Development of analytical method for ultrasensitive detection of salbutamol utilizing DNA labeled-immunoprobe.

PubMed

Lei, Yajing; Li, Xiaoyi; Akash, Muhammad Sajid Hamid; Zhou, Lifang; Tang, Xiaojing; Shi, Weixing; Liu, Zhiming; Chen, Shuqing

2015-03-25

Salbutamol (SAL) is known as quick-acting β-2 receptor agonist and its use in humans for pulmonary diseases and/or in animal feed is limited because of associated potential hazardous effects on health. Several techniques are available for the detection of SAL, but are expensive and time consuming. Here for the first time, a novel pre-assembled DNA immunoprobe-based immuno-PCR assay was developed to investigate the levels of SAL in human urine samples and compared the proposed immuno-PCR assay for the detection of SAL with that of direct competitive ELISA. Under the optimized conditions, the assay showed a linear range over seven orders of magnitude, whereas the limit of detection of SAL in buffer was found to be 21 fg/mL. Current immuno-PCR assay exhibited a 300-fold better detection limit for SAL as compared to one achieved with direct competitive ELISA using the same antibody. In conclusion, it has been found that the developed method is highly sensitive and simple method that can significantly enhance the detection sensitivity for small molecules. Moreover it can be modified and used for detecting other small molecules and/or chemicals that exist in the environment and are responsible for the environmental pollution. Copyright © 2014 Elsevier B.V. All rights reserved.

Fabrication and Evaluation of Microfluidic Immunoassay Devices with Antibody-Immobilized Microbeads Retained in Porous Hydrogel Micropillars.

PubMed

Kasama, Toshihiro; Kaji, Noritada; Tokeshi, Manabu; Baba, Yoshinobu

2017-01-01

Due to the inherent characteristics including confinement of molecular diffusion and high surface-to-volume ratio, microfluidic device-based immunoassay has great advantages in cost, speed, sensitivity, and so on, compared with conventional techniques such as microtiter plate-based ELISA, latex agglutination method, and lateral flow immunochromatography. In this paper, we explain the detection of C-reactive protein as a model antigen by using our microfluidic immunoassay device, so-called immuno-pillar device. We describe in detail how we fabricated and used the immuno-pillar devices.

Preclinical Evaluation of an Anti-Nectin-4 ImmunoPET Reagent in Tumor-Bearing Mice and Biodistribution Studies in Cynomolgus Monkeys.

PubMed

Campbell, Dean O; Noda, Akihiro; Verlinsky, Alla; Snyder, Josh; Fujita, Yuji; Murakami, Yoshihiro; Fushiki, Hiroshi; Miyoshi, Sosuke; Lacayo, Sergio; Cabral, Edward; Yang, Peng; Stover, David R; Joseph, Ingrid B J K

2016-10-01

Nectin-4 is selectively overexpressed in a variety of cancers and is currently under clinical investigation as a therapeutic target. A monoclonal antibody against nectin-4 (AGS-22M6) was evaluated as an Immuno-positron emission tomography (ImmunoPET) reagent. Its ability to assay nectin-4 expression as well as detect nectin-4 positive tumors in the liver and bone was evaluated using mouse models. The biodistribution of [(89)Zr]AGS-22M6 was evaluated in mice bearing tumors with varying levels of nectin-4 expression. An isogenic breast cancer tumor line was used to model metastatic liver and bone disease in mice. The biodistribution of [(18)F]AGS-22M6 in cynomolgus monkeys was evaluated. A positive correlation was demonstrated between tumor nectin-4 expression and [(89)Zr]AGS-22M6 uptake. Tumors in the liver and bone were detected and differentiated based on nectin-4 expression. [(18)F]AGS-22M6 showed limited uptake in cynomolgus monkey tissues. [(89)Zr]AGS-22M6 is a promising ImmunoPET reagent that can assay nectin-4 expression in both primary and metastatic lesions.

Immuno-magnetic beads-based extraction-capillary zone electrophoresis-deep UV laser-induced fluorescence analysis of erythropoietin.

PubMed

Wang, Heye; Dou, Peng; Lü, Chenchen; Liu, Zhen

2012-07-13

Erythropoietin (EPO) is an important glycoprotein hormone. Recombinant human EPO (rhEPO) is an important therapeutic drug and can be also used as doping reagent in sports. The analysis of EPO glycoforms in pharmaceutical and sports areas greatly challenges analytical scientists from several aspects, among which sensitive detection and effective and facile sample preparation are two essential issues. Herein, we investigated new possibilities for these two aspects. Deep UV laser-induced fluorescence detection (deep UV-LIF) was established to detect the intrinsic fluorescence of EPO while an immuno-magnetic beads-based extraction (IMBE) was developed to specifically extract EPO glycoforms. Combined with capillary zone electrophoresis (CZE), CZE-deep UV-LIF allows high resolution glycoform profiling with improved sensitivity. The detection sensitivity was improved by one order of magnitude as compared with UV absorbance detection. An additional advantage is that the original glycoform distribution can be completely preserved because no fluorescent labeling is needed. By combining IMBE with CZE-deep UV-LIF, the overall detection sensitivity was 1.5 × 10⁻⁸ mol/L, which was enhanced by two orders of magnitude relative to conventional CZE with UV absorbance detection. It is applicable to the analysis of pharmaceutical preparations of EPO, but the sensitivity is insufficient for the anti-doping analysis of EPO in blood and urine. IMBE can be straightforward and effective approach for sample preparation. However, antibodies with high specificity were the key for application to urine samples because some urinary proteins can severely interfere the immuno-extraction. Copyright © 2012 Elsevier B.V. All rights reserved.

Rapid immuno-analytical system physically integrated with lens-free CMOS image sensor for food-borne pathogens.

PubMed

Jeon, Jin-Woo; Kim, Jee-Hyun; Lee, Jong-Mook; Lee, Won-Ho; Lee, Do-Young; Paek, Se-Hwan

2014-02-15

To realize an inexpensive, pocket-sized immunosensor system, a rapid test devise based on cross-flow immuno-chromatography was physically combined with a lens-free CMOS image sensor (CIS), which was then applied to the detection of the food-borne pathogen, Salmonella typhimurium (S. typhimurium). Two CISs, each retaining 1.3 mega pixel array, were mounted on a printed circuit board to fabricate a disposable sensing module, being connectable with a signal detection system. For the bacterial analysis, a cellulose membrane-based immunosensing platform, ELISA-on-a-chip (EOC), was employed, being integrated with the CIS module, and the antigen-antibody reaction sites were aligned with the respective sensor. In such sensor construction, the chemiluminescent signals produced from the EOC are transferred directly into the sensors and are converted to electric signals on the detector. The EOC-CIS integrated sensor was capable of detecting a traceable amount of the bacterium (4.22 × 10(3)CFU/mL), nearly comparable to that adopting a sophisticated detector such as cooled-charge-coupled device, while having greatly reduced dimensions and cost. Upon coupling with immuno-magnetic separation, the sensor showed an additional 67-fold enhancement in the detection limit. Furthermore, a real sample test was carried out for fish muscles inoculated with a sample of 3.3CFU S. typhimurium per 10 g, which was able to be detected earlier than 6h after the onset of pre-enrichment by culture. © 2013 Elsevier B.V. All rights reserved.

Microarray evaluation of specific IgE to allergen components in elite athletes.

PubMed

Bonini, M; Marcomini, L; Gramiccioni, C; Tranquilli, C; Melioli, G; Canonica, G W; Bonini, S

2012-12-01

Allergic sensitization and diseases have been reported to have a very high and increasing prevalence in elite athletes. Over 80% of allergic athletes are poly-sensitized. This study aims at evaluating the potential diagnostic added value of a microarray technology (ImmunoCAP ISAC, Phadia AB [at present Thermo Fisher Scientific] Uppsala, Sweden which detects IgE antibodies to specific or cross-reacting allergen components. Seventy-two poly-sensitized athletes according to skin prick test (SPT) with different allergic phenotypes (asthma n = 19; rhino-conjunctivitis n = 20; food allergy and/or oral allergy syndrome n = 13; no clinical symptoms n = 20) and two different control populations (20 poly-sensitized sedentary subjects with respiratory allergy and 20 healthy athletes with negative SPT) were studied for detecting specific IgE (sIgE) both to allergen extracts (ImmunoCAPsIgE) and to allergen components (ImmunoCAP ISAC). ImmunoCAP ISAC detected the presence of sIgE in 90% of poly-sensitized athletes--in 96% with symptoms and in 75% without symptoms--and in 100% of allergic controls. The pattern of positivity towards the 103 components tested differed from subject to subject, even in those with the same sensitization to allergen extract SPT or sIgE. Based on the ISAC results, poly-sensitized athletes were classified into the following prototypical patterns, differently represented in the clinical phenotypes studied (P = 0.03): (1) One single predominant specific allergen positivity; (2) sIgE to two or more non-cross-reacting allergens; (3) sIgE to cross-reacting allergens; and (4) sIgE to components potentially responsible for severe allergic reactions. The ImmunoCAP ISAC represents a useful additional tool for diagnosis and management of poly-sensitized athletes. © 2012 John Wiley & Sons A/S.

Photocleavage-based affinity purification of biomarkers from serum: Application to multiplex allergy testing.

PubMed

Wan, Zhi; Ostendorff, Heather P; Liu, Ziying; Schneider, Lynda C; Rothschild, Kenneth J; Lim, Mark J

2018-01-01

Multiplex serological immunoassays, such as implemented on microarray or microsphere-based platforms, provide greater information content and higher throughput, while lowering the cost and blood volume required. These features are particularly attractive in pediatric food allergy testing to facilitate high throughput multi-allergen analysis from finger- or heel-stick collected blood. However, the miniaturization and microfluidics necessary for creating multiplex assays make them highly susceptible to the "matrix effect" caused by interference from non-target agents in serum and other biofluids. Such interference can result in lower sensitivity, specificity, reproducibility and quantitative accuracy. These problems have in large part prevented wide-spread implementation of multiplex immunoassays in clinical laboratories. We report the development of a novel method to eliminate the matrix effect by utilizing photocleavable capture antibodies to purify and concentrate blood-based biomarkers (a process termed PC-PURE) prior to detection in a multiplex immunoassay. To evaluate this approach, it was applied to blood-based allergy testing. Patient total IgE was purified and enriched using PC-PURE followed by multiplex microsphere-based detection of allergen-specific IgEs (termed the AllerBead assay). AllerBead was formatted to detect the eight most common pediatric food allergens: milk, soy, wheat, egg, peanuts, tree nuts, fin fish and shellfish, which account for >90% of all pediatric food allergies. 205 serum samples obtained from Boston Children's Hospital were evaluated. When PC-PURE was employed with AllerBead, excellent agreement was obtained with the standard, non-multiplex, ImmunoCAP® assay (average sensitivity above published negative predictive cutoffs = 96% and average Pearson r = 0.90; average specificity = 97%). In contrast, poor ImmunoCAP®-correlation was observed when PC-PURE was not utilized (average sensitivity above published negative predictive cutoffs = 59% and average Pearson r = 0.61; average specificity = 97%). This approach should be adaptable to improve a wide range of multiplex immunoassays such as in cancer, infectious disease and autoimmune disease.

Photocleavage-based affinity purification of biomarkers from serum: Application to multiplex allergy testing

PubMed Central

Wan, Zhi; Ostendorff, Heather P.; Liu, Ziying; Schneider, Lynda C.; Rothschild, Kenneth J.

2018-01-01

Multiplex serological immunoassays, such as implemented on microarray or microsphere-based platforms, provide greater information content and higher throughput, while lowering the cost and blood volume required. These features are particularly attractive in pediatric food allergy testing to facilitate high throughput multi-allergen analysis from finger- or heel-stick collected blood. However, the miniaturization and microfluidics necessary for creating multiplex assays make them highly susceptible to the “matrix effect” caused by interference from non-target agents in serum and other biofluids. Such interference can result in lower sensitivity, specificity, reproducibility and quantitative accuracy. These problems have in large part prevented wide-spread implementation of multiplex immunoassays in clinical laboratories. We report the development of a novel method to eliminate the matrix effect by utilizing photocleavable capture antibodies to purify and concentrate blood-based biomarkers (a process termed PC-PURE) prior to detection in a multiplex immunoassay. To evaluate this approach, it was applied to blood-based allergy testing. Patient total IgE was purified and enriched using PC-PURE followed by multiplex microsphere-based detection of allergen-specific IgEs (termed the AllerBead assay). AllerBead was formatted to detect the eight most common pediatric food allergens: milk, soy, wheat, egg, peanuts, tree nuts, fin fish and shellfish, which account for >90% of all pediatric food allergies. 205 serum samples obtained from Boston Children’s Hospital were evaluated. When PC-PURE was employed with AllerBead, excellent agreement was obtained with the standard, non-multiplex, ImmunoCAP® assay (average sensitivity above published negative predictive cutoffs = 96% and average Pearson r = 0.90; average specificity = 97%). In contrast, poor ImmunoCAP®-correlation was observed when PC-PURE was not utilized (average sensitivity above published negative predictive cutoffs = 59% and average Pearson r = 0.61; average specificity = 97%). This approach should be adaptable to improve a wide range of multiplex immunoassays such as in cancer, infectious disease and autoimmune disease. PMID:29389948

An enzyme-linked immuno-filtration assay used to compare infant and maternal antibody profiles in toxoplasmosis.

PubMed

Pinon, J M; Thoannes, H; Gruson, N

1985-02-28

Enzyme-linked immuno-filtration assay is carried out on a micropore membrane. This doubly analytical technique permits simultaneous study of antibody specificity by immunoprecipitation and characterisation of antibody isotypes by immuno-filtration with enzyme-labelled antibodies. Recognition of the same T. gondii antigenic constituent by IgG, IgA, IgM or IgE antibodies produces couplets (IgG-IgM; IgG-IgA) or triplets (IgG-IgM-IgA; IgG-IgM-IgE) which identify the functional fractions of the toxoplasmosis antigen. In acquired toxoplasmosis, the persistence of IgM antibody long after infestation puts in question the implication of recent infestation normally linked to detection of this isotype. For sera of comparable titres, comparison of immunological profiles by the method described demonstrates disparities in the composition of the specific antibody content as expressed in international units. Use of the same method to detect IgM antibodies or distinguish between transmitted maternal IgG and IgG antibodies synthesised by the foetus or neonate makes a diagnosis of congenital toxoplasmosis possible in 85% of cases during the first few days of life. With the method described the diagnosis may be made on average 5 months earlier than with classical techniques. In the course of surveillance for latent congenital toxoplasmosis, the appearance of IgM or IgE antibodies raises the possibility of complications (hydrocephalus, chorioretinitis). After cessation of treatment, a rise in IgG antibodies indicating persistence of infection is detected earlier by the present than by classical methods.

Comparing ImmunoCard with two EIA assays for Clostridium difficile toxins.

PubMed

Chan, Edward L; Seales, Diane; Drum, Hong

2009-01-01

To compare three Clostridium difficile EIA kits for the detection of C. difficile toxins from clinical specimens. A total of 287 fresh and stored stool specimens were tested using all three assays. Stools with discrepant results were sent to a reference laboratory for tissue cytotoxin assay. Trinity Medical Center, a community hospital with network hospitals. Patients with diarrhea submitted stools for detection of C. difficile toxins. Of the 287 stool specimens, 116 were positive and 171 negative for C. difficile toxins. The sensitivity, specificity, and positive and negative predictive values of Meridian EIA assay were 99.1, 97.7, 96.6, and 99.4%; ImmunoCard were 100, 98.2, 97.5, and 100%; BioStar OIA assay were 94, 98.8, 98.2, and 96% respectively. ImmunoCardprovides the best sensitivity (100%) for C. difficile toxins A and B detection. The BioStar OIA rapid test missed seven positive stool specimens possibly due to failure to detect toxin B. ImmunoCard has slightly higher predictive values, shorter turnaround time and greater convenience compared to the Meridian EIA Assay. ImmunoCard may be cost effective not only in smaller laboratories, but also in high volume laboratories, when used on a STAT basis or single request.

On-chip surface-enhanced Raman spectroscopy (SERS)-linked immuno-sensor assay (SLISA) for rapid environmental-surveillance of chemical toxins

NASA Astrophysics Data System (ADS)

Bhardwaj, Vinay; Srinivasan, Supriya; McGoron, Anthony J.

2005-05-01

The increasing threat of an intentional (attack) or accidental release of toxins, in particular chemical toxins, including chemical warfare agents (CWAs) and toxic industrial chemicals (TICs) has increased public fear. The major problem in such attacks/accidents is to detect toxins present in very low levels. Indeed, several detection techniques are currently being used for the same. However, none of them meet the most critical requirements of a RISE (Rapid, Inexpensive, Simple and Effective) detect-to-protect class of biosensors. To address this critical demand our group has developed a prototype lab-on-a-chip (LOC) using a colloidal silver-based, surface-enhanced Raman spectroscopy (SERS)-linked immuno-sensor assay (SLISA). The LOC-SLISA was tested for the measurement of RAD54, a stress-marker protein expressed by yeast in response to hydrogen peroxide (H2O2), a toxin in the EPA priority list of chemical toxins. We found SLISA has good correlation in accuracy with the traditional ELISA technique and outperforms the latter by being rapid and easy-to-use. SLISA is more sensitive, provides qualitative information on immuno-sensor's chemical characterization and antigen-antibody binding, and allows direct detection with minimal or no chance of uncertainty, which is a stringent limitation of all label-based biosensor technologies including ELISA. For translational significance of our work, we correlated our results to U.S. EPA (environmental protection agency) defined risk exposure guideline levels of H2O2 to validate the commercial potential of our on-chip SLISA. The label-free, cell-based and RISE detection offered by SERS can allow development of biomedical and environmental sensor technology (BEST) needed for direct, rapid and continuous monitoring of human health and environment

Evaluation of a novel ultra-sensitive nanoparticle probe-based assay for ricin detection.

PubMed

Yin, Hui-qiong; Jia, Min-xian; Shi, Li-jun; Liu, Jun; Wang, Rui; Lv, Mao-min; Ma, Yu-yuan; Zhao, Xiong; Zhang, Jin-gang

2014-01-01

A gold nanoparticle (GNP) probe-based assay (GNPA) modified from the bio-barcode assay (BCA) was developed for ultrasensitive detection of ricin, a potential biothreat agent. In the GNPA, a chain of ricin was captured by a GNP probe coated with polyclonal antibodies and single-stranded signal DNA. A magnetic microparticle (MMP) probe coated with ricin A chain monoclonal antibody was then added to form an immuno-complex. After being magnetically separated, the immuno-complex containing the single-stranded signal DNA was characterized by PCR and real-time PCR. A detection limit of 10(-2) fg/ml was determined for the ricin A chain; this is eight orders of magnitude more sensitive than that achieved with an ELISA and two orders more sensitive than that obtained with the BCA. The coefficients of variation (CV) of the intra- and inter-assay values ranged from 3.82-6.46%. The results here show that this novel assay is an ultrasensitive method for detection of ricin proteins and may be suitable for the ultrasensitive detection of other proteins.

Poly(dimethylsiloxane) microchip-based immunoassay with multiple reaction zones: Toward on-chip multiplex detection platform

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shao, Guocheng; Wang, Jun; Li, Zhaohui

2011-09-20

In this work, a poly(dimethylsiloxane) (PDMS) microchip-based immuno-sensing platform with integrated pneumatic micro valves is described. The microchip was fabricated with multiple layer soft lithography technology. By controlling the activation status of corresponding valves, reagent flows in the microchannel network can be well manipulated so that immuno-reactions only take place at designated reaction zones (DRZs). Four DRZs are included in the prototype microchip. Since these DRZs are all isolated from each other by micro valves, cross contamination is prevented. Using the inner surface of the all-PDMS microchannel as immunoassay substrate, on-chip sandwich format solid phase immunoassay was performed to demonstratemore » the feasibility of this immuno-sensing platform. Mouse IgG and fluorescein isothiocyanate (FITC) were used as the model analyte and the signal reporter respectively. Only 10 ul sample is needed for the assay and low detection limit of 5 ng/ml (≈33 pM) was achieved though low-cost polyclonal antibodies were used in our experiment for feasibility study only. The encouraging results from mouse IgG immunoassay proved the feasibility of our microchip design. With slight modification of the assay protocol, the same chip design can be used for multi-target detection and can provide a simple, cost-effective and integrated microchip solution for multiplex immunoassay applications.« less

Detection of hepatitis B virus core antigen by phage display mediated TaqMan real-time immuno-PCR.

PubMed

Monjezi, Razieh; Tan, Sheau Wei; Tey, Beng Ti; Sieo, Chin Chin; Tan, Wen Siang

2013-01-01

The core antigen (HBcAg) of hepatitis B virus (HBV) is one of the markers for the identification of the viral infection. The main purpose of this study was to develop a TaqMan real-time detection assay based on the concept of phage display mediated immuno-PCR (PD-IPCR) for the detection of HBcAg. PD-IPCR combines the advantages of immuno-PCR (IPCR) and phage display technology. IPCR integrates the versatility of enzyme-linked immunosorbent assay (ELISA) with the sensitivity and signal generation power of PCR. Whereas, phage display technology exploits the physical association between the displayed peptide and the encoding DNA within the same phage particle. In this study, a constrained peptide displayed on the surface of an M13 recombinant bacteriophage that interacts tightly with HBcAg was applied as a diagnostic reagent in IPCR. The phage displayed peptide and its encoding DNA can be used to replace monoclonal antibody (mAb) and chemically bound DNA, respectively. This method is able to detect as low as 10ng of HBcAg with 10(8)pfu/ml of the recombinant phage which is about 10,000 times more sensitive than the phage-ELISA. The PD-IPCR provides an alternative means for the detection of HBcAg in human serum samples. Copyright © 2012 Elsevier B.V. All rights reserved.

Ricin toxicokinetics and its sensitive detection in mouse sera or feces using immuno-PCR

USDA-ARS?s Scientific Manuscript database

Ricin (also called RCA-II or RCA60), one of the most potent toxins and documented bioweapons, is derived from castor beans of Ricinus communis. Several in vitro methods have been designed for ricin detection in complex food matrices in the event of intentional contamination. Recently, a novel Immuno...

A detection method in living plant cells for rapidly monitoring the response of plants to exogenous lanthanum.

PubMed

Cheng, Mengzhu; Wang, Lihong; Yang, Qing; Huang, Xiaohua

2018-08-30

The pollution of rare earth elements (REEs) in ecosystem is becoming more and more serious, so it is urgent to establish methods for monitoring the pollution of REEs. Monitoring environmental pollution via the response of plants to pollutants has become the most stable and accurate method compared with traditional methods, but scientists still need to find the primary response of plants to pollutants to improve the sensitivity and speed of this method. Based on the facts that the initiation of endocytosis is the primary cellular response of the plant leaf cells to REEs and the detection of endocytosis is complex and expensive, we constructed a detection method in living plant cells for rapidly monitoring the response of plants to exogenous lanthanum [La(III), a representative of REEs] by designing a new immuno-electrochemical method for detecting the content change in extracellular vitronectin-like protein (VN) that are closely related to endocytosis. Results showed that when 30 μM La(III) initiated a small amount of endocytosis, the content of extracellular VN increased by 5.46 times, but the structure and function of plasma membrane were not interfered by La(III); when 80 μM La(III) strongly initiated a large amount of endocytosis, the content of extracellular VN increased by 119 times, meanwhile, the structure and function of plasma membrane were damaged. In summary, the detection method can reflect the response of plants to La(III) via detecting the content change in extracellular VN, which provides an effective and convenient way to monitor the response of plants to exogenous REEs. Copyright © 2018. Published by Elsevier Inc.

Three-Dimensional Hierarchical Plasmonic Nano-Architecture Enhanced Surface-Enhanced Raman Scattering Immuno-Sensor for Cancer Biomarker Detection in Blood Plasma

PubMed Central

Li, Ming; Cushing, Scott K.; Zhang, Jianming; Suri, Savan; Evans, Rebecca; Petros, William P.; Gibson, Laura F.; Ma, Dongling; Liu, Yuxin; Wu, Nianqiang

2013-01-01

A three-dimensional (3D) hierarchical plasmonic nano-architecture has been designed for a sensitive surface-enhanced Raman scattering (SERS) immuno-sensor for protein biomarker detection. The capture antibody molecules are immobilized on a plasmonic gold triangle nano-array pattern. On the other hand, the detection antibody molecules are linked to the gold nano-star@Raman-reporter@silica sandwich nanoparticles. When protein biomarkers are present, the sandwich nanoparticles are captured over the gold triangle nano-array, forming a confined 3D plasmonic field, leading to the enhanced electromagnetic field in intensity and in 3D space. As a result, the Raman reporter molecules are exposed to a high density of “hot spots”, which amplifies the Raman signal remarkably, improving the sensitivity of the SERS immuno-sensor. This SERS immuno-sensor exhibits a wide linear range (0.1 pg/mL to 10 ng/mL), and a low limit of detection (7 fg/mL) toward human immunoglobulin G (IgG) protein in the buffer solution. This biosensor has been successfully used for detection of the vascular endothelial growth factor (VEGF) in the human blood plasma from clinical breast cancer patient samples. PMID:23659430

Performance of two Aspergillus IgG EIA assays compared with the precipitin test in chronic and allergic aspergillosis.

