A harmonized immunoassay with liquid chromatography-mass spectrometry analysis in egg allergen determination.

Food allergy is a serious health issue worldwide. Implementing allergen labeling regulations is extremely challenging for regulators, food manufacturers, and analytical kit manufacturers. Here we have developed an "amino acid sequence immunoassay" approach to ELISA. The new ELISA comprises of a monoclonal antibody generated via an analyte specific peptide antigen and sodium lauryl sulfate/sulfite solution. This combination enables the antibody to access the epitope site in unfolded analyte protein. The newly developed ELISA recovered 87.1%-106.4% ovalbumin from ovalbumin-incurred model processed foods, thereby demonstrating its applicability as practical egg allergen determination. Furthermore, the comparison of LC-MS/MS and the new ELISA, which targets the amino acid sequence conforming to the LC-MS/MS detection peptide, showed a good agreement. Consequently the harmonization of two methods was demonstrated. The complementary use of the new ELISA and LC-MS analysis can offer a wide range of practical benefits in terms of easiness, cost, accuracy, and efficiency in food allergen analysis. In addition, the new assay is attractive in respect to its easy antigen preparation and predetermined specificity. Graphical abstract The ELISA composing of the monoclonal antibody targeting the amino acid sequence conformed to LC-MS detection peptide, and the protein conformation unfolding reagent was developed. In ovalbumin determination, the developed ELISA showed a good agreement with LC-MS analysis. Consequently the harmonization of immunoassay with LC-MS analysis by using common target amino acid sequence was demonstrated.

Food allergen analysis for processed food using a novel extraction method to eliminate harmful reagents for both ELISA and lateral-flow tests.

Enzyme-linked immunosorbent assay (ELISA) is commonly used to determine food allergens in food products. However, a significant number of ELISAs give an erroneous result, especially when applied to highly processed food. Accordingly, an improved ELISA, which utilizes an extraction solution comprising the surfactant sodium lauryl sulfate (SDS) and reductant 2-mercaptoethanol (2-ME), has been specially developed to analyze food allergens in highly processed food by enhancing analyte protein extraction. Recently, however, the use of 2-ME has become undesirable. In the present study, a new extraction solution containing a human- and eco-friendly reductant, which is convenient to use at the food manufacturing site, has been established. Among three chemicals with different reducing properties, sodium sulfite, tris(3-hydroxypropyl)phosphine, and mercaptoethylamine sodium sulfite was selected as a 2-ME substitute. The protein extraction ability of SDS/0.1 M sodium sulfite solution was comparable to that of SDS/2-ME solution. Next, the ELISA performance for egg, milk, wheat, peanut, and buckwheat was evaluated by using model-processed foods and commercially available food products. The data showed that the SDS/0.1 M sulfite ELISA significantly correlated with the SDS/2-ME ELISA for all food allergens examined (p < 0.01), thereby establishing the validity of the SDS/0.1 M sulfite ELISA performance. Furthermore, the new SDS/0.1 M sulfite solution was investigated for its applicability to the lateral-flow (LF) test. The result demonstrated the successful analysis of food allergens in processed food, showing consistency with the SDS/0.1 M sulfite ELISA results. Accordingly, a harmonized analysis system for processed food comprising a screening LF test and a quantitative ELISA with identical extraction solution has been established. The ELISA based on the SDS/0.1 M sulfite extraction solution has now been authorized as the revised official method for food allergen analysis in Japan.

Serum gliadin monitoring extracts patients with false negative results in challenge tests for the diagnosis of wheat-dependent exercise-induced anaphylaxis.

