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.

Development of a Model Immunoassay Utilizing Monoclonal Antibodies of Different Specificities for Quantitative Determination of Dieldrin and Heptachlors in Their Mixtures.

The presence of dieldrin and heptachlor residues in cucurbitaceous crops at concentrations exceeding the limits set by the Japanese Food Sanitation Law constitutes a serious problem. To prevent accumulation of these residues in cucurbitaceous crops, development of high-throughput analysis methods for the detection of contaminants in the soil before cultivation is required. This study aimed to develop a model immunoassay using new monoclonal antibodies (MAbs) to quantitatively determine dieldrin and heptachlor contents in their mixtures. Three distinctive MAbs were obtained from mice immunized with the respective immunogens. MAb DrA-04 showed high reactivity toward dieldrin with ca. 20% cross-reactivity toward heptachlors. MAb DrC-02 displayed a similar reactivity toward dieldrin and heptachlors. The specificity and sensitivity of MAbs DrA-04 and DrC-02 were largely unaffected by the composition ratio of heptachlors in a mixture. Six standard mixtures with different dieldrin and heptachlor contents were prepared. Concentrations of dieldrin and heptachlors in standard mixtures, calculated on the basis of an immunoassay with MAbs DrA-04 and DrC-02, were 88.1-125 and 96.2-115% of the theoretical values, respectively, revealing excellent sensitivity and specificity of this assay. The developed method paves the way for a facile and rapid quantitative determination of chlorinated cyclodiene pesticides in soil.

Immunoglobulin treatment ameliorates myocardial injury in experimental autoimmune myocarditis associated with suppression of reactive oxygen species.

AIMS: We tested the hypothesis that immunoglobulin ameliorated experimental autoimmune myocarditis (EAM) in mice attributing to the suppression of reactive oxygen species (ROS)-mediated myocardial injury. METHODS: We intraperitoneally administered intact type of human immunoglobulin (Ig) or F(ab')2 fragments of human immunoglobulin, 1g/kg/day daily for 3 weeks, to male BALB/c mice with heart failure due to EAM. RESULTS: The results showed that intact type of Ig, but not F(ab')2 type, reduced the severity of myocarditis by comparing the heart weight/body weight and lung weight/body weight ratios, pericardial effusion score, macroscopic and microscopic scores. Tissue superoxide production was marked in untreated mice with EAM, which was suppressed by the treatment of immunoglobulins. The cytotoxic activities of lymphocytes in mice with EAM treated with Ig were reduced compared with untreated controls. The shift from Th1 toward Th2 cytokine balance was demonstrated by the treatment of immunoglobulins both in vitro and in vivo. CONCLUSION: ROS may be involved in the development of myocarditis. Intact Ig ameliorates myocardial damage in mice with myocarditis associated with suppression of ROS and cytotoxic activity of lymphocytes.

MCI-186 (edaravone), a novel free radical scavenger, protects against acute autoimmune myocarditis in rats.

In this study, we tested the hypothesis that MCI-186 (3-methyl-1-phenyl-2-pyrazolin-5-one; edaravone), a novel free radical scavenger, protects against acute experimental autoimmune myocarditis (EAM) in rats by the radical scavenging action associated with the suppression of cytotoxic myocardial injury. Recent evidence suggests that oxidative stress may play a role in myocarditis. We administered MCI-186 intraperitoneally at 1, 3, and 10 mg.kg(-1).day(-1) to rats with EAM for 3 wk. The results were compared with untreated rats with EAM. MCI-186 treatment did not affect hemodynamics. MCI-186 treatment (3 and 10 mg.kg(-1).day(-1)) reduced the severity of myocarditis as assessed by comparing the heart-to-body weight ratio and pathological scores. Myocardial interleukin-1beta (IL-1beta)-positive cells and myocardial oxidative stress overload with DNA damage in rats with EAM given MCI-186 treatment were significantly less compared with those of the untreated rats with EAM. In addition, MCI-186 treatment decreased not only the myocardial protein carbonyl contents but also the myocardial thiobarbituric acid reactive substance products in rats with EAM. The formation of hydroxyl radicals in MCI-186-treated heart homogenates was decreased compared with untreated heart homogenates. Furthermore, cytotoxic activities of lymphocytes of rats with EAM treated with MCI-186 were significantly lower compared with those of the untreated rats with EAM. Hydroxyl radicals may be involved in the development of myocarditis. MCI-186 protects against acute EAM in rats associated with scavenging hydroxyl free radicals, resulting in the suppression of autoimmune-mediated myocardial damage associated with reduced oxidative stress state.

