Preparation of Monoclonal Antibody and Development of Enzyme-linked Immunosorbent Assay Specific for Escherichia coli O157 in Foods

To prepare monoclonal antibodies （MAb） and antisera specific for Escherichia coli （E.coli） O157, and to develop a sandwich enzyme-linked immunosorbent assay （ELISA） to detect Eocoli O157 in foods. Methods Spleen cells from BALB/c mice immunized with the somatic antigen of E.coli O157：H7 were fused with routine Sp2/0 myeloma cells. The hybridoma cell line specific for E.coli O157 was established after having been subcloned. Antisera specific for E.coli O157 was prepared by intravenous injection into New Zealand rabbits with a stain of E.coli O157：H7. The sandwich ELISA was developed with the polyclonal antibody as the capture antibody and the MAb 3A5 as the detection antibody. The inoculated ground poultry meat and pasteurized miLk were tested to confirm efficiency of the method. Results MAb 3A5 specific for E.coli O157 and O 113：H21 belonged to subtype IgM. The ascetic titers of the antibody was 1：1× 10^6. No cross-reactivity of the MAb was observed with strains of Salmonella spp, Yersinia enterocolitica, Shigella dysenteriae, etc. The purified polyclonal antibody had a titer of 1： 1× 10^5 with E.coli O 157. The detection limit of this sandwich ELISA was 10^3- 10^4 cfu E.coli O157/mL in pure culture with a high specificity, which was characterized by every non-O157 strain with negative response. With 10h enrichment procedure, E.coli O157：H7 recovered well from inoculated ground poultry meat and pasteurized milk at levels of 0.1 cfu/g and 0.1 cfu/mL. Conclusion MAb 3A5 specific for E.coli O 157 and O 113：H21 can be produced by immunizing BALB/c mice with a strain of E.coli O157：H7. Then a sandwich ELISA can be developed with the polyclonal antibody as the capture antibody and the MAb 3A5 as the detection antibody. The method is proved to be a sensitive and specific technique to detect low number of E.coli O157 in food.

Association between Adiponectin and Metabolic Syndrome in Older Adults from Major Cities of China

Objective To investigate the association between adiponectin and metabolic syndrome （MetS） and related diseases in older adults from major cities of China. Methods A total of 2 049 adults at the age of 60-96 years from18 major cities of China were enrolled in the study. Plasma adiponectin and insulin concentrations were measured. Insulin resistance was assessed by homeostasis model assessment of insulin resistance （HOMA-IR）. The definitions proposed by International Diabetes Federation （IDF） and American Heart Association/National Heart, Lung, and Blood/nstitute （AHA/NLHBI） were used to identify MetS. Results The adiponectin concentration increased with the advance of age and was higher in women than in men. The sex specific adiponectin concentration was inversely correlated with body mass index （BMI）, waist circumference, diastolic blood pressure, triglycerides, glucose and fasting blood insulin, and positively correlated with HDL-C （P〈0.001）. The adiponectin concentration decreased with increasing MetS components. Compared with the 4th sex-specific adiponectin quartile, the odds ratio （OR） for prevalent MetS-IDF and MetS-AHA/NLHBI in subjects of the 1st quartile group was 3.25 （95% CI： 2.24, 4.71） and 3.21 （95% CI： 2.26, 4.55）, respectively. The association was independent of age, sex, life-style factors, medication, family history of chronic diseases, BMI, and HOMA-IR, The OR for MetS was much higher than those of MetS components and its related diseases. Conclusion Adiponectin is strongly associated with MetS independent of insulin resistance and obesity in older adults from major cities in China. The adiponectin concentration is a useful predictor for the risk of MetS.