BACKGROUND: Challenge testing with wheat plus exercise and/or aspirin is a gold standard for the diagnosis of wheat-dependent exercise-induced anaphylaxis (WDEIA); however, the test may often yield false-negative results. Our previous study suggested that an increase in serum wheat gliadin levels is required to induce allergic symptoms in patients with WDEIA. Based on this knowledge, we sought to extract the patients with false negative results in the challenge tests of WDEIA. METHODS: Thirty-six patients with suspected WDEIA were enrolled. First, group categorizations-Group I, challenge tests were positive; Group II, challenge tests were negative and serum gliadin were undetectable; Group III, challenge tests were negative and serum gliadin were detectable-were given according to the results of wheat plus exercise and/or aspirin challenge testing and serum gliadin levels. Second, diagnoses were made using retests and/or dietary management in Group II and III. RESULTS: Positive results for wheat plus exercise and/or aspirin challenge tests gave a diagnosis of definite WDEIA in 17 of 36 patients (Group I). Of the remaining 19 challenge negative patients, serum gliadin was undetectable in ten patients (Group II). Of the ten patients (Group II), three of them were diagnosed as definite WDEIA by retesting and six of them were diagnosed as probable WDEIA using a wheat elimination diet, whereas one patient was non-WDEIA. In the rest of the nine challenge negative patients, serum gliadin was detectable (Group III). No allergic episodes with a normal diet provided a diagnosis of non-WDEIA in seven of the nine patients, whereas the remaining two patients were probable WDEIA or had another food allergy because of repeated episodes. CONCLUSIONS: Our study revealed that serum gliadin monitoring during challenge testing is useful.

A glass fiber sheet-based electroosmotic lateral flow immunoassay for point-of-care testing.

We have developed a quantitative immunoassay chip targeting point-of-care testing. To implement a lateral flow immunoassay, a glass fiber sheet was chosen as the material for the microfluidic channel in which the negative charge on the fiber surfaces efficiently generates the electroosmotic flow (EOF). The EOF, in turn, allows controllable bound/free separation of antigen/antibody interactions on the chip and enables precise determination of the antigen concentration. In addition, the defined size of the porous matrix was suitable for the filtration of undesired large particles. We confirmed the linear relationship between the concentration of analyte and the resulting fluorescence intensity from the immunoassay of two model analytes, C-reactive protein (CRP) and insulin, demonstrating that analyte concentration was quantitatively determined within the developed chip in 20 min. The limits of detection were 8.5 ng mL(-1) and 17 ng mL(-1) for CRP and insulin, respectively.

Plasma leptin concentration in dogs with diabetes mellitus.

The plasma leptin concentration was evaluated in dogs with diabetes mellitus. Twenty normal and sixteen diabetic dogs were divided into nonobese and obese groups based on body condition score, respectively. The obese normal dogs had significantly higher plasma leptin concentrations than the nonobese normal dogs, whereas there was no significant difference between the nonobese and obese diabetic dogs. In addition, the plasma leptin concentration in the obese diabetic dogs was significantly lower than that in the obese normal dogs. In conclusion, the plasma leptin concentrations in the diabetic dogs were affected by factors other than adiposity.

Rokitamycin induces a mitochondrial defect and caspase-dependent apoptosis in human T-cell leukemia Jurkat cells.

Macrolides are a well-known family of oral antibiotics whose antibacterial spectrum of activity covers most relevant bacterial species responsible for respiratory infectious disease. In recent years, it has been reported that macrolides have not only bactericidal activity but also direct immunomodulating activity in mammals. In this study, we observed new physiological activity of macrolides and examined whether various macrolides induce apoptosis in human leukemia cell lines. We investigated the effects of 13 different macrolides on the viability of Jurkat and HL-60 cells. Among all the macrolides used in this study, rokitamycin, a semisynthetic macrolide with a 16-member ring, effectively induced cell death. Rokitamycin induced DNA fragmentation and caspase activation, resembling the progression of apoptosis. Moreover, rokitamycin reduced the mitochondrial transmembrane potential and released cytochrome c from mitochondria to the cytosol, suggesting that mitochondrial perturbation is involved in rokitamycin-induced apoptosis. These results suggest that rokitamycin possesses not only bactericidal activity but also pro-apoptotic activity in human leukemia cells.

Absorption, migration and kinetics in peripheral blood of orally administered ovalbumin in a mouse model.

