (xMAP FADA) was developed to meet analytical needs when responding to complaints by individuals with multiple food allergies and to address potential ambiguities associated with cross-reactive proteins. A single-laboratory validation (SLV) was conducted to examine the reliability of the xMAP FADA to detect 15 analytes individually or as part of a mixture at more than six concentrations in four foods. The xMAP FADA reliably detected the analytes despite the incurred dark chocolate and incurred baked muffins displaying recoveries of 10-20% and <60%, respectively. The high reliability for recoveries less than 60% in part reflects the statistical strength of the design of the xMAP FADA. Only crustacean, egg, and milk incurred in dark chocolate were not reliably detected using the PBST-buffered-detergent protocol. Following the reduced-denatured protocol, no problems were encountered in the detection of milk, although egg did not display a dynamic response in dark chocolate. The ruggedness of the xMAP FADA was ascertained by the ability of novice analysts to detect food allergens in baked rice cookies. Despite one analyst losing >80% of the beads and the count for one bead set dropping to seven, the assay displayed only a decrease in precision (increased standard deviations) and a change in the ratios between complementary antibody pairs.


Subject(s)
Allergens/analysis , Food Analysis/methods , Animals , Antibodies/immunology , Arachis/chemistry , Arachis/immunology , Cattle , Chocolate/analysis , Cross Reactions , Eggs/analysis , Food Hypersensitivity/immunology , Fruit and Vegetable Juices/analysis , Humans , Milk/chemistry , Milk/immunology , Reproducibility of Results
8.
Anal Bioanal Chem ; 410(23): 5791-5806, 2018 Sep.
Article in English | MEDLINE | ID: mdl-29916095

ABSTRACT

Food allergies affect some 15 million Americans. The only treatment for food allergies is a strict avoidance diet. To help ensure the reliability of food labels, analytical methods are employed; the most common being enzyme-linked immunosorbent assays (ELISAs). However, the commonly employed ELISAs are single analyte-specific and cannot distinguish between false positives due to cross-reactive homologous proteins; making the method of questionable utility for regulatory purposes when analyzing for unknown or multiple food allergens. Also, should the need arise to detect additional analytes, extensive research must be undertaken to develop new ELISAs. To address these and other limitations, a multiplex immunoassay, the xMAP® food allergen detection assay (xMAP FADA), was developed using 30 different antibodies against 14 different food allergens plus gluten. Besides incorporating two antibodies for the detection of most analytes, the xMAP FADA also relies on two different extraction protocols; providing multiple confirmatory end-points. Using the xMAP FADA, the cross-reactivities of 45 botanicals used in dietary supplements and spices commercially sold in the USA were assessed. Only a few displayed cross-reactivities with the antibodies in the xMAP FADA at levels exceeding 0.0001%. The utility of the xMAP FADA was exemplified by its ability to detect and distinguish between betel nut, saw palmetto, and acai which are in the same family as coconut. Other botanicals examined included allspice, amchur, anise seed, black pepper, caraway seed, cardamom, cayenne red pepper, sesame seed, poppy seed, white pepper, and wheat grass. The combination of direct antibody detection, multi-antibody profiling, high sensitivity, and a modular design made it possible for the xMAP FADA to distinguish between homologous antigens, provide multiple levels of built-in confirmatory analysis, and optimize the bead set cocktail to address specific needs.


Subject(s)
Allergens/analysis , Dietary Supplements/analysis , Food Hypersensitivity/etiology , Immunoassay/methods , Plants/chemistry , Spices/analysis , Allergens/immunology , Antibodies/chemistry , Antibodies/immunology , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Food Hypersensitivity/immunology , Humans , Plants/immunology , Reproducibility of Results
9.
J Food Prot ; 81(5): 700-704, 2018 05.
Article in English | MEDLINE | ID: mdl-29607667

