Inhibition Enzyme Immunoassay Research Articles

Prolidase could be considered a sign of inflammation associated with cigarette smoking.

Smoking causes inflammation, thickening, and narrowing of the airways. This inflammatory process is a reaction to free radicals and oxidants. Smoking affects collagen metabolism and tissue remodeling. Prolidase enzyme hydrolyzes iminodipeptides with hydroxyproline and C terminal proline. It plays a crucial role in the metabolism of collagen and the remodeling of the matrix. The present study aims to reveal the association of prolidase with inflammation caused by smoking and to compare serum prolidase levels with oxidative-antioxidative status in healthy individuals. A total of 76 participants (38 smokers and 38 nonsmokers) were involved in the present study. Serum cotinine levels were measured to show the exposure to nicotine in tobacco smoke by using the competitive inhibition enzyme immunoassay method. Serum prolidase, total oxidant status (TOS), and total antioxidant status (TAS) were determined by the enzyme-linked immunosorbent (ELISA) method, respectively. The correlation between smoking, serum prolidase levels, TOS, and TAS was investigated. TAS and serum prolidase levels of smokers were considerably lower than those in non-smokers (p < 0.001, p = 0.012 respectively). However, no differences were observed in TOS between the two groups. There was no statistically significant correlation between serum prolidase levels, TAS, and TOS. Moreover, no relationship was observed between respiratory function parameters and serum prolidase levels. To the best of our knowledge, the present study is the first study to demonstrate the role of prolidase in smoking-related inflammation. The results achieved in the present study suggest that smoking creates an imbalance in the oxidant-antioxidant activity. Smoking decreases prolidase levels, leading to decreased collagen turnover. Chronic pulmonary disease might be related to this decrease in collagen turnover.

Evaluación de los niveles de factor de necrosis tumoral alfa, interferón gamma, procalcitonina y neopterina en bovinos seropositivos para Brucella

Brucellosis is a zoonotic disease that affects a large number of people and animals, causing physical disability, workforce loss and significant economic losses in the livestock industry. In the current study, it was aimed to determine and compare the levels of tumor necrosis factor alpha (TNF–α), interferon gamma (IFN–γ), Procalcitonin (PCT) and Neopterin in the blood serums of cattle with brucellosis and vaccinated against brucellosis. The materials of this study consisted of a total 48 blood serums belonging to three basic groups, each consisting of 16 animals. Disease group (1st group) were divided into two subgrups each consisting of 8 animals that 21st day after abortion and seropositive 7 months pregnant, the vaccinated (2nd group) and the control (3rd group) groups were divided into two subgroups, each consisting of 8 animals that gave birth 21 days ago and 7 months pregnant. IFN–γ and PCT levels were determined by sandwich enzyme immunoassay, TNF–α and Neopterin levels were determined using competitive inhibition enzyme immunoassay method by using ELISA device. In this study, TNF–α, PCT and Neopterin levels measured in the blood serums of the Brucella seropositive (1st), conjunctival Brucella abortus S19 vaccine administered (2nd) and unvaccinated Brucella seronegative control groups were compared and no significant difference could be determined between the subgroups of the groups (P&gt;0.05). There were a significant differences between 1st, 2nd, and 3rd groups (P&lt;0.05). IFN–γ levels determined in the blood serums of 1st, 2nd and 3rd groups were compared and nosignificant differences were found between the subgroups of 2nd and 3rd groups (P&gt;0.05), but there were a significant differences between the subgroups of the 1st group (P&lt;0.05). Similarly, a significant differences were determined between 1st, 2nd and 3rd groups in terms of IFN–γ levels (P&lt;0.05). As a result, it was thought that detecting very high serum TNF–α, IFN–γ, neopterin levels in cattle with brucellosis would be helpful in the diagnosis and follow–up of brucellosis. However, it was concluded that there is a need for controlled studies comparing more herds with brucellosis to determine whether the relevant cytokines can be used in the diagnosis of brucellosis.