Intestinal absorption of food proteins is well known, whereas its physiological significance remains to be investigated. Various amounts (1, 10 and 50 mg) of ovalbumin were orally administered to mice and the blood kinetics were subsequently analyzed by two-site ELISA. The blood ovalbumin concentration consistently reached its maximum (7-90 ng/ml) about 20 min after the oral administration and then gradually decreased in a dose-dependent manner. Only intact (45 kDa) and truncated (40 kDa) ovalbumins were always detected in the blood independently of the administration site, intra-stomach or intra-intestine, while various fragments of the protein were observed in the gastrointestinal lumen after the oral administration. Recognition by a specific monoclonal antibody and an acidic shift of its pI value suggested that the 40-kDa truncated ovalbumin was produced by intracellular limited proteolysis at its C-terminus. Such stable absorption and blood kinetics of undigested ovalbumin in normal mice suggest some sort of physiological significance for the intestinal uptake of intact food proteins.

The IL-6 family of cytokines modulates STAT3 activation by desumoylation of PML through SENP1 induction.

Post-translational modification by small ubiquitin-like modifier (SUMO) plays an important role in the regulation of different signaling pathways and is involved in the formation of promyelocytic leukemia (PML) protein nuclear bodies following sumoylation of PML. In the present study, we found that IL-6 induces desumoylation of PML and dissociation between PML and SUMO1 in hepatoma cells. We also found that IL-6 induces mRNA expression of SENP1, a member of the SUMO-specific protease family. Furthermore, wild-type SENP1 but not an inactive SENP1 mutant restored the PML-mediated suppression of STAT3 activation. These results indicate that the IL-6 family of cytokines modulates STAT3 activation by desumoylation and inactivation PML through SENP1 induction.

LIF- and IL-6-induced acetylation of STAT3 at Lys-685 through PI3K/Akt activation.

Signal transducer and activator of transcription 3 (STAT3), which mediates biological actions in many physiological processes, is activated by cytokines and growth factors via specific tyrosine or serine phosphorylation, dimerization and nuclear translocation. A recent study has demonstrated, by using antibody to acetylated lysine, and a STAT3 mutant with Lys-685-to-Arg substitution, that STAT3 is acetylated at Lys-685 by histone acetyltransferase p300, and that acetylation at Lys-685 is critical for STAT3 activation. In the present study, we created an acetyl-specific antibody against STAT3 acetylated at Lys-685, and found that leukemia inhibitory factor (LIF) or interleukin (IL)-6 induced acetylation of STAT3 at Lys-685 in 293T and Hep3B cells. Moreover, acetylation of STAT3 at Lys-685 was suppressed by PI3K inhibitor LY294002, or a dominant negative Akt. Taken together, our findings demonstrate that endogenous STAT3 is acetylated at Lys-685 by LIF or IL-6 through PI3K/Akt activation.

Effects of chronic obesity and weight loss on plasma ghrelin and leptin concentrations in dogs.

The objective of this study was to evaluate, in dogs, the effects of obesity and weight loss on plasma total ghrelin and leptin concentrations. Twenty-four Beagle dogs, 12 control lean and 12 obese dogs of both genders and aged between 1 and 9 years, were used for the experiments. Mean body weight was 12.7+/-0.7 kg for the lean group and 21.9+/-0.8 kg for the obese group. The trial was divided into three phases. During phase 1, all 24 Beagle dogs were fed a maintenance diet. During phase 2, the obese dogs were submitted to a weight loss protocol with a high protein-low energy diet. The weight loss protocol ended once dogs reached optimal body weight. During phase 3, the dogs that were submitted to the weight loss protocol were maintained at their optimal body weight for 6 months. Plasma total ghrelin, leptin, insulin and glucose concentrations were measured to evaluate the effects of obesity and weight loss on these parameters in dogs. Body weight, body condition score, thoracic and pelvic perimeters, and ingested food amounts were also recorded during the study. Obese dogs demonstrated a significant decrease in plasma ghrelin and a significant increase in plasma leptin and insulin concentrations when compared with control dogs. During weight loss, significant increases in plasma total ghrelin and glucose and significant decreases in plasma leptin and insulin were observed. The increase in plasma ghrelin concentrations seemed to be transient. Body weight and the morphometric parameters correlated positively with leptin concentrations and negatively with total ghrelin concentrations. These results suggest that ghrelin and leptin could play a role in dogs in the adaptation to a positive or negative energy balance, as observed in humans.

