A comparison of cell death mechanisms of antioxidants, butylated hydroxyanisole and butylated hydroxytoluene.

Butylated hydroxyanisole (BHA) and the chemically similar butylated hydroxytoluene (BHT) are widely used as antioxidants. Toxicity of BHA and BHT has been reported under in vitro and in vivo experimental conditions. However, the mechanism of BHA-induced toxic effects in cells is unclear. In this study, the cytotoxic effects of BHA and differences in cell death mechanism for BHA and BHT were investigated in rat thymocytes by flow cytometric analysis using a fluorescent probe. We observed a significant increase in propidium iodide fluorescence in the population of cells treated with 100 µM and 300 µM BHA (dead cells). Thymocytes treated with 100 µM BHA showed increased intracellular Ca2+ and Zn2+ levels and depolarized cell membranes. BHA (30-100 µM) decreased non-protein thiol content of cells, indicating decreased glutathione content. Co-stimulation with 100 µM BHA and 300 µM H2O2 acted synergistically to increase cell lethality. Moreover, BHA significantly increased caspase-3 activity and the number of annexin-V-positive cells in a concentration-dependent manner, indicating apoptosis. However, BHT reduced caspase-3 activity and increased the number of annexin-V-negative dead cells, indicating non-apoptotic cell death. Our results reveal the toxicity of BHA could be attributed to increased levels of intracellular Ca2+ and Zn2+, resulting in an increased vulnerability of rat thymocytes to oxidative stress. In addition, we demonstrate that whereas BHA induced apoptosis, BHT induced non-apoptotic cell death in rat thymocytes. Therefore, these results may support the safety of BHA, but also demonstrate the importance of performing toxicity evaluation at the cellular level besides the tissue level.

Bioactivity of alginetin, a caramelization product of pectin: Cytometric analysis of rat thymic lymphocytes using fluorescent probes.

Alginetin is the major product formed from pentoses and hexurionic acids. Alginetin is producted by cooking process of food including pection, a naturally-occurring polysacharride found in many plants. However, the biological interaction and toxicity of alginetin are not known at all. The aim of the present study was to investigate the cellular actions of alginetin on rat thymic lymphocytes. The effects of alginetin on the cell were examined using flow cytometry with fluorescent probes. Alginetin increased cellular content of non-protein thiols ([NPT]i) and elevated intracellular Zn2+ levels ([Zn2+]i). Chelation of intracellular Zn2+ reduced the effect of alginetin on [NPT]i, and chelation of external Zn2+ almost completely diminished alginetin-induced elevation of [Zn2+]i, indicating that alginetin treatment increased Zn2+ influx. Increased [NPT]i and [Zn2+]i levels in response to alginetin were positively correlated. Alginetin protected cells against oxidative stress induced by hydrogen peroxide and Ca2+ overload by calcium ionophore. It is considered that the increases in [NPT]i and [Zn2+]i are responsible for the cytoprotective activity of alginetin because NPT attenuates oxidative stress and Zn2+ competes with Ca2+. Alginetin may be produced during manufacturing of jam, which may provide additional health benefits of jam.

Change in membrane potential induced by streptolysin O, a pore-forming toxin: flow cytometric analysis using a voltage-sensitive fluorescent probe and rat thymic lymphocytes.

Streptolysin O (SLO) is a bacterial pore-forming toxin that is employed to permeabilize cell membranes in some biological experiments. SLO forms various types of pores with different shapes, increasing membrane ion permeability and subsequently inducing changes in membrane potential. To characterize the pores formed by SLO, the changes in membrane potential induced by SLO in rat lymphocytes were considered using flow cytometry with a voltage-sensitive fluorescent probe, bis-(1,3-dibutylbarbituric acid)trimethine oxonol (Oxonol). SLO caused three types of membrane potential responses accessed with Oxonol. One type induces a great decrease in Oxonol fluorescence (large hyperpolarization) that may be elicited via the increase of Ca2+ -dependent K+ permeability by SLO-induced influx of external Ca2+ . A second type is an increase in Oxonol fluorescence (depolarization) that may be caused by a nonspecific increase in membrane cation permeability. The third type is a small decrease in Oxonol fluorescence (small hyperpolarization), probably via an increase in Cl- permeability. That SLO transitionally changes membrane ion permeability may have implications in the pathology of pyogenic group streptococci infections in which SLO is thought to be one of the key virulence factors.

