Competitive ELISA Method Research Articles

M-008 LOWER OXIDIZED LDL AUTOANTIBODIES PREDICT RESTENOSIS IN ACUTE MYOCARDIAL INFARCTION PATIENT AFTER PRIMARY BALLOON ANGIOPLASTY

Background Oxidized low-density lipoproteins (ox-LDLs) and their autoantibodies (OLAB) have been detected in human plasma and in atherosclerosis lesion. OLAB have a role in the clearance of oxLDLs from circulation and might be protective against the risk of atherosclerosis. However, the prognostic value of circulating OLAB in patients with acute myocardial infarction (AMI) is unknown. Methods and Results Fifty-eight AMI patients undergoing primary coronary balloon angioplasty were recruited. Plasma levels of oxLDLs and OLAB were measured using competitive ELISA method. Measurements were taken before primary angioplasty, on the 3rd day, on the 7th day and at 1 month after acute event. After 6 month follow-up, angiographic coronary artery restenosis occurred in 18 patients (31%). Plasma OLAB levels were significantly lower in restenosis group. The serial OLAB levels were presented as follows: before PCI (190.9 ± 118.5 vs. 330.8 ± 254.8 U/L, P = 0.006), on the 3rd day (155.2 ± 90.00 vs. 273.6 ± 184.1 U/L, P = 0.004), on the 7th day (189.1 ± 120.2 vs. 337.1 ± 276.0 U/L, P = 0.001) and at 30 days (310.2 ± 305.0 vs. 337.1 ± 326.0 U/L, P = 0.769). However, the serial oxLDLs levels were without significant difference between restenosis and non-restenosis group. The oxLDLs levels before PCI (66.80 ± 19.72 vs. 67.24 ± 22.48 mg/dL, P = 0.939), on the 3rd day (59.25 ± 16.17 vs. 66.33 ± 19.91 mg/dL, P = 0.407), on the 7th day (50.82 ± 12.55 vs. 53.62 ± 16.37, P = 0.514) and at 0 days (54.70 ± 22.11 vs. 60.22 ± 29.70, P = 0.476) were presented. Conclusion Lower levels of circulating OLAB in acute and subacute stage of AMI patients, can predict restenosis of infarct related artery after primary angioplasty. Our data support the notion that OLAB play a role in the maintenance of vascular health.

Comparison of the results of the ELISA, histochemical, and immunohistochemical detection of soya proteins in meat products

This work compares the commonly used immunochemical methods for soya protein detection and alternative microscopic methods. Immunochemical methods were represented by the competitive ELISA method. Histochemical and immunohistochemical methods were used for microscopical examination. From a group of 252 meat products, each sample was examined for soya proteins by ELISA, histochemical, and immunohistochemical methods. The products came from the following categories: cooked sausages, ham, dry cooked sausages, and fermented sausages. The results showed that the highest accuracy was achieved by immunohistochemical examination. However, in the category of cooked sausages, this result was not statistically significant. Since the results in the individual categories differed, our results demonstrate that one single method does not always provide reliable and completely objective results. Immunohistochemical methods seem to be the most suitable for the verification of the reference immunochemical method results and prevention of false results.

Monoclonal Antibody-Based ELISA and Colloidal Gold Immunoassay for Detecting 19-Nortestosterone Residue in Animal Tissues

This article presents the generation of monoclonal antibodies (mAbs) with high specificity against 19-nortestosterone (NT) through cell fusion techniques and the development of a mAb-based indirect competitive ELISA (icELISA) method and colloidal gold-based immuno-chromatographic assay to detect NT residues in beef and pork samples. A modified carbodiimide method was employed to synthesize the artificial antigen, and BALB/c mice were used to produce anti-NT mAbs. On the basis of the checkerboard titration, an indirect competitive ELISA standard curve was established. This assay was sensitive and had a linear range from 0.03 to 38 ng/mL in phosphate buffered saline (PBS), with IC(50) and LOD values of 0.52 ng/mL and 0.01 ng/mL, respectively. Of all the competitive analogues, the produced mAb exhibited a high cross-reactivity to 17α-nortestosterone (83.6%), the main metabolite of NT in animal tissues. Except for moderate cross-reactivities with trenbolone (22.6%) and β-boldenone (13.8%), the other interference to the assay was negligible (<0.05%). In contrast, the strip test had a visual detection limit of 1 ng/mL in PBS, 2 μg/kg in beef, and 2 μg/kg in pork, respectively, and the results can be judged within 10 min. The ELISA and GC-MS results showed close correlation in beef (R2=0.9945) and in pork (R2=0.9977). Therefore, the combination of two immunoassays provides a useful screening method for quantitative or qualitative detection of NT residues in animal-origin products.