PubMed

Baxter, C G; Denning, D W; Jones, A M; Todd, A; Moore, C B; Richardson, M D

2013-04-01

Detection of Aspergillus IgG antibodies is important in the diagnosis of chronic pulmonary aspergillosis and allergic bronchopulmonary aspergillosis. Immunoprecipitation techniques to detect these antibodies appear to lack sensitivity and accurate quantitation compared with enzyme immunoassays (EIA). This study assessed the performance of two commercial EIAs compared with counterimmunoelectrophoresis (CIE). This was a prospective cohort study of 175 adult patients with chronic or allergic pulmonary aspergillosis. Aspergillus IgG antibodies were detected using CIE, Phadia ImmunoCap Aspergillus IgG and Bio-Rad Platelia Aspergillus IgG. Inter-assay reproducibility was determined for each method and 25 patients had two serum samples analysed within a 6-month interval. When compared with CIE, both ImmunoCap and Platelia Aspergillus IgG had good sensitivity (97 and 93%, respectively) for detection of Aspergillus IgG antibodies. The level of agreement between the two EIAs for positive results was good, but the concentration of antibodies was not correlated between the tests or with CIE titre. ImmunoCap IgG inter-assay coefficient of variation was 5%, whereas Platelia IgG was 33%. Median ImmunoCap IgG values for CPA and allergic aspergillosis were 95 and 32 mg/L, respectively, whereas Platelia IgG values were >80 and 6 AU/mL. The direction of CIE titre change over 6 months was mirrored by ImmunoCap IgG levels in 92% of patients, and by Platelia IgG in 72% of patients. Both ImmunoCap and Platelia Aspergillus IgG EIAs are sensitive measures of Aspergillus IgG antibodies compared with CIE. However, ImmunoCap appears to have better reproducibility and may be more suitable for monitoring patient disease. © 2012 The Authors Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.

Applications of immunoPET: Using 124I-anti-PSCA A11 minibody for imaging disease progression and response to therapy in mouse xenograft models of prostate cancer

DOE PAGES

Knowles, Scott M.; Tavare, Richard; Zettlitz, Kirstin A.; ...

2014-10-17

Here, prostate stem cell antigen (PSCA) is highly expressed in local prostate cancers and prostate cancer bone metastases and its expression correlates with androgen receptor activation and a poor prognosis. Here in this study, we investigate the potential clinical applications of immunoPET with the anti-PSCA A11 minibody, an antibody fragment optimized for use as an imaging agent. We compare A11 minibody immunoPET to 18F-Fluoride PET bone scans for detecting prostate cancer bone tumors and evaluate the ability of the A11 minibody to image tumor response to androgen deprivation. Osteoblastic, PSCA expressing, LAPC-9 intratibial xenografts were imaged with serial 124I-anti-PSCA A11more » minibody immunoPET and 18F-Fluoride bone scans. Mice bearing LAPC-9 subcutaneous xenografts were treated with either vehicle or MDV-3100 and imaged with A11 minibody immunoPET/CT scans pre- and post-treatment. Ex vivo flow cytometry measured the change in PSCA expression in response to androgen deprivation. A11 minibody demonstrated improved sensitivity and specificity over 18F-Fluoride bone scans for detecting LAPC-9 intratibial xenografts at all time points. Finally, LAPC-9 subcutaneous xenografts showed downregulation of PSCA when treated with MDV-3100 which A11 minibody immunoPET was able to detect in vivo.« less

A novel immuno-gold labeling protocol for nanobody-based detection of HER2 in breast cancer cells using immuno-electron microscopy.

PubMed

Kijanka, M; van Donselaar, E G; Müller, W H; Dorresteijn, B; Popov-Čeleketić, D; El Khattabi, M; Verrips, C T; van Bergen En Henegouwen, P M P; Post, J A

2017-07-01

Immuno-electron microscopy is commonly performed with the use of antibodies. In the last decade the antibody fragment indicated as nanobody (VHH or single domain antibody) has found its way to different applications previously done with conventional antibodies. Nanobodies can be selected to bind with high affinity and specificity to different antigens. They are small (molecular weight ca. 15kDa) and are usually easy to produce in microorganisms. Here we have evaluated the feasibility of a nanobody binding to HER2 for application in immuno-electron microscopy. To obtain highest labeling efficiency combined with optimal specificity, different labeling conditions were analysed, which included nanobody concentration, fixation and blocking conditions. The obtained optimal protocol was applied for post-embedment labeling of Tokuyasu cryosections and for pre-embedment labeling of HER2 for fluorescence microscopy and both transmission and scanning electron microscopy. We show that formaldehyde fixation after incubation with the anti-HER2 nanobody, improves labeling intensity. Among all tested blocking agents the best results were obtained with a mixture of cold water fish gelatine and acetylated bovine serum albumin, which prevented a-specific interactions causing background labeling while preserving specific interactions at the same time. In conclusion, we have developed a nanobody-based protocol for immuno-gold labeling of HER2 for Tokuyasu cryosections in TEM as well as for pre-embedment gold labeling of cells for both TEM and SEM. Copyright © 2017. Published by Elsevier Inc.

Comparison of the Performance of Skin Prick, ImmunoCAP, and ISAC Tests in the Diagnosis of Patients with Allergy.

PubMed

Griffiths, Rebecca L M; El-Shanawany, Tariq; Jolles, Stephen R A; Selwood, Clive; Heaps, Adrian G; Carne, Emily M; Williams, Paul E

2017-01-01

Allergy is diagnosed from typical symptoms, and tests are performed to incriminate the suspected precipitant. Skin prick tests (SPTs) are commonly performed, inexpensive, and give immediate results. Laboratory tests (ImmunoCAP) for serum allergen-specific IgE antibodies are usually performed more selectively. The immuno-solid phase allergen chip (ISAC) enables testing for specific IgE against multiple allergen components in a multiplex assay. We retrospectively analysed clinic letters, case notes, and laboratory results of 118 patients attending the National Adult Allergy Service at the University Hospital of Wales who presented diagnostic difficulty, to evaluate which testing strategy (SPT, ImmunoCAP, or ISAC) was the most appropriate to use to confirm the diagnosis in these complex patients, evaluated in a "real-life" clinical service setting. In patients with nut allergy, the detection rates of SPTs (56%) and ISAC (65%) were lower than those of ImmunoCAP (71%). ISAC had a higher detection rate (88%) than ImmunoCAP (69%) or SPT (33%) in the diagnosis of oral allergy syndrome. ImmunoCAP test results identified all 9 patients with anaphylaxis due to wheat allergy (100%), whereas ISAC was positive in only 6 of these 9 (67%). In this difficult diagnostic group, the ImmunoCAP test should be the preferred single test for possible allergy to nuts, wheat, other specific foods, and anaphylaxis of any cause. In these conditions, SPT and ISAC tests give comparable results. The most useful single test for oral allergy syndrome is ISAC, and SPT should be the preferred test for latex allergy. © 2017 S. Karger AG, Basel.

A screen for over-secretion of proteins by yeast based on a dual component cellular phosphatase and immuno-chromogenic stain for exported bacterial alkaline phosphatase reporter

PubMed Central

2013-01-01

Background To isolate over-secretors, we subjected to saturation mutagenesis, a strain of P.pastoris exporting E. coli alkaline phosphatase (EAP) fused to the secretory domain of the yeast α factor pheromone through cellular PHO1/KEX2 secretory processing signals as the α-sec-EAP reporter protein. Direct chromogenic staining for α-sec-EAP activity is non-specific as its NBT/BCIP substrate cross-reacts with cellular phosphatases which can be inhibited with Levulinic acid. However, the parental E(P) strain only exports detectable levels of α-sec-EAP at 69 hours and not within the 36 hour period post-seeding required for effective screening with the consequent absence of a reference for secretion. We substituted the endogenous cellular phosphatase activity as a comparative reference for secretion rate and levels as well as for colony alignment while elevating specificity and sensitivity of detection of the exported protein with other innovative modifications of the immuno-chromogenic staining application for screening protein export mutants. Results Raising the specificity and utility of staining for α-sec-EAP activity required 5 modifications including some to published methods. These included, exploitation of endogenous phosphatase activity, reduction of the cell/protein burden, establishment of the direct relation between concentrations of transcriptional inducer and exported membrane immobilized protein and concentrations of protein exported into growth media, amplification of immuno-specificity and sensitivity of detection of α-sec-EAP reporter enzyme signal and restriction of staining to optimal concentrations of antisera and time periods. The resultant immuno-chromogenic screen allows for the detection of early secretion and as little as 1.3 fold over-secretion of α-sec-EAP reporter protein by E(M) mutants in the presence of 10 fold -216 fold higher concentrations of HSA. Conclusions The modified immuno-chromogenic screen is sensitive, specific and has led to the isolation of mutants E(M) over-secreting the α-sec-EAP reporter protein by a minimum of 50 fold higher levels than that exported by non-mutagenized E(P) parental strains. Unselected proteins were also over-secreted. PMID:23602005

ELISA and ImmunoStrip® for detection of Phytophthora ramorum, P. kernoviae, and other Phytophthora species

Treesearch

Francisco J. Avila; Barbara Schoedel; Z. Gloria Abad; Michael D. Coffey; Cheryl Blomquist

2009-01-01

The goal of this work was to develop improved tools for the detection of Phytophthora ramorum and P. kernoviae for field and the laboratory use. ImmunoStrip® and ELISA were selected as the test formats for development. Presently, the diagnosis of sudden oak death (SOD) in the national survey of P. ramorum ...

Detection of measles, mumps and rubella viruses by immuno-colorimetric assay and its application in focus reduction neutralization tests.

PubMed

Vaidya, Sunil R; Kumbhar, Neelakshi S; Bhide, Vandana S

2014-12-01

Measles, mumps and rubella are vaccine-preventable diseases; however limited epidemiological data are available from low-income or developing countries. Thus, it is important to investigate the transmission of these viruses in different geographical regions. In this context, a cell culture-based rapid and reliable immuno-colorimetric assay (ICA) was established and its utility studied. Twenty-three measles, six mumps and six rubella virus isolates and three vaccine strains were studied. Detection by ICA was compared with plaque and RT-PCR assays. In addition, ICA was used to detect viruses in throat swabs (n = 24) collected from patients with suspected measles or mumps. Similarly, ICA was used in a focus reduction neutralization test (FRNT) and the results compared with those obtained by a commercial IgG enzyme immuno assay. Measles and mumps virus were detected 2 days post-infection in Vero or Vero-human signaling lymphocytic activation molecule cells, whereas rubella virus was detected 3 days post-infection in Vero cells. The blue stained viral foci were visible by the naked eye or through a magnifying glass. In conclusion, ICA was successfully used on 35 virus isolates, three vaccine strains and clinical specimens collected from suspected cases of measles and mumps. Furthermore, an application of ICA in a neutralization test (i.e., FRNT) was documented; this may be useful for sero-epidemiological, cross-neutralization and pre/post-vaccine studies. © 2014 The Societies and Wiley Publishing Asia Pty Ltd.

A Single-Stranded DNA Aptamer That Selectively Binds to Staphylococcus aureus Enterotoxin B

PubMed Central

DeGrasse, Jeffrey A.

2012-01-01

The bacterium Staphylococcus aureus is a common foodborne pathogen capable of secreting a cocktail of small, stable, and strain-specific, staphylococcal enterotoxins (SEs). Staphylococcal food poisoning (SFP) results when improperly handled food contaminated with SEs is consumed. Gastrointestinal symptoms of SFP include emesis, diarrhea and severe abdominal pain, which manifest within hours of ingesting contaminated food. Immuno-affinity based methods directly detect, identify, and quantify several SEs within a food or clinical sample. However, the success of these assays depends upon the availability of a monoclonal antibody, the development of which is non-trivial and costly. The current scope of the available immuno-affinity based methods is limited to the classical SEs and does not encompass all of the known or emergent SEs. In contrast to antibodies, aptamers are short nucleic acids that exhibit high affinity and specificity for their targets without the high-costs and ethical concerns of animal husbandry. Further, researchers may choose to freely distribute aptamers and develop assays without the proprietary issues that increase the per-sample cost of immuno-affinity assays. This study describes a novel aptamer, selected in vitro, with affinity to staphylococcal enterotoxin B (SEB) that may be used in lieu of antibodies in SE detection assays. The aptamer, designated APTSEB1, successfully isolates SEB from a complex mixture of SEs with extremely high discrimination. This work sets the foundation for future aptamer and assay development towards the entire family of SEs. The rapid, robust, and low-cost identification and quantification of all of the SEs in S. aureus contaminated food is essential for food safety and epidemiological efforts. An in vitro generated library of SE aptamers could potentially allow for the comprehensive and cost-effective analysis of food samples that immuno-affinity assays currently cannot provide. PMID:22438927

A single-stranded DNA aptamer that selectively binds to Staphylococcus aureus enterotoxin B.

PubMed

DeGrasse, Jeffrey A

2012-01-01

The bacterium Staphylococcus aureus is a common foodborne pathogen capable of secreting a cocktail of small, stable, and strain-specific, staphylococcal enterotoxins (SEs). Staphylococcal food poisoning (SFP) results when improperly handled food contaminated with SEs is consumed. Gastrointestinal symptoms of SFP include emesis, diarrhea and severe abdominal pain, which manifest within hours of ingesting contaminated food. Immuno-affinity based methods directly detect, identify, and quantify several SEs within a food or clinical sample. However, the success of these assays depends upon the availability of a monoclonal antibody, the development of which is non-trivial and costly. The current scope of the available immuno-affinity based methods is limited to the classical SEs and does not encompass all of the known or emergent SEs. In contrast to antibodies, aptamers are short nucleic acids that exhibit high affinity and specificity for their targets without the high-costs and ethical concerns of animal husbandry. Further, researchers may choose to freely distribute aptamers and develop assays without the proprietary issues that increase the per-sample cost of immuno-affinity assays. This study describes a novel aptamer, selected in vitro, with affinity to staphylococcal enterotoxin B (SEB) that may be used in lieu of antibodies in SE detection assays. The aptamer, designated APT(SEB1), successfully isolates SEB from a complex mixture of SEs with extremely high discrimination. This work sets the foundation for future aptamer and assay development towards the entire family of SEs. The rapid, robust, and low-cost identification and quantification of all of the SEs in S. aureus contaminated food is essential for food safety and epidemiological efforts. An in vitro generated library of SE aptamers could potentially allow for the comprehensive and cost-effective analysis of food samples that immuno-affinity assays currently cannot provide.

'Nano-immuno test' for the detection of live Mycobacterium avium subspecies paratuberculosis bacilli in the milk samples using magnetic nano-particles and chromogen.

PubMed

Singh, Manju; Singh, Shoor Vir; Gupta, Saurabh; Chaubey, Kundan Kumar; Stephan, Bjorn John; Sohal, Jagdip Singh; Dutta, Manali

2018-04-26

Early rapid detection of Mycobacterium avium subspecies paratuberculosis (MAP) bacilli in milk samples is the major challenge since traditional culture method is time consuming and laboratory dependent. We report a simple, sensitive and specific nano-technology based 'Nano-immuno test' capable of detecting viable MAP bacilli in the milk samples within 10 h. Viable MAP bacilli were captured by MAP specific antibody-conjugated magnetic nano-particles using resazurin dye as chromogen. Test was optimized using true culture positive (10-bovine and 12-goats) and true culture negative (16-bovine and 25-goats) raw milk samples. Domestic livestock species in India are endemically infected with MAP. After successful optimization, sensitivity and specificity of the 'nano-immuno test' in goats with respect to milk culture was 91.7% and 96.0%, respectively. Whereas, it was 90.0% (sensitivity) and 92.6% (specificity) with respect to IS900 PCR. In bovine milk samples, sensitivity and specificity of 'nano-immuno test' with respect to milk culture was 90.0% and 93.7%, respectively. However, with respect to IS900 PCR, the sensitivity and specificity was 88.9% and 94.1%, respectively. Test was validated with field raw milk samples (goats-258 and bovine-138) collected from domestic livestock species to detect live/viable MAP bacilli. Of 138 bovine raw milk samples screened by six diagnostic tests, 81 (58.7%) milk samples were positive for MAP infection in one or more than one diagnostic tests. Of 81 (58.7%) positive bovine raw milk samples, only 24 (17.4%) samples were detected positive for the presence of viable MAP bacilli. Of 258 goats raw milk samples screened by six diagnostic tests, 141 (54.6%) were positive for MAP infection in one or more than one test. Of 141 (54.6%) positive raw milk samples from goats, only 48 (34.0%) were detected positive for live MAP bacilli. Simplicity and efficiency of this novel 'nano-immuno test' makes it suitable for wide-scale screening of milk samples in the field. Standardization, validation and re-usability of functionalized nano-particles and the test was successfully achieved in field samples. Test was highly specific, simple to perform and easy to read by naked eyes and does not require laboratory support in the performance of test. Test has potential to be used as screening test to estimate bio-load of MAP in milk samples at National level.

[Evaluation of the new ImmunoCard STAT!® CGE test for the diagnosis of Amebiasis].

PubMed

Formenti, F; Perandin, F; Bonafini, S; Degani, M; Bisoffi, Z

2015-08-01

For many years, microscopic examination of stool samples has been considered to be the "gold standard" for diagnosis of intestinal parasites although the Polymerase Chain Reaction (PCR) analysis is increasingly utilized due to its high accuracy. Recently, PCR has been approved by the World Health Organization as the current method of choice for the diagnosis of Entamoeba histolytica infection. In this study we evaluated a novel immunochromatographic antigen detection rapid test, ImmunoCardSTAT CGE (Meridian Bioscence, Milan, Italy), which has been proposed for the diagnosis of infections caused by Cryptosporidium parvum-Giardia intestinalis-Entamoeba histolytica. There is another rapid test with a similar name, the ImmunoCard STAT! Crypto/Giardia, but it is just for Cryptosporidium and Giardia. We aimed to compare E. histolytica results obtained from the rapid test with those of a rt-PCR for the detection of E. histolytica / E. dispar DNA. The new ImmunoCard rapid antigen detection test exhibited 88% sensitivity and 92% specificity (if assessed on rt-PCR negative samples) but showed a high proportion of cross-reaction between the pathogenic E. histolytica and the non pathogenic E. dispar.

Component-Resolved Diagnostic Study of Dermatophagoides Pteronyssinus Major Allergen Molecules in a Southern Chinese Cohort.

PubMed

Zeng, G; Luo, W; Zheng, P; Wei, N; Huang, H; Sun, B; Zhao, X

2015-01-01

Little is known about component-resolved diagnosis (CRD) for Dermatophagoides pteronyssinus (Der p) sensitization in the Chinese population. We aimed to evaluate sensitization to Der p components in southern China. Two-hundred immunotherapy-naïve patients with asthma and/or rhinitis positive to specific IgE (sIgE) against Der p extract, along with 20 Der p-negative nonallergic healthy controls, were tested for sIgE against Der p 1, Der p 2, and Der p 10 using ImmunoCAP 100. Seventy-five were further examined with the ImmunoCAP Immuno Solid-phase Allergen Chip (ISAC). Der p 10-positive patients were also tested for sIgE against crude extracts of cockroach, moth, and shrimp. In total, 183 (91.5%) of the 200 patients were sensitized to Der p 1 and/or Der p 2. The proportion of positive results and the median level of s1gE against Der p 1 were higher in children than in adults. Der p 1 and Der p 2 correlated with Der p in sIgE levels. ImmunoCAP ISAC demonstrated 100% specificity and 84% sensitivity in detecting Der p 1, Der p 2, and Der p 10 compared with ImmunoCAP 100. Sensitization to Der p 10 correlated well with sIgE to shrimp, moths, cockroaches, Pen m 1, Bla g 7, and Ani s 3. The detection of Der p 1 and Der p 2 provided a good reflection of atopy to Der p in a Chinese cohort. Sensitization to Der p 10 may result from cross-reactivity with seafood and cockroaches in coastal southern China. ImmunoCAP ISAC may be a useful tool for CRD, with comparable performance to ImmunoCAP 100.

Immuno-PCR assay for sensitive detection of proteins in real time

USDA-ARS?s Scientific Manuscript database

The immuno-PCR (IPCR) assay combines the versatility and robustness of immunoassays with the exponential signal amplification power of the polymerase chain reaction (PCR). Typically, IPCR allows a 10–1,000-fold increase in sensitivity over the analogous enzyme-linked immunosorbent assay (ELISA). Thi...

A sensitive immunosorbent bio-barcode assay based on real-time immuno-PCR for detecting 3,4,3',4'-tetrachlorobiphenyl.

PubMed

Yang, Guang-Xin; Zhuang, Hui-Sheng; Chen, Han-Yu; Ping, Xian-Yin; Bu, Dan

2014-02-01

A functionalized gold-nanoparticle bio-barcode assay, based on real-time immuno-PCR (IPCR), was designed for the determination of 3,4,3',4'-tetrachlorobiphenyl (PCB77). 15 nm gold nanoparticles were synthesized, and modified with thiol-capped DNA and goat anti-rabbit IgG. The nanoparticle probes were used to replace antibody-DNA conjugate in the IPCR, and were fixed on the PCR tube wall via the immune reaction. Real-time PCR was performed to quantify the DNA signal directly. Under optimized conditions, the new method was used to detect PCB77 with a linearity range from 5 pg L(-1) to 10 ng L(-1), and the limit of detection (LOD) was 1.72 pg L(-1). Real samples of Larimichthys polyactis, collected from the East China Sea, were analyzed. Recovery was from 82 % to 112 %, and the coefficient of variation (CV) was acceptable. The results were compared with GC-ECD, revealing that the method would be acceptable for providing rapid, semi-quantitative, and reliable test results for making environmental decisions.

Comparison of the skin-prick test and Phadia ImmunoCAP as tools to diagnose house-dust mite allergy.

PubMed

Jung, Yong Gi; Cho, Hyun-Jin; Park, Ga Young; Min, Jin-Young; Kim, Hyo Yeol; Dhong, Hun-Jong; Chung, Seung Kyu; Kim, Seon Woo

2010-01-01

When the skin-prick test (SPT) and in vitro test such as ImmunoCAP assay are performed simultaneously, results do not always coincide in some patients. Our objectives, therefore, were (1) to assess differences in allergic test results according to age group and (2) to establish appropriate guidelines for diagnosing mite allergy according to age. A total of 692 participants complaining of allergic rhinitis symptoms participated. Patients were divided according to age; the mean age was 32 years (range, 8-76 years). The SPT and ImmunoCAP assays were performed to detect allergies to house-dust mites (Dermatophagoides pteronyssinus and Dermatophagoides farinae). The association between age and the result of each allergy test were examined, and a cutoff age for proper application of each test was than estimated. Three hundred thirty-six patients (48.6%) were allergic to D. pteronyssinus and 350 patients (50.6%) were allergic to D. farinae. In the case of D. pteronyssinus, SPT was proved to be more useful in detecting allergy for subjects <50 years old (p < 0.0001). However in case of D. farinae, ImmunoCAP was useful for all age groups, but SPT showed decreased ratio of positive result for subjects >30 years old (p < 0.0001). This study was the first to compare results of allergy tests according to age using true allergens. For patients >50 years of age, the ImmunoCAP was found to be the preferred method for detecting allergy to house-dust mites and for patients <30 years old, SPT is the recommended first choice.

IMMUNO-SPIN TRAPPING FROM BIOCHEMISTRY TO MEDICINE: advances, challenges, and pitfalls

PubMed Central

Gomez-Mejiba, Sandra E.; Zili, Zhai; Della-Vedova, Maria C.; Muñoz, Marcos D.; Chatterjee, Saurabh; Towner, Rheal A.; Hensley, Kenneth; Floyd, Robert A.; Mason, Ronald P.; Ramirez, Dario C.