Cross-allergenicity of crustacean and the edible insect Gryllus bimaculatus in patients with shrimp allergy.

Food scarcity is a serious problem for many developing as well as developed countries. Edible insects have attracted attention recently as a novel food source. Crickets are especially high in nutritional value and easy to breed and harvest. In this study, we evaluated the risk of allergic reactions associated with cricket consumption in individuals with crustacean allergy. We evaluated food allergy risk in the consumption of Gryllus bimaculatus (cricket) in patients with shrimp allergy, using enzyme-linked immunosorbent assay (ELISA) and IgE crosslinking-induced luciferase expression assay (EXiLE). Sera from individuals with shrimp allergy (positive for shrimp-specific IgE by ImmunoCAP (>0.35 UA/mL; n = 9) or without shrimp allergy (negative for shrimp-specific IgE; n = 6) were obtained. There was a strong correlation between shrimp- and Gryllus-specific IgE levels obtained by ELISA (rs = 0.99; P < 0.001). The binding of shrimp-specific IgE on shrimp allergen was dose-dependently inhibited by Gryllus allergen (0-1.0 mg/mL). There was a strong correlation between shrimp- and Gryllus-specific IgE responses, as assessed by EXiLE assays (rs = 0.89; P < 0.001). We determined that a protein of approximately 40 kDa reacted with the positive, but not negative, sera for shrimp-specific IgE by ImmunoCAP. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified the major allergen in shrimp and Gryllus to be tropomyosin. Our data suggest that the cricket allergen has the potential to induce an allergic reaction in individuals with crustacean allergy. Therefore, allergy risk and shrimp-specific IgE levels should be considered before consumption of cricket meal.

Methylcyclopentadienyl manganese tricarbonyl increases cell vulnerability to oxidative stress on rat thymocytes.

Methylcyclopentadienyl manganese tricarbonyl (MMT) is used as a gasoline antiknock additive. However, the toxic effect of MMT is currently not well understood. In this study, we investigated the toxic effect of MMT on rat thymocytes using a flow cytometer and fluorescent probes. MMT at 100-300 µM significantly increased the population of cells exhibiting propidium fluorescence, i.e., the population of dead cells. The intensity of BES-So-AM fluorescence significantly increased when using 100 µM MMT. In addition, the intensity of oxonol fluorescence in rat thymocytes increased with the treatment with MMT in a concentration-dependent manner (10-100 µM). The toxic effect of MMT was inhibited by quercetin, antioxidant flavonoid. Moreover, co-treatment with 30-100 µM MMT and 100 µM H2O2 increased the cell lethality further. These results indicate that MMT increases cell vulnerability to oxidative stress on rat thymocytes. This study provides insight into the toxic effect of MMT on the immune system.

Cytotoxicity of benzophenone-3, an organic ultraviolet filter, caused by increased intracellular Zn2+ levels in rat thymocytes.

Benzophenone-3 (BP-3) is often used as an ultraviolet (UV) light filter in sunscreen products. Although BP-3 protects the human skin and hair from damage caused by excessive UV radiation, it is reported to exhibit toxic effects in human. However, the cytotoxicity of BP-3 on various cells is still not well understood. In the present study, the cytotoxicity of BP-3 against rat thymocytes was evaluated using a flow cytometric technique with fluorescent probes. Cell mortality increased significantly after 3 h of exposure to 300 µM BP-3, whereas the mean intensity of 5-chloromethylfluorescein diacetate (5-CMF) fluorescence and cellular content of non-protein thiols decreased significantly. However, the membrane potential of thymocytes was not change by BP-3 treatment. Moreover, intracellular Zn2+ levels increased significantly in a concentration-dependent manner in response to 30 µM BP-3 or higher. The BP-3-induced changes in intracellular Zn2+ levels and non-protein thiol content increased the vulnerability of thymocytes to oxidative stress. We concluded that BP-3-induced cytotoxicity may be caused by oxidative stress associated with an increase in intracellular Zn2+ levels.

Biphenyl-induced cytotoxicity is mediated by an increase in intracellular Zn2.

