Quantitative sandwich ELISA for determination of traces of hazelnut (Corylus avellana) protein in complex food matrixes.

A hazelnut-specific sandwich-type ELISA based on polyclonal antisera was developed for detection of hidden hazelnut protein residues in complex food matrixes. In the absence of a food matrix, extractable protein from different native and toasted hazelnuts was detected at rates of 94 +/- 13 and 96 +/- 7% applying standards prepared from native and toasted hazelnuts, respectively. From complex food matrixes, 0.001-10% of hazelnut was recovered between 67 and 132%, in average by 106 +/- 17%. Depending on the food matrix, hazelnut protein could be detected down to the ppb (ng/g) level. Intraassay precision was <6% for hazelnut >/= 0.001% and interassay precision was <15% for hazelnut >/= 0.01%. In 12 of 28 commercial food products without labeling or declaration of hazelnut components, between 2 and 421 ppm of hazelnut protein was detected, demonstrating a remarkable presence of potentially allergenic hazelnut protein "hidden" in commercial food products.     

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.     

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

Among allergenic foods, soybean is known as a food causing adverse reactions in allergenic patients. To clarify the validity of labeling, the specific and sensitive detection method for the analysis of the soybean protein would be necessary. The p34 protein, originally characterized to be p34 as an oil-body associated protein in soybean, has been identified as one of the major allergenic proteins and named Gly m Bd 30K. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of the soybean protein in processed foods was developed using polyclonal antibodies raised against p34 as a soybean marker protein and the specific extraction buffer for extract. The developed sandwich ELISA method was highly specific for the soybean protein. The limit of detection (LOD) and the limit of quantification (LOQ) of the developed ELISA were 0.47 ng/mL (equivalent to 0.19 microg/g in foods) and 0.94 ng/mL (equivalent to 0.38 microg/g in foods), respectively. The recovery ranged from 87.7 to 98.7%, whereas the intra- and interassay coefficients of variation were less than 4.2 and 7.5%, respectively. This study showed that the developed ELISA method is a specific, precise, and reliable tool for the quantitative analysis of the soybean protein in processed foods.     

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.     

Detection of potentially allergenic hazelnut (Corylus avellana) residues in food: a comparative study with DNA PCR-ELISA and protein sandwich-ELISA

Allergen detection is of increasing interest for food labeling purposes. A comparative study with a commercial hazelnut-specific PCR-ELISA and a sandwich-type ELISA detecting hazelnut protein was performed to investigate to what extent immunochemical and DNA-based techniques would correlate in the detection of trace amounts of potentially allergenic hazelnut residues. Both methods were highly sensitive and allowed the detection of even <10 ppm of hazelnut in complex food matrixes. The protein-ELISA was highly specific for hazelnut. However, some foods could lead to false-positive results at the 10 ppm level. The PCR-ELISA did not show any cross-reactions with non-hazelnut foods, thus reducing the probability of having false positives at the trace level. Forty-one commercial food products with and without hazelnut components on their labels were analyzed for the presence of hazelnut. Of the 27 products in which hazelnut components were detected, two samples were not identified by the protein-ELISA, and only one sample, namely one white chocolate having <1 ppm of hazelnut protein, was not detected by PCR-ELISA. The good correlation of the results of PCR-ELISA and protein-ELISA suggested that both PCR-based and immunochemical techniques are suitable for reliable detection of potentially allergenic hazelnut residues in foods at the trace level.     

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.     

Quantitative sandwich ELISA for the determination of tropomyosin from crustaceans in foods

The ubiquitous muscle protein tropomyosin has been identified as the major shrimp allergen and is suggested to be a cross-reacting allergen. Previously, only a few methods for the detection of tropomyosin in food have been published. A quantitative sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of tropomyosin from crustaceans in foods has been developed and validated. A polyclonal rabbit antitropomyosin capture antibody and the biotinylated conjugate of the same antibody for detection were the basis for the ELISA, which was specific for crustaceans. The ELISA was able to quantitate tropomyosin in various food matrixes, had a detection limit of 1 microg/g, and cross-reacted to some extent with cockroach. Recoveries ranged from 63 to 120%, and the intra and interassay coefficients of variation were <6 and <14%, respectively.     