Seasonal changes in serum leptin of the feral raccoon (Procyon lotor) determined by canine-leptin-specific ELISA.

Several reports have been published on blood leptin concentrations in feral animals, including members of the Carnivora, using a commercially available multi-species radioimmunoassay (RIA) kit with anti-human leptin antibody. However, we observed weak immunoreactivity between recombinant canine leptin and anti-human leptin antibody, suggesting a limitation in the applicability of the RIA kit for leptin assays in Carnivora species. We tested the applicability of RIA and sandwich enzyme-linked immunosorbent assay (ELISA) with anti-canine leptin antibody to assay blood leptin in the dog (Canis familiaris) and the raccoon (Procyon lotor). When RIA was used for recombinant canine leptin and dog sera, values were much lower than those determined by ELISA at higher concentrations (>10 ng/ml), while rather higher at lower concentrations (<2 ng/ml). A similar discrepancy between the two methods was found for serum leptin concentrations in raccoons. Clear seasonal variations were observed by ELISA, but not by RIA, with high values in autumn (3.46+/-0.45 ng/ml) and low values in spring and summer (0.71+/-0.07 ng/ml). Serum leptin concentrations in raccoons correlated positively with their body weight (r=0.753) and body mass index (r=0.755), corroborating our previous findings of a strong positive correlation between serum leptin concentrations and body fat content in dogs. Thus, the canine leptin ELISA is useful for assays of dog and raccoon leptin, and blood leptin is a good marker of nutritional condition in the species of Carnivora assayed in this study.

Selenoprotein expression in Hürthle cell carcinomas and in the human Hürthle cell carcinoma line XTC.UC1.

Hürthle cell carcinomas (HTC) are characterized by mitochondrial amplification and enhanced oxygen metabolism. To clarify if defects in enzymes scavenging reactive oxygen species are involved in the pathogenesis of HTC, we analyzed selenium (Se)-dependent expression of various detoxifying selenoproteins in the HTC cell line XTC.UC1. Glutathione peroxidase and thioredoxin reductase activity was found both in cell lysates and conditioned media of XTC.UC1 cells and was increased by Na(2)SeO(3). Western blot analysis demonstrated the presence of thioredoxin reductase both in cell lysates and conditioned media and of glutathione peroxidase 3 in conditioned media. Type I 5'-deiodinase, another selenoprotein that catalyzes thyroid hormone metabolism, was detectable only in cell lysates by enzyme assay and Western blot, and responded to stimulation by both Na(2)SeO(3) and retinoic acid. A selenoprotein P signal was detected in conditioned media by Western blot, but was not enhanced by Na(2)SeO(3) treatment. In situ hybridization revealed glutathione peroxidase mRNAs in HTC specimen; glutathione peroxidase 3 mRNA levels were reduced. These data suggest adequate expression and Se-dependent regulation of a couple of selenoproteins involved in antioxidant defense and thyroid hormone metabolism in XTC.UC1 cells, so far giving no evidence of a role of these proteins in the pathogenesis of HTCs.

Novel ELISA for the detection of raw and processed egg using extraction buffer containing a surfactant and a reducing agent.

Enzyme-linked immunosorbent assay (ELISA) has been considered extremely useful for the detection of markers of allergenic substances in food, because it is simple, offers a suitable sensitivity, and is useful in providing quantitative results. Allergenic protein present in processed food can be denatured or altered, hindering therefore their possibility to be extracted and detected. This paper reports the development of an ELISA method that can be used for the determination of allergenic proteins in buffer solutions containing SDS, a surfactant, and 2-mercaptoethanol, a reducing agent. Measurement by ELISA in solutions containing 1% SDS and 7% 2-mercaptoethanol has been made possible by using an antibody prepared through immunization with an antigen denatured with SDS and 2-mercaptoethanol. This ELISA technique can be used to measure proteins in food that have been denatured by various manufacturing processes. An example is egg white albumin, which is susceptible to heat denaturation and has been difficult to recover from food in the past. Its recovery was improved 10- to 100-fold by the new ELISA method as compared with previous methods. This means that allergenic substances in food can now be detected quantitatively. This method can be very useful in allergy prevention and control strategies.

