Quantitative detection of hazelnut (Corylus avellana) in cookies: ELISA versus real-time PCR

Hazelnuts (Corylus avellana) are used widely in the food industry, especially in confectionery, where they are used raw, roasted, or in a processed formulation (e.g., praline paste and hazelnut oil). Hazelnuts contain multiple allergenic proteins, which can induce an allergic reaction associated with symptoms ranging from mild irritation to life-threatening anaphylactic shock. To date, immunochemical (e.g., ELISA or dipstick) and PCR-based analyses are the only methods available that can be applied as routine tests. The aim of this study is to make a comparative evaluation of the effectiveness of ELISA and real-time PCR in detecting and correctly quantifying hazelnut in food model systems. To this end, the performances of two commercial ELISAs were compared to those of two commercial and one in-house-developed real-time PCR assays. The results showed that although ELISA seemed to be more sensitive compared to real-time PCR, both detection techniques suffered from matrix effects and lacked robustness with regard to food processing. As these impacts were highly variable among the different evaluated assays (both ELISA and real-time PCR), no firm conclusion can be made as to which technique is suited best to detect hazelnut in (processed) food products. In this regard, the current lack of appropriate DNA calibrators to quantify an allergenic ingredient by means of real-time PCR is highlighted.     

Immunochemical assays for direct sulfonamide antibiotic detection in milk and hair samples using antibody derivatized magnetic nanoparticles

Two direct enzyme-linked immunosorbent assays (ELISAs) have been developed for detection of sulfonamide antibiotic residues in milk samples. One of them is using magnetic nanoparticles (MNP) for target capture/enrichment (Ab-MNP-ELISA), and the second is performed using microtiter plates. Selective polyclonal antibodies, raised against 5-[6-(4-amino-benzenesulfonylamino)-pyridin-3-yl]-2-methyl-pentanoic acid (SA1), used in combination with an enzyme tracer prepared with the same hapten, has allowed us to reach a limit of detection (LOD) lower than 0.5 microg L(-1) for both ELISA formats. Sulfapyridine, sulfamethoxypyridazine, sulfathiazole, and sulfachloropyridazine are detected below the maximum residue limits established by the European Union for these antibiotics in milk (100 microg L(-1)). Matrix effects and accuracy studies performed with full-cream milk and hair extracts indicated a lack of interference from these sample matrices and very good recovery values, especially when using the Ab-MNP format. Milk samples and hair extracts can be measured without any previous treatment. The results demonstrate the high potential of these methods as screening tools for food safety and inspection controls.     

Symposium on microbiology update: old friends and new enemies. Staphylococcus aureus

The analytical methods for the detection of the staphylococcal enterotoxins can be divided into 2 categories: (1) methods for detection of enterotoxin-producing staphylococcal strains; (2) methods for detection of enterotoxin in foods. Gel diffusion methods (Ouchterlony, microslide), in which the enterotoxin produced by any given strain is compared to one of the identified enterotoxins, are used most frequently for strain testing. The sensitivity of these methods is from 0.1 to 0.5 micrograms enterotoxin/mL, which is normally adequate to determine the enterotoxigenicity of strains. The methods for the detection of enterotoxin in foods need to be much more sensitive to detect less than 1 ng of enterotoxin/g of food that may be present. The radioimmunoassay (RIA), the enzyme-linked immunosorbent assay (ELISA), and the reversed passive latex agglutination (RPLA) method have the necessary sensitivity to detect 1 ng/g of enterotoxin in foods without the use of complicated extraction-concentration procedures. Kits based on the ELISA and RPLA methods are now available commercially for the detection of enterotoxins in foods. Tests have shown that the ELISA methods are somewhat more sensitive than the RPLA method.     