2013-01-01

BACKGROUND Immuno-spin trapping (IST) is based on the reaction of a spin trap with a free radical to form a stable nitrone adduct, followed by the use of antibodies, rather than traditional electron paramagnetic resonance spectroscopy, to detect the nitrone adduct. IST has been successfully applied to mechanistic in vitro studies, and recently, macromolecule-centered radicals have been detected in models of drug-induced agranulocytosis, hepatotoxicity, cardiotoxicity, and ischemia/reperfusion, as well as in models of neurological, metabolic and immunological diseases. SCOPE OF THE REVIEW To critically evaluate advances, challenges, and pitfalls as well as the scientific opportunities of IST as applied to the study of protein-centered free radicals generated in stressed organelles, cells, tissues and animal models of disease and exposure. MAJOR CONCLUSIONS Because the spin trap has to be present at high enough concentrations in the microenvironment where the radical is formed, the possible effects of the spin trap on gene expression, metabolism and cell physiology have to be considered in the use of IST and in the interpretation of results. These factors have not yet been thoroughly dealt with in the literature. GENERAL SIGNIFICANCE The identification of radicalized proteins during cell/tissue response to stressors will help define their role in the complex cellular response to stressors and pathogenesis; however, the fidelity of spin trapping/ immuno-detection and the effects of the spin trap on the biological system should be considered. PMID:23644035

Site-specific detection of radicals on α-lactalbumin after a riboflavin-sensitized reaction, detected by immuno-spin trapping, ESR, and MS.

PubMed

Dalsgaard, Trine K; Triquigneaux, Mathilde; Deterding, Leesa; Summers, Fiona; Ranguelova, Kalina; Mortensen, Grith; Mason, Ronald P

2013-01-16

Free radicals and other oxidation products were characterized on α-lactalbumin with electron spin resonance (ESR), immuno-spin trapping, and mass spectrometry (MS) after riboflavin-mediated oxidation. Radicals were detected using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in immuno-spin trapping with both enzyme-linked immunosorbent assay (ELISA) and Western blotting and further characterized with mass spectrometry. A DMPO-trapped radical was identified at His68 and another at one of the tyrosine residues, Tyr50 or Tyr36, respectively, generated by a type II or I mechanism. Not all tyrosyl radicals were trapped, as the secondary oxidation product, 3,4-dihydroxyphenylalanine (DOPA), was detected by mass spectrometry at Tyr18 and Tyr50. A further oxidation of DOPA resulted in the DOPA o-semiquinone radical, which was characterized by ESR. Both surface exposure and the neighboring residues in the local environment of the tertiary structure of α-lactalbumin seem to play a role in the generation of DMPO trapped radicals and secondary oxidation products.

Nanobody medicated immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein.

PubMed

Chen, Jing; He, Qing-hua; Xu, Yang; Fu, Jin-heng; Li, Yan-ping; Tu, Zhui; Wang, Dan; Shu, Mei; Qiu, Yu-lou; Yang, Hong-wei; Liu, Yuan-yuan

2016-01-15

Immunoassay for cancer biomarkers plays an important role in cancer prevention and early diagnosis. To the development of immunoassay, the quality and stability of applied antibody is one of the key points to obtain reliability and high sensitivity for immunoassay. The main purpose of this study was to develop a novel immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein (AFP) based on nanobody against AFP. Two nanobodies which bind to AFP were selected from a phage display nanobody library by biopanning strategy. The prepared nanobodies are clonable, thermally stable and applied in both sandwich enzyme linked immunoassay (ELISA) and immuno-PCR assay for ultrasensitive detection of AFP. The limit detection of sandwich ELISA setup with optimized nanobodies was 0.48ng mL(-1), and the half of saturation concentration (SC50) value was 6.68±0.56ng mL(-1). These nanobodies were also used to develop an immuno-PCR assay for ultrasensitive detection of AFP, its limit detection values was 0.005ng mL(-1), and the linear range was 0.01-10,000ng mL(-1). These established immunoassays based on nanobodies were highly specific to AFP and with negligible cross reactivity with other tested caner biomarkers. Furthermore, this novel concept of nanobodies mediated immunoassay may provide potential applications in a general method for the ultrasensitive detection of various cancer biomarkers. Copyright © 2015 Elsevier B.V. All rights reserved.

[The immuno-endocrine system. A new endocrine theory: the problem of the packed transport].

PubMed

Csaba, György

2011-05-15

Since the eighties of the last century hormone content was justified in immune cells (lymphocytes, granulocytes, monocytes, macrophages and mast cells), which produce, store and secrete these hormones. Although the amount of these materials in immune cells is relatively small, the mass of the producers (immune cells) is so large, that the phenomenon must be considered from endocrinological point of view, underlying the important differences between the "classical" and immuno-endocrine systems. Cells of the classic (built-in) endocrine system are mono-producers, while immune cells can synthesize many types of hormones (polyproducers). In addition, these cells can transport the whole hormone-producing machinery to the site of need, producing a local effect. This can be observed, for example, in the case of endorphin producing immune cells during inflammation and during early pregnancy around the chorionic villi. Hormone producing immune cells also have receptors for many hormones, so that they are poly-receivers. Via hormone producing and receiving capacity there is a bidirectional connection between the neuro-endocrine and immuno-endocrine systems. In addition, there is a network inside the immuno-endocrine system. The packed transport theory attempts to explain the mechanism and importance of the immuno-endocrine system.

The interplay between pH sensitivity and label-free protein detection in immunologically modified nano-scaled field-effect transistor.

PubMed

Shalev, Gil; Rosenwaks, Yossi; Levy, Ilan

2012-01-15

We present experimental results in order to establish a correlation between pH sensitivity of immunologically modified nano-scaled field-effect transistor (NS-ImmunoFET) with their sensing capacity for label-free detection. The NS-ImmunoFETs are fabricated from silicon-on-insulator (SOI) wafers and are fully-depleted with thickness of ~20 nm. The data shows that higher sensitivity to pH entails enhanced sensitivity to analyte detection. This suggests that the mechanism of analyte detection as pure electrostatic perturbation induced by antibody-analyte interaction is over simplified. The fundamental assumption, in existing models for field-effect sensing mechanism assumes that the analyte molecules do not directly interact with the surface but rather stand 'deep' in the solution and away from the dielectric surface. Recent studies clearly provide contradicting evidence demonstrating that antibodies lie down flat on the surface. These observations led us to propose that the proteins that cover the gate area intimately interact with active sites on the surface thus forming a network of interacting sites. Since sensitivity to pH is directly correlated with the amount of amphoteric sites, we witness a direct correlation between sensitivity to pH and analyte detection. The highest and lowest threshold voltage shift for a label-free and specific detection of 6.5 nM IgG were 40 mV and 2.3 mV for NS-ImmunoFETs with pH sensitivity of 35 mV/decade and 15 mV/decade, respectively. Finally, physical modeling of the NS-ImmunoFET is presented and charge of a single IgG protein at pH 6 is calculated. The obtained value is consistent with charge of IgG protein cited in literature. Copyright © 2011 Elsevier B.V. All rights reserved.

[PREPARATION OF HUMAN TISSUE PROTEIN EXTRACTS ENRICHED WITH THE SPHINGOMYELIN SYNTHASE 1].

PubMed

Sudarkina, O Yu; Dergunova, L V

2015-01-01

Sphingomyelin synthase 1 (SMS 1) catalyzes sphingomyelin biosynthesis in eukaryotic cells. We previously studied the structure of the human SGMS1 gene, which encodes the enzyme and its numerous transcripts. The tissue-specific expression of the transcripts was also described. Analysis of the SMS1 protein expression in human tissues using immunoblotting of tissue extracts prepared in the RIPA (Radio Immuno-Precipitation Assay) buffer revealed a weak signal in renal cortex, testis, lung, and no signal in placenta and lymphatic node. In this work, a new method of preparation of the tissue protein extracts enriched with SMS1 was suggested. The method based on the consecutive extraction with a buffer containing 0.05 and 1 mg/ml of the Quillaja saponaria saponin allowed SMS1 to be detected in all tissues tested. The SMS1 content in the saponin extract of kidney cortex is about 12-fold higher compared to the RIPA extraction procedure.

Study of the temperature dependent immuno-reaction kinetics for the bio-functionalized magnetic nanoparticle assay of bio-markers of colorectal cancer

NASA Astrophysics Data System (ADS)

Yang, S. Y.; Chang, J. F.; Chen, T. C.; Yang, C. C.; Ho, C. S.

2014-01-01

By conjugating antibodies on magnetic nanoparticles, target antigens can be quantitatively detected by measuring the magnetic signals of the magnetic nanoparticles due to their association with target antigens. This method of detection is called magnetically labeled immunoassay. The assay characteristics of magnetically labeled immunoassay have been reported widely. However, the immuno-reaction kinetics of magnetically labeled immunoassay has not been studied. In this work, the reaction rates between magnetic nanoparticles and target antigens are measured at various temperatures. It is found that the temperature dependent reaction rate obeys Arrhenius's equation, which shows the collision frequency and activation energy for the immuno-reaction between antibody-functionalized magnetic nanoparticles and target antigens. The carcinoembryonic antigen, which is a regular blood bio-marker for in-vitro diagnosis of colorectal cancer, is used as a target antigen for the example.

Surface immuno-functionalisation for the capture and detection of Vibrio species in the marine environment: a new management tool for industrial facilities.

PubMed

Laczka, Olivier F; Labbate, Maurizio; Seymour, Justin R; Bourne, David G; Fielder, Stewart S; Doblin, Martina A

2014-01-01

Bacteria from the genus Vibrio are a common and environmentally important group of bacteria within coastal environments and include species pathogenic to aquaculture organisms. Their distribution and abundance are linked to specific environmental parameters, including temperature, salinity and nutrient enrichment. Accurate and efficient detection of Vibrios in environmental samples provides a potential important indicator of overall ecosystem health while also allowing rapid management responses for species pathogenic to humans or species implicated in disease of economically important aquacultured fish and invertebrates. In this study, we developed a surface immuno-functionalisation protocol, based on an avidin-biotin type covalent binding strategy, allowing specific sandwich-type detection of bacteria from the Vibrio genus. The assay was optimized on 12 diverse Vibrio strains, including species that have implications for aquaculture industries, reaching detection limits between 7×10(3) to 3×10(4) cells mL(-1). Current techniques for the detection of total Vibrios rely on laborious or inefficient analyses resulting in delayed management decisions. This work represents a novel approach for a rapid, accurate, sensitive and robust tool for quantifying Vibrios directly in industrial systems and in the environment, thereby facilitating rapid management responses.

Immuno-PCR: Achievements and Perspectives.

PubMed

Ryazantsev, D Y; Voronina, D V; Zavriev, S K

2016-12-01

The immuno-PCR (iPCR) method combines advantages of enzyme-linked immunosorbent assay and polymerase chain reaction, which is used in iPCR as a method of "visualization" of antigen-antibody interaction. The use of iPCR provides classical PCR sensitivity to objects traditionally detected by ELISA. This method could be very sensitive and allow for detection of quantities of femtograms/ml order. However, iPCR is still not widely used. The aim of this review is to highlight the special features of the iPCR method and to show the main aspects of its development and application in recent years.

Development of a magnetic bead fluorescence microscopy immunoassay to detect and quantify Leptospira in environmental water samples.

PubMed

Schreier, Stefan; Doungchawee, Galayanee; Triampo, Darapond; Wangroongsarb, Piyada; Hartskeerl, Rudi A; Triampo, Wannapong

2012-04-01

Climate change, world population growth, and poverty have led to an increase in the incidence of leptospirosis. Leptospirosis is caused by pathogenic spirochaete bacteria that belong to the genus Leptospira. The bacteria are maintained in the renal tubules of the reservoir hosts (typically a rodent), then shed into the environment via the urine. Water is key for environmental survival and transmission, as leptospires can survive for several weeks in a moist environment. Therefore, environmental epidemiological studies are needed to study the contamination of environmental water sources. However, few such studies have been performed using cultivation of the isolates and PCR assays. But, leptospira cultivation can be easily contaminated by other organisms and takes usually several weeks. Moreover, PCR is a complex and costly analysis for the underdeveloped countries that have the highest incidence of leptospirosis. In this study, we describe two modifications of a fluorescence microscopy assay based on immuno-magnetic separation (IMS) to detect leptospires in environmental water samples that mainly differ in fluorescent dye staining. The first type uses acridine orange fluorescent dye staining combined with multiplexed IMS for sample screening. The detection limit ranged from 10(2) to 10(3) organisms per mL and largely depended on the capture efficiency (CE) of the immuno-magnetic particles. The second type uses serogroup-specific immuno-particles and direct fluorescence antibody staining (DFA) to detect leptospires; the detection limit of this second assay was approximately 10(1) cells per mL. Both assay types were applied to natural and experimentally infected water samples, which were also analysed with DFM and real-time PCR. Our data show that the fluorescent microscopy immunoassay successfully identified experimental leptospire contamination and was as sensitive as PCR. This modified immune-fluorescence assay may therefore enable epidemiological studies of leptospirosis. Copyright © 2012 Elsevier B.V. All rights reserved.

Increased expression of (immuno)proteasome subunits during epileptogenesis is attenuated by inhibition of the mammalian target of rapamycin pathway.

PubMed

Broekaart, Diede W M; van Scheppingen, Jackelien; Geijtenbeek, Karlijne W; Zuidberg, Mark R J; Anink, Jasper J; Baayen, Johannes C; Mühlebner, Angelika; Aronica, Eleonora; Gorter, Jan A; van Vliet, Erwin A

2017-08-01

Inhibition of the mammalian target of rapamycin (mTOR) pathway reduces epileptogenesis in various epilepsy models, possibly by inhibition of inflammatory processes, which may include the proteasome system. To study the role of mTOR inhibition in the regulation of the proteasome system, we investigated (immuno)proteasome expression during epileptogenesis, as well as the effects of the mTOR inhibitor rapamycin. The expression of constitutive (β1, β5) and immunoproteasome (β1i, β5i) subunits was investigated during epileptogenesis using immunohistochemistry in the electrical post-status epilepticus (SE) rat model for temporal lobe epilepsy (TLE). The effect of rapamycin was studied on (immuno)proteasome subunit expression in post-SE rats that were treated for 6 weeks. (Immuno)proteasome expression was validated in the brain tissue of patients who had SE or drug-resistant TLE and the effect of rapamycin was studied in primary human astrocyte cultures. In post-SE rats, increased (immuno)proteasome expression was detected throughout epileptogenesis in neurons and astrocytes within the hippocampus and piriform cortex and was most evident in rats that developed a progressive form of epilepsy. Rapamycin-treated post-SE rats had reduced (immuno)proteasome protein expression and a lower number of spontaneous seizures compared to vehicle-treated rats. (Immuno)proteasome expression was also increased in neurons and astrocytes within the human hippocampus after SE and in patients with drug-resistant TLE. In vitro studies using cultured human astrocytes showed that interleukin (IL)-1β-induced (immuno)proteasome gene expression could be attenuated by rapamycin. Because dysregulation of the (immuno)proteasome system is observed before the occurrence of spontaneous seizures in rats, is associated with progression of epilepsy, and can be modulated via the mTOR pathway, it may represent an interesting novel target for drug treatment in epilepsy. Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.

[Brain emboli in the lungs of cattle].

PubMed

Horlacher, Sabine; Lücker, E; Eigenbrodt, E; Wenisch, Sabine

2002-01-01

There is no information whether the BSE agent is introduced into the human food chain through contamination of the lungs of cattle with central nervous system tissue (CNS). Studies in the United Kingdom and in the USA showed that CNS tissue could contaminate the lungs after using pneumatic powered air injection stunners (e.g. "The Knocker") or after pithing. Thus, pithing was forbidden in the European Union since January 2001. In German abattoirs conventional cartridge-fired stunners (e.g. model by Schermer) are usually applied. Pithing was used up to December 2000 in approx. 75% of the German abattoirs. In the present study 323 lungs of cattle were analysed for CNS. The lungs were derived from cattle exclusive stunned by use of the knocker from Schermer. 60% of the lungs contained emboli which were tested with immuno chemistry as well as immuno histochemistry to detect CNS. Two of 108 pooled samples showed a faint immuno reaction in the anti-NSE and anti-GFAP immunoblot. Further two particles showed a faint reaction for NSE and GFAP in immuno histochemistry, thus suggesting the presence of CNS. Even though CNS tissue could not be shown in the histological investigation, we used our findings to estimate the worst case scenario for human BSE exposure risk (HER) by lung contaminated by CNS emboli. The content of CNS in the samples was estimated to be about 0.11% when the respective immuno reactions were calibrated against standards containing known brain concentrations. Under the assumption that only one lung in the pooled samples was contaminated with BSE-infected central nervous tissue, the HER was calculated to reach a maximum of 2.2 x 10(-5) CoID50/consumer after consumption of a sausage with a portion of 10% lung. The results of our study suggest that the contamination of the lung with CNS after using a conventional cartridge-fired stunner cannot be excluded, however, the incidence appears to be very low. In addition, presumed CNS emboli, if at all, are microscopically small. Furthermore the incidence of BSE in Germany is very low and lungs of cattle are usually not consumed. Thus we can judge the potential for human oral exposure after consumption of lungs of cattle which were stunned in Germany to be extremely low. A final assessment, however, is impossible as there is no knowledge about the minimum infectious dose for humans.

Immuno-analysis of microparticles: probing at the limits of detection

PubMed Central

Latham, Sharissa L.; Tiberti, Natalia; Gokoolparsadh, Naveena; Holdaway, Karen; Olivier Couraud, Pierre; Grau, Georges E. R.; Combes, Valery

2015-01-01

Microparticle (MP) research is clouded by debate regarding the accuracy and validity of flow cytometry (FCM) as an analytical methodology, as it is influenced by many variables including the pre-analytical conditions, instruments physical capabilities and detection parameters. This study utilises a simplistic in vitro system for generating MP, and through comparative analysis with immuno-electron microscopy (Immuno-EM) assesses the strengths and limitations of probe selection and high-sensitivity FCM. Of the markers examined, MP were most specifically labelled with phosphatidylserine ligands, annexin V and lactadherin, although only ~60% MP are PS positive. Whilst these two ligands detect comparable absolute MP numbers, they interact with the same population in distinct manners; annexin V binding is enhanced on TNF induced MP. CD105 and CD54 expression were, as expected, consistent and enhanced following TNF activation respectively. Their labelling however accounted for as few as 30–40% of MP. The greatest discrepancies between FCM and I-EM were observed in the population solely labelled for the surface antigen. These findings demonstrate that despite significant improvements in resolution, high-sensitivity FCM remains limited in detecting small-size MP expressing low antigen levels. This study highlights factors to consider when selecting endothelial MP probes, as well as interpreting and representing data. PMID:26553743

Magnetic bead based immuno-detection of Listeria monocytogenes and Listeria ivanovii from infant formula and leafy green vegetables using the Bio-Plex suspension array system.

PubMed

Day, J B; Basavanna, U

2015-04-01

Listeriosis, a disease contracted via the consumption of foods contaminated with pathogenic Listeria species, can produce severe symptoms and high mortality in susceptible people and animals. The development of molecular methods and immuno-based techniques for detection of pathogenic Listeria in foods has been challenging due to the presence of assay inhibiting food components. In this study, we utilize a macrophage cell culture system for the isolation and enrichment of Listeria monocytogenes and Listeria ivanovii from infant formula and leafy green vegetables for subsequent identification using the Luminex xMAP technique. Macrophage monolayers were exposed to infant formula, lettuce and celery contaminated with L. monocytogenes or L. ivanovii. Magnetic microspheres conjugated to Listeria specific antibody were used to capture Listeria from infected macrophages and then analyzed using the Bio-Plex 200 analyzer. As few as 10 CFU/mL or g of L. monocytogenes was detected in all foods tested. The detection limit for L. ivanovii was 10 CFU/mL in infant formula and 100 CFU/g in leafy greens. Microsphere bound Listeria obtained from infected macrophage lysates could also be isolated on selective media for subsequent confirmatory identification. This method presumptively identifies L. monocytogenes and L. ivanovii from infant formula, lettuce and celery in less than 28 h with confirmatory identifications completed in less than 48 h. Published by Elsevier Ltd.

A low cost and high throughput magnetic bead-based immuno-agglutination assay in confined droplets.

PubMed

Teste, Bruno; Ali-Cherif, Anaïs; Viovy, Jean Louis; Malaquin, Laurent

2013-06-21

Although passive immuno-agglutination assays consist of one step and simple procedures, they are usually not adapted for high throughput analyses and they require expensive and bulky equipment for quantitation steps. Here we demonstrate a low cost, multimodal and high throughput immuno-agglutination assay that relies on a combination of magnetic beads (MBs), droplets microfluidics and magnetic tweezers. Antibody coated MBs were used as a capture support in the homogeneous phase. Following the immune interaction, water in oil droplets containing MBs and analytes were generated and transported in Teflon tubing. When passing in between magnetic tweezers, the MBs contained in the droplets were magnetically confined in order to enhance the agglutination rate and kinetics. When releasing the magnetic field, the internal recirculation flows in the droplet induce shear forces that favor MBs redispersion. In the presence of the analyte, the system preserves specific interactions and MBs stay in the aggregated state while in the case of a non-specific analyte, redispersion of particles occurs. The analyte quantitation procedure relies on the MBs redispersion rate within the droplet. The influence of different parameters such as magnetic field intensity, flow rate and MBs concentration on the agglutination performances have been investigated and optimized. Although the immuno-agglutination assay described in this work may not compete with enzyme linked immunosorbent assay (ELISA) in terms of sensitivity, it offers major advantages regarding the reagents consumption (analysis is performed in sub microliter droplet) and the platform cost that yields to very cheap analyses. Moreover the fully automated analysis procedure provides reproducible analyses with throughput well above those of existing technologies. We demonstrated the detection of biotinylated phosphatase alkaline in 100 nL sample volumes with an analysis rate of 300 assays per hour and a limit of detection of 100 pM.

High-contrast grating resonators for label-free detection of disease biomarkers

PubMed Central

Sun, Tianbo; Kan, Shu; Marriott, Gerard; Chang-Hasnain, Connie

2016-01-01

A label-free optical biosensor is described that employs a silicon-based high-contrast grating (HCG) resonator with a spectral linewidth of ~500 pm that is sensitive to ligand-induced changes in surface properties. The device is used to generate thermodynamic and kinetic data on surface-attached antibodies with their respective antigens. The device can detect serum cardiac troponin I, a biomarker of cardiac disease to 100 pg/ml within 4 mins, which is faster, and as sensitive as current enzyme-linked immuno-assays for cTnI. PMID:27265624

High-contrast grating resonators for label-free detection of disease biomarkers

NASA Astrophysics Data System (ADS)

Sun, Tianbo; Kan, Shu; Marriott, Gerard; Chang-Hasnain, Connie

2016-06-01

A label-free optical biosensor is described that employs a silicon-based high-contrast grating (HCG) resonator with a spectral linewidth of ~500 pm that is sensitive to ligand-induced changes in surface properties. The device is used to generate thermodynamic and kinetic data on surface-attached antibodies with their respective antigens. The device can detect serum cardiac troponin I, a biomarker of cardiac disease to 100 pg/ml within 4 mins, which is faster, and as sensitive as current enzyme-linked immuno-assays for cTnI.

Gold patterned biochips for on-chip immuno-MALDI-TOF MS: SPR imaging coupled multi-protein MS analysis.

PubMed

Kim, Young Eun; Yi, So Yeon; Lee, Chang-Soo; Jung, Yongwon; Chung, Bong Hyun

2012-01-21

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of immuno-captured target protein efficiently complements conventional immunoassays by offering rich molecular information such as protein isoforms or modifications. Direct immobilization of antibodies on MALDI solid support enables both target enrichment and MS analysis on the same plate, allowing simplified and potentially multiplexing protein MS analysis. Reliable on-chip immuno-MALDI-TOF MS for multiple biomarkers requires successful adaptation of antibody array biochips, which also must accommodate consistent reaction conditions on antibody arrays during immuno-capture and MS analysis. Here we developed a facile fabrication process of versatile antibody array biochips for reliable on-chip MALDI-TOF-MS analysis of multiple immuno-captured proteins. Hydrophilic gold arrays surrounded by super-hydrophobic surfaces were formed on a gold patterned biochip via spontaneous chemical or protein layer deposition. From antibody immobilization to MALDI matrix treatment, this hydrophilic/phobic pattern allowed highly consistent surface reactions on each gold spot. Various antibodies were immobilized on these gold spots both by covalent coupling or protein G binding. Four different protein markers were successfully analyzed on the present immuno-MALDI biochip from complex protein mixtures including serum samples. Tryptic digests of captured PSA protein were also effectively detected by on-chip MALDI-TOF-MS. Moreover, the present MALDI biochip can be directly applied to the SPR imaging system, by which antibody and subsequent antigen immobilization were successfully monitored.