Portal 5-hydroxytryptophan infusion enhances glucose disposal in conscious dogs.

Intraportal serotonin infusion enhances net hepatic glucose uptake (NHGU) during glucose infusion but blunts nonhepatic glucose uptake and can cause gastrointestinal discomfort and diarrhea at high doses. Whether the serotonin precursor 5-hydroxytryptophan (5-HTP) could enhance NHGU without gastrointestinal side effects during glucose infusion was examined in conscious 42-h-fasted dogs, using arteriovenous difference and tracer ([3-3H]glucose) techniques. Experiments consisted of equilibration (-120 to -30 min), basal (-30 to 0 min), and experimental (EXP; 0-270 min) periods. During EXP, somatostatin, fourfold basal intraportal insulin, basal intraportal glucagon, and peripheral glucose (to double the hepatic glucose load) were infused. In one group of dogs (HTP, n = 6), saline was infused intraportally from 0 to 90 min (P1), and 5-HTP was infused intraportally at 10, 20, and 40 microg x kg(-1) x min(-1) from 90 to 150 (P2), 150 to 210 (P3), and 210 to 270 (P4) min, respectively. In the other group (SAL, n = 7), saline was infused intraportally from 0 to 270 min. NHGU in SAL was 14.8 +/- 1.9, 18.5 +/- 2.3, 16.3 +/- 1.4, and 19.7 +/- 1.6 micromol x kg(-1) x min(-1) in P1-P4, whereas NHGU in 5-HTP averaged 16.4 +/- 2.6, 18.5 +/- 1.4, 20.8 +/- 2.0, and 27.6 +/- 2.6 micromol x kg(-1) x min(-1) (P < 0.05 vs. SAL). Nonhepatic glucose uptake (micromol x kg(-1) x min(-1)) in SAL was 30.2 +/- 4.3, 36.8 +/- 5.8, 44.3 +/- 5.8, and 54.6 +/- 11.8 during P1-P4, respectively, whereas in HTP the corresponding values were 26.3 +/- 6.8, 44.9 +/- 10.1, 47.5 +/- 11.7, and 51.4 +/- 13.2 (not significant between groups). Intraportal 5-HTP enhances NHGU without significantly altering nonhepatic glucose uptake or causing gastrointestinal side effects, raising the possibility that a related agent might have a role in reducing postprandial hyperglycemia.

[Inter-laboratory evaluation studies for development of notified ELISA methods for allergic substances (milk)].

Extracts of sausage, sauce, cookie, cereal and pasta sauce spiked with milk standard protein at a level of 5-20 ng/mL as sample solutions were analyzed in replicate in 10 laboratories. Coefficients of variation (CVs) of the three ELISA methods using a Milk Protein Casein ELISA Kit (Casein kit), a Milk Protein Beta-Lactoglobulin ELISA Kit (Beta-Lactoglobulin kit) and a FASTKIT Milk ELISA Kit (Milk ELISA kit) were mostly below 10%. Mean recoveries of the milk standard protein from the food extracts were over 40% in the three ELISA methods with a few excertions. The recoveries of milk standard protein from the sauce extract in Casein kit were improved by adjusting the extract to neutrality before the Casein kit assay. The recoveries of milk standard protein from cookie, cereal and pasta sauce were improved by the increasing the amount of antibody coated in the Milk ELISA kit. The detection limits of all the ELISA methods were 1 ng/mL in sample solutions. These results suggested that the notified ELISA methods are reliable and reproducible for the inspection of milk protein levels in extracts of sausage, sauce, cookie, cereal and pasta sauce.