Early effects of erythropoietin on serum hepcidin and serum iron bioavailability in healthy volunteers

Hepcidin regulates plasma iron bioavailability and subsequently iron availability for erythropoiesis. rHuEPO has been reported to decrease hepcidin expression in case of repeated subcutaneous injections. Thus, hepcidin level measurement could be a candidate marker for detection of rHuEPO abuse. However, when used for doping, rHuEPO can be injected intravenously and the scheme of injection is unknown. Our aim was to evaluate the early effects of a single intravenous rHuEPO injection on serum hepcidin levels. Fourteen male healthy volunteers received one intravenous injection of 50U/Kg of rHuEPO during a placebo-controlled, randomized, double-blind, cross-over study. Serum hepcidin, quantified by a competitive ELISA method and iron parameters was then evaluated for 24h. Serum levels of hepcidin were significantly increased 4h after rHuEPO injection when compared with placebo injection (78.3±55.5 vs. 57.5±34.6ng/ml, respectively; +36%, p<0.05), whereas iron and transferrin saturation dramatically decreased 12h after rHuEPO injection when compared with placebo injection (9.2±3.5 vs. 15.8±4.2μg/l, respectively; -42%, p<0.05 and 14.8±5.0 vs. 26.3±6.4%, respectively; -44%, p<0.05). In addition, 12 and 24h after rHuEPO injection serum hepcidin levels were lower compared with placebo injection (41.6±27.4 vs. 56.6±28.1ng/ml after 12h; -27%, p<0.05 and 26.0±29.6 vs. 81.2±29.4ng/ml after 24h; -68%, p<0.05). Intravenous injection of recombinant EPO induces a precocious and transient increase of serum hepcidin leading to a transient decrease of iron bioavailability. The transitory increase and dynamics of its concentration make difficult the practical use of hepcidin to detect rHuEPO doping.