Determination of florfenicol amine residues in animal edible tissues by an indirect competitive ELISA

Florfenicol (FF) is a broad-spectrum antibiotic used increasingly in aquaculture, livestock, and poultry to treat diseases. To avoid using labor-intensive instrumental methods to detect residues of FF in food and food products, a simple and convenient indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method for florfenicol's major metabolite, florfenicol amine (FFA), was developed using a polyclonal antibody prepared in this study. FFA was covalently attached to carrier protein as immunogen by using the glutaraldehyde method. The antibodies obtained were characterized by an ELISA method and showed excellent specificity and sensitivity with the 50% inhibition values (IC 50) of 3.34 microg/L for FFA in PBS buffer. In the ELISA, sample extractions were performed by ethyl acetate/ammonium hydroxide (90 + 10, v/v) following combined acid hydrolysis of FF and its known metabolites. The limits of detection (LOD) calculated from the analysis of 20 known negative swine muscle, chicken muscle, and fish samples were 3.08, 3.3, and 3.86 microg/kg (mean + 3 SD), respectively. Recoveries of FFA fortified at the levels of 5, 50, 100, and 300 microg/kg ranged from 64.6 to 124.7%, with coefficients of variation of 11.3-25.8% over the range of FFA concentrations studied. Validation of the ELISA method with FFA-fortified swine muscle at the levels of 10, 50, 100, and 200 microg/kg was carried out using GC, resulting in a similar correlation in swine muscle ( r = 0.97). The results suggest that this ELISA is a specific, accurate, and sensitive method, which is suitable for use as a screening method to detect residues of FFA in animal edible tissues.     

Development of a microtiter plate ELISA and a dipstick ELISA for the determination of the organophosphorus insecticide fenthion

In previous studies, polyclonal antibodies against the organophosphorus insecticide fenthion were obtained and an indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed for this pesticide. In this study, using these antibodies and an enzyme tracer, direct competitive ELISAs for fenthion in microtiter plate and dipstick formats were developed. The microtiter plate ELISA showed an IC(50) value of 1.2 microg/L with a detection limit of 0.1 microg/L. The antibodies showed negligible cross-reactivity with other organophosphorus pesticides. The use of the dipstick format using Immunodyne as a support membrane allowed the quick visual detection of fenthion in concentrations >10 microg/L. The IC(50) value of the dipstick format using reflectance detection was 15 microg/L with a detection limit of 0.5 microg/L. The recoveries of fenthion from spiked vegetable samples using the two formats without any prior enrichment or cleanup steps were 87-116%.     

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.     

Multiplex DNA detection of food allergens on a digital versatile disk

The development of a DNA microarray method on a digital versatile disk (DVD) is described for the simultaneous detection of traces of hazelnut ( Corylus avellana L.), peanut ( Arachis hypogaea ), and soybean ( Glycine max ) in foods. After DNA extraction, multiplex PCR was set up using 5'-labeled specific primers for Cor a 1, Ar h 2, and Le genes, respectively. Digoxin-labeled PCR products were detected by hybridization with 5'-biotinylated probes immobilized on a streptavidin-modified DVD surface. The reaction product attenuates the signal intensity of the laser that reached the DVD drive used as detector, correlating well with the amount of amplified sequence. Analytical performances showed a detection limit of 1 μg/g and good assay reproducibility (RSD 8%), suitable for the simultaneous detection of the three targeted allergens. The developed methodology was tested with several commercially available foodstuffs, demonstrating its applicability. The results were in good agreement, in terms of sensitivity and reproducibility, with those obtained with ELISA, PCR-gel agarose electrophoresis, and RT-PCR.     