ABSTRACT

A problem often encountered in the detection and identification of undeclared tree nut food allergens is the lack of analytical methods. This problem is accentuated by the current trend, whereby the primary methods used to detect food allergens are antibody-based enzyme-linked immunosorbent assays (ELISAs) and the development of analyte-specific antibodies takes months. The recently developed xMAP food allergen detection assay (xMAP FADA) has the ability to generate multiantigen profiles with tree nuts, thereby providing a potential solution to this problem. The xMAP FADA includes 22 antibodies targeting peanut, soy, and nine tree nuts. The high number of antibodies to a diverse group of tree nuts and legumes and the propensity of tree nuts to cross-react have enabled the development of multiantigen profiling, whereby an analyte reacts with the various antibodies to generate a profile. Recently, a question arose regarding the possible presence of pecan dust at a manufacturer of pecan products that also stored fresh produce. The lack of suitable pecan ELISAs created an analytical challenge that was resolved using multiantigen profiling with the xMAP FADA. Pecan was detected on swab samples by using multiantigen profiling and confirmed by DNA analysis. The use of multiantigen profiling provided an analytical capability beyond what was possible with an analyte-specific analytical method.


Subject(s)
Allergens , Antibodies/immunology , Carya , Allergens/analysis , Allergens/immunology , Arachis , Carya/immunology , Humans , Nut Hypersensitivity/diagnosis , Nuts
10.
Anal Bioanal Chem ; 409(25): 5999-6014, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28801713

ABSTRACT

The homology between proteins in legumes and tree nuts makes it common for individuals with food allergies to be allergic to multiple legumes and tree nuts. This propensity for allergenic and antigenic cross-reactivity means that commonly employed commercial immunodiagnostic assays (e.g., dipsticks) for the detection of food allergens may not always accurately detect, identify, and quantitate legumes and tree nuts unless additional orthogonal analytical methods or secondary measures of analysis are employed. The xMAP® Multiplex Food Allergen Detection Assay (FADA) was used to determine the cross-reactivity patterns and the utility of multi-antibody antigenic profiling to distinguish between legumes and tree nuts. Pure legumes and tree nuts extracted using buffered detergent displayed a high level of cross-reactivity that decreased upon dilution or by using a buffer (UD buffer) designed to increase the stringency of binding conditions and reduce the occurrence of false positives due to plant-derived lectins. Testing for unexpected food allergens or the screening for multiple food allergens often involves not knowing the identity of the allergen present, its concentration, or the degree of modification during processing. As such, the analytical response measured may represent multiple antigens of varying antigenicity (cross-reactivity). This problem of multiple potential analytes is usually unresolved and the focus becomes the primary analyte, the antigen the antibody was raised against, or quantitative interpretation of the content of the analytical sample problematic. The alternative solution offered here to this problem is the use of an antigenic profile as generated by the xMAP FADA using multiple antibodies (bead sets). By comparing the antigenic profile to standards, the allergen may be identified along with an estimate of the concentration present. Cluster analysis of the xMAP FADA data was also performed and agreed with the known phylogeny of the legumes and tree nuts being analyzed. Graphical abstract The use of cluster analysis to compare the multi-antigen profiles of food allergens.


Subject(s)
Allergens/analysis , Allergens/immunology , Fabaceae/immunology , Immunoassay/methods , Nuts/immunology , Antibodies/immunology , Cluster Analysis , Cross Reactions , Fabaceae/chemistry , Food Hypersensitivity/diagnosis , Food Hypersensitivity/immunology , Humans , Nuts/chemistry , Reagent Strips/analysis
12.
J Agric Food Chem ; 64(5): 1202-11, 2016 Feb 10.
Article in English | MEDLINE | ID: mdl-26769163

ABSTRACT

Beginning in the autumn of 2014, millions of dollars of food and over 675 products were recalled in the United States due to the presence of undeclared peanut, attributed to cumin used in the manufacture of the products. Initial analyses also indicated the presence of almond. Subsequent research showed that the presence of peanut and almond did not fully explain the analytical results for the cumin samples. Using a combination of mass spectrometry, DNA-based methods (i.e., PCR and Sanger DNA Sequencing), microscopy, and antibody-based technologies (i.e., ELISA, Western blot analysis, and a novel xMAP multiplex assay) the presence of peanut was confirmed. Screening for secondary sources of adulteration (e.g., tree nuts, mahleb, peach, and cherry) supported the assessment that the cumin contained multiple contaminants. These results demonstrate the limitations of single analyte-specific assays and the need for orthogonal multiplex methods to detect food allergens irrespective of varietal or other differences.