ImmunoPET/MR imaging allows specific detection of Aspergillus fumigatus lung infection in vivo

PubMed Central

Rolle, Anna-Maria; Hasenberg, Mike; Thornton, Christopher R.; Solouk-Saran, Djamschid; Männ, Linda; Weski, Juliane; Maurer, Andreas; Fischer, Eliane; Spycher, Philipp R.; Schibli, Roger; Boschetti, Frederic; Stegemann-Koniszewski, Sabine; Bruder, Dunja; Severin, Gregory W.; Autenrieth, Stella E.; Krappmann, Sven; Davies, Genna; Pichler, Bernd J.; Gunzer, Matthias; Wiehr, Stefan

2016-01-01

Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease caused by the fungus Aspergillus fumigatus, and is a leading cause of invasive fungal infection-related mortality and morbidity in patients with hematological malignancies and bone marrow transplants. We developed and tested a novel probe for noninvasive detection of A. fumigatus lung infection based on antibody-guided positron emission tomography and magnetic resonance (immunoPET/MR) imaging. Administration of a [64Cu]DOTA-labeled A. fumigatus-specific monoclonal antibody (mAb), JF5, to neutrophil-depleted A. fumigatus-infected mice allowed specific localization of lung infection when combined with PET. Optical imaging with a fluorochrome-labeled version of the mAb showed colocalization with invasive hyphae. The mAb-based newly developed PET tracer [64Cu]DOTA-JF5 distinguished IPA from bacterial lung infections and, in contrast to [18F]FDG-PET, discriminated IPA from a general increase in metabolic activity associated with lung inflammation. To our knowledge, this is the first time that antibody-guided in vivo imaging has been used for noninvasive diagnosis of a fungal lung disease (IPA) of humans, an approach with enormous potential for diagnosis of infectious diseases and with potential for clinical translation. PMID:26787852

Immunochemical and biological properties of a mouse monoclonal antibody reactive to prunus necrotic ringspot ilarvirus.

PubMed

Aebig, J A; Jordan, R L; Lawson, R H; Hsu, H T

1987-01-01

A monoclonal antibody reacting with prunus necrotic ringspot ilarvirus was tested in immunochemical studies, neutralization of infectivity assays, and by immuno-electron microscopy. The antibody was able to detect the 27,000 Mr coat protein of prunus necrotic ringspot ilarvirus in western blots and also detected all polypeptide fragments generated after incubation of whole virus with proteolytic enzymes. In neutralization of infectivity studies, the antibody blocked virus infectivity, although it did not precipitate the antigen in agar gel Ouchterlony double diffusion tests. Immuno-electron microscopy confirmed that the antibody coats virions but does not cause clumping. The antibody may be a useful tool for investigating coat protein-dependent initiation of ilarvirus infection.

Employing immuno-affinity for the analysis of various microbial metabolites of the mycotoxin deoxynivalenol.

PubMed

Zhu, Yan; Hassan, Yousef I; Shao, Suqin; Zhou, Ting

2018-06-29

Deoxynivalenol (DON) is a type B trichothecene mycotoxin that is commonly detected in grains infested with Fusarium species. The maximum tolerated levels of DON in the majority of world's countries are restricted to 0.75 mg kg -1 within the human food chain and to less than 1-5 mg kg -1 in animal feed depending on the feed material and/or animal species due to DON's short and long-term adverse effects on human health and animal productivity. The ability to accurately analyze DON and some of its fungal/bacterial metabolites is increasingly gaining a paramount importance in food/feed analysis and research. In this study, we used the immuno-affinity approach to enrich and detect DON and three of its bacterial metabolites, namely 3-epi-DON, 3-keto-DON, and deepoxy-DON (DOM-1). The optimized enrichment step coupled with high performance liquid chromatography can accurately and reproducibly quantify the aforementioned metabolites in feed matrixes (silage extract as an example in this case). It minimizes any background interface and provides a fast and easy-to-operate protocol for the analytical determination of such metabolites. More importantly, the presented data demonstrates the ability of the utilized monoclonal antibody, generated originally to capture DON in Enzyme-Linked Immunosorbent Assays (ELISA), to cross react with three less/non-toxic DON metabolites. This raises the concerns about the genuine need to account for such cross-reactivity when DON contamination is assessed through an immuno-affinity based analyses using the investigated antibody. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.

Sensitivity, specificity and comparison of three commercially available immunological tests in the diagnosis of Cryptosporidium species in animals.

PubMed

Danišová, Olga; Halánová, Monika; Valenčáková, Alexandra; Luptáková, Lenka

The study was conducted to compare the specificity of immunological diagnostic methods used for the diagnosis of Cryptosporidium species capable of causing life-threatening infection in both immunosuppressed and immunocompetent patients. For the detection of Cryptosporidium species in 79 animals with diarrhoea, we used three Copro-antigen tests: RIDASCREEN ® Cryptosporidium test, Cryptosporidium 2nd Generation (ELISA) and RIDA ® QUICK Cryptosporidium. For immunoassays we used positive and negative samples detected by means of polymerase chain reaction and validated by sequencing and nested polymerase chain reaction to confirm the presence six different species of Cryptosporidium species. Prevalence of cryptosporidiosis in the entire group determined by enzyme immunoassay, enzyme linked immunosorbent assay, immuno-chromatographic test and polymerase chain reaction was 34.17%, 27.84%, 6.33% and 27.84%, respectively. Sensitivity of animal samples with enzyme immunoassay, enzyme linked immunosorbent assay, and immuno-chromatographic test was 63.6%, 40.9% and 22.7%, resp., when questionable samples were considered positive, whereas specificity of enzyme immunoassay, enzyme linked immunosorbent assay and immuno-chromatographic test was 75.9%, 78.9% and 100%, respectively. Positive predictive values and negative predictive values were different for all the tests. These differences results are controversial and therefore reliability and reproducibility of immunoassays as the only diagnostic method is questionable. The use of various Cryptosporidium species in diagnosis based on immunological testing and different results obtained by individual tests indicate potential differences in Copro-antigens produced by individual Cryptosporidium species. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Technical Advances in Intracellular Detection Using Immuno-Gold Particles: Simple Cryofixation with Metal Contact Quick Freezing.

PubMed

Song, Chihong; Lee, Ju Huck; Jun, Sangmi; Chung, Jeong Min; Hyun, Jaekyung; Jung, Hyun Suk

2016-05-01

The preparation of biological specimens using cryofixation techniques ensures excellent visibility of intracellular structures and preserves the antigenic sites of subcellular molecules. Hence, cryofixation is an effective method of preparing samples for analyses using antibodies conjugated to gold nanoparticles that are designed to detect the localization of specific target molecules within cells. However, cryofixation cannot be utilized easily because it requires expensive equipment and skilled technologists, resulting in a high level of expense for researchers. Here, we describe a simple technical approach to cryofixation that uses metal contact quick freezing followed by a modified freeze substitution technique and immuno-gold labeling electron microscopy. Micrograph images of cells prepared using this modified cryofixation method demonstrated its superiority over chemical fixation for high contrast visualization of the morphologies of cellular components and preservation of antigenicity for immuno-gold labeling. This report provides valuable technical information related to the advancement of metal contact quick freezing techniques, which can be used to visualize biomedical events of interest in an easy, simple, and rapid manner.

Six rapid tests for direct detection of Clostridium difficile and its toxins in fecal samples compared with the fibroblast cytotoxicity assay.

PubMed

Turgeon, David K; Novicki, Thomas J; Quick, John; Carlson, LaDonna; Miller, Pat; Ulness, Bruce; Cent, Anne; Ashley, Rhoda; Larson, Ann; Coyle, Marie; Limaye, Ajit P; Cookson, Brad T; Fritsche, Thomas R

2003-02-01

Clostridium difficile is one of the most frequent causes of nosocomial gastrointestinal disease. Risk factors include prior antibiotic therapy, bowel surgery, and the immunocompromised state. Direct fecal analysis for C. difficile toxin B by tissue culture cytotoxin B assay (CBA), while only 60 to 85% sensitive overall, is a common laboratory method. We have used 1,003 consecutive, nonduplicate fecal samples to compare six commercially available immunoassays (IA) for C. difficile detection with CBA: Prima System Clostridium difficile Tox A and VIDAS Clostridium difficile Tox A II, which detect C. difficile toxin A; Premier Cytoclone A/B and Techlab Clostridium difficile Tox A/B, which detect toxins A and B; and ImmunoCard Clostridium difficile and Triage Micro C. difficile panels, which detect toxin A and a species-specific antigen. For all tests, Triage antigen was most sensitive (89.1%; negative predictive value [NPV] = 98.7%) while ImmunoCard was most specific (99.7%; positive predictive value [PPV] = 95.0%). For toxin tests only, Prima System had the highest sensitivity (82.2%; NPV = 98.0%) while ImmunoCard had the highest specificity (99.7%; PPV = 95.0%). Hematopoietic stem cell transplant (HSCT) patients contributed 44.7% of all samples tested, and no significant differences in sensitivity or specificity were noted between HSCT and non-HSCT patients. IAs, while not as sensitive as direct fecal CBA, produce reasonable predictive values, especially when both antigen and toxin are detected. They also offer significant advantages over CBA in terms of turnaround time and ease of use.

Normalization of test and evaluation of biothreat detection systems: overcoming microbial air content fluctuations by using a standardized reagent bacterial mixture.

PubMed

Berchebru, Laurent; Rameil, Pascal; Gaudin, Jean-Christophe; Gausson, Sabrina; Larigauderie, Guilhem; Pujol, Céline; Morel, Yannick; Ramisse, Vincent

2014-10-01

Test and evaluation of engineered biothreat agent detection systems ("biodetectors") are a challenging task for government agencies and industries involved in biosecurity and biodefense programs. In addition to user friendly features, biodetectors need to perform both highly sensitive and specific detection, and must not produce excessive false alerts. In fact, the atmosphere displays a number of variables such as airborne bacterial content that can interfere with the detection process, thus impeding comparative tests when carried out at different times or places. To overcome these bacterial air content fluctuations, a standardized reagent bacterial mixture (SRBM), consisting in a collection of selected cultivable environmental species that are prevalent in temperate climate bioaerosols, was designed to generate a stable, reproducible, and easy to use surrogate of bioaerosol sample. The rationale, design, and production process are reported. The results showed that 8.59; CI 95%: 8.46-8.72 log cfu distributed into vials underwent a 0.95; CI 95%: 0.65-1.26 log viability decay after dehydration and subsequent reconstitution, thus advantageously mimicking a natural bioaerosol sample which is typically composed of cultivable and uncultivable particles. Dehydrated SRBM was stable for more than 12months at 4°C and allowed the reconstitution of a dead/live cells aqueous suspension that is stable for 96h at +4°C, according to plate counts. Specific detection of a simulating biothreat agent (e.g. Bacillus atrophaeus) by immuno-magnetic or PCR assays did not display any significant loss of sensitivity, false negative or positive results in the presence of SRBM. This work provides guidance on testing and evaluating detection devices, and may contribute to the establishment of suitable standards and normalized procedures. Copyright © 2014 Elsevier B.V. All rights reserved.

Chemiluminescent microparticle immunoassay based detection and prevalence of HCV infection in district Peshawar Pakistan

PubMed Central

2014-01-01

Background Due to the high rate of asymptomatic infections an advanced screening assay is of prompt importance to be used for the clinical diagnosis of HCV. Early detection of anti HCV is the first step in the management of chronic hepatitis and in the selection of patients needing treatments. In the current study we have first time used the advanced serological diagnostic technique i.e. Chemiluminescent Microparticle Immuno Assay (CMIA) for the detection of HCV infection in Peshawar Pakistan. Methods A total number of 982 samples were collected among the general public belongs to the different areas of district Peshawar. The samples were centrifuged at high speed to obtain a clear supernatant serum. All the samples were run on Architect system a fully automated immuno analyzer CMIA base technology. Results Out of 982 blood samples analyzed in this study, 160 (15.9%) were confirmed to be positive for active HCV infection. The overall prevalence was found to be 13.4%. Gender wise prevalence was recorded to be higher in male (19.1%) than female (12.7%). The age group 21-30 years was identified as the highest risk group among the studied population. Conclusion Among the tested samples, overall prevalence of active HCV infection was found to be 13.4% in the general population of Peshawar Pakistan. The young middle aged population of this region was at higher risk of HCV ailments compared to the other age groups. PMID:25016473

Detection of CEA in human serum using surface-enhanced Raman spectroscopy coupled with antibody-modified Au and γ-Fe₂O₃@Au nanoparticles.

PubMed

Lin, Yan; Xu, Guanhong; Wei, Fangdi; Zhang, Aixia; Yang, Jing; Hu, Qin

2016-03-20

In this present work, a rapid and simple method to detect carcinoembryonic antigen (CEA) was developed by using surface-enhanced Raman spectroscopy (SERS) coupled with antibody-modified Au and γ-Fe2O3@Au nanoparticles. First, Au@Raman reporter and γ-Fe2O3@Au were prepared, and then modified with CEA antibody. When CEA was present, the immuno-Au@Raman reporter and immuno-γ-Fe2O3@Au formed a complex through antibody-antigen-antibody interaction. The selective and sensitive detection of CEA could be achieved by SERS after magnetic separation. Under the optimal conditions, a linear relationship was observed between the Raman peak intensity and the concentration of CEA in the range of 1-50 ng mL(-1) with an excellent correlation coefficient of 0.9942. The limit of detection based on two times ratio of signal to noise was 0.1 ng/mL. The recoveries of CEA standard solution spiked with human serum samples were in the range of 88.5-105.9% with the relative standard deviations less than 17.4%. The method built was applied to the detection of CEA in human serum, and the relative deviations of the analysis results between the present method and electrochemiluminescence immunoassay were all less than 16.6%. The proposed method is practical and has a potential for clinic test of CEA. Copyright © 2016 Elsevier B.V. All rights reserved.

Immuno-SPET/CT and immuno-PET/CT: a step ahead to translational imaging.

PubMed

Pecking, Alain P; Bellet, Dominique; Alberini, Jean Louis

2012-10-01

Malignant tumours have the remarkable property to express cell surface antigens. Pressman was first reporting that radiolabeled antibodies were capable of organ localization. It was a promising challenge but the expected success and the development of this imaging method was limited by a poor imaging resolution despite a rather good specificity of the antibodies used. Identification of key cell surface markers is opening a new era as potential molecular imaging biomarkers in oncologic applications. Antibodies production has been promoted by the development of engineered fragments with preserved immunological properties and pharmacokinetics optimized for molecular imaging. A good compromise has to be obtained between the biological properties of the antibody and the physical half-life of the radionuclide. Several positron emission tomography (PET) radionuclides such as iodine-124, copper-64, yttrium-86 or zirconium-89 have been the focus of recent immuno-PET studies with interesting informative images in preclinical and clinical studies. Thanks to the development of more sensitive new detectors and specific software, molecular imaging methods, particularly PET imaging, allow nowadays the detection of lesions smaller than 5 mm in human. Immuno-PET can potentially be used for tumour detection and identification at diagnosis, staging and restaging, for treatment selection and monitoring, and during follow-up. Moreover the availability of matched imaging or therapeutic radionuclide pairs, such as (124)I/(131)I, (64)Cu/(67)Cu and (86)Y/(90)Y, make easier the quantification of tissue uptake and dosimetry calculation for radioimmunotherapy.

Unexpected Listeria monocytogenes detection with a dithiothreitol-based device during an aseptic hip revision.

PubMed

Banche, Giuliana; Bistolfi, Alessandro; Allizond, Valeria; Galletta, Claudia; Iannantuoni, Maria Rita; Marra, Elisa Simona; Merlino, Chiara; Massè, Alessandro; Cuffini, Anna Maria

2018-06-18

Prosthetic joint infection diagnosis is often difficult since biofilm-embedded microorganisms attach well to the prosthetic surfaces and resist their detection by conventional methods. DL-dithiothreitol has been described as a valid method for biofilm detachment on orthopedic devices. We report the case of an occasional detection of Listeria monocytogenes in a non immuno-compromised patient with a preoperative diagnosis of aseptic loosening. The infection diagnosis due to such rare bacteria was made postoperatively, thanks to a DL-dithiothreitol-based device. This may be considered a feasible approach for the microbiological analysis of prosthetic joint infection, considering that a prompt diagnosis of such biofilm-associated infections could bring some advantages, such as an early and appropriate antibiotic therapy administration and a reduction of undiagnosed infections.

Comparison of premier CAMPY enzyme immunoassay (EIA), ProSpecT Campylobacter EIA, and ImmunoCard STAT! CAMPY tests with culture for laboratory diagnosis of Campylobacter enteric infections.

PubMed

Granato, Paul A; Chen, Li; Holiday, Iris; Rawling, Russell A; Novak-Weekley, Susan M; Quinlan, Tammy; Musser, Kimberlee A

2010-11-01

Campylobacter enteritis is a food-borne or waterborne illness caused almost exclusively by Campylobacter jejuni and, to a lesser extent, by Campylobacter coli. These organisms produce indistinguishable clinical diseases and together represent the second most common cause of bacterial diarrhea in the United States and the leading cause of enteric infection throughout the world. The conventional approach to the laboratory diagnosis of Campylobacter enteritis is based on the recovery of the organism from a stool specimen, which requires the use of a specialized medium incubated at 42°C for several days in an artificially created microaerophilic environment. Recently, several commercially available enzyme immunoassays (EIAs) have been developed for the direct detection of C. jejuni and C. coli in stool specimens. This study compared conventional culture with three EIA methods, the Premier CAMPY EIA (Meridian Bioscience, Cincinnati, OH), the ProSpecT Campylobacter EIA (Remel, Lenexa, KS), and the ImmunoCard STAT! CAMPY test (Meridian Bioscience, Cincinnati, OH), for the detection of C. jejuni and C. coli in 485 patient stool samples. Discordant results were arbitrated by using an in-house, real-time PCR assay that was developed and validated by a public health reference laboratory. Following analyses of the discrepant specimens by PCR, the sensitivity and specificity of both the Premier CAMPY and ProSpecT Campylobacter EIAs were 99.3% and 98%, respectively, while the ImmunoCard STAT! CAMPY test had a sensitivity of 98.5% and a specificity of 98.2%. By use of the PCR test as the reference standard, culture detected 127 of 135 Campylobacter-positive stool specimens, yielding a sensitivity of 94.1%. These results showed that the three EIAs evaluated in this study provide a rapid and reliable alternative for the laboratory diagnosis of enteric infections with C. jejuni and C. coli and that conventional culture may no longer be recognized as the "gold standard" for diagnosis.

Detection of ovomucoid-specific low-affinity IgE in infants and its relationship to eczema.

PubMed

Kawamoto, Norio; Kamemura, Norio; Kido, Hiroshi; Fukao, Toshiyuki

2017-06-01

Allergen-specific low-affinity IgE was previously detected in cord blood by a highly sensitive densely carboxylated protein (DCP) chip, but not by ImmunoCAP. Here, we investigated the presence of low-affinity IgE during the early life of infants and observed its relationship with eczema. We conducted a birth cohort study, collecting sera at birth and 6 and 14 months of age (n = 110). We monitored the ovomucoid (OM)- and egg white (EW)-specific IgE (sIgE) by ImmunoCAP or DCP chip and analyzed the antigen affinity of sIgE by binding inhibition assays in the presence or absence of a mild chaotropic agent, diethyl amine (DEA). The low- and high-affinity OM-sIgEs and sensitization risk factors were analyzed by a multivariate logistic analysis. The OM-sIgE measured by DCP chip significantly correlated with that measured by ImmunoCAP, but some samples assessed as OM-sIgE positive by DCP chip were considered OM-sIgE negative by ImmunoCAP. Binding inhibition analysis after DEA treatment was performed for participants judged as OM-sIgE positive by DCP chip at 14 M. The group assessed as negative for OM- and EW-sIgE by ImmunoCAP at 6 and 14 months showed a larger binding inhibition curve shift after DEA treatment than did the group assessed as positive at these times, indicating the presence of low-affinity sIgE antibodies at 14 months. The logistic regression analysis found that persistent eczema from 6 to 14 months is a significant risk factor for developing high-affinity, but not low-affinity, sIgE. Human infant peripheral blood contains allergen-specific low-affinity sIgE. Persistent eczema is related to the development of high-affinity, but not low-affinity, IgE. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A novel enzyme-linked immuno-sorbent assay (ELISA) for the quantification of total and free polysaccharide in Haemophilus influenzae b-Tetanus toxoid conjugate vaccines in monovalent and combined vaccine formulations.

PubMed

Saydam, Manolya; Rigsby, Peter; Mawas, Fatme

2014-01-01

Current Haemophilus influenzae b conjugate vaccines (Hib), which are made of purified capsular polysaccharide (poly-ribosyl-ribitol-phosphate; PRP) conjugated to a carrier protein, are almost completely evaluated by physico-chemical methods to ensure the integrity and stability of the vaccine and consistency of manufacture of batches. The absence of a potency assay makes the quantification of total PRP content (in SI units) and of % free polysaccharide in final fills or bulk components of Hib vaccines critical release tests for both manufacturers and national control authorities. Here we describe a simple and sensitive Enzyme-Linked Immuno-sorbent Assay (ELISA) which has been developed to quantify total and free PRP content in Hib-TT vaccine alone or when in combination with other vaccines. The assay is robust, specific and highly sensitive. Copyright © 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Allergen Chip Diagnosis for Soy-Allergic Patients: Gly m 4 as a Marker for Severe Food-Allergic Reactions to Soy

PubMed Central

Berneder, M.; Bublin, M.; Hoffmann-Sommergruber, K.; Hawranek, T.; Lang, R.

2016-01-01

Background Gly m 5 and Gly m 6 are known to induce severe reactions in soy-allergic patients. For birch pollen (BP)-allergic patients, the Bet v 1 homologous allergen Gly m 4 is also a potential trigger of generalized severe reactions upon soy consumption. Therefore, reliable component-resolved diagnosis of soy allergy is needed. Methods IgE reactivity from sera of 20 patients from a BP environment with reported soy allergy was assessed. Skin prick tests (SPT) with BP and soy drink were performed. Specific IgE for BP, soy, Bet v 1 and Gly m 4 was analyzed by ImmunoCAP. In addition, ISAC microarray profiling was performed. Results Nineteen of 20 patients were BP allergic (positive SPT and/or CAP results for BP extract and Bet v 1). Eighteen soy-allergic patients were tested positive with soy drink in SPT. Soy CAP results were negative in the majority of tests (15/20), whereas 19/20 sera had specific IgE to Gly m 4. In the microarray approach, 14/20 sera displayed Gly m 4-specific IgE, the additional 6 sera had IgE levels below 0.3 ISAC standardized units. The BP-negative serum had Gly m 5- and Gly m 6-specific IgE which correlated with positive soy ImmunoCAP. Conclusions Soy sensitization detected by SPT and Gly m 4 ImmunoCAP were in good qualitative agreement with ISAC results. Soy ImmunoCAP was only specific for Gly m 5 and Gly m 6 sensitization. Gly m 4 ImmunoCAP has a higher sensitivity than ImmunoCAP ISAC. In this patient cohort, Gly m 4 sensitization was linked to the development of severe and generalized allergic reactions upon soy consumption. PMID:23548307

Immuno-biosensor for Detection of CD20-Positive Cells Using Surface Plasmon Resonance.

PubMed

Shanehbandi, Dariush; Majidi, Jafar; Kazemi, Tohid; Baradaran, Behzad; Aghebati-Maleki, Leili; Fathi, Farzaneh; Ezzati Nazhad Dolatabadi, Jafar

2017-06-01

Purpose: Surface plasmon resonance (SPR) sensing confers a real-time assessment of molecular interactions between biomolecules and their ligands. This approach is highly sensitive and reproducible and could be employed to confirm the successful binding of drugs to cell surface targets. The specific affinity of monoclonal antibodies (MAb) for their target antigens is being utilized for development of immuno-sensors and therapeutic agents. CD20 is a surface protein of B lymphocytes which has been widely employed for immuno-targeting of B-cell related disorders. In the present study, binding ability of an anti-CD20 MAb to surface antigens of intact target cells was investigated by SPR technique. Methods: Two distinct strategies were used for immobilization of the anti-CD20 MAb onto gold (Au) chips. MUA (11-mercaptoundecanoic acid) and Staphylococcus aureus protein A (SpA) were the two systems used for this purpose. A suspension of CD20-positive Raji cells was injected in the analyte phase and the resulting interactions were analyzed and compared to those of MOLT-4 cell line as CD20-negative control. Results: Efficient binding of anti-CD20 MAb to the surface antigens of Raji cell line was confirmed by both immobilizing methods, whereas this MAb had not a noticeable affinity to the MOLT-4 cells. Conclusion: According to the outcomes, the investigated MAb had acceptable affinity and specificity to the target antigens on the cell surface and could be utilized for immuno-detection of CD20-positive intact cells by SPR method.