Validated method for quantification of genetically modified organisms in samples of maize flour

Sensitive and accurate testing for trace amounts of biotechnology-derived DNA from plant material is the prerequisite for detection of 1% or 0.5% genetically modified ingredients in food products or raw materials thereof. Compared to ELISA detection of expressed proteins, real-time PCR (RT-PCR) amplification has easier sample preparation and detection limits are lower. Of the different methods of DNA preparation CTAB method with high flexibility in starting material and generation of sufficient DNA with relevant quality was chosen. Previous RT-PCR data generated with the SYBR green detection method showed that the method is highly sensitive to sample matrices and genomic DNA content influencing the interpretation of results. Therefore, this paper describes a real-time DNA quantification based on the TaqMan probe method, indicating high accuracy and sensitivity with detection limits of lower than 18 copies per sample applicable and comparable to highly purified plasmid standards as well as complex matrices of genomic DNA samples. The results were evaluated with ValiData for homology of variance, linearity, accuracy of the standard curve, and standard deviation.     

Improved Protocols for ELISA Determination of Gliadin in Glucose Syrups

Simple modifications of existing protocols for high‐sensitivity detection of gluten proteins by immunochemical methods allowed rapid and sensitive determination of residual gluten in highly viscous samples of glucose and maltose syrups obtained from processing wheat starch. Dilution of the original syrup to no less than 15–20% in solids allowed retention of gluten proteins in a soluble form so that ELISA determination of gliadin was possible without an extraction step in aqueous ethanol. An ultrafiltration step may be added to concentrate residual gluten proteins in the diluted syrup samples and allow a further increase in sensitivity. The results are relevant for quality assessment of wheat starch derived syrups as raw materials for use in gluten‐free foods for celiac individuals.     

Development and comparison of three diagnostic immunoassay formats for the detection of azoxystrobin

The currently accepted method of detection for azoxystrobin, a strobilurin fungicide, involves a labor-intensive organic solvent extraction and gas chromatography analysis. Three diagnostic assay formats, i.e., enzyme-linked immunosorbent assay (ELISA), fluorescence polarization (FP), and time-resolved fluorescence (TR-FIA), were developed and compared with regard to detection and quantification of azoxystrobin in grape extract and river, lake, and well water samples. These three assay formats require no initial sample extraction and were not affected by any of the environmental matrices tested, and each had a linear working range of 0-400 pg/mL. The polyclonal antibodies used for each of the immunoassays were specific to azoxystrobin; that is, the highest cross-reactivity to other pesticides observed was 5.7%. The limits of detection of the immunoassays were similar at 3 (ELISA), 46 (FP), and 28 (TR-FIA) pg/mL, as were the respective IC50 values of 306, 252, and 244 pg/mL. Each of the three immunoassays developed was less labor-intensive and approximately 100-fold more sensitive than the gas chromatographic method. While the three formats were comparable in terms of performance, the fluorescence polarization assay was the least labor-intensive and required the least time to perform.     

Detecting and quantifying the adventitious presence of transgenic seeds in safflower, Carthamus tinctorius L

Safflower ( Carthamus tinctorius L.) is currently being developed as a platform for the production of novel proteins. Methods for detecting and quantifying transgenic safflower are needed to ensure seed quality and to monitor for its adventitious presence. We developed and compared three methods of assaying for transgenic safflower presence in conventional seedlots: field bioassays, enzyme-linked immunosorbent assays (ELISA), and quantitative polymerase chain reaction (Q-PCR). Limits for reliable quantification for both ELISA and Q-PCR are approximately 0.1%, although levels at least as low as 0.02% can be detected by Q-PCR. Levels of quantification for the field bioassay are limited only by space and resources available. Multiple sampling methods to detect and quantify transgenic safflower presence at levels lower than 0.1% were used on field collected samples from a pollen outcrossing experiment to quantify the adventitious presence of transgenic safflower. Taking into account the potential utility and relative advantages or disadvantages of each detection method, it is recommended that the initial testing for the adventitious presence of transgenic seed be carried out using an antibody-based test if available and that Q-PCR-based assays to quantify transgenic proportion be used when it is necessary to identify specific transgenic constructs or if antibody-based assays are not readily available.     

Compliance with Gluten‐Free Labelling Regulation in the Brazilian Food Industry

Gluten is an important protein complex for baking products found in wheat, rye, barley, and some oat varieties. However, some people need to avoid these grains and their products because they result in gluten‐related disorders. The only treatment for these individuals is to engage in a gluten‐free diet. The objective of this work was to verify if the gluten content of several commercial food products sold in Brazil complied with their labeling. The Méndez ELISA R5 sandwich method was used to analyze 437 samples, and of these, 70% were labeled as gluten‐free, 26% as containing gluten, and 4% not labeled in relation to gluten. The results indicated that 89% of the products labeled as gluten‐free were correctly labeled and 11% were not, which represented a risk for celiac people.     