Antioxidant Defense Expressed as Glutathione Status and Keap1-Nrf2 System Action in Relation to Anthropometric Parameters and Body Composition in Young Women with Polycystic Ovary Syndrome

Metabolic disorders present in women with polycystic ovary syndrome (PCOS) and the associated risk of obesity may result in increased oxidative stress and reproductive failure. Therefore, we evaluated the concentrations of reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione peroxidase (GPx), and reductase (GR), as well as nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associating protein1 (Keap1) in the serum of 56 women with PCOS divided according to the visceral to subcutaneous fat surface ratio (VAT/SAT) and waist-to-hip ratio (WHR) values. Antioxidant parameter levels were measured by competitive inhibition enzyme immunoassay technique. As the VAT/SAT ratio and WHR increased, we observed significantly higher concentrations of GSSG and Keap1 protein and a lower value of the GSSG/GSH ratio (R-index), which is considered an index of cellular redox (p < 0.05). Negative correlations were found between the R-index and body weight, BMI, WHR, subcutaneous and visceral fat surface and the VAT/SAT ratio, and total body fat; positive links were found with fat free mass and total body water. Opposite associations were noted between GSSG level and the aforementioned body composition parameters. Oxidative stress characterized by a depleted reduced-to-oxidized glutathione index is associated with anthropometric and body composition parameters in women with PCOS. In particular, abdominal obesity expressed by the VAT/SAT ratio and/or WHR seems to have a negative impact on glutathione status, which may lead to a disruption of many biological cell processes. The observed negative association of Keap1 with R-index suggests that the elevated oxidative changes dependent on the VAT/SAT ratio may lead to Nrf2 activation to promote antioxidant enzyme expression. Although the GSH/GSSG index as well as the VAT/SAT ratio appear to be good indicators of oxidative status, studies on a larger group of patients should continue to confirm these links among women with PCOS.

Assessment of circulating immune complexes in canine leishmaniosis and dirofilariosis.

Canine leishmaniosis (CanL) by Leishmania infantum (L.i.) and heartworm disease by Dirofilaria immitis (D.i.) are common zoonotic vector-borne diseases (VBDs) characterized by a variety of pathological and clinical signs. The immunopathology in both VBDs is extremely complex, and their clinical manifestations are strongly dependent on the type of immune response elicited by the parasites. In particular, the formation of circulating immune complexes (CICs) plays an important role in the pathogenesis of these VBDs. Based on the international guidelines, dogs with high anti-L. infantum antibody titres and one or more clinical and/or laboratory signs related to CanL require anti-Leishmania treatment. Consequently, the CICs measurement could be used for improving the clinical staging process of CanL. The aim of the study was to assess the CICs level by a competitive inhibition enzyme immunoassay, in healthy or sick dogs seropositive to L.i. and in healthy dogs positive to D.i.. Out of 51 enrolled dogs, 11 were included in Group A (seronegative to L.i., D.i. negative and healthy), 15 in Group B (exposed to L.i., D.i. negative and healthy), 12 in Group C (seropositive to L.i., D.i. negative and sick) and 13 in Group D (seronegative to L.i, D.i. positive and healthy). The comparison of CIC level in canine sera revealed a significant difference among groups (P < 0.001), with the highest concentration (i.e., median = 104.6µg/mL) in dogs with CanL. The findings of the study highlight the CICs measurement as a useful tool in the clinical staging of CanL for avoiding misclassification of dogs as leishmaniotic, thus not requiring anti-Leishmania therapy, as well as the possibility of results misuse in geographical areas where both leishmaniosis and heart-worm disease are endemic.

Body mass index is associated with Helicobacter pylori infection and increased oxidative DNA damage in an obese population.