Immuno-biosensor for Detection of CD20-Positive Cells Using Surface Plasmon Resonance

PubMed Central

Shanehbandi, Dariush; Majidi, Jafar; Kazemi, Tohid; Baradaran, Behzad; Aghebati-Maleki, Leili; Fathi, Farzaneh; Ezzati Nazhad Dolatabadi, Jafar

2017-01-01

Purpose: Surface plasmon resonance (SPR) sensing confers a real-time assessment of molecular interactions between biomolecules and their ligands. This approach is highly sensitive and reproducible and could be employed to confirm the successful binding of drugs to cell surface targets. The specific affinity of monoclonal antibodies (MAb) for their target antigens is being utilized for development of immuno-sensors and therapeutic agents. CD20 is a surface protein of B lymphocytes which has been widely employed for immuno-targeting of B-cell related disorders. In the present study, binding ability of an anti-CD20 MAb to surface antigens of intact target cells was investigated by SPR technique. Methods: Two distinct strategies were used for immobilization of the anti-CD20 MAb onto gold (Au) chips. MUA (11-mercaptoundecanoic acid) and Staphylococcus aureus protein A (SpA) were the two systems used for this purpose. A suspension of CD20-positive Raji cells was injected in the analyte phase and the resulting interactions were analyzed and compared to those of MOLT-4 cell line as CD20-negative control. Results: Efficient binding of anti-CD20 MAb to the surface antigens of Raji cell line was confirmed by both immobilizing methods, whereas this MAb had not a noticeable affinity to the MOLT-4 cells. Conclusion: According to the outcomes, the investigated MAb had acceptable affinity and specificity to the target antigens on the cell surface and could be utilized for immuno-detection of CD20-positive intact cells by SPR method. PMID:28761820

[A VALIDATION STUDY OF THE IMPROVED PRODUCT FOR MEASURING JAPANESE CYPRESS POLLEN-SPECIFIC IgE (THERMO SCIENTIFIC™ ImmunoCAP™ ImmunoCAP JAPANESE CYPRESS POLLEN-SPECIFIC IgE)].

PubMed

Yonekura, Syuji; Okamoto, Yoshitaka; Nakayama, Satoshi

Japanese cypress pollen is a major causative allergen of seasonal allergic rhinitis in Japan. Although ImmunoCAP-specific immunoglobulin E (IgE) reagent Japanese cypress pollen has been widely used as a diagnostic aid, its sensitivity requires enhancement. This study evaluated an improved version of this reagent. Serum samples from 61 subjects who underwent Japanese cypress pollen exposure testing in an environmental challenge chamber in Chiba University were assessed using the conventional ImmunoCAPspecific IgE Japanese cypress pollen product and the improved product. In addition, specific IgE for Cha o 1 and Cha o 2, the primary allergen components of Japanese cypress pollen, was evaluated and their reactivity to specific IgE was compared between the conventional and improved products. The antibody titer of the improved product was approximately 1.8-fold that of the conventional product. In addition, higher correlations with Cha o 1 and Cha o 2 were observed for the improved product than for the conventional product. The clinical sensitivity (≥class 2) in 56 exposure test-positive subjects was better for the improved product (80.4%) than for the conventional product (71.4%). An improvement of the ImmunoCAP-specific IgE reagent Japanese cypress pollen resulted in enhanced Japanese cypress pollen-specific IgE sensitivity. The primary reason for this appeared to be an improved Cha o 1- and Cha o 2-specific IgE detectability.

Single particle electrochemical sensors and methods of utilization

DOEpatents

Schoeniger, Joseph [Oakland, CA; Flounders, Albert W [Berkeley, CA; Hughes, Robert C [Albuquerque, NM; Ricco, Antonio J [Los Gatos, CA; Wally, Karl [Lafayette, CA; Kravitz, Stanley H [Placitas, NM; Janek, Richard P [Oakland, CA

2006-04-04

The present invention discloses an electrochemical device for detecting single particles, and methods for using such a device to achieve high sensitivity for detecting particles such as bacteria, viruses, aggregates, immuno-complexes, molecules, or ionic species. The device provides for affinity-based electrochemical detection of particles with single-particle sensitivity. The disclosed device and methods are based on microelectrodes with surface-attached, affinity ligands (e.g., antibodies, combinatorial peptides, glycolipids) that bind selectively to some target particle species. The electrodes electrolyze chemical species present in the particle-containing solution, and particle interaction with a sensor element modulates its electrolytic activity. The devices may be used individually, employed as sensors, used in arrays for a single specific type of particle or for a range of particle types, or configured into arrays of sensors having both these attributes.

Transmission of a Viral Disease (AIDS) Detected by a Modified ELISA Reaction: A Laboratory Simulation.

ERIC Educational Resources Information Center

Grimes, William J.; Chambers, Linda; Kubo, Kenneth M.; Narro, Martha L.

1998-01-01

Describes a laboratory exercise that simulates the spread of an infectious agent among students in a classroom. Uses a modified Enzyme Linked ImmunoSorbent Assay (ELISA) to provide students with experience using an authentic diagnostic tool for detecting human infections. (DDR)

Label-Free Immuno-Sensors for the Fast Detection of Listeria in Food.

PubMed

Morlay, Alexandra; Roux, Agnès; Templier, Vincent; Piat, Félix; Roupioz, Yoann

2017-01-01

Foodborne diseases are a major concern for both food industry and health organizations due to the economic costs and potential threats for human lives. For these reasons, specific regulations impose the research of pathogenic bacteria in food products. Nevertheless, current methods, references and alternatives, take up to several days and require many handling steps. In order to improve pathogen detection in food, we developed an immune-sensor, based on Surface Plasmon Resonance imaging (SPRi) and bacterial growth which allows the detection of a very low number of Listeria monocytogenes in food sample in one day. Adequate sensitivity is achieved by the deposition of several antibodies in a micro-array format allowing real-time detection. This label-free method thus reduces handling and time to result compared with current methods.

Comparative diagnostics of allergy using quantitative immuno-PCR and ELISA.

PubMed

Simonova, Maria A; Pivovarov, Victor D; Ryazantsev, Dmitry Y; Dolgova, Anna S; Berzhets, Valentina M; Zavriev, Sergei K; Svirshchevskaya, Elena V

2018-05-01

Estimation of specific IgE is essential for the prevention of allergy progression. Quantitative immuno-PCR (qiPCR) can increase the sensitivity of IgE detection. We aimed to develop qiPCR and compare it to the conventional ELISA in identification of IgE to Alt a 1 and Fel d 1 allergens. Single stranded 60-mer DNA conjugated to streptavidin was used to detect antigen-IgE-biotin complex by qiPCR. In semi-logarithmic scale qiPCR data were linear in a full range of serum dilutions resulting in three- to ten-times higher sensitivity of qiPCR in comparison with ELISA in IgE estimation in low titer sera. Higher sensitivity of qiPCR in identification of low titer IgE is a result of a higher linearity of qiPCR data.

Environmental effects on the production of Shiga-like toxins by Escherichia coli O157:H7 as revealed by sandwiched immuno-chemiluminescence detection

NASA Astrophysics Data System (ADS)

Tu, Shu-I.; Uknalis, Joseph; He, Yiping

2009-05-01

We have developed a sandwiched immuno assay to detect sensitively Shiga-like toxins (SLTs) produced by Escherichia coli O157:H7. The method involved the capture of toxins by specific immuno magnetic beads followed by tagging the toxins with peroxidase-labeled anti E. coli O157:H7 antibody. Upon addition of proper substrate, peroxidase induced luminescence was used to measure the presence of SLTs. We have previously demonstrated that co-incubation of shiga toxin (SLT) producing E. coli O157:H7 with certain other bacteria can inhibit toxin production but does not affect the growth of the E. coli. We show here that media in which the cells have grown been centrifuged from (conditioned media) have similar effects on cell growth and SLT production. Adjusting the pH and adding nutrients to the conditioned media did not have any effect on the reduction of SLT produced. Bacteria communicate with each other via secreted sensing molecules. Several types of the molecules have been identified. However, the mechanisms of control remain to be established. This pattern for bacteria growth and toxin production is also observed when quorum-sensing molecules of homoserine lactone and indole are added to the media prior to inoculation.

Rapid, sensitive, and simultaneous detection of three foodborne pathogens using magnetic nanobead-based immunoseparation and quantum dot-based multiplex immunoassay.

PubMed

Wang, Hong; Li, Yanbin; Wang, Andrew; Slavik, Michael

2011-12-01

Losses caused by foodborne diseases are enormous in terms of human life, illness, medical costs, and food product recalls. Rapid detection of multiple bacterial pathogens in foods is extremely important to ensure food safety. The objective of this research was to develop a multiplex immunoassay by integrating magnetic nanobeads (MNBs) for immunoseparation with quantum dots (QDs) as fluorescent labels for rapid, sensitive, and simultaneous detection of three major pathogenic bacteria, Salmonella Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes, in food products. In this research, both streptavidin-conjugated MNBs (30- and 150-nm diameter) and QDs (530-, 580-, and 620-nm emission wavelength) were separately coated with biotinylated anti-Salmonella, anti-E. coli, and anti-Listeria antibodies. The immuno-MNBs were mixed with a food sample to capture the three target bacteria. After being magnetically separated from the sample, the MNB-cell conjugates were mixed with the immuno-QDs to form the MNB-cell-QD complexes, and unattached QDs were removed. The fluorescence intensity of the MNB-cell-QD complexes was measured at wavelengths of 530, 580, and 620 nm to determine the populations of Salmonella Typhimurium, E. coli O157:H7, and L. monocytogenes, respectively. This multiplex immunoassay simultaneously detected Salmonella Typhimurium, E. coli O157:H7, and L. monocytogenes at levels as low as 20 to 50 CFU/ml in food samples in less than 2 h without enrichment. The change in fluorescence intensity was linearly correlated (R(2) > 0.96) with the logarithmic value of bacterial level in the range of 10 to 10(3) CFU/ml. More than 85% of the three target pathogens could be simultaneously separated from food samples. The multiplex immunoassay could be expanded to detect more target pathogens, depending on the availability of specific antibodies and QDs with different emission wavelengths.

Immunohistochemical Detection of a Unique Protein within Cells of Snakes Having Inclusion Body Disease, a World-Wide Disease Seen in Members of the Families Boidae and Pythonidae

PubMed Central

Chang, Li-Wen; Fu, Ann; Wozniak, Edward; Chow, Marjorie; Duke, Diane G.; Green, Linda; Kelley, Karen; Hernandez, Jorge A.; Jacobson, Elliott R.

2013-01-01

Inclusion body disease (IBD) is a worldwide disease in captive boa constrictors (boa constrictor) and occasionally in other snakes of the families Boidae and Pythonidae. The exact causative agent(s) and pathogenesis are not yet fully understood. Currently, diagnosis of IBD is based on the light microscopic identification of eosinophilic intracytoplasmic inclusion bodies in hematoxylin and eosin stained tissues or blood smears. An antigenically unique 68 KDa protein was identified within the IBD inclusion bodies, called IBD protein. A validated immuno-based ante-mortem diagnostic test is needed for screening snakes that are at risk of having IBD. In this study, despite difficulties in solubilizing semi-purified inclusion bodies, utilizing hybridoma technology a mouse anti-IBD protein monoclonal antibody (MAB) was produced. The antigenic specificity of the antibody was confirmed and validated by western blots, enzyme-linked immunosorbent assay, immuno-transmission electron microscopy, and immunohistochemical staining. Paraffin embedded tissues of IBD positive and negative boa constrictors (n=94) collected from 1990 to 2011 were tested with immunohistochemical staining. In boa constrictors, the anti-IBDP MAB had a sensitivity of 83% and specificity of 100% in detecting IBD. The antibody also cross-reacted with IBD inclusion bodies in carpet pythons (Morelia spilota) and a ball python (python regius). This validated antibody can serve as a tool for the development of ante-mortem immunodiagnostic tests for IBD. PMID:24340066

Immunohistochemical detection of a unique protein within cells of snakes having inclusion body disease, a world-wide disease seen in members of the families Boidae and Pythonidae.

PubMed

Chang, Li-Wen; Fu, Ann; Wozniak, Edward; Chow, Marjorie; Duke, Diane G; Green, Linda; Kelley, Karen; Hernandez, Jorge A; Jacobson, Elliott R

2013-01-01

Inclusion body disease (IBD) is a worldwide disease in captive boa constrictors (boa constrictor) and occasionally in other snakes of the families Boidae and Pythonidae. The exact causative agent(s) and pathogenesis are not yet fully understood. Currently, diagnosis of IBD is based on the light microscopic identification of eosinophilic intracytoplasmic inclusion bodies in hematoxylin and eosin stained tissues or blood smears. An antigenically unique 68 KDa protein was identified within the IBD inclusion bodies, called IBD protein. A validated immuno-based ante-mortem diagnostic test is needed for screening snakes that are at risk of having IBD. In this study, despite difficulties in solubilizing semi-purified inclusion bodies, utilizing hybridoma technology a mouse anti-IBD protein monoclonal antibody (MAB) was produced. The antigenic specificity of the antibody was confirmed and validated by western blots, enzyme-linked immunosorbent assay, immuno-transmission electron microscopy, and immunohistochemical staining. Paraffin embedded tissues of IBD positive and negative boa constrictors (n=94) collected from 1990 to 2011 were tested with immunohistochemical staining. In boa constrictors, the anti-IBDP MAB had a sensitivity of 83% and specificity of 100% in detecting IBD. The antibody also cross-reacted with IBD inclusion bodies in carpet pythons (Morelia spilota) and a ball python (python regius). This validated antibody can serve as a tool for the development of ante-mortem immunodiagnostic tests for IBD.

Method for estimating protein binding capacity of polymeric systems.

PubMed

Sharma, Vaibhav; Blackwood, Keith A; Haddow, David; Hook, Lilian; Mason, Chris; Dye, Julian F; García-Gareta, Elena

2015-01-01

Composite biomaterials made from synthetic and protein-based polymers are extensively researched in tissue engineering. To successfully fabricate a protein-polymer composite, it is critical to understand how strongly the protein binds to the synthetic polymer, which occurs through protein adsorption. Currently, there is no cost-effective and simple method for characterizing this interfacial binding. To characterize this interfacial binding, we introduce a simple three-step method that involves: 1) synthetic polymer surface characterisation, 2) a quick, inexpensive and robust novel immuno-based assay that uses protein extraction compounds to characterize protein binding strength followed by 3) an in vitro 2D model of cell culture to confirm the results of the immuno-based assay. Fibrinogen, precursor of fibrin, was adsorbed (test protein) on three different polymeric surfaces: silicone, poly(acrylic acid)-coated silicone and poly(allylamine)-coated silicone. Polystyrene surface was used as a reference. Characterisation of the different surfaces revealed different chemistry and roughness. The novel immuno-based assay showed significantly stronger binding of fibrinogen to both poly(acrylic acid) and poly(allylamine) coated silicone. Finally, cell studies showed that the strength of the interaction between the protein and the polymer had an effect on cell growth. This novel immuno-based assay is a valuable tool in developing composite biomaterials of synthetic and protein-based polymers with the potential to be applied in other fields of research where protein adsorption onto surfaces plays an important role.

Optical diamagnetic biosensor for immunocomplexes on beads

NASA Astrophysics Data System (ADS)

Norina, Svetlana B.

2000-12-01

In the present work, diamagnetic separation parameters for the porous beads are studied using optical video recording microscopy. The possible direct amount determination of single or double macromolecular layers immobilized in the meshes of the porous beads is demonstrated for the concentrations' range used in heterogenic immunotest and the affinity chromatography, where the direct rapid detection of ligands within sorbent particles is known to be the actual task. A gradient diamagnetic biosensor is described as suitable for rapid quantitative detection of single or double macromolecular layers in porous nonmagnetic beads. Measurements of capture traveling time or accumulation radius in gradient magnetic field have shown that it is possible to determine 0.20 mg/ml of macromolecular amount within several seconds. The portative devices were made on the base of the fabre optic technique to detect accumulation radius of collected beads in two gradient magnetic positions: diamagnetic and paramagnetic zones of magnetized wire with 55 μm in diameter and to registrate with a lot of fabre wires having 30 μm in diameters. The successive procedures of the present method can be described by: the obtaining of agarose immuno-beads, the incubation of beads with the ligand sample or the injection of sample through affinity mini-column, the submerging of the loaded beads into the glass cell containing Ni-wire or the narrow gap of magnetic poles; the computational obtaining of immuno- parameters; binding constants, accumulation radius. Several biotechnological applications of the biosensor are presented on sorbent beads, human lymphocytes.

Comparison of Premier CAMPY Enzyme Immunoassay (EIA), ProSpecT Campylobacter EIA, and ImmunoCard STAT! CAMPY Tests with Culture for Laboratory Diagnosis of Campylobacter Enteric Infections ▿ †

PubMed Central

Granato, Paul A.; Chen, Li; Holiday, Iris; Rawling, Russell A.; Novak-Weekley, Susan M.; Quinlan, Tammy; Musser, Kimberlee A.

2010-01-01

Campylobacter enteritis is a food-borne or waterborne illness caused almost exclusively by Campylobacter jejuni and, to a lesser extent, by Campylobacter coli. These organisms produce indistinguishable clinical diseases and together represent the second most common cause of bacterial diarrhea in the United States and the leading cause of enteric infection throughout the world. The conventional approach to the laboratory diagnosis of Campylobacter enteritis is based on the recovery of the organism from a stool specimen, which requires the use of a specialized medium incubated at 42°C for several days in an artificially created microaerophilic environment. Recently, several commercially available enzyme immunoassays (EIAs) have been developed for the direct detection of C. jejuni and C. coli in stool specimens. This study compared conventional culture with three EIA methods, the Premier CAMPY EIA (Meridian Bioscience, Cincinnati, OH), the ProSpecT Campylobacter EIA (Remel, Lenexa, KS), and the ImmunoCard STAT! CAMPY test (Meridian Bioscience, Cincinnati, OH), for the detection of C. jejuni and C. coli in 485 patient stool samples. Discordant results were arbitrated by using an in-house, real-time PCR assay that was developed and validated by a public health reference laboratory. Following analyses of the discrepant specimens by PCR, the sensitivity and specificity of both the Premier CAMPY and ProSpecT Campylobacter EIAs were 99.3% and 98%, respectively, while the ImmunoCard STAT! CAMPY test had a sensitivity of 98.5% and a specificity of 98.2%. By use of the PCR test as the reference standard, culture detected 127 of 135 Campylobacter-positive stool specimens, yielding a sensitivity of 94.1%. These results showed that the three EIAs evaluated in this study provide a rapid and reliable alternative for the laboratory diagnosis of enteric infections with C. jejuni and C. coli and that conventional culture may no longer be recognized as the “gold standard” for diagnosis. PMID:20810765

Highly sensitive optical detection of specific protein in breast cancer cells using microstructured fiber in extremely low sample volume

NASA Astrophysics Data System (ADS)

Padmanabhan, Saraswathi; Shinoj, Vengalathunadakal K.; Murukeshan, Vadakke M.; Padmanabhan, Parasuraman

2010-01-01

A simple optical method using hollow-core photonic crystal fiber for protein detection has been described. In this study, estrogen receptor (ER) from a MCF-7 breast carcinoma cell lysates immobilized inside a hollow-core photonic crystal fiber was detected using anti-ER primary antibody with either Alexa™ Fluor 488 (green fluorescent dye) or 555 (red Fluorescent dye) labeled Goat anti-rabbit IgG as the secondary antibody. The fluorescence fingerprints of the ERα protein were observed under fluorescence microscope, and its optical characteristics were analyzed. The ERα protein detection by this proposed method is based on immuno binding from sample volume as low as 50 nL. This method is expected to offer great potential as a biosensor for medical diagnostics and therapeutics applications.

A new paper-based platform technology for point-of-care diagnostics.

PubMed

Gerbers, Roman; Foellscher, Wilke; Chen, Hong; Anagnostopoulos, Constantine; Faghri, Mohammad

2014-10-21

Currently, the Lateral flow Immunoassays (LFIAs) are not able to perform complex multi-step immunodetection tests because of their inability to introduce multiple reagents in a controlled manner to the detection area autonomously. In this research, a point-of-care (POC) paper-based lateral flow immunosensor was developed incorporating a novel microfluidic valve technology. Layers of paper and tape were used to create a three-dimensional structure to form the fluidic network. Unlike the existing LFIAs, multiple directional valves are embedded in the test strip layers to control the order and the timing of mixing for the sample and multiple reagents. In this paper, we report a four-valve device which autonomously directs three different fluids to flow sequentially over the detection area. As proof of concept, a three-step alkaline phosphatase based Enzyme-Linked ImmunoSorbent Assay (ELISA) protocol with Rabbit IgG as the model analyte was conducted to prove the suitability of the device for immunoassays. The detection limit of about 4.8 fm was obtained.

Immunoanalysis Methods for the Detection of Dioxins and Related Chemicals

PubMed Central

Tian, Wenjing; Xie, Heidi Qunhui; Fu, Hualing; Pei, Xinhui; Zhao, Bin

2012-01-01

With the development of biotechnology, approaches based on antibodies, such as enzyme-linked immunosorbent assay (ELISA), active aryl hydrocarbon immunoassay (Ah-I) and other multi-analyte immunoassays, have been utilized as alternatives to the conventional techniques based on gas chromatography and mass spectroscopy for the analysis of dioxin and dioxin-like compounds in environmental and biological samples. These screening methods have been verified as rapid, simple and cost-effective. This paper provides an overview on the development and application of antibody-based approaches, such as ELISA, Ah-I, and multi-analyte immunoassays, covering the sample extraction and cleanup, antigen design, antibody preparation and immunoanalysis. However, in order to meet the requirements for on-site fast detection and relative quantification of dioxins in the environment, further optimization is needed to make these immuno-analytical methods more sensitive and easy to use. PMID:23443395

Predicting the response to CTLA-4 blockade by longitudinal noninvasive monitoring of CD8 T cells

PubMed Central

Whang, Katherine A.; LeGall, Camille; Cragnolini, Juan J.; Bierie, Brian; Gostissa, Monica; Grotenbreg, Gijsbert M.; Bhan, Atul; Weinberg, Robert A.

2017-01-01

Immunotherapy using checkpoint-blocking antibodies against targets such as CTLA-4 and PD-1 can cure melanoma and non–small cell lung cancer in a subset of patients. The presence of CD8 T cells in the tumor correlates with improved survival. We show that immuno–positron emission tomography (immuno-PET) can visualize tumors by detecting infiltrating lymphocytes and, through longitudinal observation of individual animals, distinguish responding tumors from those that do not respond to therapy. We used 89Zr-labeled PEGylated single-domain antibody fragments (VHHs) specific for CD8 to track the presence of intratumoral CD8+ T cells in the immunotherapy-susceptible B16 melanoma model in response to checkpoint blockade. A 89Zr-labeled PEGylated anti-CD8 VHH detected thymus and secondary lymphoid structures as well as intratumoral CD8 T cells. Animals that responded to CTLA-4 therapy showed a homogeneous distribution of the anti-CD8 PET signal throughout the tumor, whereas more heterogeneous infiltration of CD8 T cells correlated with faster tumor growth and worse responses. To support the validity of these observations, we used two different transplantable breast cancer models, yielding results that conformed with predictions based on the antimelanoma response. It may thus be possible to use immuno-PET and monitor antitumor immune responses as a prognostic tool to predict patient responses to checkpoint therapies. PMID:28666979

Decalcification by ascorbic acid for immuno- and affinohistochemical techniques on the inner ear.