Estrogen Receptor α and β Expressions in Hypothalamus-pituitary-ovary Axis in Rats Exposed Lactationally to Soy Isoflavones and Bisphenol A

Objectives This paper aims to investigate the uterotrophic activities of lactational exposure to combination of soy isoflavones （SIF） and bisphenol A （BPA） and to examine estrogen receptor α （ERα） and estrogen receptor β （ERβ） expressions in hypothalamus-pituitary-ovary axis and uterus.Methods Maternal rats that were breeding about 8 litters were randomly divided into four groups with seven dams in each group.Dams in different treatment groups received corn oil （control）,150 mg/kg BW of SIF,150 mg/kg BW of BPA or combination of 150 mg/kg BW of SIF and 150 mg/kg BW of BPA,respectively,from postnatal day 5 to 11 （PND5-11） by gavage.On PND12 and PND70,10 female litters were killed and hypothalamus,pituitary,ovary and uterus were collected.ERα and ERβ expressions in these organs were detected with Western blotting assay.And vaginal opening time and estrus cycle were examined in animals fed for PND70.Results On PND12,the relative uterine weight of rats treated with ISF or BPA or their combination was significantly higher than that of untreated rats （P〈0.05）.But the relative uterine weight of rats in the co-exposure group was slightly lower than that in the group only exposed to SIF or BPA.On PND 70,however,the relative uterine weight in each treatment group was not statistically different from that in the control group （P〈0.05）.Vaginal opening time and estrus cycle in groups treated with SIF or BPA or their combination were similar to those in the control group （P〈0.05）.Exposure to SIF or BPA or their combination could up-regulate or down-regulate ERα and ERβ expressions in hypothalamus,pituitary,ovary and uterus on PND12 and PND70.These regulation patterns for ERα and ERβ were different in different organs at different time points.Conclusion Lactational exposure to ISF or BPA or their combination could induce uterotrophic responses in neonate rats,which disappeared in later life.But these data fail to suggest a possibility for synergic actions between SIF and BPA.It was also demonstrated that the uterotrophic effects of SIF and BPA exposure might,at least,involve modification of ERα or ERβ expressions in the hypothalamus-pituitary-ovary axis.

Preparation of Monoclonal Antibody Against HPT and Its Application to Detecting Marker Protein in Genetically Modified Rice

To produce the monoclonal antibodies （mAbs） against hygromycin B phosphotransferase （HPT） and to develop immunoassay based on mAbs for biosafety assessment of HPT in genetically modified rice （GM rice）. Methods BALB/c mice were immunized with purified recombinant 6His. HPT protein, and the conventional hybridoma technology was used to generate the monoclonal hybridoma cells. ELISA and Western blot were used to analyze the specificity of mAbs recognizing HPT and the cross reaction with other proteins. A double-Ab sandwich ELISA method was established to detect HPT expression level in the sck gene-modified rice plants. Results Four hybridomas, named F1,D4-2, D4-4, and D4-5, producing the mAbs against HPT were successfully obtained with the titer of ascetic mAbs ranging from 10×10^-4 to10×10^-5. Identification of subclass showed that all the produced mAbs belonged to IgG1. Western blot showed specific binding reaction between the mAbs to the HPT proteins expressed in the GM rice. A double sandwich ELISA coated with anti-HPT polyclonal antibody was established with mAbs as sandwich antibody, which showed a sensitivity of 30ng/mL and did not crossreact with other proteins. The expression level of HPT in the leaves of sck-transformed lines was detected （80-150ng/mL）. But HPT protein in the grain and seed of GM rice could not be detected using this ELISA assay, Conclusion Anti-HPT mAbs prepared herein have a high specificity and can be used for rapid assay of HPT antigen. The expression level of HPT in the GM rice grain and seed is lower than our ELISA detection limit.