ObjectiveTo determine the prevalence of Helicobacter pylori infection in a Saudi Arabian population and its association with the body mass index (BMI) and serum 8-hydroxy deoxyguanine (8-OHdG) levels as biomarker for oxidative stress.MethodsThis cross-sectional study enrolled patients that had experienced epigastric discomfort or dyspepsia for > 1 month and had undergone diagnostic upper endoscopy. Patients with a body mass index (BMI) ≥30 kg/m2 were defined as obese. The presence of anti-H. pylori antibodies was confirmed using an H. pylori immunoglobulin G (IgG) antibody enzyme-linked immunosorbent assay. The levels of 8-OHdG were measured using a competitive inhibition enzyme immunoassay.ResultsA total of 298 patients were enrolled in the study. Of these, 186 (62.4%) patients were H. pylori-positive and 112 (37.6%) patients were H. pylori-negative. The mean ± SD age of the overall study cohort was 47.17 ± 9.27 years. The H. pylori-positive patients had significantly higher levels of H. pylori IgG antibodies than H. pylori-negative patients. H. pylori prevalence linearly correlated with BMI quantile. The 8-OHdG levels were strongly associated with the BMI of the patients in the H. pylori-positive group.ConclusionObese individuals exhibited higher H. pylori prevalence than individuals with a lean BMI (BMI < 25.00 kg/m2).

Influence of Oxidative Stress Generated by Smoking during Pregnancy on Glutathione Status in Mother-Newborn Pairs.

Glutathione plays a key role in maintaining a physiological balance between prooxidants and antioxidants in the human body. Therefore, we examined the influence of maternal smoking as a source of oxidative stress measured by total oxidant capacity (TOC) on reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione peroxidase (GPx-3), and reductase (GR) amount in maternal and umbilical cord blood in 110 (45 smoking and 65 non-smoking) mother-newborn pairs. Concentrations of glutathione status markers and TOC were evaluated by competitive inhibition enzyme immunoassay technique. Plasma TOC levels were significantly higher and the GSH/GSSG ratio, which is considered an index of the cell’s redox status, were significantly lower in smoking women and their offspring than in non-smoking pairs. Decreased GR levels were found in smoking mothers and their newborns compared with similar non-smoking groups. Although plasma GPx-3 concentrations were similar in both maternal groups, in the cord blood of newborns exposed to tobacco smoke in utero they were reduced compared with the levels observed in children of tobacco abstinent mothers. Oxidative stress generated by tobacco smoke impairs glutathione homeostasis in both the mother and the newborn. The severity of oxidative processes in the mother co-existing with the reduced potential of antioxidant systems may have a negative effect on the oxidative-antioxidant balance in the newborn.

Evaluation of spore inoculum and confirmation of pathway genetic blueprint of T13\u03b1H and DBAT from a Taxol-producing endophytic fungus

Taxol (paclitaxel), a plant-derived anticancer drug, has been among the most successful anticancer drugs of natural origin. Endophytic fungi have been proposed as a prominent alternative source for Taxol and its intermediate Baccatin III, however the very low yields remain a hinderance to their commercial utilization. Significant research efforts towards this end are underway globally. Here, we report the results on our earlier reported Taxol-producing endophytic fungus, Fusarium solani from the standpoint of spores as seed inoculum and media selection for enhanced Taxol and baccatin III yields. Spores produced on M1D medium with 94.76% viability were used for further media optimization for Taxol and Baccatin III production in five different liquid media under static and shaker condition at different cultivation days. Taxol and Baccatin III when quantified through competitive inhibition enzyme immunoassay (CIEIA), showed maximum production at 136.3 µg L−1 and 128.3 µg L−1, respectively in the modified flask basal broth (MFBB) under shaking condition. Further, two important genes of this pathway, namely taxane 13α-hydroxylase (T13αH) and 10-deacetylbaccatin III-10-β-O-acetyltransferase (DBAT) have been identified in this fungus. These findings are hoped to assist in further manipulation and metabolic engineering of the parent F. solani strain towards the enhanced production of Taxol and baccatin III.