Synthesis of haptens for development of antibodies to alkylphenols and evaluation and optimization of a selected antibody for ELISA development

The development of an enzyme-linked immunosorbent assay (ELISA) based on polyclonal antibodies for a class of endocrine disrupting compounds, 4-nonylphenol, is described. The parent molecule was derivatized at the ortho position of the free phenolic hydroxyl group to obtain the hapten, NP1, and it was conjugated with keyhole limpet hemocyanin, which was used as an immunogen. Four antisera were generated and screened against three coating antigens. The most sensitive ELISA from the screening tests (antiserum NP03As, 1/1000, and coating antigen NP1-BSA, 1 microg/mL) was further optimized and characterized. The influence of various physicochemical factors (organic solvent, pH, ion strength) was investigated. Methanol as the additive organic solvent was found to be the best organic solvent for the ELISA, with optimal sensitivity observed at a concentration of 5%. The ELISA parameters were changed at more acidic or basic pH values, whereas higher ionic strengths strongly suppressed the I(50) value and the maximum absorbance. The most sensitive ELISA for 4-nonylphenol exhibited an I(50) value of 38.6 +/- 5.5 microg/L, with a dynamic range from 12 to 350 microg/L, and the lower limit of detection was 7.7 +/- 1.3 microg/L. The optimized ELISA displayed no significant cross-reaction against the parent compounds, nonylphenol ethoxylates, degradation products, carboxylates, and bisphenol A, except in 4-octylphenol.     

Monoclonal antibody-based ELISA and colloidal gold immunoassay for detecting 19-nortestosterone residue in animal tissues

This article presents the generation of monoclonal antibodies (mAbs) with high specificity against 19-nortestosterone (NT) through cell fusion techniques and the development of a mAb-based indirect competitive ELISA (icELISA) method and colloidal gold-based immuno-chromatographic assay to detect NT residues in beef and pork samples. A modified carbodiimide method was employed to synthesize the artificial antigen, and BALB/c mice were used to produce anti-NT mAbs. On the basis of the checkerboard titration, an indirect competitive ELISA standard curve was established. This assay was sensitive and had a linear range from 0.03 to 38 ng/mL in phosphate buffered saline (PBS), with IC(50) and LOD values of 0.52 ng/mL and 0.01 ng/mL, respectively. Of all the competitive analogues, the produced mAb exhibited a high cross-reactivity to 17α-nortestosterone (83.6%), the main metabolite of NT in animal tissues. Except for moderate cross-reactivities with trenbolone (22.6%) and β-boldenone (13.8%), the other interference to the assay was negligible (<0.05%). In contrast, the strip test had a visual detection limit of 1 ng/mL in PBS, 2 μg/kg in beef, and 2 μg/kg in pork, respectively, and the results can be judged within 10 min. The ELISA and GC-MS results showed close correlation in beef (R2=0.9945) and in pork (R2=0.9977). Therefore, the combination of two immunoassays provides a useful screening method for quantitative or qualitative detection of NT residues in animal-origin products.     

Single-tube nested real-time PCR as a new highly sensitive approach to trace hazelnut

Hazelnut is one of the most commonly consumed tree nuts, being largely used by the food industry in a wide variety of processed foods. However, it is a source of allergens capable of inducing mild to severe allergic reactions in sensitized individuals. Hence, the development of highly sensitive methodologies for hazelnut traceability is essential. In this work, we developed a novel technique for hazelnut detection based on a single-tube nested real-time PCR system. The system presents high specificity and sensitivity, enabling a relative limit of detection of 50 mg/kg of hazelnut in wheat material and an absolute limit of detection of 0.5 pg of hazelnut DNA (1 DNA copy). Its application to processed food samples was successfully achieved, detecting trace amounts of hazelnut in chocolate down to 60 mg/kg. These results highlight the adequacy of the technique for the specific detection and semiquantitation of hazelnut as potential hidden allergens in foods.     

Poly- and monoclonal antibody-based ELISAs for fipronil

An enzyme-linked immunosorbent assay (ELISA) for fipronil was developed by using polyclonal antibodies (pABs) or monoclonal antibodies (mABs), and its suitability of the determination of this analyte in spiked water samples was studied. The pABs-based assay showed I50 = 17.95 ppb, I90 = 203.40 ppb, and I10 = 0.066 ppb, whereas the mABs-based assay showed I50 = 5.99 ppb, I90 = 485.40 ppb, and I10 = 0.074 ppb. The recoveries of fipronil from tap water samples by pABs-based ELISA were 93.00-124.00% in the range of 0-500 ng/mL, and those obtained from the samples by mABs-based ELISA were 94.70-108.00%. Different types of water from pool, river, and sea were spiked at different levels (ranging form 0.1 to 10 microg/L) and were assayed by the indirect ELISA with mABs. The recoveries of fipronil by this ELISA were in the range of 80-120%. The results demonstrate that this assay is suitable for the quantitative detection of fipronil at trace levels in water samples.     