Biphenyl is found both in natural and anthropogenic sources and is used as a fungistat in the packaging of citrus fruits. Acute exposure to high levels of biphenyl has been observed to cause skin irritation and toxic effects on the liver and kidneys. However, the mechanisms of cytotoxicity induced by biphenyl are not yet well understood. In the present study, the cytotoxicity of biphenyl was studied by flow cytometry with fluorescent probes. Biphenyl at 100 µM significantly increased cell lethality after 3 h in rat thymocytes. In addition, biphenyl at 100 µM or more elevated intracellular Zn2+ levels. N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), an intracellular and extracellular Zn2+ chelator, but not diethylenetriamine-N,N,N',N″,N″-pentaacetic acid (DTPA), a membrane-impermeable Zn2+ chelator, attenuated the biphenyl-induced increase in intracellular Zn2+ levels and cell death. These results suggested that biphenyl-induced cytotoxicity caused an increase in intracellular Zn2+ levels, which was dependent on internal Zn2+. Moreover, biphenyl led to an increase in sensitivity to oxidative stress, while TPEN inhibited this biphenyl-induced increase. Our findings revealed that biphenyl caused an increase in the intracellular free Zn2+ concentration, inducing cytotoxicity, cell death, and an increase in sensitivity to oxidative stress.

Captan-induced increase in the concentrations of intracellular Ca2+ and Zn2+ and its correlation with oxidative stress in rat thymic lymphocytes.

Captan, a phthalimide fungicide, is considered to be relatively nontoxic to mammals. There is a possibility that captan affects membrane and cellular parameters of mammalian cells, resulting in adverse effects, because of high residue levels. To test the possibility, we examined the effects of captan on rat thymic lymphocytes using flow-cytometry with appropriate fluorescent probes. Treatment with 10 and 30 µM captan induced apoptotic and necrotic cell death. Before cell death occurred, captan elevated the intracellular concentrations of Ca2+ and Zn2+ and decreased the concentration of cellular thiol compounds. These captan-induced phenomena are shown to cause cell death and are similar to those caused by oxidative stress. Captan also elevated the cytotoxicity of hydrogen peroxide. Results indicate that 10 and 30 µM captan cause cytotoxic effects on mammalian cells. Despite no report on the significant environmental toxicity hazard of captan in humans, it may exhibit adverse effects, described above, on wild organisms.

Change in plasma membrane potential of rat thymocytes by tert-butylhydroquinone, a food additive: Possible risk on lymphocytes.

Tertiary butylhydroquinone (TBHQ) is a food additive and has various beneficial actions under in vitro and in vivo experimental conditions. Therefore, it is necessary to collect additional data on the toxicity of TBHQ in order to avoid adverse effects during clinical applications. Changes in plasma membrane potential are associated with changes in physiological functions even in non-excitable cells such as lymphocytes. Thus, compounds that affect membrane potential may modify some lymphocytic functions. The effect of TBHQ on plasma membrane potential was examined in rat thymocytes using flow cytometric techniques. Treatment of rat thymocytes with TBHQ caused hyperpolarization and then depolarization. The TBHQ-induced hyperpolarization was due to the activation of Ca2+-dependent K+ channels. TBHQ elevated intracellular Ca2+ levels. The depolarization by TBHQ was caused by a nonspecific increase in membrane ionic permeability. Both the sustained depolarization and elevation of intracellular Ca2+ level by TBHQ are thought to be adverse for thymocytes because such changes disturb membrane and intracellular signaling. The thymus is most active during neonatal and pre-adolescent periods. If TBHQ exerts adverse actions on thymocytes, it may result in an immunotoxic effect in neonates and adolescents.

Detection of ovomucoid-specific low-affinity IgE in infants and its relationship to eczema.