Olmesartan, a novel AT1 antagonist, suppresses cytotoxic myocardial injury in autoimmune heart failure.

Some ANG II receptor type 1 (AT(1)) antagonists are reported to inhibit proinflammatory cytokine production in vitro and in vivo. However, the effects of the drugs on autoimmune diseases are unknown. We tested the hypothesis that olmesartan, a novel AT(1) antagonist, ameliorated experimental autoimmune myocarditis (EAM) in rats attributed to the suppression of inflammatory cytokines as well as to the immunomodulatory action of the heart. We administered olmesartan orally at does of 1, 3, and 10 mg.kg(-1).day(-1) to rats with EAM for 3 wk. The results showed that olmesartan decreased blood pressure significantly compared with the untreated group and markedly reduced the severity of myocarditis associated with the decrease of myocardial macrophage, CD4(+), and CD8(+) T-cell expression by comparison of heart wt-to-body wt ratios, pericardial effusion scores, and macroscopic and microscopic scores. Numbers of myocardial interleukin-1beta (IL-1beta)-positive-staining cells (obtained by immunohistochemistry) and quantities of IL-1beta expression (obtained by Western blotting) were significantly lower in rats with EAM given olmesartan treatment compared with rats given vehicle. Cardiac myosin-specific, delayed-type hypersensitivity was significantly lower in olmesartan-treated rats than in control rats. The cytotoxic activities of lymphocytes in rats with EAM treated with olmesartan were reduced compared with untreated control rats. In vitro study showed that both olmesartan and its active metabolite RNH-6270 suppressed IL-1beta production in U-937 cells and cultured myocytes. Olmesartan ameliorates acute EAM in rats. The cardioprotection of olmesartan may be due to suppression of inflammatory cytokines as well as to suppressive effects of cytotoxic myocardial injury in addition to hemodynamic modifications.

Thioredoxin, a redox-regulating protein, is expressed in spontaneous myocarditis in inbred strains of mice.

Redox-regulating mechanisms may be involved in the pathogenesis of aging. Thioredoxin (TRX) is a small multifunctional protein which contains a redox active sequence. Spontaneous myocarditis is often observed in aged mice. In this study, we examined the histopathology and characteristics of TRX expression in spontaneous myocarditis in inbred strains of mice. No spontaneous myocarditis was found in adult 4-week-old inbred strains of mice. High incidence of spontaneous myocarditis was found in aged 8-week-old DBA/2 mice, and low incidence was in 8-week-old BALB/c or C57BL/6 mice. The lesions, limited to the right ventricle, were most severe in DBA/2 mice. TRX was upregulated, and the expression was correlated with the severity of the disease in these strains. Also, 8-hydroxy-2'-deoxyguanosine (8-OHdG), which was an established marker for oxidative stress, was concomitantly positive in necrotic lesions among them. In addition, the long-term anti-oxidant treatment with N-acetylcysteine (NAC) suppressed the development of spontaneous myocarditis. Thus, TRX may be induced by the spontaneously developed myocarditis, and the redox-regulating system may play an important role in the development of aging-related myocarditis.

Beneficial effects of olmesartan, a novel angiotensin II receptor type 1 antagonist, upon acute autoimmune myocarditis.

Excess amount of cytokine produced by inflammatory stimuli contributes to the progression of myocardial damage in myocarditis. Some angiotensin II receptor type 1 antagonists are reported to inhibit proinflammatory cytokine production in vitro and in vivo. We tested the hypothesis that olmesartan, a novel angiotensin II receptor type 1 antagonist, ameliorated experimental autoimmune myocarditis (EAM) in rats attributing to the suppression of inflammatory cytokines in the heart. We orally administered olmesartan 1, 3, and 10 mg/kg/day to rats with EAM for 3 weeks. The results showed that olmesartan decreased blood pressure significantly compared with the untreated group, but markedly reduced the severity of myocarditis by comparing the heart weight/body weight ratio, pericardial effusion scores, macroscopic scores and microscopic scores. Myocardial interleukin (IL)- 1beta expression by western blotting and IL-1beta-positive staining cells by immunohistochemistry were significantly lower in rats with EAM given olmesartan treatment compared with those of rats given vehicle. We conclude that Olmesartan ameliorates acute EAM in rats. The cardioprotection of olmesartan may be due to suppression of inflammatory cytokines dependent of the hemodynamic modifications.