Total Energy Expenditure of 16 Chinese Young Men Measured by the Doubly Labeled Water Method

Objective Doubly labeled water （DLW） method is the gold standard for measuring total energy expenditure （TEE）. We used this method to measure TEE in Chinese young men. Methods Sixteen healthy young men age 23±1 recruited. TEE was measured by the DLW method, by indirect calorimetry. We also conducted 24-h estimate energy requirements of the subjects. years with body mass index 22.0±1.4 kg/m2 were and basal energy expenditure （BEE） was determined activity, energy balance and factorial approach to Results TEE of subjects by DLW method was 9.45±0.57 M J/day （2258±180 kcal/day）. The 24-h activity was 10.80±0.33 M J/day （2582±136 kcal/day）. The energy requirement, derived from energy balance observations, was 9.93±1.32 M J/day （2373±315 kcal/day）. The BEE of 6.65±0.28 M J/day （1589±67 kcal/day）, calculated by the adjusted Schofield equation, was significantly higher （P〈O.O01） than that measured by indirect calorimetry, 5.99-±0.66 M J/day （1433-±158 kcal/day）. The TEE derived from the factorial approach was 10.31-±0.43 M J/day （2463±104 kcal/day）. Conclusion The TEE of Chinese young men measured by the DLW method was about 10% lower than the current recommended nutrient intake （RNI）, suggesting that the RNI for Chinese men maybe overestimated. Further studies are warranted to determine the value of the estimated energy requirement.

Effects of inorganic arsenic on growth and microcystin production of a Microcystis strain isolated from an algal bloom in Dianchi Lake,China

Our previous data have shown that inorganic arsenic concentrations were high in Dianchi Lake,China,where Microcystis blooms often occur.To explore the relationship between arsenic and the growth of Microcystis,the effects of arsenite[As(III)]and arsenate[As(V)]on the growth and toxin production of M.aeruginosa strain FACHB 905 were tested.Results showed that M.aeruginosa FACHB 905 was tolerant to inorganic arsenic and its growth was not inhibited when the concentration of As(III)was below 10-5 mol L-1 or that of As(V)below 10-3 mol L-1.Total microcystin production was stimulated in the presence of 10-7 mol L-1 As(III)and the response of this M.aeruginosa strain to As(III)seemed to be a typical inverted U-shaped hormesis.The content increase of microcystin-LR per cell indicated that the toxicity was enhanced as M.aeruginosa FACHB 905 was exposed to As(V).Considering the relatively high concentration of inorganic arsenic in Dianchi Lake(139μg L-1 in epilimnetic water),the origin of the M.aeruginosa strain,inorganic arsenic favors survival of M.aeruginosa FACHB 905 and may stimulate its microcystin production and cellular toxicity.

Dietary Iodine Intake in the Chinese Population

Objective To evaluate dietary iodine intake and its potential risks among the Chinese population Methods Individual dietary iodine intake was calculated using food consumption data multiplying by iodine concentration in foods, table salt and drinking water, followed by summing, and then compared with the corresponding age-specific reference values, including Upper Intake Level （UL） and Recommended Nutrient Intake （RNI）. Results In areas with water iodine concentration （Wl） lower than 150 ug/L, 80.8% of residents had iodine intake between the RNI and UL, 5.8% higher than UL, and the remaining （13.4%） lower than RNI if iodized salt was consumed. However, in the uniodized salt consumption scenario, only 1.0% of residents between RNI and UL, 1.4% higher than UL, and a large part of residents （97.6%） lower than RNI. In areas with Wl higher than 150 ug/L ,all residents had iodine intake between RNI and UL if iodized salt was consumed, except 10.5% and 24.9% of residents higher than UL in areas with Wl at 150-300 ug/L and higher than 300 ug/L respectively. However, in the uniodized salt consumption scenario, only 1.5% and 1.7% of residents had higher iodine intake than UL respectively. Conclusion The findings suggested that in general, the dietary iodine intake by the Chinese population was appropriate and safe at the present stage. People in areas with WI lower than 150 ug/L were more likely to have iodine deficiency. While people in areas with WI higher than 150 ug/L were more likely to have excessive iodine intake if iodized salt was consumed.