Vitamin C가 방사선과 Aflatoxin B1을 투여한 흰쥐의 혈청 중 Aflatoxin B1잔류량에 미치는 영향

Atlatoxin은 Aspergillus속 곰팡이로부터 생성되며 사람에게 있어서 간독성, 및 간암을 유발하는 잠재력을 가진 대표적 곰팡이 독소이며, 생체에 섭취된 <TEX>$AFB_1$</TEX>은 여러 가지 대사경로를 거쳐 동물 조직 내에 축적되는 것으로 알려져 있다. 방사선은 수술, 항암약물요법과 더불어 임상 시 중요한 치료방법이나 정상세포에 방사선을 조사하였을 때 반응성이 높은 활성산소와 과산화라디칼(<TEX>$OH{\bullet}$</TEX>)을 생성하여 세포막의 불포화 지방산을 지질 과산화물로 변성시켜 세포 산화적 손상을 일으킨다. 본 연구는 곡류를 주식으로 하는 우리나라 사람들의 경우 <TEX>$AFB_1$</TEX>에 노출되기 쉽고 이들 중 일부는 위, 간, 담도 암과 같은 상복부 암으로 진단되어 간을 포함하는 부위에 대해 방사선 치료를 받을 수 있는 경우를 생각하여 흰쥐에게 <TEX>$AFB_1$</TEX>을 투여하거나 방사선과 <TEX>$AFB_1$</TEX>을 병합처리하여 vitamin C가 흰쥐의 혈청중의 <TEX>$AFB_1$</TEX> 잔류량에 미치는 영향을 조사하기 위하여 수행되었다. X-ray 조사는 실험기간 내 단 1회로 실험사육기간 첫 일에 조사 하였고 X-ray 조사 후 vitamin C를 투여하였으며 vitamin C 투여 1시간 후 <TEX>$AFB_1$</TEX>을 투여하였다. vitamin C와 AFB은 모두 복강투여로 실험 사육 첫 일부터 1회 시작하여 3일에 한번씩, 5회 반복 투였으며 실험동물 사육기간은 총 15일로 하였다. 실험이 끝난 마우스 혈청 중 <TEX>$AFB_1$</TEX>은 indirect competitive ELISA와 HPLC법으로 측정 후 비교하였다. ELISA 법에 의한 흰쥐의 혈청 중 <TEX>$AFB_1$</TEX> 의 함량은 ELISA 실험에서 AFB1 단독 투여한 2군에서는 <TEX>$5.17{\pm}0.34$</TEX> ng/mL 였으며, 2군에 vitamin C를 투여한 3군에서는 <TEX>$3.23{\pm}0.76$</TEX> ng/mL로 감소하는 경향이었다. 또한 X선 조사와 <TEX>$AFB_1$</TEX> 병합처리한 4군의 혈청 중 <TEX>$AFB_1$</TEX> 함량은 <TEX>$5.62{\pm}0.44$</TEX> ng/mL 이었으며 여기에 vitamin C를 함께 투여 한 5군에서는 <TEX>$4.84{\pm}0.36$</TEX> ng/mL로 약간 감소하는 결과를 나타내었다. 한편 HPLC 결과에서도 ELISA와 같은 결과를 나타내었으며, <TEX>$AFB_1$</TEX> 단독 투여군(2군)과 X선 조사와 <TEX>$AFB_1$</TEX> 병합처리군(4군)에서의 혈청 중 <TEX>$AFB_1$</TEX> 함량은 각각 <TEX>$4.82{\pm}0.17$</TEX> ng/mL, <TEX>$5.36{\pm}0.42$</TEX> ng/mL 이었으나 여기에 vitamin C를 함께 투여한 3군과 5군에서는 각각 <TEX>$2.77{\pm}0.23$</TEX> ng/mL, <TEX>$4.33{\pm}0.14$</TEX> ng/mL로 유의적인 감소를 보여 vitamin C가 흰쥐의 혈청 중 <TEX>$AFB_1$</TEX> 잔류량을 감소시키는 것으로 나타났다. Aflatoxin (<TEX>$AFB_1$</TEX>) is a potent hepatotoxic and hepatocarcinogenic mycotoxin in humans. It is also well-known to be accumulated in animal tissues via various metabolic pathways. This study was conducted to determine the effects of vitamin C on the residual <TEX>$AFB_1$</TEX> in rat sera that were treated with radiation and <TEX>$AFB_1$</TEX>. Six week-old male Sprague-Dawley rats were randomly divided into five groups: a control group, <TEX>$AFB_1$</TEX>-treated group, the group treated with <TEX>$AFB_1$</TEX> and vitamin C, the group treated with X-ray and AFB1, and the group treated with X-ray and <TEX>$AFB_1$</TEX> with vitamin C. On the first day of the experiment, only one dose of X-rays was exposed to the entire liver at 1,500 cGy. Next, vitamin C was injected at 10 mg/kg body weight via intraperitoneal injection, followed 1 hr later by the administration of 0.4 mg/kg of <TEX>$AFB_1$</TEX> via intraperitoneal injection. These treatments were then administered every three days over a period of 15 days. On the 16th day of treatments, the animals were sacrificed. The contents of <TEX>$AFB_1$</TEX> in rat sera were determined via indirect competitive ELISA and HPLC method. In the quantitative analysis of <TEX>$AFB_1$</TEX> in rat sera via ELISA, <TEX>$5.17{\pm}0.34$</TEX> ng/mL of <TEX>$AFB_1$</TEX> was detected in the <TEX>$AFB_1$</TEX>-treated groups, but the amount more significantly decreased to <TEX>$3.23{\pm}0.76$</TEX> ng/mL in the groups treated with <TEX>$AFB_1$</TEX> and vitamin C (p<0.01) than in the <TEX>$AFB_1$</TEX>-treated groups. The <TEX>$AFB_1$</TEX> contents of the rat sera of the groups treated with X-ray and <TEX>$AFB_1$</TEX> did not significantly decreased with the administration of vitamin C. The <TEX>$AFB_1$</TEX> content of the rat sera that was analyzed via HPLC showed a tendency similar to that of the content that was analyzed via ELISA. With regard to these data, vitamin C was very effective in reducing <TEX>$AFB_1$</TEX> residue in rat sera.

