A micro-ELISA system for the rapid and sensitive measurement of total and specific immunoglobulin E and clinical application to allergy diagnosis.

We have developed a novel, practical micro-ELISA system for sensitive and rapid allergy diagnosis. The enzymatic reactions occurred under stopped-flow conditions, resulting in both high precision and high sensitivity. A BSA-biotin-avidin linker was introduced for the immobilization of water-soluble allergens on polystyrene microbeads, enabling immobilization of allergens in sufficient density to provide high sensitivity. Evaluation of the system's performance showed a good detection limit (2 ng/mL) for total IgE measurement. In addition, a good correlation with a conventional method (CAP method) was demonstrated using human serum samples from 85 allergy patients. Importantly, sample volumes (5 microL) were 10 times smaller and analysis time (12 min) was >20 times faster than the conventional method. All procedures were automatically regulated with our simple microfluidic system, and all the fluidic, optic and electronic components were integrated for portability. We believe that our system has the potential to become a very powerful tool, particularly for point-of-care diagnosis.

Wogonin, a plant flavone, potentiates etoposide-induced apoptosis in cancer cells.

Etoposide, a podophylotoxin anticancer agent, induces apoptotic cell death in normal and cancer cells. Etoposide-induced apoptosis plays a role in not only anticancer effect but also adverse reaction, such as myelosuppression. Since we have found that wogonin, a flavone found in Scutellaria baicalensis Georgi, prevents thymocyte apoptosis induced by various compounds including etoposide, we examined the effect of this flavone on etoposide-induced apoptosis in cancer cells. Although 100 muM wogonin itself significantly increased DNA fragmentation in HL-60 cells, this change was not observed in Jurkat cells. On the other hand, this flavone significantly potentiated etoposide-induced apoptosis in Jurkat and HL-60 cells. Similarly, wogonin accelerated etoposide-induced cell death in lung cancer cells. Since wogonin had no effect on the action of other anticancer agents, such as 5-FU and cisplatin, this flavone seems to accelerate only etoposide-induced apoptotic cell death in cancer cells. These results suggest that the modification of etoposide-induced apoptosis by wogonin may be available to reduce the adverse reaction of this agent.

A challenge for monitoring iodine deficiency disorders in rural Nepal.

Developing a strategy for monitoring iodine deficiency disorders (IDD) remains a big challenge in rural Nepal where great variations could exist in IDD status. To explore the possibility of variation in urinary iodine excretion (UIE) level in rural settings, we carried out a detailed study of UIE among 586 school children of 20 schools in five villages. Our data revealed statisitically significant differences in UIE values among rural villages and schools in the same villages. The policy-makers should keep such variations in mind for a successful monitoring of IDD in Nepal and other countries where such variations may exist.

Changes in serum iodine concentration, urinary iodine excretion and thyroid function after hysterosalpingography using an oil-soluble iodinated contrast medium (lipiodol).

OBJECTIVE: Reports of hypothyroidism after hysterosalpingography (HSG) using lipiodol are emerging. The present study was designed to investigate the changes in serum iodine concentration (SIC), urinary iodine concentration/creatinine excretion (UI/Cr), and thyroid function before and after HSG using lipiodol. METHODS: The prospective observation study included 22 infertile euthyroid women with no previous history of thyroid disease. All underwent HSG between April 2007 and August 2008 at our institution. We examined SIC, UI/Cr, and thyroid function before HSG, and at 4, 8, 12, and 24 weeks, and 9-12 months after HSG. RESULTS: The median value of SIC and UI/Cr peaked at 4 weeks after HSG and remained at significantly high levels at 8, 12, and 24 weeks post-HSG compared with pre-HSG. In sync with the increase of iodine, the mean level of TSH significantly increased at 4, 8, 12, and 24 weeks post-HSG compared with pre-HSG. After 24 weeks, differences in SIC, UI/Cr, and TSH levels before and after HSG became nonsignificant. The mean value of free triiodothyronine and free thyroxine showed no significant difference at any of the time points compared with pre-HSG. Three cases (13.6%) showed transient high TSH (>5 µIU/L) with normal thyroid hormones at 4 or 8 weeks after HSG. CONCLUSION: Thyroid monitoring should be conducted in the first 4-8 weeks after HSG using lipiodol and attention to thyroid dysfunction should be paid for up to 6 months after the procedure due to the possibility of excess iodine.