Development of a real-time PCR and a sandwich ELISA for detection of potentially allergenic trace amounts of peanut (Arachis hypogaea) in processed foods

Hidden allergens in food products are, especially for peanut-allergic consumers, a serious problem because even low amounts (approximately 200 microg) of peanut can elicit allergic reactions. Undeclared peanut traces can be found in processed food products, because contaminations with peanut during production processes are frequent. To minimize the risk of such cross-contaminations, it is necessary to develop sensitive analytical methods for the detection of hidden allergens in foods. For this approach we developed two peanut-specific assays based on the detection of peanut protein by specific antibodies (sandwich ELISA) and by the detection of peanut-specific DNA (part of the coding region of Ara h 2) by a real-time PCR. Both tests did not show any cross-reactivity with 22 common food ingredients (cereals, nuts, legumes), and the limit of detection is <10 ppm peanut in processed foods. Thirty-three random samples of food products were tested for the presence of peanut to compare both assay types with each other and to evaluate the percentage of foods on the German market that are contaminated with peanut traces. We found that four products (13.3%) without peanut in the list of ingredients contained peanut protein in a range from 1 to 74 ppm peanut protein and that the results of both tests correlated well. The real-time PCR was able to detect one more positive sample than the sandwich ELISA. In conclusion, both assays are sensitive and specific tools for the detection of hidden allergens in processed foods.     

施肥对砂姜黑土基础肥力及强筋小麦产量、品质的影响

为探讨不同施肥方式对砂姜黑土的培肥效果和不同土壤基础肥力水平对优质强筋小麦产量及品质的影响,在22年长期定位试验的基础上,研究了不同施肥方式下砂姜黑土基础肥力的变化,并分析了土壤主要养分性状与强筋小麦产量和品质的关系.结果表明,长期单施有机肥或有机肥与化肥配合施用均能较单施化肥处理显著提高土壤全氮、有机质、碱解氮、速效磷及速效钾的含量;同一施氮水平,有机肥与化肥配施处理的基础肥力产量(不施肥时的产量)较单施化肥的高2715.0kg·hm-2.土壤有机质、全氮、全磷及速效磷含量与籽粒产量、蛋白质、湿面筋和沉降值均呈正相关.有机肥与化肥配施不仅是培肥地力的主要途径,同时还是确保优质小麦保优栽培,实现可持续发展的有效措施.     

Determination of spinosad and its metabolites in food and environmental matrices. 1. High-performance liquid chromatography with ultraviolet detection

Spinosad is an insect control agent that is derived from a naturally occurring soil bacterium and is effective on several classes of insects, especially Lepidoptera larvae. Spinosad is registered in many countries for use on a variety of crops, including cotton, corn, soybeans, fruits, and vegetables. Residue methods utilizing high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection have been described for determining spinosad and its metabolites in environmental and food matrices. These residue methods typically involve an extraction with organic solvents, followed by purification using liquid-liquid partitioning and/or solid phase extraction prior to measurement by HPLC-UV. The residue methods determine the active ingredients (spinosyns A and D) and up to three minor metabolites (spinosyn B, spinosyn K, and N-demethylspinosyn D). The methods have validated limits of quantitation ranging from 0.010 to 0.040 microgram/g. This paper briefly reviews the residue methodology for spinosad and metabolites in food and environmental matrices and provides a summary of method validation results for 61 different sample types, including newly published results for 37 additional crop matrices and processed commodities.     