Pigment epithelium-derived factor inhibits advanced glycation end-products-induced cytotoxicity in retinal pericytes

Aim This study investigated the effects of pigment epithelium-derived factor (PEDF) on advanced glycation end-product (AGE)-induced cytotoxicity in porcine retinal pericytes and the signalling mechanism involved. Methods Retinal pericytes were isolated from porcine eyes and characterized by immunocytochemistry. The effect of AGEs and PEDF on cell proliferation was determined by bromodeoxyuridine (BrdU) assay. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was analyzed by luminescence assay. Reactive oxygen species (ROS), nitric oxide (NO), superoxide dismutase (SOD) and glutathione peroxidase (GSH) were determined by biochemical assays. Induction of apoptosis was determined by caspase-3 colorimetric assay and DNA fragmentation analysis. Src activity was assessed by transient transfection analysis, and the status of Src phosphorylation at Y419 was analyzed by a competitive ELISA method. Results AGEs significantly increased intracellular ROS generation in pericytes via NADPH oxidase and induced cell death via caspase-3 enzyme activation, whereas PEDF increased cell proliferation in a dose-dependent manner. In addition, PEDF inhibited AGE-induced ROS generation by increasing levels of SOD and GSH, and also blocked the activation of caspase-3. Furthermore, PEDF induced cell survival via the Src pathway by Src phosphorylation at Y419, as evidenced by a pharmacological inhibitor and Src mutants. Conclusion These results suggest that PEDF abrogates AGE-induced oxidative stress and apoptosis in retinal pericytes via the Src pathway, thereby suggesting that PEDF is an effective therapeutic agent for the treatment of loss of pericytes in early diabetic retinopathy.

Development of a sensitive competitive indirect ELISA method for determination of ochratoxin A levels in cereals originating from Nanjing, China

A monoclonal antibody against ochratoxin A (OTA) was produced and a simple, rapid and reliable competitive indirect ELISA (ci-ELISA) for detection of OTA was developed. The 50% inhibitory concentration (IC 50) was 1.70 ng/mL, with a detectable range of 0.55–6.75 ng/mL, and a limit of determination (LOD) at 0.15 ng/mL. The cross-reactivity of the antibody against ochratoxin B was 17%, and <10% against deoxynivalenol, fumonisin B 1 or aflatoxin B 1. A survey of 65 cereal samples was carried out in Nanjing, the capital of Jiangsu Province, China. The frequencies of OTA contamination were 36.36%, 26.08% and 15.00%, and the mean concentrations of positive samples were 4.248 ng/g, 7.360 ng/g and 3.382 ng/g for wheat, corn and rice, respectively. The results of this study suggest that the ci-ELISA developed can be used to determine OTA in agricultural commodities such as cereals, and a survey might be needed to investigate OTA contamination in food and feed to prevent their harmful health effects.

Development and Optimization of a mAb-Based Indirect Competitive ELISA for Enrofloxacin Residue in Duck

This paper presents the generation of monoclonal antibodies (mAbs) with high specificity against enrofloxacin (ENR) through cell fusion procedures, and the development of a mAb-based indirect competitive ELISA (icELISA) method to detect ENR residue using one of these Hybridomas (clone 4B5-D6). Under the optimal experimental conditions, this assay exhibited a working range of 0.004-38 ng/mL with IC50 and LOD values of 0.4 and 0.002 ng/mL, respectively. Except for a high cross-reactivity (105.2%) to Ciprofloxacin, negligible cross-reactivity to the other compounds was observed. After optimization, 10% of methanol was used in the assay buffer and this ELISA system can tolerate acetonitrile not higher than 10%. Recovery studies indicate that an excellent correlation between concentration spiked and concentration determined was found, and the results also suggest this assay has the potential to be incorporated into a quantitative monitoring program for the rapid screening of ENR residue in poultry tissues.