Determination of cattle foot-and-mouth disease virus by micro-ELISA method.

The development of foot-and-mouth disease virus (FMDV) detection methods is crucial for animal food security, tackling regional FMDV epidemic, and global FMDV prognostic control. For these purposes, a fast and sensitive analysis method is required. In this study, we developed a microchip-based ELISA (enzyme-linked immunosorbent assay), micro-ELISA, to realize FMDV detection. Nickel(II) chelating chemistry was utilized to immobilize recombinant protein (antigen) on polystyrene micro-beads in order to determine FMDV antibodies in cattle serum samples. In addition, reaction protocol and conditions were investigated. As a result, the FMDV detection was successfully demonstrated with only a 10-µL sample volume in 25-minute assay time. Analytical sensitivity was evaluated by a maximum nominal positiveness percentage value (NPPV) of 303 and a dilution factor of 32×. The method's inter-run and intra-run CV (coefficients of variance) values were 15.5 and 17.1%, respectively, which were fully compatible with the OIE (World Organization for Animal Health) principle of validation of diagnosis assays for infectious diseases. The developed method should become a powerful tool for determining other animal contagious diseases and/or zoonosis.

Thermal lens detection device.

A thermal lens detection device was developed to realize an easy-to-use, portable and sensitive detector for nonfluorescent molecules. Two laser diodes (658 nm for excitation and 785 nm for probe) were made coaxial in an optical unit and were coupled to a single-mode optical fiber. On a microfluidic chip, a small holder for the optical fiber was fixed, and micro-lenses (numerical aperture of 0.2) were also integrated inside the holder. The micro-lenses were designed to realize an adequate chromatic aberration (50 µm), which was essential for sensitive thermal lens detection. Compared with conventional thermal lens detection systems which required very laborious and accurate optical alignment with the microchannel, the new device needed just attachment-detachment of the optical fiber, which was important for practical application. The lower limit of detection was 10 nM for nickel(II) phthalocyaninetetrasulfonic acid tetrasodium salt solutions (model sample), and the absorbance was 9 × 10(-6) AU. The absolute number of molecules detected was less than 200 zmol. The coefficient of variance for 5-time attachment-detachment of the optical probe was as small as 3.6%. The technical development allowed integration of the thermal lens detection devices inside a microsystem (e.g. enzyme-linked immuno-sorbent assay system), and practical microsystems were realized with sensitivities several-orders higher than absorptiometry.

Iodine status of pregnant and postpartum Japanese women: effect of iodine intake on maternal and neonatal thyroid function in an iodine-sufficient area.