Значение комплексной лабораторной диагностики ОРВИ крупного рогатого скота для ветеринарной практики

Abstract. This article summarizes the results of laboratory studies of acute respiratory viral infections of cattle in agricultural enterprises of the Ural Federal district and the Udmurt Republic. The purpose of the research was to show the spread of respiratory viral infections in cattle in various regions and the significance of laboratory diagnostics in this pathology. According to research data 815 samples for the period 2018–2020 a variety of combinations of mixed respiratory viruses in animals was detected using biomaterials from different age patients and patients with respiratory diseases of cattle from agricultural enterprises of the Ural Federal district, the Volga Federal District and the Udmurt Republic using polymerase chain reaction, indirect hemagglutination reaction, hemagglutination inhibition reaction, Elisa , and enzyme immunoassay. Scientific novelty. The etiological structure of mixed acute respiratory viral infections in cattle in agricultural enterprises in four regions of the Ural Federal District, the Volga Federal District and the Udmurt Republic in 2018–2020 was studied using modern diagnostic technologies. Results. In the Ural Federal District, the Volga Federal district and the Udmurt Republic, 80 % of cases of acute respiratory diseases were caused by bovine viral infections, mainly representatives of 2 families of viruses whose genome is represented by an RNA molecule (paramixoviruses, togaviruses) and a family of viruses whose genome is represented by a DNA molecule (herpesviruses,). The leading place among acute respiratory infections was occupied by infectious rhinotracheitis of cattle.

Influence of dental extraction on patient’s stress and anxiety levels by assessing the salivary cortisol concentration at different time points during the extraction procedure

The aim of our study was to analyze the stress and anxiety in patients undergoingroutine dental extraction, by the assessment of cortisol in saliva, which can beconsidered as one of the major hormones that is released during stressful events.After obtaining the ethical approval and the patients were consented for their rolein this study, we examined 26 patients (13 males, and 13 females).The age rangedfrom 29-42 with mean age of 36.2. We collected three salivary samples from eachpatient after asking him to chew on a cotton roll to stimulate salivary secretion. Thefirst sample was obtained 10 minutes prior to local anesthetic injection, the secondsample collected 6-8 minutes after the local anesthetic injection, and third sampleacquired 10-15 minutes post-extraction. The samples were assessed and analyzedusing ELISA immunoassay (competitive inhibition enzyme immunoassay technique).Statistical analysis was performed using SPSS statistical software (version19).ANOVA and t-test were applied to obtain the significance and correlationbetween the examined samples.Significant difference was shown among the three samples (P-Value =0.002).The mean value in the third sample (post-extraction salivary sample) wasconsiderably high when compared to the other samples (Mean ± SD = 0.184± 0.184,0.307 ± 0.121 and 0.357 ± 0.141) respectively, which positively correlates with thepatient’s level of stress. Cortisol concentration in μg/dl in percentages between eachsample and the percentages, 50%, were0.105 μg/dl, 0.295 μg/dl and 0.410 μg/dl, (75%) were0.348 μg/dl, 0.410 μg/dl and 0.443 μg/dl and (90%), 0.443 μg/dl, 0.468 μg/dland 0.518 μg/dl.Our study revealed that salivary cortisol is within its highest levels after extractionin 15 minutes.

An inhibition enzyme immuno assay exploring recombinant invariant surface glycoprotein and monoclonal antibodies for surveillance of surra in animals

The present study is aimed at the development of inhibition ELISA (I-ELISA) exploring monoclonal antibodies (MAbs) and recombinant invariant surface glycoprotein. The extracellular domain (ED) of invariant surface glycoprotein (ISG-75) from Trypanosoma evasni has been heterologously expressed in Pichia pastoris (X-33). The recombinant ISG-75 (rISG-75ED) was characterized by immunoblot and ELISA, followed by the production of MAbs against rISG-75ED. The MAbs were characterized by immunoblot and then explored in the development of I-ELISA for the detection of surra. The diagnostic potential of the developed test has been evaluated using 1192 field sera sample including cattle, buffalo, donkey, horse and camel. The statistical analysis of the data showed optimum combination of diagnostic sensitivity and specificity at 98.8% and 99.2% respectively, with cut-off percentage inhibition (PI) value of >45. The Cohen's kappa coefficient of agreement was found to be 0.98. Hence, the diagnostic test developed in the present study can be exploited as a potential and reliable tool in the serodiagnosis and surveillance of surra in animals.