BACKGROUND: Allergen-specific low-affinity IgE was previously detected in cord blood by a highly sensitive densely carboxylated protein (DCP) chip, but not by ImmunoCAP. Here, we investigated the presence of low-affinity IgE during the early life of infants and observed its relationship with eczema. METHODS: We conducted a birth cohort study, collecting sera at birth and 6 and 14 months of age (n = 110). We monitored the ovomucoid (OM)- and egg white (EW)-specific IgE (sIgE) by ImmunoCAP or DCP chip and analyzed the antigen affinity of sIgE by binding inhibition assays in the presence or absence of a mild chaotropic agent, diethyl amine (DEA). The low- and high-affinity OM-sIgEs and sensitization risk factors were analyzed by a multivariate logistic analysis. RESULTS: The OM-sIgE measured by DCP chip significantly correlated with that measured by ImmunoCAP, but some samples assessed as OM-sIgE positive by DCP chip were considered OM-sIgE negative by ImmunoCAP. Binding inhibition analysis after DEA treatment was performed for participants judged as OM-sIgE positive by DCP chip at 14 M. The group assessed as negative for OM- and EW-sIgE by ImmunoCAP at 6 and 14 months showed a larger binding inhibition curve shift after DEA treatment than did the group assessed as positive at these times, indicating the presence of low-affinity sIgE antibodies at 14 months. The logistic regression analysis found that persistent eczema from 6 to 14 months is a significant risk factor for developing high-affinity, but not low-affinity, sIgE. CONCLUSIONS: Human infant peripheral blood contains allergen-specific low-affinity sIgE. Persistent eczema is related to the development of high-affinity, but not low-affinity, IgE.

Type II cGMP-dependent protein kinase negatively regulates fibroblast growth factor signaling by phosphorylating Raf-1 at serine 43 in rat chondrosarcoma cells.

Although type II cGMP-dependent protein kinase (PKGII) is a major downstream effector of cGMP in chondrocytes and attenuates the FGF receptor 3/ERK signaling pathway, its direct target proteins have not been fully explored. In the present study, we attempted to identify PKGII-targeted proteins, which are associated with the inhibition of FGF-induced MAPK activation. Although FGF2 stimulation induced the phosphorylation of ERK1/2, MEK1/2, and Raf-1 at Ser-338 in rat chondrosarcoma cells, pretreatment with a cell-permeable cGMP analog strongly inhibited their phosphorylation. On the other hand, Ser-43 of Raf-1 was phosphorylated by cGMP in a dose-dependent manner. Therefore, we examined the direct phosphorylation of Raf-1 by PKGII. Wild-type PKGII phosphorylated Raf-1 at Ser-43 in a cGMP-dependent manner, but a PKGII D412A/R415A mutant, which has a low affinity for cGMP, did not. Finally, we found that a phospho-mimic mutant, Raf-1 S43D, suppressed FGF2-induced MAPK pathway. These results suggest that PKGII counters FGF-induced MEK/ERK activation through the phosphorylation of Raf-1 at Ser-43 in chondrocytes.

Diverse cellular actions of tert-butylhydroquinone, a food additive, on rat thymocytes.

Tertiary butylhydroquinone (TBHQ) is a food additive that possesses antioxidant activity. Its alternative applications have been explored in recent studies. However, there is controversy regarding safety. In this study using rat thymocytes, the cellular actions of TBHQ at sublethal concentrations were examined. TBHQ at concentrations of 3 µM or more elevated intracellular Zn2+ concentration ([Zn2+]i) in a dose-dependent manner, by increasing membrane Zn2+ permeability and releasing Zn2+ from cellular stores. TBHQ at 30 µM significantly increased side scatter (cell density) and the exposure of phosphatidylserine (PS) on cell membrane surfaces. It also decreased cellular glutathione (GSH) content without affecting cell lethality. Forward scatter was attenuated by 100 µM TBHQ. Thus, it is considered that TBHQ at sublethal concentrations (30 µM or less) exerts some adverse actions on cells. TBHQ at 10-30 µM attenuated the increase in cell lethality induced by hydrogen peroxide (H2O2), while potentiation of H2O2 cytotoxicity by 100 µM TBHQ was observed. The range of concentrations of TBHQ from benefit to toxicity under in vitro conditions may be 10-30 µM. Although TBHQ exhibits antioxidative actions at concentrations that are lower than those which elicit adverse cellular effects, sublethal levels of TBHQ cause some adverse actions that may be clinically concerned.

Differential response in allergen-specific IgE, IgGs, and IgA levels for predicting outcome of oral immunotherapy.