BACKGROUND: Iodine deficiency in pregnant and lactating women results in serious damage to their fetuses, newborns, and weaning infants. The effect of dietary iodine intake on maternal and infantile thyroid function has not been well studied in iodine-sufficient areas, and there are few data on appropriate gestational age-specific reference ranges for urinary iodine excretion during pregnancy and lactation. OBJECTIVES: The aim of the study was to characterize the gestational change of urinary iodine excretion in Japanese women and to assess the effects of iodine status on thyroid function in mother and infant. METHODS: A total of 934 Japanese women and their 722 newborn infants were enrolled in the study. Iodine and creatinine concentrations were determined in spot urine samples in the three trimesters of pregnancy and the postpartum period at 34.0 d after delivery. Serum thyroperoxidase antibody and thyroglobulin antibody, TSH, and free T(4) were measured in each trimester, and neonatal TSH was measured on postnatal d 4. RESULTS: The overall median urinary iodine concentration (UIC) during pregnancy was 219.0 µg/liter, higher than that in postpartum women (135.0 µg/liter). The prevalence of pregnant women with low UIC less than 100 µg/liter or high UIC greater than 500 µg/liter was 16.1 and 22.2%, respectively. Urinary iodine excretion decreased from 221.0 µg/liter in the first trimester to 208.0 µg/liter in the second trimester to 193.0 µg/liter in the third trimester, and then remained at 135.0 µg/liter postpartum. [corrected]. The maternal UIC correlated positively with serum TSH during pregnancy. There was no significant difference in UIC between subjects with positive thyroid autoantibodies and those with negative antibodies. CONCLUSIONS: Iodine intake assessed by UIC in Japanese pregnant women is regarded as sufficient and not excessive according to World Health Organization criteria. Although the data are local, our results provide additional information on the reference range for UIC throughout gestation in iodine-sufficient areas.

Rapid screening swine foot-and-mouth disease virus using micro-ELISA system.

In order to tackle both regional and global foot-and-mouth disease virus (FMDV) epdimics, we hereby develop a rapid microfluidic thermal lens microscopic method to screen swine type O FMDV with good efficiency. The scheme has great merits in terms of field portability, sample volume, assay time, analytical sensitivity, and test reproducibility.

On-chip antibody immobilization for on-demand and rapid immunoassay on a microfluidic chip.

Immunoassay is one of the important applications of microfluidic chips and many methodologies were reported for decreasing sample∕reagent volume, shortening assay time, and so on. Micro-enzyme-linked immunosorbent assay (micro-ELISA) is our method that utilizes packed microbeads in the microfluidic channel and the immunoreactions are induced on the beads surface. Due to the large surface-to-volume ratio and small analytical volume, excellent performances have been verified in assay time and sample∕reagent volume. In order to realize the micro-ELISA, one of the important processes is the immobilization of antibody on the beads surface. Previously, the immobilization process was performed in a macroscale tube by physisorption of antibody, and long time (2 h) and large amount of antibody (or high concentration) were required for the immobilization. In addition, the processes including the reaction and washing were laborious, and changing the analyte was not easy. In this research, we integrated the immobilization process into a microfluidic chip by applying the avidin-biotin surface chemistry. The integration enabled very fast (1 min) immobilization with very small amount of precious antibody consumption (100 ng) for one assay. Because the laborious immobilization process can be automatically performed on the microfluidic chip, ELISA method became very easy. On-demand immunoassay was also possible just by changing the antibodies without using large amount of precious antibodies. Finally, the analytical performance was investigated by measuring C-reactive protein and good performance (limit of detection <20 ng∕ml) was verified.

Rapid analysis of methamphetamine in hair by micropulverized extraction and microchip-based competitive ELISA.

An automated full-range quantitation method for identifying d-methamphetamine in human hair using a microchip-based ELISA system (microELISA) in combination with a micropulverized extraction method for sample preparation has been developed. An antibody and a peroxidase-linked methamphetamine, both are commercially available, were used for the competitive ELISA assay. Method validation was carried out using doped hair samples, and segmental analyses of real-case specimens were carried out by both microELISA and LC/MS/MS to verify the reliability and applicability of this new method. Due to the small size of the system and the lack of an evaporation process, sample preparation and quantitation can be accomplished easily and quickly (less than 30 min) in small-scale contamination-free environments.

Iodine prophylaxis around the Semipalatinsk Nuclear Testing Site, Republic of Kazakstan.

