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Quantitation of gluten in gluten-free products is a great challenge as it is hindered by several factors including the lack of certified reference materials. To resolve this problem, our research group, in cooperation with other international experts, started a series of experiments with the goal of the production of a suitable gluten reference material. As a part of this research, several different wheat cultivars and their isolated gluten proteins were characterized by different methods, including enzyme-linked immunosorbent assay (ELISA). However, we need to know the performance of the ELISA methods used for this special area of research. During the present work we investigated the accuracy and precision of two different ELISA methods for our own laboratory conditions and special sample matrices (wheat flours and gliadin isolate). We have found that the tested performance characteristics of the methods seem to be appropriate on a case-by-case basis, but the long-term measurement uncertainty is higher, which makes it difficult to evaluate the results obtained with the ELISA method for these types of samples.
BOOK REVIEW Substance and seduction Elisa Guerra Doce Department of Prehistoria, Arqueología
Zachary, A. A., Ratner, L. E., Graziani, J. A. és mtsai: Characterization of HLA class I specific antibodies by ELISA using solubilized antigen targets: II. Clinical relevance. Human Immunology, 2001, 62 , 236
The early detection of pregnancy and the determination of fetal numbers have economic benefits in sheep production because of the seasonal breeding patterns where missing a breeding opportunity means the loss of one productive year. The purpose of this study was to evaluate the efficacy of the B6-HRP ELISA for ovine pregnancy-specific protein B (oPSPB) measurement in the detection of pregnancy and estimation of fetal numbers in different sheep breeds. BioPRYN® ELISA assay kit was used for the detection of pregnancy in the experimental animals. Ninety-three ewes of three breeds (British Milksheep – BM, Lacaune – L and Transylvanian Racka – TR), each from three farms in Hungary, were included in the study. BM and L ewes were artificially inseminated (AI). Thirty-five days after AI, all ewes were examined by transabdominal ultrasound. The TR flock was mated naturally over a six-week period. At the end of the mating period, the ewes were similarly examined by ultrasound. Blood samples were taken from all pregnant ewes twice (35 and 65 days after AI), and serum samples were assayed by the BioPRYN test. It can be concluded that the detection of serum PSPB by ELISA is a much easier, safer, less expensive and highly accurate method for the detection of ovine pregnancy. Although some breed-related differences were detectable at 35 and 65 days post breeding, no differences in oPSPB levels were found in pregnant ewes carrying different numbers of fetuses.
Ear rots of maize caused by Fusarium spp. reduce grain yield and produce mycotoxins, which are harmful to humans and animals. To breed maize cultivars resistant to Fusarium spp., reliable large-scale phenotyping is essential. Our objectives were to (i) examine the precision of the ELISA method for determination of important mycotoxins, namely deoxynivalenol (DON) and fumonisins (FUM), (ii) evaluate the potential of near-infrared reflectance spectroscopy (NIRS) to estimate concentrations of DON and FUM in grain produced in inoculated maize plants, and (iii) compare the efficiency of ELISA, NIRS, and visual rating of disease severity for estimation of mycotoxin concentrations. Insignificant variation was observed between duplicate evaluations of DON and FUM by ELISA, showing the high repeatability of this method. DON and FUM determinations by ELISA were more closely correlated with mycotoxin concentrations predicted through NIRS than with visual rating of disease severity. For the prediction of DON, NIRS had very high magnitude of the coefficients of determination of calibration and cross validation (R 2 = 0.90–0.88). Thus, NIRS has a promising potential to predict DON concentration in grain samples of inoculated maize genotypes evaluated in resistance breeding programs.
Enzyme-linked immunosorbent assays (ELISAs) are widely used to determine gluten contamination in gluten-free and low gluten food samples. ELISA assays developed using monoclonal antibodies against known toxic peptides have an advantage in the identification of toxic prolamin content in protein extracts of different food samples, as well as raw materials. R5 and G12 monoclonal antibodies specific for two known toxic peptides used in commercially available gluten ELISA assays were applied to test toxic peptide contents in wheat relatives and wild wheat species with different genome composition and complexity. Although the R5 peptide content showed some correlation with ploidy levels in Triticum species, there was a high variance among Aegilops species. Some of the analysed diploid Aegilops species showed extremely high R5 peptide contents. Based on the bioinformatics analyses, the R5 peptide was present in most of the sulphur rich prolamins in all the analysed species, whereas the G12 epitope was exclusively present in alpha gliadins. High variation was detected in the position and frequency of epitopes in sequences originating from the same species, thus highlighting the importance of genotypic variation within species. Identification of new prolamin alleles of wheat relatives and wild wheat species is of great importance in order to find germplasm for special end-use quality purposes as well as development of food with reduced toxicity.