BACKGROUND: Oral immunotherapy (OIT) induces desensitization and/or tolerance in patients with persistent food allergy, but the biomarkers of clinical outcomes remain obscure. Although OIT-induced changes in serum allergen-specific IgE and IgG4 levels have been investigated, the response of other allergen-specific IgG subclasses and IgA during OIT remains obscure. METHODS: A pilot study was conducted to investigate egg OIT-induced changes in allergen-specific IgE, IgG subclasses, and IgA levels and search for possible prediction biomarkers of desensitization. We measured serum levels of egg white-, ovomucoid-, and ovalbumin-specific IgE, IgA, and IgG subclasses by high-sensitivity allergen microarray in 26 children with egg allergy who received rush OIT. RESULTS: Allergen-specific IgE gradually decreased while IgG4 increased during 12-month OIT. Serum levels of IgG1, IgG3, and IgA increased significantly after the rush phase, then decreased during the maintenance phase. IgG2 levels changed in a manner similar to that of IgG4. In particular, significantly high fold increases in egg white-specific IgG1, relative to baseline, after the rush phase and high IgA levels before OIT were observed in responders, compared with low-responders to OIT. Patients who could not keep desensitization showed relatively small changes in all immunoglobulin levels during OIT. CONCLUSION: The response to OIT was associated with significant increases in serum allergen-specific IgG1 levels after rush phase and high baseline IgA levels, compared with small changes in immunoglobulin response in low-responders. The characteristic IgG1 changes and IgA levels in the responders could be potentially useful biomarkers for the prediction of positive clinical response to OIT.

Measurement of allergen-specific secretory IgA in stool of neonates, infants and toddlers by protection against degradation of immunoglobulins and allergens.

BACKGROUND AND AIMS: Measurement of secretory immunoglobulin A (SIgA) level is important to monitor various disease conditions. However SIgA in gut mucosa is degraded by pancreatic proteases and proteolytic enzymes from enteric microbiota. Currently, there is no reliable quantitation method that measures allergen-specific SIgA levels in stool of neonates, infants and toddlers. METHODS: Allergen-specific SIgA levels in stool of 10 healthy neonates, infants and toddlers aged 0 to 36 months were measured by our new allergen microarray with densely carboxylated arms on a glass slide chip. RESULTS: Protease activities in stool of 3-day-old neonates were low and no degradation of SIgA, IgA and allergens was detected. However, immunofluorescence signals of SIgA, IgA and allergen on the chip were markedly reduced by stool extracts obtained from infants and toddlers aged more than one month in dose- and time-dependent manners. Such reduction was almost completely inhibited by serine protease inhibitors, phenylmethylsulfonyl fluoride (PMSF) and partly by leupeptin, but not by a variety of other protease inhibitors tested. CONCLUSION: Allergen-specific SIgA levels in stool of neonates, infants and toddlers under 36 months of age could be analyzed using protease inhibitors, including PMSF and leupeptin.

Application of moisturizer to neonates prevents development of atopic dermatitis.

BACKGROUND: Recent studies have suggested that epidermal barrier dysfunction contributes to the development of atopic dermatitis (AD) and other allergic diseases. OBJECTIVE: We performed a prospective, randomized controlled trial to investigate whether protecting the skin barrier with a moisturizer during the neonatal period prevents development of AD and allergic sensitization. METHODS: An emulsion-type moisturizer was applied daily during the first 32 weeks of life to 59 of 118 neonates at high risk for AD (based on having a parent or sibling with AD) who were enrolled in this study. The onset of AD (eczematous symptoms lasting >4 weeks) and eczema (lasting >2 weeks) was assessed by a dermatology specialist on the basis of the modified Hanifin and Rajka criteria. The primary outcome was the cumulative incidence of AD plus eczema (AD/eczema) at week 32 of life. A secondary outcome, allergic sensitization, was evaluated based on serum levels of allergen-specific IgE determined by using a high-sensitivity allergen microarray of diamond-like carbon-coated chips. RESULTS: Approximately 32% fewer neonates who received the moisturizer had AD/eczema by week 32 than control subjects (P = .012, log-rank test). We did not show a statistically significant effect of emollient on allergic sensitization based on the level of IgE antibody against egg white at 0.34 kUA/L CAP-FEIA equivalents. However, the sensitization rate was significantly higher in infants who had AD/eczema than in those who did not (odds ratio, 2.86; 95% CI, 1.22-6.73). CONCLUSION: Daily application of moisturizer during the first 32 weeks of life reduces the risk of AD/eczema in infants. Allergic sensitization during this time period is associated with the presence of eczematous skin but not with moisturizer use.

Intrauterine sensitization of allergen-specific IgE analyzed by a highly sensitive new allergen microarray.