Enhancement of the Immunogenicity of Synthetic Carbohydrate Vaccines by Chemical Modifications of STn Antigen

The abnormal glycans expressed on the surface of tumor cells, known as tumor-associated carbohydrate antigens, increase the chance to develop carbohydrate-based anticancer vaccines. However, carbohydrate antigens pose certain difficulties, and the major drawback is their weak immunogenicity. To tackle this problem, numerous structurally modified STn antigens were designed and synthesized in this work. These synthetic antigens were screened in vitro by using competitive ELISA method, and the antigens with positive response were conjugated to the protein carrier for vaccination. The vaccination results on mice showed that some fluorine-containing modifications on the STn antigen can significantly increase the anti-STn IgG titers and improve the ratios of anti-STn IgG/IgM. The antisera can recognize the tumor cells expressing the native STn antigen.

Aflatoxin M1 levels in retail yoghurt and ayran in Erzurum in Turkey

This study was carried out to determine the presence and levels of aflatoxin M1 (AFM1) in yoghurt and ayran consumed in the province of Erzurum, Turkey. For this purpose, a total of 80 yoghurt samples and 80 ayran samples were randomly obtained from markets. The samples' AFM1 content and concentrations were examined by competitive ELISA method. AFM1 at detectable level (5 ng/kg) was found in 87.5% of the yoghurt samples and in 90.0% of the ayran samples. AFM1 levels in 16 (20%) yoghurt and in 11 (13.6%) ayran samples exceeded the maximum tolerable limit of the Turkish Food Codex. The positive incidence of AFM1 in the yoghurt and ayran samples ranged from 10 to 475 ng/kg and from 6 to 264 ng/kg, respectively. It was concluded that high AFM1 level is an important problem threatening public health in Turkey. Therefore, dairy products have to be controlled routinely for presence of AFM1 contamination by public health authorities.

Development of an Immunoassay for Determination of Fluoroquinolones Pollutant in Environmental Water Samples

An immunoassay method for determination of fluoroquinolones (FQs) residues in water samples has been developed. For this purpose, NHS ester technology was employed to synthesize the immunogen and coating antigen of sarafloxacin (SAR). SDS-PAGE, UV-visible spectra and infrared spectra identification showed that the artificial antigen was conjugated successfully. Based on the square matrix titration, an indirect competitive ELISA (icELISA) method was established. The dynamic range in assay buffer was from 0.048 to 62.4 ng/mL, with LOD and IC 50 value of 0.018 and 2.5 ng/mL, respectively. This assay showed a high cross-reactivity to difloxacin (86.7%), norfloxacin (63.2%), and pefloxacin (33.8%). When applied in authentic tap water, river water and fishing water samples, the recoveries for sarafloxacin, difloxacin, norfloxacin and pefloxacin were 89.5-119.5%, 90.5-121.2%, 92.1-118.6%, and 88.2-116.8%, respectively, with coefficient of variation (CV) values all < 10%. The results suggest this immunoassay can be used for simultaneous detecting sarafloxacin, difloxacin, norfloxacin, and pefloxacin residues in water samples.

Multiresidue Determination of Sarafloxacin, Difloxacin, Norfloxacin, and Pefloxacin in Fish using an Enzyme-Linked Immunosorbent Assay

An indirect competitive ELISA (icELISA) method for multiresidue determination of Fluoroquinolones (FQs) residues in fish samples has been developed. For this purpose, Sarafloxacin (SAR) was employed to synthesize the immunogen and coating antigen through EDC conjugation method, and cell fusion technology was used to produce anti-SAR monoclonal antiobdy. Based on the square matrix titration, an icELISA method was established. The dynamic range for Sarafloxacin in assay buffer was from 0.004 to 18 ng/mL, with LOD and IC50 value of 0.002 ng/mL and 0.32 ng/mL, respectively. After optimization, the physiological pH (7.4) was selected for the immunoassays, and this assay could tolerate up to 10% acetonitrile. The results of this assay showed a high cross- reactivity to Difloxacin (85.5%), Norfloxacin (61.7%), and Pefloxacin (34.8%). Under the 10-fold dilution in authentic fish samples, the regression curve equations for Sarafloxacin, Difloxacin, Norfloxacin and Pefloxacin were y=1.0114x - 0.4003, R2=0.9901; y=0.9782x + 0.2754, R2=0.9807; y=0.9892x +0.0489, R2=0.9843; and y=0.9797x +0.8017, R2=0.9844, respectively. The results suggest this immunoassay can be used for simultaneous detecting four kinds of FQs in fish samples.