PubMed

Merchán-Pérez, A; Gil-Loyzaga, P; Bartolomé, M V; Remezal, M; Fernández, P; Rodríguez, T

1999-08-01

An ascorbic acid decalcifying solution was applied to immuno- and affinohistochemical studies on the inner ear. Rat inner ears fixed in 4% paraformaldehyde in PBS or in 2% acetic acid in ethanol solutions were adequately decalcified in an ascorbic acid solution, at a temperature of 4 degrees C. The decalcifying solution was prepared with 1% ascorbic acid and 0.84% sodium chloride in distilled water (pH 2.5-2.6). The decalcification time was in a direct relationship to the specimen calcification. In this study, two neuroactive substances (gamma-aminobutyric acid and calcitonin gene-related peptide), neurofilaments, and the galectine endogenous lectin were successfully detected immunohistochemically.

A Nanoparticle Based Sp17 Peptide Vaccine Exposes New Immuno-Dominant and Species Cross-reactive B Cell Epitopes

PubMed Central

Xiang, Sue D.; Gao, Qian; Wilson, Kirsty L.; Heyerick, Arne; Plebanski, Magdalena

2015-01-01

Sperm protein antigen 17 (Sp17), expressed in primary as well as in metastatic lesions in >83% of patients with ovarian cancer, is a promising ovarian cancer vaccine candidate. Herein we describe the formulation of nanoparticle based vaccines based on human Sp17 (hSp17) sequence derived peptides, and map the immuno-dominant T cell and antibody epitopes induced using such formulations. The primary T and B cell immuno-dominant region within Sp17 was found to be the same when using biocompatible nanoparticle carriers or the conventional “mix-in” pro-inflammatory adjuvant CpG, both mapping to amino acids (aa) 111–142. However, delivery of hSp17111–142 as a nanoparticle conjugate promoted a number of new properties, changing the dominant antibody isotype induced from IgG2a to IgG1 and the fine specificity of the B cell epitopes within hSp17111–142, from an immuno-dominant region 134–142 aa for CpG, to region 121–138 aa for nanoparticles. Associated with this change in specificity was a substantial increase in antibody cross-reactivity between mouse and human Sp17. These results indicate conjugation of antigen to nanoparticles can have major effects on fine antigen specificity, which surprisingly could be beneficially used to increase the cross-reactivity of antibody responses. PMID:26529027

Profiling the immunotoxicity of chemicals based on in vitro evaluation by a combination of the Multi-ImmunoTox assay and the IL-8 Luc assay.

PubMed

Kimura, Yutaka; Fujimura, Chizu; Ito, Yumiko; Takahashi, Toshiya; Terui, Hitoshi; Aiba, Setsuya

2018-06-01

We established a luciferase reporter assay system, the Multi-ImmunoTox Assay (MITA), which can evaluate the effects of chemicals on the promoter activities of four cytokines: IL-2, IFN-γ, IL-1β, and IL-8. We previously reported that MITA correctly reflected the change in mRNA of human whole-blood cells treated with dexamethasone, cyclosporine, FK506, or several other immunosuppressive drugs. In this study, we combined MITA with the IL-8 Luc assay to detect skin sensitization chemicals (OECD 442E) (modified MITA: mMITA) and established a data set of 60 chemicals examined by mMITA. Using the mMITA results, chemicals can be classified based on the lowest observed effect level (LOEL) of chemicals in suppressing or augmenting the promoter activities of the four cytokines. Moreover, we demonstrated that K-means clustering and hierarchical clustering of the 60 chemicals based on the LOEL for their effects on IL-2 and IL-8 promoter activities and the judgment by the IL-8 Luc assay resulted in the same 6-cluster solution: cluster 1 with preferential suppression of IL-8, cluster 2 with suppression of IL-2 and a positive IL-8 Luc assay result, cluster 3 with suppression of both IL-2 and IL-8, cluster 4 with no effects on IL-2 or IL-8 and a negative IL-8 Luc assay result, cluster 5 with suppression of both IL-2 and IL-8 and a negative IL-8 Luc assay result, and cluster 6 with preferential suppression of IL-8. These data suggest that mMITA is a promising novel high-throughput approach for detecting unrecognized immunological effects of chemicals and for profiling their immunotoxic effects.

Simple Objective Detection of Human Lyme Disease Infection Using Immuno-PCR and a Single Recombinant Hybrid Antigen

PubMed Central

Halpern, Micah D.; Molins, Claudia R.; Schriefer, Martin

2014-01-01

A serology-based tiered approach has, to date, provided the most effective means of laboratory confirmation of clinically suspected cases of Lyme disease, but it lacks sensitivity in the early stages of disease and is often dependent on subjectively scored immunoblots. We recently demonstrated the use of immuno-PCR (iPCR) for detecting Borrelia burgdorferi antibodies in patient serum samples that were positive for Lyme disease. To better understand the performance of the Lyme disease iPCR assay, the repeatability and variability of the background of the assay across samples from a healthy population (n = 36) were analyzed. Both of these parameters were found to have coefficients of variation of <3%. Using eight antigen-specific iPCR assays and positive call thresholds established for each assay, iPCR IgM and/or IgG diagnosis from Lyme disease patient serum samples (n = 12) demonstrated a strong correlation with that of 2-tier testing. Furthermore, a simplified iPCR approach using a single hybrid antigen and detecting only IgG antibodies confirmed the 2-tier diagnosis in the Lyme disease patient serum samples (n = 12). Validation of the hybrid antigen IgG iPCR assay using a blinded panel of Lyme disease and non-Lyme disease patient serum samples (n = 92) resulted in a sensitivity of 69% (95% confidence interval [CI], 50% to 84%), compared to that of the 2-tier analysis at 59% (95% CI, 41% to 76%), and a specificity of 98% (95% CI, 91% to 100%) compared to that of the 2-tier analysis at 97% (95% CI, 88% to 100%). A single-tier hybrid antigen iPCR assay has the potential to be an improved method for detecting host-generated antibodies against B. burgdorferi. PMID:24899074

Comparison of two commercial rapid in-clinic serological tests for detection of antibodies against Leishmania spp. in dogs.

PubMed

Athanasiou, Labrini V; Petanides, Theodoros A; Chatzis, Manolis K; Kasabalis, Dimitrios; Apostolidis, Kosmas N; Saridomichelakis, Manolis N

2014-03-01

Antibodies against Leishmania spp. are detected in most dogs with clinical signs of leishmaniasis due to Leishmania infantum. Accurate, rapid in-clinic serological tests may permit immediate confirmation of the diagnosis and implementation of therapeutic measures. The aim of the current study was to evaluate the diagnostic accuracy of 2 commercial, rapid in-clinic serological tests for the detection of anti-Leishmania antibodies in sera of dogs, the Snap Canine Leishmania Antibody Test kit (IDEXX Laboratories Inc., Westbrook, Maine) and the ImmunoRun Antibody Detection kit (Biogal Galed Labs, Kibbutz Galed, Israel), using indirect fluorescent antibody test (IFAT) as the reference method. A total of 109 sera collected from 65 seropositive and 44 seronegative dogs were used. The sensitivities of the Snap and ImmunoRun kits were 89.23% (95% confidence interval: 79.05-95.54%) and 86.15% (95% confidence interval: 75.33-93.45%), respectively, and the specificity of both tests was 100%. A good agreement between each of the rapid in-clinic serological tests and IFAT and between the 2 rapid in-clinic serological tests was witnessed. Both rapid in-clinic serological tests showed an adequate diagnostic accuracy and can be used for the fast detection of antibodies against L. infantum in dogs.

Electrochemical Affinity Biosensors Based on Disposable Screen-Printed Electrodes for Detection of Food Allergens

PubMed Central

Vasilescu, Alina; Nunes, Gilvanda; Hayat, Akhtar; Latif, Usman; Marty, Jean-Louis

2016-01-01

Food allergens are proteins from nuts and tree nuts, fish, shellfish, wheat, soy, eggs or milk which trigger severe adverse reactions in the human body, involving IgE-type antibodies. Sensitive detection of allergens in a large variety of food matrices has become increasingly important considering the emergence of functional foods and new food manufacturing technologies. For example, proteins such as casein from milk or lysozyme and ovalbumin from eggs are sometimes used as fining agents in the wine industry. Nonetheless, allergen detection in processed foods is a challenging endeavor, as allergen proteins are degraded during food processing steps involving heating or fermentation. Detection of food allergens was primarily achieved via Enzyme-Linked Immuno Assay (ELISA) or by chromatographic methods. With the advent of biosensors, electrochemical affinity-based biosensors such as those incorporating antibodies and aptamers as biorecognition elements were also reported in the literature. In this review paper, we highlight the success achieved in the design of electrochemical affinity biosensors based on disposable screen-printed electrodes towards detection of protein allergens. We will discuss the analytical figures of merit for various disposable screen-printed affinity sensors in relation to methodologies employed for immobilization of bioreceptors on transducer surface. PMID:27827963

Electrochemical Affinity Biosensors Based on Disposable Screen-Printed Electrodes for Detection of Food Allergens.

PubMed

Vasilescu, Alina; Nunes, Gilvanda; Hayat, Akhtar; Latif, Usman; Marty, Jean-Louis

2016-11-05

Food allergens are proteins from nuts and tree nuts, fish, shellfish, wheat, soy, eggs or milk which trigger severe adverse reactions in the human body, involving IgE-type antibodies. Sensitive detection of allergens in a large variety of food matrices has become increasingly important considering the emergence of functional foods and new food manufacturing technologies. For example, proteins such as casein from milk or lysozyme and ovalbumin from eggs are sometimes used as fining agents in the wine industry. Nonetheless, allergen detection in processed foods is a challenging endeavor, as allergen proteins are degraded during food processing steps involving heating or fermentation. Detection of food allergens was primarily achieved via Enzyme-Linked Immuno Assay (ELISA) or by chromatographic methods. With the advent of biosensors, electrochemical affinity-based biosensors such as those incorporating antibodies and aptamers as biorecognition elements were also reported in the literature. In this review paper, we highlight the success achieved in the design of electrochemical affinity biosensors based on disposable screen-printed electrodes towards detection of protein allergens. We will discuss the analytical figures of merit for various disposable screen-printed affinity sensors in relation to methodologies employed for immobilization of bioreceptors on transducer surface.

ImmunoRatio: a publicly available web application for quantitative image analysis of estrogen receptor (ER), progesterone receptor (PR), and Ki-67

PubMed Central

2010-01-01

Introduction Accurate assessment of estrogen receptor (ER), progesterone receptor (PR), and Ki-67 is essential in the histopathologic diagnostics of breast cancer. Commercially available image analysis systems are usually bundled with dedicated analysis hardware and, to our knowledge, no easily installable, free software for immunostained slide scoring has been described. In this study, we describe a free, Internet-based web application for quantitative image analysis of ER, PR, and Ki-67 immunohistochemistry in breast cancer tissue sections. Methods The application, named ImmunoRatio, calculates the percentage of positively stained nuclear area (labeling index) by using a color deconvolution algorithm for separating the staining components (diaminobenzidine and hematoxylin) and adaptive thresholding for nuclear area segmentation. ImmunoRatio was calibrated using cell counts defined visually as the gold standard (training set, n = 50). Validation was done using a separate set of 50 ER, PR, and Ki-67 stained slides (test set, n = 50). In addition, Ki-67 labeling indexes determined by ImmunoRatio were studied for their prognostic value in a retrospective cohort of 123 breast cancer patients. Results The labeling indexes by calibrated ImmunoRatio analyses correlated well with those defined visually in the test set (correlation coefficient r = 0.98). Using the median Ki-67 labeling index (20%) as a cutoff, a hazard ratio of 2.2 was obtained in the survival analysis (n = 123, P = 0.01). ImmunoRatio was shown to adapt to various staining protocols, microscope setups, digital camera models, and image acquisition settings. The application can be used directly with web browsers running on modern operating systems (e.g., Microsoft Windows, Linux distributions, and Mac OS). No software downloads or installations are required. ImmunoRatio is open source software, and the web application is publicly accessible on our website. Conclusions We anticipate that free web applications, such as ImmunoRatio, will make the quantitative image analysis of ER, PR, and Ki-67 easy and straightforward in the diagnostic assessment of breast cancer specimens. PMID:20663194

ImmunoRatio: a publicly available web application for quantitative image analysis of estrogen receptor (ER), progesterone receptor (PR), and Ki-67.

PubMed

Tuominen, Vilppu J; Ruotoistenmäki, Sanna; Viitanen, Arttu; Jumppanen, Mervi; Isola, Jorma

2010-01-01

Accurate assessment of estrogen receptor (ER), progesterone receptor (PR), and Ki-67 is essential in the histopathologic diagnostics of breast cancer. Commercially available image analysis systems are usually bundled with dedicated analysis hardware and, to our knowledge, no easily installable, free software for immunostained slide scoring has been described. In this study, we describe a free, Internet-based web application for quantitative image analysis of ER, PR, and Ki-67 immunohistochemistry in breast cancer tissue sections. The application, named ImmunoRatio, calculates the percentage of positively stained nuclear area (labeling index) by using a color deconvolution algorithm for separating the staining components (diaminobenzidine and hematoxylin) and adaptive thresholding for nuclear area segmentation. ImmunoRatio was calibrated using cell counts defined visually as the gold standard (training set, n = 50). Validation was done using a separate set of 50 ER, PR, and Ki-67 stained slides (test set, n = 50). In addition, Ki-67 labeling indexes determined by ImmunoRatio were studied for their prognostic value in a retrospective cohort of 123 breast cancer patients. The labeling indexes by calibrated ImmunoRatio analyses correlated well with those defined visually in the test set (correlation coefficient r = 0.98). Using the median Ki-67 labeling index (20%) as a cutoff, a hazard ratio of 2.2 was obtained in the survival analysis (n = 123, P = 0.01). ImmunoRatio was shown to adapt to various staining protocols, microscope setups, digital camera models, and image acquisition settings. The application can be used directly with web browsers running on modern operating systems (e.g., Microsoft Windows, Linux distributions, and Mac OS). No software downloads or installations are required. ImmunoRatio is open source software, and the web application is publicly accessible on our website. We anticipate that free web applications, such as ImmunoRatio, will make the quantitative image analysis of ER, PR, and Ki-67 easy and straightforward in the diagnostic assessment of breast cancer specimens.

A two-step enzymatic modification method to reduce immuno-reactivity of milk proteins.

PubMed

Damodaran, Srinivasan; Li, Yan

2017-12-15

A two-step enzymatic approach to reduce immuno-reactivity of whey protein isolate and casein has been studied. The method involves partial hydrolysis of proteins with proteases, followed by repolymerization with microbial transglutaminase. Whey protein isolate partially hydrolyzed with chymotrypsin, trypsin, or thermolysin retained about 80%, 30%, and 20% of the original immuno-reactivity, respectively. Upon repolymerization the immuno-reactivity decreased to 45%, 35%, and 5%, respectively. The immuno-reactivity of hydrolyzed and repolymerized casein was negligible compared to native casein. The repolymerized products were partially resistant to in vitro digestion. Peptides released during digestion of repolymerized thermolysin-whey protein hydrolysate had less than 5% immuno-reactivity, whereas those of whey protein control exhibited a sinusoidal immuno-reactivity ranging from 5 to 20%. Peptides released during digestion of repolymerized thermolysin-casein hydrolysates had no immuno-reactivity. These results indicated that it is possible to produce hypoallergenic milk protein products using the two-step enzymatic modification method involving thermolysin and transglutaminase. Copyright © 2017. Published by Elsevier Ltd.

Magnetic Electrochemical Sensing Platform for Biomonitoring of Exposure to Organophosphorus Pesticides and Nerve Agents Based on Simultaneous Measurement of Total Enzyme Amount and Enzyme Activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, Dan; Wang, Jun; Wang, Limin

We report a new approach for electrochemical quantification of enzymatic inhibition and phosphorylation for biomonitoring of exposure to organophosphorus (OP) pesticides and nerve agents based on a magnetic beads (MBs) immunosensing platform. The principle of this approach is based on the combination of MBs immuno-capture based enzyme activity assay and competitive immunoassay of total amount of enzyme for simultaneous detection of enzyme inhibition and phosphorylation in biological fluids. Butyrylcholinesterase (BChE) was chosen as a model enzyme. In competitive immunoassay, the target total BChE in a sample (mixture of OP-inhibited BChE and active BChE) competes with the BChE modified on themore » MBs to bind to the limited anti-BChE antibody labeled with quantum dots (QDs-anti-BChE), and followed by electrochemical stripping analysis of the bound QDs conjugate on the MBs. This assay shows a linear response over the total BChE concentration range of 0.1~20 nM. Simultaneously, real time BChE activity was measured on an electrochemical carbon nanotube-based sensor coupled with microflow injection system after immuno-capture by MBs-anti-BChE conjugate. Therefore, the formed phosphorylated adduct (OP-BChE) can be estimated by the difference values of the total amount BChE (including active and OP-inhibited) and active BChE from established calibration curves. This approach not only eliminates the difficulty in screening of low-dose OP exposure (less than 20% inhibition of BChE) because of individual variation of BChE values, but also avoids the drawback of the scarce availability of OP-BChE antibody. It is sensitive enough to detect 0.5 nM OP-BChE, which is less than 2% BChE inhibition. This method offers a new method for rapid, accurate, selective and inexpensive quantification of phosphorylated adducts and enzyme inhibition for biomonitoring of OP and nerve agent exposures.« less

IMMUNO-DETECTION OF SIGNALING INTERMEDIATES IN AN INTACT LUNG PREPARATION FOLLOWING METALLIC EXPOSURE

EPA Science Inventory

Residual oil fly ash (ROFA) is a particulate pollutant produced during the combustion of fuel oil. ROFA exposure causes adverse respiratory effects in humans and induces lung inflammation in animals and inflammatory mediator expression in cultured human airway epithelial cells....

Generation and purification of highly-specific antibodies for detecting post-translationally modified proteins in vivo

PubMed Central

Arur, Swathi; Schedl, Tim

2014-01-01

Post-translational modifications alter protein structure, affecting activity, stability, localization and/or binding partners. Antibodies that specifically recognize post-translationally modified proteins have a number of uses including immuno-cytochemistry and immuno-precipitation of the modified protein to purify protein-protein and protein-nucleic acid complexes. However, antibodies directed at modified sites on individual proteins are often non-specific. Here we describe a protocol to purify polyclonal antibodies that specifically detect the modified protein of interest. The approach uses iterative rounds of subtraction and affinity purification, using stringent washes to remove antibodies that recognize the unmodified protein and low sequence complexity epitopes containing the modified amino acid. Dot and western blots assays are employed to assess antibody preparation specificity. The approach is designed to overcome the common occurrence that a single round of subtraction and affinity purification is not sufficient to obtain a modified protein specific antibody preparation. One full round of antibody purification and specificity testing takes 6 days of discontinuous time. PMID:24457330

Rapid screening test for detection of oxytetracycline residues in milk using lateral flow assay.

PubMed

Naik, Laxmana; Sharma, Rajan; Mann, Bimlesh; Lata, Kiran; Rajput, Y S; Surendra Nath, B

2017-03-15

A rapid, semi-quantitative lateral flow assay (LFA) was developed to screen the oxytetracycline (OTC) antibiotics residues in milk samples. In this study a competitive immuno-assay format was established. Colloidal gold nano-particles (GNP) were prepared and used as labelling material in LFA. Polyclonal antibodies were generated against OTC molecule (anti-OTC), purified and the quality was assessed by enzyme linked immuno sorbet assay. For the first time membrane components required for LFA in milk system was optimized. GNP and anti-OTC stable conjugate preparation method was standardized, and then these components were placed over the conjugate pad. OTC coupled with carrier protein was placed on test line; species specific secondary antibodies were placed on the control line of the membrane matrix. Assay was validated by spiking OTC to antibiotic free milk samples and results could be accomplished within 5min. without need of any equipment. The visual detection limit was 30ppb. Copyright © 2016 Elsevier Ltd. All rights reserved.

Application of immuno-PCR assay for the detection of serum IgE specific to Bermuda allergen.

PubMed

Rahmatpour, Samine; Khan, Amjad Hayat; Nasiri Kalmarzi, Rasoul; Rajabibazl, Masoumeh; Tavoosidana, Gholamreza; Motevaseli, Elahe; Zarghami, Nosratollah; Sadroddiny, Esmaeil

2017-04-01

In vivo and in vitro tests are the two major ways of identifying the triggering allergens in sensitized individuals with allergic symptoms. Both methods are equally significant in terms of sensitivity and specificity. However, in certain circumstances, in vitro methods are highly preferred because they circumvent the use of sensitizing drugs in patients. In current study, we described a highly sensitive immuno-PCR (iPCR) assay for serum IgE specific to Bermuda allergens. Using oligonucleotide-labelled antibody, we used iPCR for the sensitive detection of serum IgE. The nucleotide sequence was amplified using conventional PCR and the bands were visualized on 2.5% agarose gel. Results demonstrated a 100-fold enhancement in sensitivity of iPCR over commercially available enzyme-linked immunosorbent assay (ELISA) kit. Our iPCR method was highly sensitive for Bermuda-specific serum IgE and could be beneficial in allergy clinics. Copyright © 2016 Elsevier Ltd. All rights reserved.

21 CFR 864.9285 - Automated cell-washing centrifuge for immuno-hematology.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Automated cell-washing centrifuge for immuno... Establishments That Manufacture Blood and Blood Products § 864.9285 Automated cell-washing centrifuge for immuno-hematology. (a) Identification. An automated cell-washing centrifuge for immuno-hematology is a device used...

21 CFR 864.9285 - Automated cell-washing centrifuge for immuno-hematology.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Automated cell-washing centrifuge for immuno... Establishments That Manufacture Blood and Blood Products § 864.9285 Automated cell-washing centrifuge for immuno-hematology. (a) Identification. An automated cell-washing centrifuge for immuno-hematology is a device used...

21 CFR 864.9285 - Automated cell-washing centrifuge for immuno-hematology.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Automated cell-washing centrifuge for immuno... Establishments That Manufacture Blood and Blood Products § 864.9285 Automated cell-washing centrifuge for immuno-hematology. (a) Identification. An automated cell-washing centrifuge for immuno-hematology is a device used...

21 CFR 864.9285 - Automated cell-washing centrifuge for immuno-hematology.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Automated cell-washing centrifuge for immuno... Establishments That Manufacture Blood and Blood Products § 864.9285 Automated cell-washing centrifuge for immuno-hematology. (a) Identification. An automated cell-washing centrifuge for immuno-hematology is a device used...

21 CFR 864.9285 - Automated cell-washing centrifuge for immuno-hematology.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Automated cell-washing centrifuge for immuno... Establishments That Manufacture Blood and Blood Products § 864.9285 Automated cell-washing centrifuge for immuno-hematology. (a) Identification. An automated cell-washing centrifuge for immuno-hematology is a device used...

DETECTION OF GIARDIA IN ENVIRONMENTAL WATERS BY IMMUNO-PCR AMPLIFICATION METHODS

EPA Science Inventory

Genomic DNA was extracted either directly from Giardia muris cysts seeded into environmental surface waters or from cysts isolated by immunomagnetic beads (IMB).A 0.171-kbp segment of the giardin gene was PCR-amplified following "direct extraction" of Giardia DNA from seeded Caha...

DETECTION OF GIARDIA IN ENVIRONMENTAL WATERS BY IMMUNO-PCR AMPLIFICATION METHODS

EPA Science Inventory

Genomic DNA was extracted either directly from Giardia muris cysts seeded into environmental surface waters or from cysts isolated by immunomagnetic beads (IMB}. A 0.171-kbp segment of the giardin gene was PCR-amplified following "direct extraction" of Giardia DNA from seeded Cah...

Identification of liver protein targets modified by tienilic acid metabolites using a two-dimensional Western blot-mass spectrometry approach

NASA Astrophysics Data System (ADS)

Methogo, Ruth Menque; Dansette, Patrick M.; Klarskov, Klaus

2007-12-01

A combined approach based on two-dimensional electrophoresis-immuno-blotting and nanoliquid chromatography coupled on-line with electrospray ionization mass spectrometry (nLC-MS/MS) was used to identify proteins modified by a reactive intermediate of tienilic acid (TA). Liver homogenates from rats exposed to TA were fractionated using ultra centrifugation; four fractions were obtained and subjected to 2D electrophoresis. Following transfer to PVDF membranes, modified proteins were visualized after India ink staining, using an anti-serum raised against TA and ECL detection. Immuno-reactive spots were localized on the PVDF membrane by superposition of the ECL image, protein spots of interest were excised, digested on the membrane with trypsin followed by nLC-MS/MS analysis and protein identification. A total of 15 proteins were identified as likely targets modified by a TA reactive metabolite. These include selenium binding protein 2, senescence marker protein SMP-30, adenosine kinase, Acy1 protein, adenosylhomocysteinase, capping protein (actin filament), protein disulfide isomerase, fumarylacetoacetase, arginase chain A, ketohexokinase, proteasome endopeptidase complex, triosephosphate isomerase, superoxide dismutase, dna-type molecular chaperone hsc73 and malate dehydrogenase.