Weak cation exchange magnetic beads coupled with matrix-assisted laser desorption ionization-time of flight-mass spectrometry in screening serum protein markers in osteopenia.

The present study aimed at investigating the weak cation magnetic separation technology and matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) in screening serum protein markers of osteopenia from ten postmenopausal women and ten postmenopausal women without osteopenia as control group, to find a new method for screening biomarkers and establishing a diagnostic model for primary type I osteoporosis. Serum samples were collected from postmenopausal women with osteopenia and postmenopausal women with normal bone mass. Proteins were extracted from serum samples by weak cation exchange magnetic beads technology, and mass spectra acquisition was done by MALDI-TOF-MS. The visualization and comparison of data sets, statistical peak evaluation, model recognition, and discovery of biomarker candidates were handled by the proteinchip data analysis system software(ZJU-PDAS). The diagnostic models were established using genetic arithmetic based support vector machine (SVM). The SVM result with the highest Youden Index was selected as the model. Combinatorial Peaks having the highest accuracy in distinguishing different samples were selected as potential biomarker. From the two group serum samples, a total of 133 differential features were selected. Ten features with significant intensity differences were screened. In the pair-wise comparisons, processing of MALDI-TOF spectra resulted in the identification of ten differential features between postmenopausal women with osteopenia and postmenopausal women with normal bone mass. The difference of features by Youden index showed that the highest features had a mass to charge ratio of 1699 and 3038 Da. A diagnosis model was established with these two peaks as the candidate marker, and the specificity of the model is 100 %, the sensitivity was 90 % by leave-one-out cross validation test. The two groups of specimens in SVM results on the scatter plot could be clearly distinguished. The peak with m/z 3038 in the SVM model was suggested as Secretin by TagIdent tool. To provide further validation, the secretin levels in serum were analyzed using enzyme-linked immunosorbent assays that is a competitive inhibition enzyme immunoassay technique for the in vitro quantitative measurement of secretin in human serum.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-2276-4) contains supplementary material, which is available to authorized users.

Analysis of cross-reactivity between the Fatty Acid Binding Proteins from Blomia tropicalis and Litopenaeus vannamei

Analysis of cross-reactivity between the Fatty Acid Binding Proteins from Blomia tropicalis and Litopenaeus vannamei

Determinants of house dust, endotoxin, and β-(1→3)-D-glucan in homes of Danish children.

Little is known about the geographic variation and determinants of bacterial endotoxin and β-(1,3)-D-glucan in Danish house dust. In a population of 317 children, we: (i) described loads and concentrations of floor dust, endotoxin, and β-(1→3)-D-glucan and (ii) their correlations and (iii) assessed their determinants; (iv) Finally, we compared our findings with previous European studies. Bedroom floor dust was analyzed for endotoxin content by the kinetic limulus amoebocyte lysate assay and for β-(1→3)-D-glucan by the inhibition enzyme immunoassay. The parents answered questions regarding potential determinants. We found: geometric means (geometric standard deviations) 186 mg/m(2) (4.3) for dust; 5.46 × 10(3) EU/m(2) (8.0) and 31.1 × 10(3) EU/g (2.6) for endotoxin; and 142 μg/m(2) (14.3) and 0.71 × 10(3) μg/g (7.3) for β-(1→3)-D-glucan. High correlations (r > 0.75) were found between floor dust and endotoxin and β-(1→3)-D-glucan loads, while endotoxin and β-(1→3)-D-glucan concentrations were moderately correlated (r = 0.36-0.41) with the dust load. Having a carpet was positively associated with dust load and with endotoxin and β-(1→3)-D-glucan concentrations. Pet keeping, dwelling type, and dwelling location were determinants of endotoxin concentrations. No other determinants were associated with β-(1→3)-D-glucan concentrations. Compared with other European studies, we found lower β-(1→3)-D-glucan loads and concentrations but higher endotoxin loads and concentrations suggesting a geographically determined different composition of Danish floor dust compared with other European regions.