Subject(s)
Cuminum/chemistry , Food Analysis/methods , Food Contamination/analysis , Antigens, Plant/analysis , Antigens, Plant/genetics , Cuminum/genetics , Food Hypersensitivity/immunology , Humans
13.
J Agric Food Chem ; 63(48): 10525-35, 2015 Dec 09.
Article in English | MEDLINE | ID: mdl-26548701

ABSTRACT

The effectiveness of a proline endopeptidase (PEP) in hydrolyzing gluten and its putative immunopathogenic sequences was examined using antibody-based methods and mass spectrometry (MS). Based on the results of the antibody-based methods, fermentation of wheat gluten containing sorghum beer resulted in a reduction in the detectable gluten concentration. The addition of PEP further reduced the gluten concentration. Only one sandwich ELISA was able to detect the apparent low levels of gluten present in the beers. A competitive ELISA using a pepsin-trypsin hydrolysate calibrant was unreliable because the peptide profiles of the beers were inconsistent with that of the hydrolysate calibrant. Analysis by MS indicated that PEP enhanced the loss of a fragment of an immunopathogenic 33-mer peptide in the beer. However, Western blot results indicated partial resistance of the high molecular weight (HMW) glutenins to the action of PEP, questioning the ability of PEP in digesting all immunopathogenic sequences present in gluten.


Subject(s)
Beer/analysis , Enzyme-Linked Immunosorbent Assay/methods , Food Contamination/analysis , Glutens/analysis , Mass Spectrometry/methods , Sorghum/chemistry , Antibodies/analysis , Beer/microbiology , Enzyme-Linked Immunosorbent Assay/instrumentation , Fermentation , Glutens/metabolism , Prolyl Oligopeptidases , Serine Endopeptidases/analysis , Sorghum/microbiology , Triticum/chemistry , Triticum/microbiology
14.
J Food Prot ; 78(6): 1167-77, 2015 Jun.
Article in English | MEDLINE | ID: mdl-26038908

ABSTRACT

In 2013 the U.S. Food and Drug Administration (FDA) defined the term ''gluten-free'' and identified a gap in the analytical methodology for detection and quantification of gluten in foods subjected to fermentation and hydrolysis. To ascertain the ability of current enzyme-linked immunosorbent assays (ELISAs) to detect and quantify gluten in fermented and hydrolyzed products, sorghum beer was spiked in the initial phases of production with 0, 20, and 200 µg/ml wheat gluten, and samples were collected throughout the beer production process. The samples were analyzed using five sandwich ELISAs and two competitive ELISAs and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Western analysis employing four antibodies (MIoBS, R5, G12, and Skerritt). The sensitivity of the MIoBS ELISA (0.25 ppm) enabled the reliable detection of gluten throughout the manufacturing process, including fermentation, when the initial concentration of 20 µg/ml dropped to 2 µg/ml. The R5 antibody-based and G12 antibody-based sandwich ELISAs were unable to reliably detect gluten, initially at 20 µg/ml, after the onset of production. The Skerritt antibody-based sandwich ELISA overestimated the gluten concentration in all samples. The R5 antibody-based and G12 antibody-based competitive ELISAs were less sensitive than the sandwich ELISAs and did not provide accurate results for quantifying gluten concentration. The Western analyses were able to detect gluten at less than 5 µg/ml in the samples and confirmed the results of the ELISAs. Although further research is necessary before all problems associated with detection and quantification of hydrolyzed and fermented gluten are resolved, the analytical methods recommended by the FDA for regulatory samples can detect ≥ 20 µg/ml gluten that has undergone brewing and fermentation processes associated with the manufacture of beer.


Subject(s)
Beer/analysis , Fermentation , Food Analysis/methods , Food Technology/methods , Glutens/analysis , Antibodies , Edible Grain/chemistry , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Hydrolysis , Reproducibility of Results , Sorghum/chemistry
15.
Anal Bioanal Chem ; 407(14): 4195-206, 2015 May.
Article in English | MEDLINE | ID: mdl-25814275