Elevated urinary total sialic acid and increased oxidative stress in patients with bladder cancer

Abstract Background: Increased production and release of sialic acid have been reported in many malignant conditions including bladder cancer. 8-hydroxydeoxyguanosine (8-OHdG) and malondialdehyde (MDA) have been widely used as oxidative stress biomarkers. Objective: Determine urinary levels of total sialic acid (TSA), 8-OHdG, and MDA in patients with urinary bladder cancer, and evaluate their clinical relevance. Patients and methods: Forty-five patients with histologically proven bladder cancer and 41 healthy subjects were recruited for the study. Morning urine samples were collected from all participants for measurements of TSA, 8-OHdG and MDA using thiobarbituric assay, competitive ELISA and spectrophotometry methods, respectively. Histological examination was performed for all patients. Results: Bladder cancer patients excreted urinary TSA, 8-OHdG, and MDA significantly higher than healthy controls. Based on receiver operating characteristic curve analysis, urinary TSA had adequate diagnostic potential to distinguish patients from healthy populations, and its cutoff value was chosen at 95.26 μg/g creatinine. Sensitivity, specificity, and accuracy of urinary TSA determination were 75.6%, 75.6%, and 75.6%, respectively. Both in patient and healthy groups, urinary TSA was linearly correlated with urinary 8-OHdG. Patients with highseverity grade (n=27) excreted urinary TSA significantly greater than those with low-severity grade (n=18). Conclusion: Urinary TSA, 8-OHdG, and MDA increased in patients with bladder cancer. The elevated urinary TSA was associated with enhanced oxidative stress. In addition, urinary TSA increased with progressiveness of the tumor.

High DNA Oxidative Damage in Systemic Sclerosis

Several lines of evidence suggest that the generation of reactive oxygen species (ROS) is of major importance in the pathogenesis of SSc. Protein and lipid damage have previously been demonstrated, but scarce data are available on oxidative damage to DNA. In patients with SSc, we evaluated levels of 8-hydroxy-2'-deoxyguanosine (8-oxodG), the main validated biomarker of endogenous oxidative damage to DNA, compared to levels of F2-isoprostane, a product of free radical-mediated peroxidation of arachidonic acid. Urinary levels of 8-oxodG and 8-isoprostaglandin-F(2α) (8-iso-PGF(2α)) were determined by competitive ELISA method in consecutive SSc patients and controls matched for age and sex. We included 80 unrelated SSc patients (72 women, mean age 56 ± 11 yrs) and 39 controls (33 women, mean age 64 ± 8 yrs). Urinary levels of 8-oxodG/creat and 8-iso-PGF(2α)/creat in SSc patients were found to be higher than in controls (6.5 ng/mg vs 3.7 ng/mg, p = 0.0001; and 11.4 ng/mg vs 4.2 ng/mg, p < 0.0001). In multivariate analysis, 8-oxodG levels were associated with the presence of pulmonary fibrosis on computerized tomography scan, decreased forced vital capacity, and decreased DLCO/alveolar volume. In patients with the diffuse cutaneous subset, a modified Rodnan skin score > 14 was independently associated with 8-oxodG levels. In SSc, 8-oxodG and 8-iso-PGF(2α) values were correlated (r = 0.32; p = 0.005). Our study confirmed marked oxidative stress in SSc. We also found increased values of 8-oxodG in SSc patients and a relevant association with a fibrotic phenotype. The predictive value of this marker and its potential influence on fibrotic disturbances remain to be determined.