Efficacy of the HPV-16/18 vaccine: Final according to protocol results from the blinded phase of the randomized Costa Rica HPV-16/18 vaccine trial

BackgroundA community-based randomized trial was conducted in Costa Rica to evaluate the HPV-16/18 AS04-adjuvanted vaccine (NCT00128661). The primary objective was to evaluate efficacy of the vaccine to prevent cervical intraepithelial neoplasia 2 or more severe disease (CIN2+) associated with incident HPV-16/18 cervical infections. Secondary objectives were to evaluate efficacy against CIN2+ associated with incident cervical infection by any oncogenic HPVs and to evaluate duration of protection against incident cervical infection with HPV-16/18. Vaccine safety and immunogenicity over the 4-year follow-up were also evaluated. MethodsWe randomized (3727 HPV arm; 3739 control arm), vaccinated (HPV-16/18 or Hepatitis A) and followed (median 53.8 months) 7466 healthy women aged 18–25 years. 5312 women (2635 HPV arm; 2677 control arm) were included in the according to protocol analysis for efficacy. The full cohort was evaluated for safety. Immunogenicity was considered on a subset of 354 (HPV-16) and 379 (HPV-18) women. HPV type was assessed by PCR on cervical specimens. Immunogenicity was assessed using ELISA and inhibition enzyme immunoassays. Disease outcomes were histologically confirmed. Vaccine efficacy and 95% confidence intervals (95%CI) were computed. ResultsVaccine efficacy was 89.8% (95% CI: 39.5–99.5; N=11 events total) against HPV-16/18 associated CIN2+, 59.9% (95% CI: 20.7–80.8; N=39 events total) against CIN2+ associated with non-HPV-16/18 oncogenic HPVs and 61.4% (95% CI: 29.5–79.8; N=51 events total) against CIN2+ irrespective of HPV type. The vaccine had an acceptable safety profile and induced robust and long-lasting antibody responses. ConclusionsOur findings confirm the high efficacy and immunogenicity of the HPV-16/18 vaccine against incident HPV infections and cervical disease associated with HPV-16/18 and other oncogenic HPV types. These results will serve as a benchmark to which we can compare future findings from the ongoing extended follow-up of participants in the Costa Rica trial. Trial registration: Registered with clinicaltrials.gov: NCT00128661.

Comparison of Hemagglutination Inhibition Assay and Enzyme Immunoassay for Determination of Mumps and Rubella Immune Status in Health Care Personnel

BackgroundScreening tests are available to determine immunity to vaccine‐preventable diseases, such as mumps and rubella. We aimed to define better assay for detecting immune status of health care personnel to vaccine‐preventable diseases.MethodsMumps and rubella antibodies of health care personnel at Shimane University Hospital were examined by hemagglutination inhibition assay (HI), comparing with those by enzyme immunoassay (EIA).ResultsA total of 910 sera from health care personnel were tested. There was poor correlation between HI and EIA in detecting mumps antibodies with correlation coefficient values (r) = 0.190 (P < 0.001), but in rubella antibodies HI and EIA were relatively well correlated (r = 0.930, P < 0.001). Seropositivity rate of HI versus EIA was found to be 65.7 versus 93.2, and 89.5 versus 86.5% for mumps and rubella, respectively. As compared with EIA, HI identified sixfold larger seronegative subjects in mumps. Moreover, in mumps, 88.8% of seronegative subjects detected by HI were seropositive by EIA, while 3.7% of seropositive subjects detected by HI were seronegative by EIA. In rubella, 2.1% of seronegative subjects detected by HI were seropositive by EIA, and 1.7% of seropositive by HI was seronegative by EIA.ConclusionConsiderable difference between HI and EIA in determining immune status of health care personnel to mumps and rubella suggests beneficial use of EIA for the identification of accurate susceptible personnel who subsequently undergo an effective vaccination programs. Seroprevalence survey of health care personnel by using appropriate assay is essential for prevention and infection control strategies in health care settings.