The Biochemical Basis of Celiac Disease

Celiac disease (CD) is an inflammatory disorder of the upper small intestine triggered by the ingestion of wheat, rye, barley, and possibly oat products. The clinical feature of CD is characterized by a flat intestinal mucosa with the absence of normal villi, resulting in a generalized malabsorption of nutrients. The prevalence of CD among Caucasians is now thought to be in a range of 1:100–300. There is a strong genetic association with human leukocyte antigens (HLA‐)DQ2 and DQ8 and currently unknown non‐HLA genes. During the last decade, intense biochemical studies have contributed to substantial progress in understanding the general principles that determine the pathogenesis of CD. The precipitating factors of toxic cereals are the storage proteins, termed gluten in the field of CD (gliadins and glutenins of wheat, secalins of rye, and hordeins of barley). There is still disagreement about the toxicity of oat avenins. The structural features unique to all CD toxic proteins are sequence domains rich in Gln and Pro. The high Pro content renders these proteins resistant to complete proteolytic digestion by gastrointestinal enzymes. Consequently, large Pro‐ and Gln‐rich peptides are cumulated in the small intestine and reach the subepithelial lymphatic tissue. Depending on the amino acid sequences, these peptides can induce two different immune responses. The rapid innate response is characterized by the secretion of the cytokine interleukin‐15 and the massive increase of intraepithelial lymphocytes. The slower adaptive response includes the binding of gluten peptides (native or partially deamidated by tissue transglutaminase) to HLA‐DQ2 or ‐DQ8 of antigen presenting cells and the subsequent stimulation of T‐cells accompanied by the release of proinflammatory cytokines such as interferon‐γ and the activation of matrix metalloproteinases. Both immune responses result in mucosal destruction and epithelial apoptosis. Additionally, stimulated T‐cells activate B‐cells that produce serum IgA and IgG antibodies against gluten proteins (antigen) and tissue transglutaminase (autoantigen). These antibodies can be used for noninvasive screening tests to diagnose CD. The current essential therapy of CD is a strict lifelong adherence to gluten‐free diet. Dietetic gluten‐free foods produced for CD patients underlie the regulations of the Codex Alimentarius Standard for Gluten‐Free Foods. The “Draft Revised Codex Standard” edited in March 2006 proposes a maximum level of 20 mg of gluten/kg for naturally gluten‐free foods (e.g., based on rice or corn flour) and 200 mg/kg for foods rendered gluten‐free (e.g., wheat starch). Numerous analytical methods for gluten determination have been developed, mostly based on immunochemical assays, mass spectrometry, or polymerase chain reaction. So far, only two enzyme‐linked immunosorbent assays have been successfully ring‐tested and are commercially available. During the last decade, future strategies for prevention and treatment of CD have been proposed. They are based on the removal of toxic epitopes by enzymatic degradation or gene engineering and on blocking parts of the immune system. However, any alternative treatment should have a safety profile competitive with gluten‐free diet.     

Rapid monoclonal antibody-based enzyme-linked immunosorbent assay for detection of Listeria in food products

A rapid diagnostic test for the detection of Listeria in food products has been created. This test, known as Listeria-Tek, uses 2 monoclonal antibodies specific for Listeria in an enzyme-linked immunosorbent assay (ELISA) format. The test requires only 40 h of broth enrichment with no culturing on solid media. It is extremely simple to perform and easy to interpret, and is at least as sensitive and accurate as the best of the culture methods. The test can be used with dairy products, meat products, and environmental samples. The ELISA test is safely performed on the open bench of the laboratory because no live cultures, no radioactivity, no phage, etc., are necessary. There is no need for special licenses or reserved laboratory space, and no waste disposal problems are encountered. If necessary, one technician could easily perform hundreds of assays per day. A printed data sheet is available for permanent records.     

Label‐Free Proteomic Analysis of Wheat Gluten Proteins and Their Immunoreactivity to ELISA Antibodies

Enzyme‐linked immunosorbent assay (ELISA) methods are currently the most widely used for gluten quantification. However, the lack of comparable measurements among commercial kits has caused much concern. Here, we studied the immunoreactivity of five commercial ELISA kits to wheat gluten fractionated by reversed‐phase high‐performance liquid chromatography and identified the proteins and peptides in the resulting fractions by mass spectrometry to understand the extent to which these may be contributing to the lack of comparability. The investigated monoclonal antibodies clearly demonstrated divergent responses to the fractioned wheat gluten proteins and sometimes to their initial intended targets. To make comparable gluten measurements a reality, the analytical measurement community requires a set of agreed peptide markers, known conversion factors from these markers to total gluten content, and appropriately characterized (certified) reference materials representative of gluten.     