[Inter-laboratory evaluation studies for development of notified ELISA methods for allergic substances (wheat)].

Extracts of sausage, sauce, pasta sauce, fish paste and cereal spiked with wheat standard protein at a level of 5-20 ng/mL as sample solutions were analyzed in replicate in 10 laboratories. Coefficients of variation (CVs) of both ELISA methods using a Wheat Protein ELISA Kit (Gliadin kit) and a FASTKIT Wheat ELISA Kit (Wheat ELISA kit) were mostly below 10%. Mean recoveries of the wheat standard protein from the food extracts were over 40% in the two ELISA methods except those from cereal extract determined using the Wheat ELISA kit. Repeatability relative standard deviations of wheat standard protein in the five food extracts were in the ranges of 16-26.9% and 3.7-36.2% for the Gliadin kit and the Wheat ELISA kit, respectively. Reproducibility relative standard deviations of wheat standard protein in the five food extracts were 21.6-38.5%, 29.7-53.8% for the Gliadin kit and the Wheat ELISA kit, respectively. The recoveries of wheat standard protein from the cereal extract were improved by the increasing the amount of antibody coated on the plate in the Wheat ELISA kit. The detection limits of both ELISA methods were 1 ng/mL in sample solutions. These results suggested that the notified ELISA methods are reliable and reproducible for the inspection of wheat protein levels in extracts of sausage, sauce, pasta sauce, fish paste and cereal.

[Inter-laboratory evaluation studies for establishment of notified ELISA methods for allergic substances (buckwheat)].

Inter-laboratory evaluation studies were conducted for the ELISA methods for allergic substances (buckwheat). Extracts of snack, bun and udon spiked with buckwheat standard protein at a level of 5-20 ng/mL as sample solutions were analyzed in replicate at 10 laboratories. Coefficients of variation (CVs) of the ELISA methods using a Buckwheat Protein ELISA Kit (Buckwheat kit) and a FASTKIT Buckwheat ELISA kit (Buckwheat ELISA kit) were mostly below 10%. Mean recoveries of the buckwheat standard protein from the food extracts were over 40% in the two ELISA methods. Repeatability relative standard deviations of buckwheat standard protein in three food extracts were in the ranges of 6.8-78.5% and 5.0-33.9% for the Buckwheat kit and the Buckwheat ELISA kit, respectively. Reproducibility relative standard deviations of buckwheat standard protein in three food extracts were 11.9-69.5% and 16.5-34.1% for the Buckwheat kit and the Buckwheat ELISA kit, respectively. The detection limits of both ELISA methods were 1 ng/mL in sample solutions. These results suggest that the notified ELISA methods are reliable and reproducible for the inspection of buckwheat protein levels in extracts of snack, bun and udon.

[Inter-laboratory evaluation studies for establishment of notified ELISA methods for allergic substances (peanuts)].

Inter-laboratory evaluation studies were conducted for ELISA methods for allergic substances (peanuts). Extracts of biscuit, sauce, chocolate and butter spiked with peanut standard protein at a level of 5-20 ng/mL as sample solutions were analyzed in replicate in 10 laboratories. Coefficients of variation (CVs) of the ELISA methods using a Peanut Protein ELISA Kit (Peanut kit) and a FASTKIT Peanut ELISA kit (Peanut ELISA kit) were mostly below 10%. Mean recoveries of the peanut standard protein from the food extracts were over 40% in the two ELISA methods. Repeatability relative standard deviations of peanut standard protein in four food extracts were in the ranges of 15.2-49.7% and 3.0-28.3% for the Peanut kit and the Peanut ELISA kit, respectively. Reproducibility relative standard deviations of peanut standard protein in four food extracts were 23.5-44.4%, 9.6-28.4% for the Peanut kit and the Peanut ELISA kit, respectively. The detection limits of both ELISA methods were 2-2.5 ng/mL in sample solutions. These results suggested that the notified ELISA methods are reliable and reproducible for the inspection of peanut protein levels in extracts of biscuit, sauce, chocolate and butter.