Anaphylactic reaction to probiotics. Cow's milk and hen's egg allergens in probiotic compounds.

PubMed

Martín-Muñoz, María Flora; Fortuni, Monserrat; Caminoa, Magdalena; Belver, Teresa; Quirce, Santiago; Caballero, Teresa

2012-12-01

Probiotics are used in the treatment of allergic diseases. We investigated the safety of probiotics for subjects with food allergy. Labels of probiotics commercially available in Spain were examined to assess their content of cow's milk or hen's egg. Skin prick tests with these compounds (20 mg/ml) were performed in five children allergic to cow's milk, five children allergic to hen's white egg, and five control subjects non-allergic to food. Three serum pools: I (positive-specific IgE to cow's milk and hen's egg white proteins), II (positive-specific IgE to cow's milk and negative to hen's egg white proteins), and III (negative-specific IgE to cow's milk and positive to hen's egg white proteins) were used to detect cow's milk and hen's egg white allergens in probiotics. ImmunoCAP(®) (Phadia), in-house ELISA, SDS-PAGE immunoblotting, and inhibition studies of these assays were performed. Proteins were quantified by enzyme-immunoassay. Eleven probiotics were studied. No label advertised about egg content, eight labels warned about lactose, lactic acid or cow's milk, one label claimed to be milk-free, and two gave no information. Cow's milk proteins were detected, by at least one lab technique, in 10/11 probiotics, three over 2.5 mg/kg (21, 52, 112 mg/kg). Hen's egg white proteins were detected in 3/11 probiotics, only one had more than 2.5 mg/kg (47 mg/kg). Probiotic compounds may contain hidden allergens of food and may not be safe for subjects with allergy to cow's milk or hen's egg. © 2012 John Wiley & Sons A/S.

What could be the role of molecular-based allergy diagnostics in detecting the risk of developing allergic sensitization to furry animals?

PubMed

Liccardi, Gennaro; Bilò, M B; Manzi, F; Piccolo, A; Di Maro, E; Salzillo, A

2015-09-01

Although this highly refined diagnostic approach has been used in several fields of allergy diagnosis, we noticed the scarcity of data on the role of CDR in detecting current sensitization to the allergens of common pets (cat / dog) and, especially, its potential usefulness in predicting the risk of sensitization to other furry animals. Reported data suggest that cross-reacting mechanisms might play an important role in a significant proportion of allergic sensitizations to furry animals (common pets and unusual / exotic mammals) especially in the absence of any possible direct / indirect contact. In this context an evaluation of specific IgE by using the micro-array technique ImmunoCAP ISAC (Thermofisher Scientific - Immuno-Diagnostics, Sweden) for lipocalins (Can f 1, Can f 2, Equ c 1, Fel d 4, Mus m 1) and albumins (Bos d 6, Can f 3, Equ c 3, Fel d 2) might be very useful to evaluate the possibility of cross-reactions between the allergens of different animals. In fact, allergic sensitization without animal exposure is a relevant risk for patients, because they are not aware about the possibility that even severe respiratory symptoms may develop after an occasional animal contact. This aspect should be taken into account by susceptible individuals before acquiring new pets, after removal of common pets or beginning a contact for working / leisure activity with a common as well as uncommon animal.

Ferrocenemonocarboxylic-HRP@Pt nanoparticles labeled RCA for multiple amplification of electro-immunosensing.

PubMed

Su, Huilan; Yuan, Ruo; Chai, Yaqin; Mao, Li; Zhuo, Ying

2011-07-15

A multiple amplification immunoassay was proposed to detect alpha-fetoprotein (AFP), which was based on ferrocenemonocarboxylic-HRP conjugated on Pt nanoparticles as labels for rolling circle amplification (RCA). Firstly, the capture antibody (anti-AFP) was immobilized on glass carbon electrode (GCE) deposited nano-sized gold particles. After a typical immuno-sandwich protocol, primary DNA was immobilized by labeling secondary antibody, which acted as a precursor to initiate RCA. The products of RCA provide large amount of sites to link detection DNAs, which were labeled by signal probes (ferrocenemonocarboxylic) and horseradish peroxidase (HRP). Moreover, the enzymatic amplification signals could be produced by the catalysis of HRP and Pt nanoparticles with the addition of H₂O₂. These lead to multiple amplification signals monitoring by electrochemical instrument and further resulted in high sensitivity of the immunoassay with the detection limit of 1.7 pg/mL. Copyright © 2011 Elsevier B.V. All rights reserved.

ImmunoCAP assays: Pros and cons in allergology.

PubMed

van Hage, Marianne; Hamsten, Carl; Valenta, Rudolf

2017-10-01

Allergen-specific IgE measurements and the clinical history are the cornerstones of allergy diagnosis. During the past decades, both characterization and standardization of allergen extracts and assay technology have improved. Here we discuss the uses, advantages, misinterpretations, and limitations of ImmunoCAP IgE assays (Thermo Fisher Scientific/Phadia, Uppsala, Sweden) in the field of allergology. They can be performed as singleplex (ImmunoCAP) and, for the last decade, as multiplex (Immuno Solid-phase Allergen Chip [ISAC]). The major benefit of ImmunoCAP is the obtained quantified allergen-specific IgE antibody level and the lack of interference from allergen-specific IgG antibodies. However, ImmunoCAP allergen extracts are limited to the composition of the extract. The introduction of allergen molecules has had a major effect on analytic specificity and allergy diagnosis. They are used in both singleplex ImmunoCAP and multiplex ImmunoCAP ISAC assays. The major advantage of ISAC is the comprehensive IgE pattern obtained with a minute amount of serum. The shortcomings are its semiquantitative measurements, lower linear range, and cost per assay. With respect to assay performance, ImmunoCAP allergen extracts are good screening tools, but allergen molecules dissect the IgE response on a molecular level and put allergy research on the map of precision medicine. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Evaluation of instant cup noodle, irradiated for immuno-compromised patients

NASA Astrophysics Data System (ADS)

Lee, Ji-Hye; Kim, Jae-Kyung; Park, Jae-Nam; Yoon, Young-Min; Sung, Nak-Yun; Kim, Jae-Hun; Song, Beom-Seok; Yook, Hong-Sun; Kim, Byeong-Keun; Lee, Ju-Woon

2012-08-01

In the present study, initial microbial load of instant cup noodle (ICN) was investigated and gamma irradiation applied to develop immuno-compromised patients food for their safe consumption. The initial microbial population of dried vegetable and meat, and noodle was below the detection limit (1 log CFU/g); however, that of seasoning powder was just above 4 log CFU/g. Moreover, rehydrated-ICN with water at 100 °C still show above 3 log CFU/g of microbial load, which indicates the need for an additional process to control microbial safety of the seasoning powder. The total aerobic bacteria in seasoning powder and rehydrated-ICN could be controlled with 17 kGy gamma irradiation. This result referred 17 kGy gamma irradiation could reach 'practical sterility' of ICN. The overall difference in sensory properties between the non-irradiated and irradiated ICN was insignificant. Thus, gamma irradiation could improve the microbial quality of ICN, and reduce the risk of infection posed by the seasoning powder, without any adverse effects on their sensory quality. These results suggest that gamma-irradiated ICN can be used as a snack food for immuno-compromised patients.

Integrating Allergen Analysis Within a Risk Assessment Framework: Approaches to Development of Targeted Mass Spectrometry Methods for Allergen Detection and Quantification in the iFAAM Project.

PubMed

Nitride, Chiara; Lee, Victoria; Baricevic-Jones, Ivona; Adel-Patient, Karine; Baumgartner, Sabine; Mills, E N Clare

2018-01-01

Allergen analysis is central to implementing and monitoring food allergen risk assessment and management processes by the food industry, but current methods for the determination of allergens in foods give highly variable results. The European Union-funded "Integrated Approaches to Food Allergen and Allergy Risk Management" (iFAAM) project has been working to address gaps in knowledge regarding food allergen management and analysis, including the development of novel MS and immuno-based allergen determination methods. Common allergenic food ingredients (peanut, hazelnut, walnut, cow's milk [Bos domesticus], and hen's egg [Gallus domesticus]) and common food matrixes (chocolate dessert and cookie) have been used for both clinical studies and analytical method development to ensure that the new methods are clinically relevant. Allergen molecules have been used as analytical targets and allergenic ingredients incurred into matrixes at levels close to reference doses that may trigger the use of precautionary allergen labeling. An interlaboratory method comparison has been undertaken for the determination of peanut in chocolate dessert using MS and immuno-based methods. The iFAAM approach has highlighted the need for methods to report test results in allergenic protein. This will allow food business operators to use them in risk assessments that are founded on clinical study data in which protein has been used as a measure of allergenic potency.

Free digital image analysis software helps to resolve equivocal scores in HER2 immunohistochemistry.

PubMed

Helin, Henrik O; Tuominen, Vilppu J; Ylinen, Onni; Helin, Heikki J; Isola, Jorma

2016-02-01

Evaluation of human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) is subject to interobserver variation and lack of reproducibility. Digital image analysis (DIA) has been shown to improve the consistency and accuracy of the evaluation and its use is encouraged in current testing guidelines. We studied whether digital image analysis using a free software application (ImmunoMembrane) can assist in interpreting HER2 IHC in equivocal 2+ cases. We also compared digital photomicrographs with whole-slide images (WSI) as material for ImmunoMembrane DIA. We stained 750 surgical resection specimens of invasive breast cancers immunohistochemically for HER2 and analysed staining with ImmunoMembrane. The ImmunoMembrane DIA scores were compared with the originally responsible pathologists' visual scores, a researcher's visual scores and in situ hybridisation (ISH) results. The originally responsible pathologists reported 9.1 % positive 3+ IHC scores, for the researcher this was 8.4 % and for ImmunoMembrane 9.5 %. Equivocal 2+ scores were 34 % for the pathologists, 43.7 % for the researcher and 10.1 % for ImmunoMembrane. Negative 0/1+ scores were 57.6 % for the pathologists, 46.8 % for the researcher and 80.8 % for ImmunoMembrane. There were six false positive cases, which were classified as 3+ by ImmunoMembrane and negative by ISH. Six cases were false negative defined as 0/1+ by IHC and positive by ISH. ImmunoMembrane DIA using digital photomicrographs and WSI showed almost perfect agreement. In conclusion, digital image analysis by ImmunoMembrane can help to resolve a majority of equivocal 2+ cases in HER2 IHC, which reduces the need for ISH testing.

Nanometal particle reagents for sensitive, MEMS based fiber-optic, multi-analyte, immuno-biosensing

NASA Astrophysics Data System (ADS)

Hong, Bin

Integration of nanotechnology to medical diagnostics has brought a new era to public health practice. An excellent example is the utilization of unique optoelectronic properties of nanoparticles to develop highly sensitive biosensing devices for point-of-care (POC) disease diagnosis/prognosis. Fluorophore mediated, immuno-biosensors are important disease detection tools. The property of intra-molecular fluorescence quenching of most fluorophores, however, limits the sensitivity of this type of sensors. A plasmon-rich nanometal particle (NMP) can transfer the lone pair electrons of a fluorophore, which normally participate in the fluorescence self-quenching, to its surface plasmon field, resulting in artificial fluorescence enhancement. The enhancement was found to depend on the metal type, the particle size, the distance between a particle and a fluorophore, and the quantum yield of a fluorophore. Some biocompatible solvents were also found to increase the fluorescence emission efficiency via effective dipole coupling between the fluorophore and the solvent molecule. The application of solvents in inmuno-sensing could additionally improve the fluorescence light retrieval by the conformational change of the protein complexes in solvent. The mixture of the NMP and the solvent, which we defined as nanometal particle reagent (NMPR), provided even higher enhancements. Cardiovascular diseases (CVDs) kill 1 person in every 6 seconds. Among the CVDs, acute myocardial infarction (AMI; commonly known as heart attack) is the most dangerous and time-sensitive killer. A rapid and accurate AMI diagnosis is crucial for saving many lives. For this purpose, a fluorophore mediated, immuno-reaction based, multi-cardiac-marker sensing device was developed, to quantify four myocardium-specific proteins simultaneously, accurately, rapidly, and user-friendly. The four cardiac markers of our choice were myoglobin (MG), C-reactive protein (CRP), cardiac troponin I (cTnI), and B-type natriuretic peptide (BNP). Two of these cardiac markers, cTnI and BNP, were rather difficult to sense due to their low concentrations (tens of picomolar) in blood plasma immediately after the AMI. The NMPRs that we have developed enhanced the sensor signals as high as 8 times, accomplishing a sensitive and accurate quantification of all four markers in 7 minutes with an average signal-to-noise ratio of 35. As a promising POC sensing device, system portability, sensing reliability, and user-friendly operation are important. Micro-electro-mechanical system (MEMS) technology was, therefore, integrated to the immuno-sensing device with microfluidic control and microfabrication. A LabVIEW(TM) computer code was also written for a "one-click" automated immunoassay and a real-time data analysis. Our four-cardiac-marker immuno-sensing device incorporated with nanotechnology and MEMS technology is capable of simultaneous quantification at a near-real time, which can save many lives. This device can be also used for measuring any disease-representing biomarkers (e.g., cancer markers) for rapid and accurate disease diagnosis/prognosis.

Multicenter evaluation of the Bayer Immuno I CA 15-3 assay.

PubMed

Cheli, C D; Morris, D L; Kish, L; Goldblatt, J; Neaman, I; Allard, W J; Yeung, K K; Wu, A H; Moore, R; Chan, D W; Fritsche, H A; Schwartz, M K; Very, D L

1998-04-01

We conducted a multicenter evaluation of the analytical and clinical features of the automated Bayer Immuno 1 CA 15-3 assay and compared assay performance to two manual tests. Results of the 10-day imprecision study of the Bayer Immuno 1 assay pooled across four evaluation sites and three lots of reagent produced total CV < or = 4%. Lot-to-lot reproducibility for 26 different lots of reagents and calibrators manufactured over a 2-year period was demonstrated (CV, 1.1%). Results for the Bayer Immuno 1 assay correlated well with the Biomira TRUQUANT BR 27.29 and Centocor CA 15-3 RIAs (r > or = 0.94). The upper limit of the reference interval for the Bayer Immuno 1 assay was 35.9 kilounits/L (35.9 units/mL); values were similar for all methods. Longitudinal monitoring of healthy women yielded assay values with an average CV of 11% and 21% for the Bayer Immuno 1 and Biomira assays, respectively. The Bayer Immuno 1 assay demonstrated the analytical features, intermethod correlation, and long-term performance characteristics that are essential for longitudinal monitoring of breast cancer patients.

The Escherichia coli O157:H7 cattle immuno-proteome includes outer membrane protein A (OmpA), a modulator of adherence to bovine recto-anal junction squamous epithelial (RSE) cells

PubMed Central

Kudva, Indira T.; Krastins, Bryan; Torres, Alfredo G.; Griffin, Robert W.; Sheng, Haiqing; Sarracino, David A.; Hovde, Carolyn J.; Calderwood, Stephen B.; John, Manohar

2015-01-01

SUMMARY Building on previous studies, we defined the repertoire of proteins comprising the immuno-proteome of E. coli O157:H7 (O157) cultured in DMEM supplemented with norepinephrine (NE; O157 immuno-proteome), a β-adrenergic hormone that regulates E. coli O157 gene expression in the gastrointestinal tract, using a variation of a novel proteomics-based platform proteome mining tool for antigen discovery, called Proteomics-based Expression Library Screening (PELS; Kudva et al., 2006). The E. coli O157 immuno-proteome (O157-IP) comprised 91 proteins, and included those identified previously using PELS, and also proteins comprising DMEM- and bovine rumen fluid- proteomes. Outer membrane protein A (OmpA), a common component of the above proteomes, and reportedly a contributor to E. coli O157 adherence to cultured Hep-2 epithelial cells, was interestingly found to be a modulator rather than a contributor to E. coli O157 adherence to bovine recto-anal junction squamous epithelial (RSE) cells. Our results point to a role for yet to be identified members of the O157-IP in E. coli O157 adherence to RSE-cells, and additionally implicate a possible role for the OmpA regulator, TdcA, in the expression of such adhesins. Our observations have implications for development of efficacious vaccines for preventing E. coli O157 colonization of the bovine gastrointestinal tract. PMID:25643951

An enzyme-linked immuno-mass spectrometric assay with the substrate adenosine monophosphate.

PubMed

Florentinus-Mefailoski, Angelique; Soosaipillai, Antonius; Dufresne, Jaimie; Diamandis, Eleftherios P; Marshall, John G

2015-02-01

An enzyme-linked immuno-mass spectrometric assay (ELIMSA) with the specific detection probe streptavidin conjugated to alkaline phosphatase catalyzed the production of adenosine from the substrate adenosine monophosphate (AMP) for sensitive quantification of prostate-specific antigen (PSA) by mass spectrometry. Adenosine ionized efficiently and was measured to the femtomole range by dilution and direct analysis with micro-liquid chromatography, electrospray ionization, and mass spectrometry (LC-ESI-MS). The LC-ESI-MS assay for adenosine production was shown to be linear and accurate using internal (13)C(15)N adenosine isotope dilution, internal (13)C(15)N adenosine one-point calibration, and external adenosine standard curves with close agreement. The detection limits of LC-ESI-MS for alkaline phosphatase-streptavidin (AP-SA, ∼190,000 Da) was tested by injecting 0.1 μl of a 1 pg/ml solution, i.e., 100 attograms or 526 yoctomole (5.26E-22) of the alkaline-phosphatase labeled probe on column (about 315 AP-SA molecules). The ELIMSA for PSA was linear and showed strong signals across the picogram per milliliter range and could robustly detect PSA from all of the prostatectomy patients and all of the female plasma samples that ranged as low as 70 pg/ml with strong signals well separated from the background and well within the limit of quantification of the AP-SA probe. The results of the ELIMSA assay for PSA are normal and homogenous when independently replicated with a fresh standard over multiple days, and intra and inter diem assay variation was less than 10 % of the mean. In a blind comparison, ELIMSA showed excellent agreement with, but was more sensitive than, the present gold standard commercial fluorescent ELISA, or ECL-based detection, of PSA from normal and prostatectomy samples, respectively.

Serodiagnostic potential of immuno-PCR using a cocktail of mycobacterial antigen 85B, ESAT-6 and cord factor in tuberculosis patients.

PubMed

Singh, Netrapal; Sreenivas, Vishnubhatla; Sheoran, Abhishek; Sharma, Suman; Gupta, Krishna B; Khuller, Gopal K; Mehta, Promod K

2016-01-01

A novel indirect immuno-polymerase chain reaction (I-PCR) assay was developed for the detection of circulating anti-Ag85B (antigen 85B, Rv1886c), anti-ESAT-6 (early secretory antigenic target-6, Rv3875) and anti-cord factor (trehalose 6,6'-dimycolate) antibodies from the sera samples of pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis (EPTB) patients and the results were compared with an analogous enzyme-linked immunosorbent assay (ELISA). We covalently attached the amino-modified reporter DNA to the dithiothreitol (DTT)-reduced anti-human IgG antibody through a chemical linker succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC). The detection of cocktail of anti-Ag85B, anti-ESAT-6 and anti-cord factor antibodies was found to be superior to the detection of individual antibodies. The sensitivities of 89.5% and 77.5% with I-PCR and 70.8% and 65% with ELISA were observed in smear-positive and smear-negative PTB cases, respectively with high specificity (90.9%). On the other hand, a sensitivity of 77.5% with I-PCR and 65% with ELISA was observed in EBTB cases. The detection of cocktail of antibodies by I-PCR is likely to improve the utility of existing algorithms for TB diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

Electrochemical impedance immunosensor based on gold nanoparticles and aryl diazonium salt functionalized gold electrodes for the detection of antibody.

PubMed

Liu, Guozhen; Liu, Jingquan; Davis, Thomas P; Gooding, J Justin

2011-04-15

Electrodes modified with passivating organic layers have been shown to, here and previously, to exhibit good Faradaic electrochemistry upon attachment of gold nanoparticles (AuNP). Due to their low background capacitances these constructs have good potential in electrochemical sensing. Herein is reported the application of these electrode constructs for impedance based immunosensing. The immunosensor was constructed by modifying a gold electrode with 4-thiophenol (4-TP) passivating layers by diazonium salt chemistry. Subsequently, the attachment of AuNP and then a biotin derivative as a model epitope to detect anti-biotin IgG were carried out. The interfacial properties of the modified electrodes were evaluated in the presence of Fe(CN)(6)(4-/3-) redox couple as a probe by cyclic voltammetry and electrochemical impedance spectroscopy. The impedance change, due to the specific immuno-interaction at the immunosensor surface was utilized to detect anti-biotin IgG. The increase in charge-transfer resistance (R(ct)) was linearly proportional to the concentration of anti-biotin IgG in the range of 5-500 ng mL(-1), with a detection limit of 5 ng mL(-1). Copyright © 2011 Elsevier B.V. All rights reserved.

Correlation analysis of two serum-specific immunoglobulin E test systems and skin-prick test in allergic rhinitis patients from northeast China.

PubMed

Jiang, Xiao-Dan; Li, Guang-Yu; Dong, Zhen; Zhu, Dong-Dong

2011-01-01

Skin-prick testing (SPT) is the most common screening method for allergy evaluation. The detection of serum-specific immunoglobulin E (sIgE) is also commonly used. The sensitivity and specificity of these testing methods may vary due to type of causative allergen and type of allergic manifestation. The purpose of this study was to evaluate the correlation between two methods of measuring sIgE (AllergyScreen [Mediwiss Analytic GmbH, Moers, Germany] and ImmunoCAP [Pharmacia, Uppsala, Sweden]) and SPT for the diagnosis of allergic rhinitis (AR). All 216 patients who were referred to the allergist for suspected AR from June to October 2009 had SPT and the two serological tests. One hundred fifty-eight patients had a positive clinical history and a related positive SPT. The SPT was used as reference standard, and we selected three allergens (Dermatophagoides pteronyssinus, mugwort, and ragweed), which were common in fall in northeast China, to analyze the correlation of the two serum tests and SPT. Compared with the SPT, the diagnostic indexes (accuracy, sensitivity and specificity) of the AllergyScreen system and the ImmunoCAP system were 0.819 versus 0.810, 0.780 versus 0.872, and 0.862 versus 0.741, respectively. The accuracy was similar between the two systems (p > 0.05). The ImmunoCAP system method had a higher sensitivity (p < 0.01). The AllergyScreen system had a higher specificity (p < 0.01). These data support that the AllergyScreen system and ImmunoCAP system can identify potentially significant allergens in the diagnosis of AR in patients from northeastern China.

Detection and quantitation of Escherichia coli O157:H7 in raw milk by direct qPCR

USDA-ARS?s Scientific Manuscript database

There has been a shift in US food consumption patterns toward unprocessed foods perceived to be healthier. Part of this trend is increasing consumption of raw, unpasteurized milk. Raw milk can be a source of pathogens that cause serious or fatal illness, particularly in immuno-compromised individ...

Immuno-detection of OCTN1 (SLC22A4) in HeLa cells and characterization of transport function.

PubMed

Pochini, Lorena; Scalise, Mariafrancesca; Indiveri, Cesare

2015-11-01

OCTN1 was immuno-detected in the cervical cancer cell HeLa, in which the complete pattern of acetylcholine metabolizing enzymes is expressed. Comparison of immuno-staining intensity of HeLa OCTN1 with the purified recombinant human OCTN1 allowed measuring the specific OCTN1 concentration in the HeLa cell extract and, hence calculating the HeLa OCTN1 specific transport activity that was about 10 nmol×min(-1)×mg protein(-1), measured as uptake of [(3)H]acetylcholine in proteoliposomes reconstituted with HeLa extract. This value was very similar to the specific activity of the recombinant protein. Acetylcholine transport was suppressed by incubation of the protein or proteoliposomes with the anti-OCTN1 antibody and was strongly inhibited by PLP and MTSEA, known inhibitors of OCTN1. The absence of ATP in the internal side of proteoliposomes strongly impaired transport function of both the HeLa and, as expected, the recombinant OCTN1. HeLa OCTN1 was inhibited by spermine, NaCl (Na(+)), TEA, γ-butyrobetaine, choline, acetylcarnitine and ipratropium but not by neostigmine. Besides acetylcholine, choline was taken up by HeLa OCTN1 proteoliposomes. The transporter catalyzed also acetylcholine and choline efflux which, differently from uptake, was not inhibited by MTSEA. Time course of [(3)H]acetylcholine uptake in intact HeLa cells was measured. As in proteoliposomes, acetylcholine transport in intact cells was inhibited by TEA and NaCl. Efflux of [(3)H]acetylcholine occurred in intact cells, as well. The experimental data concur in demonstrating a role of OCTN1 in transporting acetylcholine and choline in HeLa cells. Copyright © 2015 Elsevier B.V. All rights reserved.