BACKGROUND: To design a rational allergy prevention program, it is important to determine whether allergic sensitization starts in utero under the maternal immune system. OBJECTIVE: To investigate the origin of allergen-specific IgE antibodies in cord blood (CB) and maternofetal transfer of immunoglobulins. METHODS: The levels of food and inhalant allergen-specific IgE, IgA, IgG, and IgG(4) antibodies in CB and maternal blood (MB) from 92 paired neonates and mothers were measured by using a novel allergen microarray of diamond-like-carbon-coated chip, with high-sensitivity detection of allergen-specific antibodies and allergen profiles. RESULTS: The levels of allergen-specific IgE antibodies against food and inhalant allergens and allergen profiles were identical in CB and newborn blood, but the levels and profiles, specifically against inhalant allergens, were different from those in MB. The level of allergen-specific IgA antibodies was below the detection levels in CB despite clear detection in MB. Therefore, contamination with MB in CB was excluded on the basis of extremely low levels of IgA antibodies in CB and the obvious mismatch of the allergen-specific IgE and IgA profiles between CB and MB. However, the levels of allergen-specific IgG and IgG(4) antibodies and their allergen profiles were almost identical in both MB and CB. CONCLUSION: Allergen-specific levels of IgE and IgA antibodies and their allergen profiles analyzed by the diamond-like-carbon allergen chip indicate that IgE antibodies in CB are of fetal origin. Food-allergen specific IgE antibodies were detected more often than inhalant-allergen specific IgE antibodies in CB, the reason of which remains unclarified.

L-tryptophan suppresses rise in blood glucose and preserves insulin secretion in type-2 diabetes mellitus rats.

Ample evidence indicates that a high-protein/low-carbohydrate diet increases glucose energy expenditure and is beneficial in patients with type-2 diabetes mellitus (T2DM). The present study was designed to investigate the effects of L-tryptophan in T2DM. Blood glucose was measured by the glucose dehydrogenase assay and serum insulin was measured with ELISA in both normal and hereditary T2DM rats after oral glucose administration with or without L-D-tryptophan and tryptamine. The effect of tryptophan on glucose absorption was examined in the small intestine of rats using the everted-sac method. Glucose incorporation in adipocytes was assayed with [(3)H]-2-deoxy-D-glucose using a liquid scintillation counter. Indirect computer-regulated respiratory gas-assay calorimetry was applied to assay energy expenditure in rats. L-Tryptophan suppressed both serum glucose and insulin levels after oral glucose administration and inhibited glucose absorption from the intestine. Tryptamine, but not L-tryptophan, enhanced insulin-stimulated [(3)H]-glucose incorporation into differentiated adipocytes. L-Tryptophan increased glucose-associated energy expenditure in rats in vivo. L-Tryptophan-rich chow consumed from a young age preserved the secretion of insulin and delayed the progression of T2DM in hereditary diabetic rats. The results suggested that L-tryptophan suppresses the elevation of blood glucose and lessens the burden associated with insulin secretion from ß-cells.

Allergen diagnosis microarray with high-density immobilization capacity using diamond-like carbon-coated chips for profiling allergen-specific IgE and other immunoglobulins.

The diagnosis of antibody-mediated allergic disorders is based on clinical findings, skin prick tests and detection of allergen-specific IgE in serum. Here, we present a new microarray technique of high-density antigen immobilization using carboxylated arms on the surface of a diamond-like carbon (DLC)-coated chip. High immobilization capacity of antigen on DLC chip at (0.94-7.82)×10(9) molecules mm(-2) allowed the analysis of allergen-specific immunoglobulins against not only purified proteins but also natural allergen extracts with wide assay dynamic range. The higher sensitivity of the allergen-specific IgE detection on DLC chip was observed for comparison with the UniCAP system: the DLC chip allowed lowering the limit of dilution rate in UniCAP system to further dilution at 4-8-fold. High correlations (ρ>0.9-0.85) of allergen-specific IgE values determined by the DLC chip and UniCAP were found in most of 20 different allergens tested. The DLC chip was useful to determine allergen-induced antibodies of IgA, IgG, IgG1, and IgG4 in sera, apart from IgE, as well as secretory IgA in saliva against the same series of allergens on the chip in a minimal amount (1-2 µL) of sample.