Development and clinical application of an enzyme immunoassay for the determination of midregional proadrenomedullin

Adrenomedullin (ADM) is a 52-amino acid peptide with a variety of physiologic functions such as immunomodulating activity, direct bactericidal activity, maintenance of renal homeostasis, and vasodilatory activity. Midregional proADM (MR-proADM) is derived from a larger 185-amino acid precursor peptide, prepro-adrenomedulin (preproADM), by posttranslational processing. It is suggested to be co-synthesized with ADM in equimolar amounts and has the advantages over ADM in having a longer half-life, no bioactivity, and no binding to protein. Therefore, MR-proADM serves as a surrogate for ADM secretion. In this study, we attempted to develop an enzyme immunoassay (EIA) for quantifying MR-proADM-like immunoreactive substance (IS), which is applicable for monitoring plasma MR-proADM levels. By using β-d-galactosidase-labeled preproADM(83-94) as a marker antigen, anti-rabbit IgG-coated immunoplate as a bound/free separator, and 4-methylumbelliferyl-β-d-galactopyranoside as a fluorogenic substrate, a sensitive and specific EIA was developed for the quantification of MR-proADM-IS in human plasma. The lower limit of quantification was 0.032 pmol/well, and the steep competitive inhibition EIA calibration curve obtained was linear between 0.16 and 10 nmol/L. By using human plasma samples containing 0.2 and 2.0 nmol/L of MR-proADM, the interassay coefficients of variation (reproducibility) were 10.78% and 8.83%, respectively, and intraassay coefficients were 3.91% and 7.81%. Plasma MR-proADM-IS level was significantly higher in patients with chronic renal failure (1.39 ± 0.50 nmol/L) compared with healthy subjects (0.19 ± 0.07 nmol/L). These results suggest that our EIA may be useful to evaluate plasma MR-proADM levels as a biomarker in various clinical settings.

A new endophytic taxol- and baccatin III-producing fungus isolated from Taxus chinensis var. mairei

120 endophytic fungi were isolated from the old inner bark of Taxus chinensis var. mairei and only a fungus was detected to produce Taxol and related taxanes in potato dextrose agar (PDA) medium. The presence of taxol and baccatin III was confirmed by high performance liquid chromatography combined with mass spectrometry (LC-MS) and competitive inhibition enzyme immunoassay (CIEIA). The fungal compound showed cytotoxic activity on liver cancer cell line BEL7402 in vitro. The strain was identified as one of Didymostilbe sp. (designated as DF110) according to its morphological characteristics. The isolation of such fungi provided a promising alternative approach to producing taxol in the near future. Key words : Taxol, baccatin III, endophytic fungus, Didymostilbe sp.

Temporal and Spatial Patterns of West Nile Virus Transmission in Saginaw County, Michigan, 2003–2006