Preparation of Monoclonal Antibody for Melamine and Development of an Indirect Competitive ELISA for Melamine Detection in Raw Milk, Milk Powder, and Animal Feeds

Melamine (MEL) has been involved in several food recalls after the discovery of severe kidney damages in children and pets poisoned by melamine-adulterated food. To detect MEL residue in foods and animal feeds, an indirect competitive ELISA (cELISA) method was developed in this study based on preparation of monoclonal antibodies (MAbs) to MEL. The immunogen was prepared by linking MEL hapten with carrier protein via carbodiimide method. The method is applicable in the range of 5.0-135.0 microg L(-1) MEL in buffer solution, with an IC(50) value of 22.6 +/- 1.9 microg L(-1). The MAbs showed high specificity with low cross-reactivity (< or =1%) toward cyanurate, ammelide, and ammeline. The method was utilized in the detection of MEL in raw milk, milk powder, and animal feeds, with detection limits of 0.1 mg L(-1) for milk, 0.2 mg kg(-1) for milk powder, and 0.5 mg L(-1) for feeds. The recovery ratio was 79-110% for all matrices. The intra-assay and interassay coefficients of variation were <12.0 and <13.0%, respectively. Finally, the application of the cELISA in quantity evaluation of MEL in various feeds from local markets was evaluated and discussed.

Generation of anti-NAG-2 mAb from patients' memory B cells: implications for a novel therapeutic strategy in systemic sclerosis

We have previously reported that antibodies directed against the cytomegalovirus-derived protein UL94 cross react with the cell surface tetraspanin transmembrane 4 superfamily member 7 (TM4SF7 or NAG-2) molecule inducing apoptosis of endothelial cells and activation of fibroblasts in patients with systemic sclerosis (SSc). We aimed at generating a non-functional mAb directed against NAG-2 from patients’ memory B cells. Direct and competitive ELISA methods have been used to evaluate the binding of antibodies from scleroderma patients’ and controls’ sera to the NAG-2 peptide. IgG memory B cells were sorted, EBV transformed and cloned to obtain NAG-2-specific mAbs. Endothelial cells and fibroblasts were cultured under standard conditions and used for functional assays. Anti-NAG-2-purified antibodies obtained from patients’ Ig induce endothelial cell apoptosis and fibroblast proliferation. Patients’ Igs depleted of the anti-NAG-2 fraction do not exert such functional activity. Therefore, the NAG-2 molecule represents a potential novel candidate for therapeutic intervention in SSc. Here, we describe the generation of a human mAb directed against the NAG-2 molecule. Such mAb does not retain any functional property and is able to block the effect of serum pathogenetic anti-NAG-2 antibodies. The majority of SSc patients present antibodies directed against tetraspanin NAG-2 and mediate both endothelial cell apoptosis and fibroblast proliferation, features of the disease. The anti-NAG-2 human mAb we have obtained blocks signal transduction and therefore may be a potential candidate for a new treatment in SSc, a disease where the current biological therapies have little or no efficacy.

The research of MPT64 antibodies aptamer of Mycobacterium tuberculosis in the serological diagnosis

Objective To establish mixed-sandwich ELISA detection system by screening aptam-ers of MPT64 antibodies with SELEX to detect clinical serum samples, and explore the potential laboratory diagnosis value of this method. Methods To detect the affinity of the final round ssDNA library to MPT64 antibodies inhibited by MPT64 antigen with the competitive ELISA method, optimize the mixed-sandwich ELISA detection method that was aptamer-serum-horseradish peroxidase labeled goat anti-human IgG anti-body detection system to detect 230 cases of clinical serum samples as well as the lowest concentration of MPT64 antibodies and the linear range. Results In competitive ELISA test results, the percentage of inhi-bition effect of MPT64 antigen to final round ssDNA library is from 0.25% to 80% when the MPT64 antigen concentration rised from 2 μg/ml to 256 μg/ml. The Optimized detection system of mixed-sandwich ELISA was constitute of the concentration of ssDNA coated with 0.1μg/hole, serum dilution of 1/200, horseradish peroxidase labeled goat anti-human IgG antibody concentration of 1/40 000. The lowest concentration of MPT64 antibody is 3 mg/L and the linear range is between 10 mg/L and 1000 mg/L. The serum samples of 100 cases of tuberculosis patients, 100 healthy individuals and 30 cases of non-tuberculesis were tested in this system and the test result was analyzed with Graphpad Prism, the difference of tuberculosis group and healthy group was statistically significant (P<0.001 ), the difference of TB group and non-TB control group was also statistically significant (P<0.001). The specificity and the sensitivity was 96.1% and 31.0% re-spectively. Conclusion The aptamer mixed-sandwich ELISA method will play an important role in the sero-logical diagnosis of tuberculosis. Key words: MPT64 antibodies; SELEX; Aptamer; ssDNA; Serological detection