ABSTRACT

To help safeguard the food supply and detect the presence of undeclared food allergens and gluten, most producers and regulatory agencies rely on commercial test kits. Most of these are ELISAs with a few being PCR-based. These methods are very sensitive and analyte specific, requiring different assays to detect each of the different food allergens. Mass spectrometry offers an alternative approach whereby multiple allergens may be detected simultaneously. However, mass spectrometry requires expensive equipment, highly trained analysts, and several years before a quantitative approach can be achieved. Using multianalyte profiling (xMAP®) technology, a commercial multiplex test kit based on the use of established antibodies was developed for the simultaneous detection of up to 14 different food allergens plus gluten. The assay simultaneously detects crustacean seafood, egg, gluten, milk, peanut, soy, and nine tree nuts (almond, Brazil nut, cashew, coconut, hazelnut, macadamia, pine nut, pistachio, and walnut). By simultaneously performing multiple tests (typically two) for each analyte, this magnetic bead-based assay offers built-in confirmatory analyses without the need for additional resources. Twenty-five of the assays were performed on buffer extracted samples, while five were conducted on samples extracted using reduced-denatured conditions. Thus, complete analysis for all 14 allergens and gluten requires only two wells of a 96-well microtiter plate. This makes it possible to include in a single analytical run up to 48 samples. All 30 bead sets in this multiplex assay detected 5 ng/mL of food allergen and gluten with responses greater than background. In addition, 26 of the bead sets displayed signal/noise ratios of five or greater. The bead-based design makes this 30-plex assay expandable to incorporate new antibodies and capture/detector methodologies by ascribing these new detectors to any of the unassigned bead sets that are commercially available.


Subject(s)
Allergens/chemistry , Glutens/chemistry , Immunoassay/methods , Cuminum/chemistry , Enzyme-Linked Immunosorbent Assay , Food Analysis/methods , Food Hypersensitivity , Magnets
16.
J Food Prot ; 72(4): 903-6, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19435248

ABSTRACT

A recently developed bead-based deadenylase electrochemiluminescence assay for ricin is simple and sensitive in its ability to detect ricin, based on the catalytic activity of the toxin subunit, ricin A chain. The assay was modified to work in a 96-well plate format and evaluated by using juice samples. The plate-based assay, unlike the bead-based assay, includes wash steps that enable the removal of food particles. These steps minimize matrix effects and improve the signal-to-noise ratios and limits of detection (LOD). The LOD values for ricin in apple juice, vegetable juice, and citrate buffer by using the bead-based assay were 0.4, 1, and 0.1 microg/ml, respectively. In contrast, the LOD values for ricin by using the plate-based assay were 0.04, 0.1, and 0.04 microg/ml in apple juice, vegetable juice, and citrate buffer, respectively. The plate-based assay displayed three- to 10-fold lower LOD values than did the bead-based assay. Signal-to-noise ratios for the plate-based assay were comparable to those for the bead-based assay for ricin in citrate buffer, but 2- to 4.5-fold higher when the plate-based assay was used for analysis of juice samples.


Subject(s)
Electrochemical Techniques/methods , Fluorescence , Food Analysis/methods , Food Contamination/analysis , Ricin/chemistry
17.
Anal Biochem ; 378(1): 87-9, 2008 Jul 01.
Article in English | MEDLINE | ID: mdl-18394420

ABSTRACT

A simple electrochemiluminescence-based assay for RNA N-glycosidase activity has been modified to permit its use with authentic extracts of Ricinus communis (castor beans) and Abrus precatorius (jequirity seeds)--the natural sources of ricin and abrin. Modifications include the addition of an RNase inactivator to the reaction mixture, elimination of a signal-enhancing monoclonal antibody, and optimization of the incubation temperature. Concurrent testing with two substrates provides a diagnostic tool enabling castor bean toxins to be differentiated from a larger selection of N-glycosidase toxins than was previously examined.


Subject(s)
Luminescent Measurements/methods , Ribosome Inactivating Proteins/analysis , Ribosome Inactivating Proteins/metabolism , Ricin/metabolism , Ricinus communis/enzymology , Electrochemistry , Enzyme Activation , Indicator Dilution Techniques , Plant Extracts/metabolism
18.
Biochim Biophys Acta ; 1762(4): 398-403, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16483750

ABSTRACT

The list of cancer markers of current interest has grown considerably, but none of the markers used in clinical work is a true tumor marker. These cancer biomarkers are based on the determination of tumor antigens. Here, we report a single method of autoantibody enzyme immunoassay (EIA) screens for a spectrum of serum tumor markers. A comparison of the autoantibody-based EIA to conventional antigen EIA kits, using receiver operating characteristic (ROC) plots, showed that the autoantibody EIA can significantly enhance the sensitivity and specificity of tumor markers. The detection of serum autoantibodies for a spectrum of serum tumor markers, as demonstrated here, suggests that most, if not all, serum cancer biomarkers produce autoantibodies. A unique autoantibody biomarker screening method, as presented here, might therefore facilitate achieving the accurate and early diagnosis of cancer.