The dynamics of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) infection in mosquitoes, sentinel pheasants, and wild dead birds were evaluated during 2003-2006 in Saginaw Co., MI. Mosquitoes were collected by New Jersey Light Traps at 22 sites during May-September, pooled by species and sample location, and tested for presence of WNV RNA by using a real-time reverse transcription-polymerase chain reaction assay. Oral swabs from wild dead birds submitted by the public were tested by Vec-Test assay. Sentinel pheasants were bled weekly, and serum was tested for antibodies with an inhibition enzyme immunoassay. In total, 37,225 mosquitoes [Aedes vexans (Meigen), Culex pipiens L., and Culex restuans Theobald] were tested in 5,429 pools, of which 59 (1.1%) were positive. Ae. vexans was most abundant but had a comparatively low infection rate (0.06-2.11) compared with Cx. pipiens (1.75-4.59) and Cx. restuans (1.22-15.67). Mosquito abundances were temporally related to variations in 2-wk average weather variables. Infected dead crows appeared earlier each transmission season than blue jays, but infection prevalence for both peaked approximately mid-August. Space-time clusters were found in different locations each year. Sentinel pheasant seroprevalence was 19.3% (16/83), 12.7% (10/79), and 7.7% (5/65) during 2003-2005, respectively. We demonstrated temporal patterns of WNV activity in corvid birds and Culex spp. mosquitoes during the study period, suggesting virus transmission within an enzootic cycle. Despite the absence of human case reports nearby, this surveillance system demonstrated WNV transmission and possible human risk. Maintained surveillance using more appropriate gravid traps and CDC CO2 light traps could improve sensitivity of vector collection and virus detection.

Design and synthesis of immunoconjugates and development of competition inhibition enzyme-linked immunosorbent assay (CIEIA) for the detection of O-isopropyl methylphosphonofluoridate (sarin): An organophosphorous toxicant

Three haptens of the organophosphorus (OP) toxicant ‘sarin’ having different spacer arm were designed and synthesized. Haptens were conjugated with BSA (bovine serum albumin) and ovalbumin (OVA) for raising antibody and coating antigen. High antibody titer with higher specificity was obtained from 4-(4-(isopropoxy(methyl)phosphoryloxy)phenylamino)-4-oxobutanoic acid (hapten B) having reasonable long spacer arm. For the standard curve, an IC 50 (inhibitory concentration) of free antigen was found to be 0.415 μg mL −1 on the basis of indirect competitive ELISA. The study revealed that heterology in competition inhibition enzyme immunoassay (CIEIA) produced remarkable improvement in the sensitivity and specificity of the assay. Under the optimized conditions, the quantitative working range was found to be 0.19–1.56 μg mL −1 with a limit of detection (LOD) of 0.05 μg mL −1. The antibodies showed negligible cross reactivity (CR) with other OP toxicants and pesticides, which makes the assay suitable for the selective detection of sarin.

Detection of autoantibodies against glycosylated-DNA in diabetic subjects: Its possible correlation with HbA(1C).

Aims: The vascular tissues have a long memory of their previous glycemic control and intervention studies have demonstrated that microvascular complications are highly correlated with mean glycemic control as measured by glycolated hemoglobin A1C (HbA1c). The present study was carried out to evaluate the autoantibodies against glycosylated DNA (DNAAGEs) in diabetic sera to see if the level of HbA1c has correlation with the activity of DNA-AGEs, another marker of chronic glycemia. Methods: Glucose-6-phosphate induced glycosylation of native DNA was studied in 150 diabetic sera (T1DM = 9, T2DM = 141) by spectroscopic techniques (UV and fluorescence) and agarose gel electrophoresis. Direct binding and inhibition enzyme immunoassays were carried out to evaluate binding and specificity of anti-glycated-DNA autoantibodies (anti-DNA-AGE autoantibodies) in sera of diabetes patients. A quantitative estimation of HbA1c was carried out in normal and diabetes sera.Results: Anti-DNA-AGEs autoantibodies in 55% of diabetic sera (85/150) showed more binding with glycated-DNA compared to native DNA which was subjected to inhibition ELISA indicating true interaction of these autoantibodies in diabetes with glycated DNA. Higher binding with glycosylated-DNA against native-DNA was observed in subjects with HbA1c of 9.8 ± 3.3% compared to those with HbA1c of 7.7 ± 1.7% (p < 0.001). Linear correlation analysis showed that mean absorbance difference was significantly related to HbA1c (r = 0.486, p < 0.001), nephropathy (r = 0.239, p < 0.003), retinopathy (r = 0.165, p < 0.05). Conclusions: Autoantibodies against glycosylated DNA were correlated with HbA1c and microvascular complications and may be useful as another biomarker for assessment of chronic glycemia.