OBJECTIVE: This study aimed to clarify the iodine deficiency status in the Semipalatinsk region that has been contaminated by radioactive fallout from nuclear testing during the period of the former USSR. DESIGN: Based on the Japan-Kazakstan joint project of adult cancer screening around the Semipalatinsk Nuclear Testing Site (SNTS), from May to October 2002 spot urine specimens were collected at random in each village. Separately, children aged 5-15 years from around the SNTS were chosen at random and spot urine specimens were collected from them. SETTING: Area contaminated by radioactive fallout around the SNTS, Republic of Kazakstan. SUBJECTS: A total of 2609 adults aged >40 years from 16 settlements in three regions and one city, and 298 children aged 5-15 years from two regions and one city. RESULTS: Median urinary iodine concentrations of adults and children in all regions were in the range of 116.0-381.7 and 127.7-183.0 microg l(-1), respectively. The highest prevalence of values <50 microg l(-1) (14.1%) did not exceed 20%. Distributions within each group, adults and children, showed almost the same pattern, except for one region where more than 50% of adults had urinary iodine concentration >100 microg l(-1). CONCLUSIONS: In agreement with our previous studies, the urinary iodine concentration data showed no clear evidence of iodine deficiency around the SNTS. Kazakstan is geographically and nutritionally at moderate risk of iodine deficiency disorders without fortification or iodine replacement by iodised salt. The socio-medical prophylaxis against iodine deficiency has been successfully maintained in East Kazakstan.

Urinary iodine kinetics after oral loading of potassium iodine.

We studied the kinetics of iodine in various forms, in order to establish appropriate guidelines of iodine prophylaxis for thyroid blockade at nuclear emergency in "iodine rich areas", such as Japan. First, the effect of equivalent dose of potassium iodide (KI) (solution vs. tablet) was evaluated with excretions of urinary iodine (UI) at Nagasaki, Japan, and it was revealed that there was no difference of iodine kinetics between solution and tablet. We also performed the same study at Gomel, Belarus, which was known to be an iodine deficient area, and obtained the same results. Second, the kinetics of iodine included in "iodine rich" food was also evaluated with excretions of UI. Interestingly, the ratio of UI excretion with iodine rich food was significantly lower than that with KI tablets until 6 h after the intake (ANOVA, p = 0.02). These results proved that 1) KI solution as well as its tablet is useful for prophylaxis and 2) prophylaxis by iodine rich food is not effective for rapid blockade of thyroid gland at nuclear emergency. Finally, we emphasized that the approach from social medicine is definitely important to establish an effective iodine prophylaxis.

No evidence of radiation risk for thyroid gland among schoolchildren around Semipalatinsk Nuclear Testing Site.

To assess thyroid status among the schoolchildren around Semipalatinsk Nuclear Testing Site (SNTS), Kazakhstan, and to evaluate the current status of iodine deficiency in this area, we performed medical screening of schoolchildren in two villages, Kaynar and Karaul villages, East Kazakhstan Region, Republic of Kazakhstan, located within 100 km of SNTS. A total of 196 schoolchildren were chosen at random. Control groups comprised 250 schoolchildren from Nagasaki, an iodine-rich area, and 100 schoolchildren from Gomel, an iodine-deficient area contaminated by the Chernobyl Nuclear Power Plant accident. Ultrasound screening of thyroid revealed three cases of benign thyroid disease (two cases of goiter and one single cyst), but no cases suspicious of malignancy. The urinary iodine (UI) concentrations of subjects in Kaynar and Karaul ranged from 21.8 to 735.8 microg/L, 4.3% of whom showed low UI concentrations (<50 microg/L), compared with 0% in the Nagasaki group and 52% in the Gomel group. The median UI concentration in Kaynar and Karaul was 153.2 microg/L, which was significantly lower than that in Nagasaki (366.3 microg/L, p<0.0001) but higher than that in Gomel (47.3 microg/L, p<0.0001). In conclusion, there was a low incidence of morphological abnormalities in the thyroid, and no evidence for severe iodine deficiency among the Kazakhstani children studied. These results suggest that there is no transgenerational risk for schoolchildren born from parents irradiated as a result of tests carried out in SNTS.