Cardioprotective effects of carvedilol on acute autoimmune myocarditis.

We investigated whether carvedilol protects against experimental autoimmune myocarditis (EAM) attributing to antioxidant properties. Acute EAM was induced by porcine cardiac myosin in Lewis rats. We orally administered a vehicle, various dosages of carvedilol, metoprolol, or propranolol to rats with EAM for 3 weeks. Three beta-blockers decreased heart rates to the same extent. Carvedilol, but not metoprolol or propranolol, markedly reduced the severity of myocarditis at the two different dosages. Only carvedilol decreased the myocardial protein carbonyl contents, and also decreased the myocardial thiobarbituric acid reactive substance products in rats with EAM. Accordingly, carvedilol protects against acute EAM in rats, and this superior cardioprotective effect of carvedilol to metoprolol and propranolol may be due to the antioxidant properties in addition to the hemodynamic modifications.

Cardioprotective effects of 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186), a novel free radical scavenger, on acute autoimmune myocarditis in rats.

BACKGROUND: Recent evidence suggests that oxidative stress may play a role in myocarditis. PURPOSE: To test the hypothesis that 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone or MCI-186), a novel free radical scavenger, protects against acute experimental autoimmune myocarditis (EAM) in rats. METHODS: MCI-186 was administered intraperitoneally (1 mg/kg/day, 3 mg/kg/day or 10 mg/kg/day) in rats with EAM for three weeks. The results were compared with those from untreated rats with EAM. RESULTS: MCI-186 treatment did not affect the hemodynamics of the rats, but did reduce the severity of myocarditis when the heart weight:body weight ratio and the pathological scores were compared. There were significantly fewer myocardial interleukin-1beta-positive cells in rats with EAM treated with MCI-186 (at 3 mg/kg/day and 10 mg/kg/day) than in the untreated rats with EAM. CONCLUSION: MCI-186 ameliorated acute EAM in rats, suggesting that free radicals may be involved in the development of myocarditis.

Upregulation of redox-regulating protein, thioredoxin, in endomyocardial biopsy samples of patients with myocarditis and cardiomyopathies.

An important role of redox regulation in myocardial diseases and heart failure has been postulated. Thioredoxin (TRX) is a redox-regulating protein. Recent studies indicated a possible association between plasma TRX concentrations and the severity of heart failure. Accordingly, we investigated the myocardial expression of TRX in patients with myocarditis and cardiomyopathies. Four cases of hypertrophic cardiomyopathy (HCM), 10 of dilated cardiomyopathy (DCM), 6 of myocarditis, and 5 of controls were studied. Right and left ventricular endomyocardial biopsy samples were obtained at the diagnostic cardiac catheterization. The samples were processed for immunohistological staining for TRX, which was done by the indirect immunoperoxidase technique. 8-hydoxy-2'-deoxyguanosine (8-OHdG), one of the major DNA base-modified products, was also detected for an established marker for oxidative stress. TRX immunoreactivity was none or trivial in control specimens. Positive TRX staining was found in 6 cases; 3 in active myocarditis and 3 in DCM. The positive staining was found in infiltrating cells and damaged myocytes in the perinecrotic lesions. Damaged myocytes were also positive for 8-OHdG All the 3 cases of DCM positive for TRX stain showed severe left ventricular hypertrophy on electrocardiogram and highly elevated left ventricular end-diastolic pressure (> 24 mmHg), suggesting the overload of oxidative stress by hemodynamic impairment. Myocardial TRX was upregulated in myocarditis and cardiomyopathies with active necrotic stage associated with DNA damage, which may reflect the oxidative stress overload in hemodynamically uncontrolled status.