Immunochemical and structural analysis of pepsin-digested egg white ovomucoid

Ovomucoid, an egg protein comprising approximately 10% egg white, was digested using the enzyme pepsin, and fragments were isolated by anion-exchange and reverse phase HPLC. Four distinct fragments were identified by analysis with SDS-PAGE, including three large fragments with molecular weights of around 24, 18, and 14 kDa. N- and C-terminal and amino acid sequencing analyses identified the fragments as V134-C186 (domain 3), V21-A133, and A1-A133 (domain 1+2). Further separation and sequencing of the fraction composed of small peptides, to determine their exact makeup and location in the protein, remained to be carried out and identified a peptide G51-Y73. All four fragments showed IgE-binding activity, as measured by ELISA, using human sera from egg-allergic individuals. Little change in the digestibility of ovomucoid by trypsin and chymotrypsin was observed following digestion with pepsin, indicating that pepsin-digested ovomucoid retains its trypsin (protease) inhibitor activities. Reduced carboxymethylated ovomucoid was prepared, and digestion with pepsin produced significantly more peptides than did the digestion of the native ovomucoid, indicating that the disulfide bonds play a significant role in the digestive resistance of ovomucoid. The reduction of ovomucoid enhanced its digestibility and lower allergenicity of the protein.     

Preparation of specific monoclonal antibodies (MAbs) against heavy metals: MAbs that recognize chelated cadmium ions

Monoclonal antibodies (MAbs) were produced against chelated Cd (2+). Since Cd (2+) ions are too small to elicit an immune response, the metal was coupled to protein carrier (keyhole limpet hemocyanin, KLH) using a bifunctional chelator 1-(4-isothiocyanobenzyl)ethylenediamine N, N, N', N'-tetraacetic acid (ITCBE). Several mice were immunized with this Cd (2+)-ITCBE-KLH immunoconjugate. Spleen cells of two immunized mice were fused with myeloma cells, and the resulting hybridomas were screened using protein conjugates with covalently bound metal-free ITCBE (ethylenediamine tetraacetic acid) or Cd (2+)-ITCBE. Four hybridoma cell lines that produced MAbs with high selectivity and sensitivity (Aa4, Aa6, Ac4, and Ba2) were expanded for further study. Cross-reactivities with other metals were below 1% except for Hg (2+), which showed a slight cross-reactivity in competitive ELISA. These antibodies were used to construct competitive ELISAs for ionic cadmium; the IC 50 of the four antibodies (Aa4, Aa6, Ac4, and Ba2) were 10.59, 4.19, 29.45, and 6.63 microg/L, respectively. The detection range and the lowest detection limit for cadmium, using the Aa6 antibody, were 2.19-86.38 microg/L and 0.313 microg/L, respectively. Spike-recovery studies in tap and stream water showed that the most sensitive antibody can be used for cadmium detection in drinking water.     

A highly specific enzyme-linked immunosorbent assay for the detection of Cry1Ac insecticidal crystal protein in transgenic WideStrike cotton

A highly selective enzyme-linked immunosorbent assay (ELISA) has been developed for the quantitative detection of the Cry1Ac protein expressed in transgenic cotton. Two Cry1Ac-specific monoclonal antibodies (MAb), Kbt and 158E6, were developed and selected to form a sandwich format ELISA. The MAb Kbt was used as a capture antibody, and 158E6 was conjugated with horseradish peroxidase and served as a detection antibody. The assay was optimized and validated with different cotton matrices. Tissues were extracted with phosphate-buffered saline containing 0.05% Tween 20 and 1% polyvinylpyrrolidone. The extract was then treated with trypsin to truncate full-length Cry1Ac into the core toxin for quantitation. The resulting assay has good accuracy and precision with a validated limit of quantitation ranging from 0.1 to 0.375 mug/g dry weight of cotton tissues. This assay is highly specific for Cry1Ac protein and has no cross-reactivity with the nontarget proteins tested such as Cry1Ab and Cry1F.     