Subject(s)
Antibodies, Neoplasm/blood , Antigens, Neoplasm/immunology , Autoantibodies/blood , Biomarkers, Tumor/blood , Cyclic AMP-Dependent Protein Kinases/immunology , Neoplasms/diagnosis , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique, Indirect , Humans , Immunoglobulin G/blood , Neoplasms/immunology , Protein Subunits/immunology , ROC Curve , Sensitivity and Specificity
19.
Curr Pharm Des ; 11(22): 2811-23, 2005.
Article in English | MEDLINE | ID: mdl-16101439

ABSTRACT

DNA (antisense and other oligonucleotides) drug design represents a direct genetic approach for cancer treatment. Such an approach takes advantage of mechanisms that activate genes known to confer a growth advantage to neoplastic cells. The ability to block the expression of these genes allows exploration of normal growth regulation. Progress in DNA drug technology has been rapid, and the traditional antisense inhibition of gene expression is now viewed on a genomic scale. This global view has led to a new vision in antisense technology, the elimination of nonspecific and undesirable side effects, and ultimately the generation of more effective and less toxic nucleic acid medicines. Several antisense oligonucleotides are in clinical trials, are well tolerated, and are potentially active therapeutically. DNA drugs are promising molecular medicines for treating human cancer in the near future.


Subject(s)
Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , DNA/chemistry , Drug Design , Animals , Clinical Trials as Topic , Combined Modality Therapy , Genetic Therapy , Humans , Molecular Mimicry , Oligonucleotides/chemical synthesis , Oligonucleotides/pharmacology , Oligonucleotides, Antisense/chemical synthesis , Oligonucleotides, Antisense/pharmacology
20.
J Med Genet ; 41(12): 923-31, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15591278

ABSTRACT

BACKGROUND: Inactivation of the human type Ialpha regulatory subunit (RIalpha) of cyclic AMP dependent protein kinase (PKA) (PRKAR1A) leads to altered kinase activity, primary pigmented nodular adrenocortical disease (PPNAD), and sporadic adrenal and other tumours. METHODS AND RESULTS: A transgenic mouse carrying an antisense transgene for Prkar1a exon 2 (X2AS) under the control of a tetracycline responsive promoter (the Tg(Prkar1a*x2as)1Stra, Tg(tTAhCMV)3Uh or tTA/X2AS line) developed thyroid follicular hyperplasia and adenomas, adrenocortical hyperplasia and other features reminiscent of PPNAD, including late onset weight gain, visceral adiposity, and non-dexamethasone suppressible hypercorticosteronaemia, with histiocytic, epithelial hyperplasias, lymphomas, and other mesenchymal tumours. These lesions were associated with allelic losses of the mouse chromosome 11 Prkar1a locus, an increase in total type II PKA activity, and higher RIIbeta protein levels; the latter biochemical and protein changes were also documented in Carney complex tumours associated with PRKAR1A inactivating mutations and chromosome 17 PRKAR1A locus changes. CONCLUSION: We conclude that the tTA/X2AS mouse line with a downregulated Prkar1a gene replicates several of the findings in Carney complex patients and their affected tissues, supporting the role of RIalpha as a candidate tumour suppressor gene.


Subject(s)
Endocrine Gland Neoplasms/enzymology , Proteins/physiology , Adrenal Cortex Diseases/enzymology , Adrenal Cortex Diseases/genetics , Adrenal Gland Neoplasms/enzymology , Adrenal Gland Neoplasms/genetics , Alleles , Animals , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit , Cyclic AMP-Dependent Protein Kinase Type II , Cyclic AMP-Dependent Protein Kinases/metabolism , Down-Regulation , Endocrine Gland Neoplasms/genetics , Gene Deletion , Genes, Tumor Suppressor , Humans , Loss of Heterozygosity , Mice , Mice, Transgenic , Mutation , Neoplastic Syndromes, Hereditary/enzymology , Neoplastic Syndromes, Hereditary/genetics , Phenotype , Proteins/genetics , Thyroid Neoplasms/enzymology , Thyroid Neoplasms/genetics
SELECTION OF CITATIONS
SEARCH DETAIL
...