COMPETITIVE ENZYME-LINKED IMMUNOSORBENT ASSAY FOR A SELECTIVE AND SENSITIVE DETERMINATION OF DOPAMINE IN THE PRESENCE OF ASCORBIC ACID AND URIC ACID

Dopamine (DA) is one of the most important catecholamine neurotransmitter molecules in the central nervous system (CNS). An abnormal level of DA in vivo can cause CNS diseases such as Parkinsonism and Schizophrenia. Thus, it is essential to develop an accurate and easy to use method for determining the level of DA in biological fluids such as urine and serum as a tool for clinical diagnostics as well as for pathological research. This work is the first ELISA application to detect DA in the presence of a high concentration of ascorbic acid (AA) and uric acid (UA) which are endogenous in urine. The LOD value of 1.26 × 10−9 M and dynamic ranges of 3.16 × 10−3 M to 3.16 × 10−7 M were observed. It shows a good sensitivity with a broad dynamic range. Also, the competitive ELISA method for DA developed here showed no interference effect due to AA and UA, which are found in urine. The presence of AA and UA caused interference for DA determination by an electrochemical method because UA, AA, and DA have similar oxidation potential. Also, this selective and sensitive ELISA method can be made into an ELISA test kit which can be used to quantify the level of DA with ease and simplicity in clinics and laboratories.

Mycobiota and Concentration of Ochratoxin A in Concentrated Poultry Feed from Venezuela

The main objective of this study was to evaluate the frequency distribution of mycobiota and the concentration of Ochratoxin A (OTA) in 50 samples from one company of commercial brand poultry feed produced in Venezuela. The concentration of OTA in the samples analyzed was determined using the competitive ELISA method. The most frequently isolated genera of moulds were Aspergillus (36%) and Penicillium (20%). Of these genera, the most frequently isolated species were Aspergillus flavus, Aspergillus terreus and Penicillium citrinum. Ochratoxigenic species such as Eurotium herbariorum, Aspergillus niger, Aspergillus ochraceus and Aspergillus glaucus, were also found with lower frequency. Rhodotorula mucilaginosa was the only yeast isolated. 94% of the samples presented contamination by OTA in a range between 2.558 and 31.978 microg kg(-1) feed and 42% of them presented OTA levels from 10 up to 20 microg kg(-1). The findings of this investigation show that 84% of the samples of concentrated feed for meat poultry surpass the maximum permitted limit for OTA of 5 microg kg(-1), established in the majority of countries in which regulations are placed.

Determination of Aflatoxin M1 Levels in Some Cheese Types Consumed in Erzurum - Turkey

Summary In this study the total 304 cheese samples (85 white cheese, 75 kashar cheese, 62 civil cheese, 82 cream cheese) were examined in terms of AFM1. The AFM1 content and concentrations of the samples were researched by competitive ELISA method. Determinable limit was 50 ng/kg and it was determined that white cheese samples included 82.4% AFM1, kashar cheese samples 80%, civil cheese samples 19.4% and cream cheese samples 84.2%. According to European Commission limit (250 ng/kg), the sample incidence exceeding the acceptable limits were 27.1%, 34.7%, 17.1% in white cheese, kashar cheese and cream cheese samples, respectively. The sample ratio exceeding the limits regulated by Turkish Food Codex (500 ng/kg) was determined in white cheese, kashar cheese and cream cheese samples as 16.5% (14/85), 14.7% (11/75) and 6.1% (5/82) respectively, any sample exceeding these limits was not met in civil cheese samples. As understood from these results, high AFM1 level determined in some cheese types is an important problem threatening the public health in Turkey.