Enzyme-linked immunosorbent assays based on rabbit polyclonal and rat monoclonal antibodies against isoproturon

This work describes the production and characterization of rabbit polyclonal antisera (pAb) and rat monoclonal antibodies (mAb) against isoproturon. Coating antigen and enzyme-tracer formats were developed. Standard curves for isoproturon were conducted either in 40 mM phosphate buffered saline (PBS) or in Milli-Q water. PAb 352 together with the best enzyme tracer revealed in the optimized ELISA (enzyme tracer format) a test midpoint of 1.06 +/- 0.34 microg/L (n = 19, standard set up in Milli-Q water) with a detection limit of about 0.1 microg/L. The comparable ELISA with mAb IOC 7E1 had test midpoints of 0.07 +/- 0.04 microg/L (n = 7, standards in Milli-Q water) and 0.11 +/- 0.08 microg/L (n = 33; standards in 40 mM PBS). The limits of detection were about 0.003 and 0.01 microg/L in Milli-Q water and PBS, respectively. Noticeable cross reactivities (CRs) were seen with the major metabolites, namely 4-isopropylaniline, 4-isopropylphenylurea, and 1-(4-isopropylphenyl)-3-methylurea. With pAb 352, these CRs were 5%, 7%, and 31%, respectively, and with mAb IOC 7E1, they were 3%, 5%, and ca. 19%, respectively. All arylurea herbicides had only minor CRs, which ranged from no CR (e.g., chlorosulfuron) to a maximum of 3.3% (chlortoluron). Influences of organic solvents (methanol, ethanol, acetonitrile, and acetone) were evaluated. Both pAb- and mAb-based immunoassays showed the highest tolerance for methanol, up to 5%. Ethanol and acetonitrile could not be used above 2% without an influence on the assays. The same was true for acetone, although tested only in the mAb-based assay. Water samples of different origins and matrices were spiked and analyzed with these pAb and mAb ELISAs. The results demonstrated that these immunoassays are useful screening tools.     

Determination of sulfonylurea herbicides in water and food samples using sol-gel glass-based immunoaffinity extraction and liquid chromatography-ultraviolet/diode array detection or liquid chromatography-tandem mass spectrometry

Immunoaffinity supports (IAS) were prepared using broad specific polyclonal anti-sulfonylurea (SU) antibodies immobilized in sol-gel glass. Two different kinds of supports were applied, crushed sol-gel monoliths and sol-gel-coated highly porous silica particles. Both were used for the quantitative enrichment of SUs in natural water and food samples followed by high-performance liquid chromatography-ultraviolet/diode array detection (HPLC-UV/DAD) and tandem mass spectrometry (LC-MS/MS), respectively. Loading, washing, and elution conditions of IAS were optimized. The capacity of supports was determined for 30 SUs and compared with the cross-reactivity pattern of the direct competitive enzyme-linked immunosorbent assay. The capacities correlated well with the affinity to individual SU compounds. Even analytes to which the polyclonal antibodies showed only a lower cross-reactivity could be enriched to a certain degree, if a sufficient capacity of IAS was provided. The IAS could be reused at least 10 times without a loss of effectiveness. Recovery of 16 selected SUs extracted from spiked water and food samples was dependent on the affinity of both immobilized antibodies to single compounds and matrix interferences. In water, 13 SUs showed recoveries higher than 80% when immunoaffinity extraction was used in combination with LC-UV/DAD. On the basis of the enrichment of 200 mL of aqueous sample, corresponding limit of detection (LOD) values ranged between 20 and 100 ng/L. The recoveries of 10 SUs, which were extracted from 10 g of potato spiked at a 10 microg/kg level, were higher than 75%. For grain samples, recoveries were at the same order for at least five SU herbicides. The LOD of LC-MS/MS measurements was about 1 order of magnitude higher, i.e., gave LODs between 1.1 and 6.9 microg/kg of food sample, depending on the compound and extraction procedure. These LODs provide evidence that the main advantage of the prepared IAS is their high selectivity for group specific recognition of SUs as compared to other nonspecific solid phase extraction materials.