In special dietary products for people intolerant to gluten, gluten content is not supposed to exceed the regulatory thresholds. Enzyme-linked immunosorbent assays (ELISAs) are routinely used to quantitate gluten in these products. They measure gliadin/gluten with high specificity and sensitivity, but they have some limitations. Quantitative and qualitative variability of the target proteins among wheat cultivars is a factor that may cause inaccurate results. One of the aims of this work was to characterize the protein composition of five wheat cultivars grown in multiple harvest years and their blends by reversed-phase high-performance liquid chromatography (RP-HPLC). The gliadin/gluten content of these wheat flours was also analysed with two commercial ELISA kits. The effect of differences in protein profiles between the flours from an individual cultivar and the blend of five cultivars, harvest years, as well as processing procedures (dough forming and baking) on the results of two ELISA kits was investigated and their relative magnitude was determined. Among the factors investigated, the differences between flours had the greatest impact on gliadin recoveries.
Twenty-two serum samples of healthy bitches were tested with the frozen and lyophilised version of the same ELISA kit (Quanticheck, Faculty of Veterinary Science, Budapest, Hungary). Samples were chosen on the basis of their progesterone (P4) concentrations, which were between 1.00 and 20.00 ng/mL. As it is well known, this range has the highest clinical relevance in ovulation diagnosis. Both types of microplates were read at 15-min intervals from the 15th until the 90th minute (min) of incubation, and the results were compared with those of frozen plates at 60 min of incubation as 100 percent. Lyophilised microplates gave on average 18 percent higher results than the frozen version at equal incubation times. The highest difference between lyophilised and frozen samples was observed at 45 and 60 min of incubation. Ninety-four percent of the reaction in the frozen microplate occurred in the first 15 min, and during the subsequent 30 min the reaction seemingly stopped. After the 45th min of incubation, this 94 percent increased to 108 percent in the subsequent 30 min, which remained the final approximate result at the end of the 90 min of incubation. In contrast to the frozen microplate, the measured concentration increased continuously in the lyophilised version and reached the highest level at the 60th min. The results of the lyophilised microplate reached the same level at 30 min of incubation as those of the frozen version at 60 min. In conclusion, a mechanical increase or decrease of the incubation time does not generate a linear change in the test results. This study demonstrated that the results of a series of samples collected from the same bitch cannot be compared if they are measured with different laboratory methods or different ELISA kits.
needed to ensure compliance to the 20 mg kg −1 threshold. While there are no reference methods for gluten analysis, the Codex Alimentarius recommends the enzyme-linked immunosorbent assay (ELISA) using the R5 antibody, but any other with similar
Ochratoxin A, is a well-known nephrotoxic, hepatotoxic and carcinogenic mycotoxin, produced by some species of mould genera such as Aspergillus spp. and Penicillium spp. under various environmental conditions, such as moisture and temperature. The main sources of Ochratoxin A intake for humans are cereals and cereal derived products, when they are consumed in large quantities, as in the case of breakfast cereals and cereal based baby foods principally consumed by babies. In this study, a total of 150 samples (50 infant formulas, 50 follow-on formulas, and 50 cereal based supplementary foods for infants and children) were obtained randomly from various supermarkets and pharmacies in Istanbul, and 52 out of 150 (34.7%) analysed samples were contaminated with Ochratoxin A. None of the examined baby food samples were above the Turkish Food Codex maximum limit of Ochratoxin A in baby, infant, and young children foods (0.5 μg kg−1). These results reinforce the idea of strict and routine quality controls and good hygiene practices have to be performed in every step of production to minimize the potential risk of Ochratoxin A contamination.