Sandwich immunoassays for the determination of peanut and hazelnut traces in foods

People suffering from food allergies are dependent on accurate food labeling, as an avoidance diet is the only effective countermeasure. Even a small amount of allergenic protein can trigger severe reactions in highly sensitized patients. Therefore, sensitive and reliable tests are needed to detect potential cross-contamination. In this paper two fast sandwich immunoassays are described for the determination of peanut (Arachis hypogaea) and hazelnut (Corylus avellana) traces in complex food matrices. Mouse monoclonal antibodies were used as capture antibodies, and labeled rabbit polyclonal antibodies were used as detection antibodies in both assays. The assay time was 30 min in total, and cross-reactivities against a variety of fruits and seeds were found to be in the low 10(-4)% (ppm) level or in some cases not detectable. The recoveries in all tested food matrices ranged from 86 to 127%, and the limits of detection were in the range of 0.2-1.2 mg/kg (ppm) in food for both peanut and hazelnut, respectively.     

Detection of allergenic ingredients using real-time PCR: a case study on hazelnut (Corylus avellena) and soy (Glycine max)

Compliance with the European allergen labeling legislation (Directive 2007/68/EC) is only possible when coupled with appropriate methods to detect allergens in food. The aim of the current study was to develop new real-time PCR assays for the detection of hazelnut and soy and evaluate these assays via comparison with commercially available kits. Although the new assays were not as sensitive as the commercial qualitative assays, they proved to be more specific. Moreover, the cross-reactivity study indicated contamination of some of the food products used with either hazelnut or soy, which presents a risk for the allergic consumer. The assays were able to quantify as few as 5-15 genome copies. This unit, used to express analytical results for allergen detection by means of PCR, needs to be converted to a unit expressing the amount of allergenic ingredient in order to be informative. This study emphasizes that the use of real-time PCR for allergen quantification is complicated by the lack of appropriate reference materials for allergens.     

我国北方冬小麦籽粒中的戊聚糖含量及其相关分析

戊聚糖是小麦面粉加工和食品品质重要的影响因子。本研究测定了我国北方冬麦区77个品种(系)籽粒中的戊聚糖含量和蛋白质、湿面筋、沉降值、千粒重等性状。结果表明,不同品种的戊聚糖含量变幅为4.82%~7.28%,戊聚糖含量与籽粒饱满度、湿面筋含量和沉淀值之间存在着极显著的正相关关系,与千粒重具有极显著的负相关关系。同时对“间苯三酚-冰醋酸法”测定小麦戊聚糖的含量进行了讨论。     

Reliable enzyme-linked immunosorbent assay for the determination of coconut milk proteins in processed foods

This study was designed to develop a novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of coconut milk proteins in processed foods. The developed sandwich ELISA was able to detect coconut milk proteins from various coconut milk products and did not show any cross-reactivity with 41 of 42 kinds of popularly used food ingredients, thus reflecting great specificity for coconut milk proteins. In addition, the established ELISA is highly sensitive and allowed the detection of 0.31 μg/g of coconut milk protein in complex food matrices. This proposed assay could serve as a useful tool for the detection of the presence of hidden coconut milk proteins in processed foods.     

Effect of processing on recovery and variability associated with immunochemical analytical methods for multiple allergens in a single matrix: sugar cookies

Among the major food allergies, peanut, egg, and milk are the most common. The immunochemical detection of food allergens depends on various factors, such as the food matrix and processing method, which can affect allergen conformation and extractability. This study aimed to (1) develop matrix-specific incurred reference materials for allergen testing, (2) determine whether multiple allergens in the same model food can be simultaneously detected, and (3) establish the effect of processing on reference material stability and allergen detection. Defatted peanut flour, whole egg powder, and spray-dried milk were added to cookie dough at seven incurred levels before baking. Allergens were measured using five commercial enzyme-linked immunosorbent assay (ELISA) kits. All kits showed decreased recovery of all allergens after baking. Analytical coefficients of variation for most kits increased with baking time, but decreased with incurred allergen level. Thus, food processing negatively affects the recovery and variability of peanut, egg, and milk detection in a sugar cookie matrix when using immunochemical methods.