21 CFR 866.5400 - Alpha-globulin immuno-logical test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5400 Alpha-globulin immuno-logical test system. (a) Identification. An alpha-globulin immunological... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Alpha-globulin immuno-logical test system. 866...

21 CFR 866.5380 - Free secretory component immuno-logical test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5380 Free secretory component immuno-logical test system. (a) Identification. A free... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Free secretory component immuno-logical test...

21 CFR 866.5350 - Fibrinopeptide A immuno-logical test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5350 Fibrinopeptide A immuno-logical test system. (a) Identification. A fibrinopeptide A immunological... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Fibrinopeptide A immuno-logical test system. 866...

21 CFR 866.5270 - C-reactive protein immuno-logical test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5270 C-reactive protein immuno-logical test system. (a) Identification. A C-reactive protein... 21 Food and Drugs 8 2010-04-01 2010-04-01 false C-reactive protein immuno-logical test system. 866...

21 CFR 866.5360 - Cohn fraction IV immuno-logical test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5360 Cohn fraction IV immuno-logical test system. (a) Identification. A Cohn fraction IV immunological... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cohn fraction IV immuno-logical test system. 866...

21 CFR 866.5580 - Alpha-1-lipoprotein immuno-logical test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5580 Alpha-1-lipoprotein immuno-logical test system. (a) Identification. An alpha-1-lipoprotein... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Alpha-1-lipoprotein immuno-logical test system...

21 CFR 866.5775 - Rheumatoid factor immuno-logical test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

.... Measurement of rheumatoid factor may aid in the diagnosis of rheumatoid arthritis. (b) Classification. Class... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Rheumatoid factor immuno-logical test system. 866....5775 Rheumatoid factor immuno-logical test system. (a) Identification. A rheumatoid factor...

21 CFR 866.5775 - Rheumatoid factor immuno-logical test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

.... Measurement of rheumatoid factor may aid in the diagnosis of rheumatoid arthritis. (b) Classification. Class... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Rheumatoid factor immuno-logical test system. 866....5775 Rheumatoid factor immuno-logical test system. (a) Identification. A rheumatoid factor...

21 CFR 866.5775 - Rheumatoid factor immuno-logical test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

.... Measurement of rheumatoid factor may aid in the diagnosis of rheumatoid arthritis. (b) Classification. Class... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Rheumatoid factor immuno-logical test system. 866....5775 Rheumatoid factor immuno-logical test system. (a) Identification. A rheumatoid factor...

21 CFR 866.5580 - Alpha-1-lipoprotein immuno-logical test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Alpha-1-lipoprotein immuno-logical test system....5580 Alpha-1-lipoprotein immuno-logical test system. (a) Identification. An alpha-1-lipoprotein... the alpha-1-lipoprotein (high-density lipoprotein) in serum and plasma. Measurement of alpha-1...

21 CFR 866.5580 - Alpha-1-lipoprotein immuno-logical test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Alpha-1-lipoprotein immuno-logical test system....5580 Alpha-1-lipoprotein immuno-logical test system. (a) Identification. An alpha-1-lipoprotein... the alpha-1-lipoprotein (high-density lipoprotein) in serum and plasma. Measurement of alpha-1...

21 CFR 866.5580 - Alpha-1-lipoprotein immuno-logical test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Alpha-1-lipoprotein immuno-logical test system....5580 Alpha-1-lipoprotein immuno-logical test system. (a) Identification. An alpha-1-lipoprotein... the alpha-1-lipoprotein (high-density lipoprotein) in serum and plasma. Measurement of alpha-1...

21 CFR 866.5580 - Alpha-1-lipoprotein immuno-logical test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Alpha-1-lipoprotein immuno-logical test system....5580 Alpha-1-lipoprotein immuno-logical test system. (a) Identification. An alpha-1-lipoprotein... the alpha-1-lipoprotein (high-density lipoprotein) in serum and plasma. Measurement of alpha-1...

21 CFR 866.5775 - Rheumatoid factor immuno-logical test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

.... Measurement of rheumatoid factor may aid in the diagnosis of rheumatoid arthritis. (b) Classification. Class... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Rheumatoid factor immuno-logical test system. 866....5775 Rheumatoid factor immuno-logical test system. (a) Identification. A rheumatoid factor...

21 CFR 866.5775 - Rheumatoid factor immuno-logical test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

.... Measurement of rheumatoid factor may aid in the diagnosis of rheumatoid arthritis. (b) Classification. Class... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Rheumatoid factor immuno-logical test system. 866....5775 Rheumatoid factor immuno-logical test system. (a) Identification. A rheumatoid factor...

An Immuno-epidemiological Model of Paratuberculosis

NASA Astrophysics Data System (ADS)

Martcheva, M.

2011-11-01

The primary objective of this article is to introduce an immuno-epidemiological model of paratuberculosis (Johne's disease). To develop the immuno-epidemiological model, we first develop an immunological model and an epidemiological model. Then, we link the two models through time-since-infection structure and parameters of the epidemiological model. We use the nested approach to compose the immuno-epidemiological model. Our immunological model captures the switch between the T-cell immune response and the antibody response in Johne's disease. The epidemiological model is a time-since-infection model and captures the variability of transmission rate and the vertical transmission of the disease. We compute the immune-response-dependent epidemiological reproduction number. Our immuno-epidemiological model can be used for investigation of the impact of the immune response on the epidemiology of Johne's disease.

A bioanalytical platform for simultaneous detection and quantification of biological toxins.

PubMed

Weingart, Oliver G; Gao, Hui; Crevoisier, François; Heitger, Friedrich; Avondet, Marc-André; Sigrist, Hans

2012-01-01

Prevalent incidents support the notion that toxins, produced by bacteria, fungi, plants or animals are increasingly responsible for food poisoning or intoxication. Owing to their high toxicity some toxins are also regarded as potential biological warfare agents. Accordingly, control, detection and neutralization of toxic substances are a considerable economic burden to food safety, health care and military biodefense. The present contribution describes a new versatile instrument and related procedures for array-based simultaneous detection of bacterial and plant toxins using a bioanalytical platform which combines the specificity of covalently immobilized capture probes with a dedicated instrumentation and immuno-based microarray analytics. The bioanalytical platform consists of a microstructured polymer slide serving both as support of printed arrays and as incubation chamber. The platform further includes an easy-to-operate instrument for simultaneous slide processing at selectable assay temperature. Cy5 coupled streptavidin is used as unifying fluorescent tracer. Fluorescence image analysis and signal quantitation allow determination of the toxin's identity and concentration. The system's performance has been investigated by immunological detection of Botulinum Neurotoxin type A (BoNT/A), Staphylococcal enterotoxin B (SEB), and the plant toxin ricin. Toxins were detectable at levels as low as 0.5-1 ng · mL(-1) in buffer or in raw milk.

A Bioanalytical Platform for Simultaneous Detection and Quantification of Biological Toxins

PubMed Central

Weingart, Oliver G.; Gao, Hui; Crevoisier, François; Heitger, Friedrich; Avondet, Marc-André; Sigrist, Hans

2012-01-01

Prevalent incidents support the notion that toxins, produced by bacteria, fungi, plants or animals are increasingly responsible for food poisoning or intoxication. Owing to their high toxicity some toxins are also regarded as potential biological warfare agents. Accordingly, control, detection and neutralization of toxic substances are a considerable economic burden to food safety, health care and military biodefense. The present contribution describes a new versatile instrument and related procedures for array-based simultaneous detection of bacterial and plant toxins using a bioanalytical platform which combines the specificity of covalently immobilized capture probes with a dedicated instrumentation and immuno-based microarray analytics. The bioanalytical platform consists of a microstructured polymer slide serving both as support of printed arrays and as incubation chamber. The platform further includes an easy-to-operate instrument for simultaneous slide processing at selectable assay temperature. Cy5 coupled streptavidin is used as unifying fluorescent tracer. Fluorescence image analysis and signal quantitation allow determination of the toxin’s identity and concentration. The system’s performance has been investigated by immunological detection of Botulinum Neurotoxin type A (BoNT/A), Staphylococcal enterotoxin B (SEB), and the plant toxin ricin. Toxins were detectable at levels as low as 0.5–1 ng·mL−1 in buffer or in raw milk. PMID:22438766

Tools for Visualizing HIV in Cure Research.

PubMed

Niessl, Julia; Baxter, Amy E; Kaufmann, Daniel E

2018-02-01

The long-lived HIV reservoir remains a major obstacle for an HIV cure. Current techniques to analyze this reservoir are generally population-based. We highlight recent developments in methods visualizing HIV, which offer a different, complementary view, and provide indispensable information for cure strategy development. Recent advances in fluorescence in situ hybridization techniques enabled key developments in reservoir visualization. Flow cytometric detection of HIV mRNAs, concurrently with proteins, provides a high-throughput approach to study the reservoir on a single-cell level. On a tissue level, key spatial information can be obtained detecting viral RNA and DNA in situ by fluorescence microscopy. At total-body level, advancements in non-invasive immuno-positron emission tomography (PET) detection of HIV proteins may allow an encompassing view of HIV reservoir sites. HIV imaging approaches provide important, complementary information regarding the size, phenotype, and localization of the HIV reservoir. Visualizing the reservoir may contribute to the design, assessment, and monitoring of HIV cure strategies in vitro and in vivo.

Field-effect amperometric immuno-detection of protein biomarker.

PubMed

Wang, Jiapeng; Yau, Siu-Tung

2011-11-15

The field-effect enzymatic detection technique has been applied to the amperometric immunoassay of the cancer biomarker, carcinoma antigen 125 (CA 125). The detection adopted a reagentless approach, in which the analyte, CA 125, was immobilized on the detecting electrode, which was modified using carbon nanotubes, and the detection signal was obtained by measuring the reduction peak current of the enzyme that was used to label the antibody. A gating voltage was applied to the detecting electrode, inducing increase in the signal current and therefore providing amplification of the detection signal. The voltage-controlled signal amplification of the detection system has increased the sensitivity and lowered the detection limit of the system. A detection limit of 0.9U/ml was obtained in the work. Copyright © 2011 Elsevier B.V. All rights reserved.

[The expression and significance of IgE in anaphylactic shock guinea-pigs].

PubMed

Gong, Zhi-qiang; Xiao, Feng; Feng, Qiong; Xu, Xiao-ming; Zheng, Jian

2006-02-01

To seek the pathomorphological targets for forensic expertise in anaphylactic shock. The expression of IgE in hearts, lungs, livers, spleens, kidneys, gastrics, intestinals, tracheas and tonsils of anaphylactic shock guinea-pigs was observed at 0, 6, 12 h and 24 h respectively by tissue chip S-P immuno-histochemical method. Positive expression of IgE presented in lungs and tracheas in the test group with the peak at 0 hour and it declined as time advanced, and also there were significant differences at different times (P<0.05). The immuno-histochemical method of detecting the expression of IgE in lungs, tracheas and spleens can be supposed to be the pathomorphological targets for forensic expertise in anaphylactic shock. The weakening of the positive expression of IgE in lungs and tracheas as the time advanced suggested that in this kind of case the autopsy should be arried out as early as possible.

Bladder cancer screening in aluminum smelter workers.

PubMed

Taiwo, Oyebode A; Slade, Martin D; Cantley, Linda F; Tessier-Sherman, Baylah; Galusha, Deron; Kirsche, Sharon R; Donoghue, A Michael; Cullen, Mark R

2015-04-01

To present results of a bladder cancer screening program conducted in 18 aluminum smelters in the United States from January 2000 to December 2010. Data were collected on a cohort of workers with a history of working in coal tar pitch volatile exposed areas including urine analysis for conventional cytology and ImmunoCyt/uCyt+ assay. ImmunoCyt/uCyt+ and cytology in combination showed a sensitivity of 62.30%, a specificity of 92.60%, a negative predictive value of 99.90%, and a positive predictive value of 2.96%. Fourteen cases of bladder cancer were detected, and the standardized incidence ratio of bladder cancer was 1.18 (95% confidence interval, 0.65 to 1.99). Individuals who tested positive on either test who were later determined to be cancer free had undergone expensive and invasive tests. Evidence to support continued surveillance of this cohort has not been demonstrated.

Bladder Cancer Screening in Aluminum Smelter Workers

PubMed Central

Taiwo, Oyebode A.; Slade, Martin D.; Cantley, Linda F.; Tessier-Sherman, Baylah; Galusha, Deron; Kirsche, Sharon R.; Donoghue, A. Michael

2015-01-01

Objective: To present results of a bladder cancer screening program conducted in 18 aluminum smelters in the United States from January 2000 to December 2010. Methods: Data were collected on a cohort of workers with a history of working in coal tar pitch volatile exposed areas including urine analysis for conventional cytology and ImmunoCyt/uCyt+ assay. Results: ImmunoCyt/uCyt+ and cytology in combination showed a sensitivity of 62.30%, a specificity of 92.60%, a negative predictive value of 99.90%, and a positive predictive value of 2.96%. Fourteen cases of bladder cancer were detected, and the standardized incidence ratio of bladder cancer was 1.18 (95% confidence interval, 0.65 to 1.99). Individuals who tested positive on either test who were later determined to be cancer free had undergone expensive and invasive tests. Conclusions: Evidence to support continued surveillance of this cohort has not been demonstrated. PMID:25525927

High-throughput, dual probe biological assays based on single molecule detection

DOEpatents

Hollars, Christopher W [Brentwood, CA; Huser, Thomas R [Livermore, CA; Lane, Stephen M [Oakland, CA; Balhorn, Rodney L [Livermore, CA; Bakajin, Olgica [San Leandro, CA; Darrow, Christopher [Pleasanton, CA; Satcher, Jr., Joe H.

2006-07-11

A method and apparatus with the sensitivity to detect and identify single target molecules through the localization of dual, fluorescently labeled probe molecules. This can be accomplished through specific attachment of the taget to a surface or in a two-dimensional (2D) flowing fluid sheet having approximate dimensions of 0.5 .mu.m.times.100 .mu.m.times.100 .mu.m. A device using these methods would have 10.sup.3 10.sup.4 greater throughput than previous one-dimensional (1D) micro-stream devices having 1 .mu.m.sup.3 interrogation volumes and would for the first time allow immuno- and DNA assays at ultra-low (femtomolar) concentrations to be performed in short time periods (.about.10 minutes). The use of novel labels (such as metal or semiconductor nanoparticles) may be incorporated to further extend the sensitivity possibly into the attomolar range.

21 CFR 866.5330 - Factor XIII, A, S, immuno-logical test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5330 Factor XIII, A, S, immuno-logical test system. (a) Identification. A factor XIII, A, S... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Factor XIII, A, S, immuno-logical test system. 866...

In vivo detection of free radicals in mouse septic encephalopathy using molecular MRI and immuno-spin trapping.

PubMed

Towner, Rheal A; Garteiser, Philippe; Bozza, Fernando; Smith, Nataliya; Saunders, Debra; d' Avila, Joana C P; Magno, Flora; Oliveira, Marcus F; Ehrenshaft, Marilyn; Lupu, Florea; Silasi-Mansat, Robert; Ramirez, Dario C; Gomez-Mejiba, Sandra E; Mason, Ronald P; Castro Faria-Neto, Hugo C

2013-12-01

Free radicals are known to play a major role in sepsis. Combined immuno-spin trapping and molecular magnetic resonance imaging (MRI) was used to detect in vivo and in situ levels of free radicals in murine septic encephalopathy after cecal ligation and puncture (CLP). DMPO (5,5-dimethyl pyrroline N-oxide) was injected over 6h after CLP, before administration of an anti-DMPO probe (anti-DMPO antibody bound to albumin-gadolinium-diethylene triamine pentaacetic acid-biotin MRI targeting contrast agent). In vitro assessment of the anti-DMPO probe in oxidatively stressed mouse astrocytes significantly decreased T1 relaxation (p < 0.0001) compared to controls. MRI detected the presence of anti-DMPO adducts via a substantial decrease in %T1 change within the hippocampus, striatum, occipital, and medial cortex brain regions (p < 0.01 for all) in septic animals compared to shams, which was sustained for over 60 min (p < 0.05 for all). Fluorescently labeled streptavidin was used to target the anti-DMPO probe biotin, which was elevated in septic brain, liver, and lungs compared to sham. Ex vivo DMPO adducts (qualitative) and oxidative products, including 4-hydroxynonenal and 3-nitrotyrosine (quantitative, p < 0.05 for both), were elevated in septic brains compared to shams. This is the first study that has reported on the detection of in vivo and in situ levels of free radicals in murine septic encephalopathy. Copyright © 2013 Elsevier Inc. All rights reserved.

Targeting MT1-MMP as an ImmunoPET-Based Strategy for Imaging Gliomas

PubMed Central

Oteo, M.; Romero, E.; Cámara, J. A.; de Martino, A.; Arroyo, A. G.; Morcillo, M. Á.; Squatrito, M.; Martinez-Torrecuadrada, J. L.; Mulero, F.

2016-01-01

Background A critical challenge in the management of Glioblastoma Multiforme (GBM) tumors is the accurate diagnosis and assessment of tumor progression in a noninvasive manner. We have identified Membrane-type 1 matrix metalloproteinase (MT1-MMP) as an attractive biomarker for GBM imaging since this protein is actively involved in tumor growth and progression, correlates with tumor grade and is closely associated with poor prognosis in GBM patients. Here, we report the development of an immunoPET tracer for effective detection of MT1-MMP in GBM models. Methods An anti-human MT1-MMP monoclonal antibody (mAb), LEM2/15, was conjugated to p-isothiocyanatobenzyl-desferrioxamine (DFO-NCS) for 89Zr labeling. Biodistribution and PET imaging studies were performed in xenograft mice bearing human GBM cells (U251) expressing MT1-MMP and non-expressing breast carcinoma cells (MCF-7) as negative control. Two orthotopic brain GBM models, patient-derived neurospheres (TS543) and U251 cells, with different degrees of blood-brain barrier (BBB) disruption were also used for PET imaging experiments. Results 89Zr labeling of DFO-LEM2/15 was achieved with high yield (>90%) and specific activity (78.5 MBq/mg). Biodistribution experiments indicated that 89Zr-DFO-LEM2/15 showed excellent potential as a radiotracer for detection of MT1-MMP positive GBM tumors. PET imaging also indicated a specific and prominent 89Zr-DFO-LEM2/15 uptake in MT1-MMP+ U251 GBM tumors compared to MT1-MMP- MCF-7 breast tumors. Results obtained in orthotopic brain GBM models revealed a high dependence of a disrupted BBB for tracer penetrance into tumors. 89Zr-DFO-LEM2/15 showed much higher accumulation in TS543 tumors with a highly disrupted BBB than in U251 orthotopic model in which the BBB permeability was only partially increased. Histological analysis confirmed the specificity of the immunoconjugate in all GBM models. Conclusion A new anti MT1-MMP-mAb tracer, 89Zr-DFO-LEM2/15, was synthesized efficiently. In vivo validation showed high-specific-contrast imaging of MT1-MMP positive GBM tumors and provided strong evidence for utility of MT1-MMP-targeted immunoPET as an alternate to nonspecific imaging of GBM. PMID:27462980

Usefulness of antigen-specific IgE probability curves derived from the 3gAllergy assay in diagnosing egg, cow's milk, and wheat allergies.

PubMed

Sato, Sakura; Ogura, Kiyotake; Takahashi, Kyohei; Sato, Yasunori; Yanagida, Noriyuki; Ebisawa, Motohiro

2017-04-01

Specific IgE (sIgE) antibody detection using the Siemens IMMULITE ® 3gAllergy™ (3gAllergy) assay have not been sufficiently examined for the diagnosis of food allergy. The aim of this study was to evaluate the utility of measuring sIgE levels using the 3gAllergy assay to diagnose allergic reactions to egg, milk, and wheat. This retrospective study was conducted on patients with diagnosed or suspected allergies to egg, milk and wheat. Patients were divided into two groups according to their clinical reactivity to these allergens based on oral food challenge outcomes and/or convincing histories of immediate reaction to causative food(s). The sIgE levels were measured using 3gAllergy and ImmunoCAP. Predicted probability curves were estimated using logistic regression analysis. We analyzed 1561 patients, ages 0-19 y (egg = 436, milk = 499, wheat = 626). The sIgE levels determined using 3gAllergy correlated with those of ImmunoCAP, classifying 355 patients as symptomatic: egg = 149, milk = 123, wheat = 83. 3gAllergy sIgE levels were significantly higher in symptomatic than in asymptomatic patients (P < 0.0001). Predictive probability for positive food allergy was significantly increased and correlated with increased sIgE levels. The cut-offs for allergic reaction with 95% predictive probability as determined by the 3gAllergy probability curves were different from those of ImmunoCAP. Measurements of sIgE against egg, milk, and wheat as determined by 3gAllergy may be used as a tool to facilitate the diagnosis of food allergy in subjects with suspected food allergies. However, these probability curves should not be applied interchangeably between different assays. Copyright © 2016 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.

Sensitive Quantification of Aflatoxin B1 in Animal Feeds, Corn Feed Grain, and Yellow Corn Meal Using Immunomagnetic Bead-Based Recovery and Real-Time Immunoquantitative-PCR

PubMed Central

Babu, Dinesh; Muriana, Peter M.

2014-01-01

Aflatoxins are considered unavoidable natural mycotoxins encountered in foods, animal feeds, and feed grains. In this study, we demonstrate the application of our recently developed real-time immunoquantitative PCR (RT iq-PCR) assay for sensitive detection and quantification of aflatoxins in poultry feed, two types of dairy feed (1 and 2), horse feed, whole kernel corn feed grains, and retail yellow ground corn meal. Upon testing methanol/water (60:40) extractions of the above samples using competitive direct enzyme linked immunosorbent assay, the aflatoxin content was found to be <20 μg/kg. The RT iq-PCR assay exhibited high antigen hook effect in samples containing aflatoxin levels higher than the quantification limits (0.1–10 μg/kg), addressed by comparing the quantification results of undiluted and diluted extracts. In testing the reliability of the immuno-PCR assay, samples were spiked with 200 μg/kg of aflatoxin B1, but the recovery of spiked aflatoxin was found to be poor. Considering the significance of determining trace levels of aflatoxins and their serious implications for animal and human health, the RT iq-PCR method described in this study can be useful for quantifying low natural aflatoxin levels in complex matrices of food or animal feed samples without the requirement of extra sample cleanup. PMID:25474493

Sensitive quantification of aflatoxin B1 in animal feeds, corn feed grain, and yellow corn meal using immunomagnetic bead-based recovery and real-time immunoquantitative-PCR.

PubMed

Babu, Dinesh; Muriana, Peter M

2014-12-02

Aflatoxins are considered unavoidable natural mycotoxins encountered in foods, animal feeds, and feed grains. In this study, we demonstrate the application of our recently developed real-time immunoquantitative PCR (RT iq-PCR) assay for sensitive detection and quantification of aflatoxins in poultry feed, two types of dairy feed (1 and 2), horse feed, whole kernel corn feed grains, and retail yellow ground corn meal. Upon testing methanol/water (60:40) extractions of the above samples using competitive direct enzyme linked immunosorbent assay, the aflatoxin content was found to be <20 μg/kg. The RT iq-PCR assay exhibited high antigen hook effect in samples containing aflatoxin levels higher than the quantification limits (0.1-10 μg/kg), addressed by comparing the quantification results of undiluted and diluted extracts. In testing the reliability of the immuno-PCR assay, samples were spiked with 200 μg/kg of aflatoxin B1, but the recovery of spiked aflatoxin was found to be poor. Considering the significance of determining trace levels of aflatoxins and their serious implications for animal and human health, the RT iq-PCR method described in this study can be useful for quantifying low natural aflatoxin levels in complex matrices of food or animal feed samples without the requirement of extra sample cleanup.

Local IL-23 expression in murine vaginal candidiasis and its relationship with infection and immune status.

PubMed

Wu, Yan; Tan, Zhijian; Liu, Zhixiang; Xia, Dechao; Li, Jiawen

2006-01-01

To investigate the expression of vaginal IL-23 and its role in experimental murine vaginal candidiasis and its relationship with infection and immune status, immuno-competent (group A) and immuno-suppressed (group B) murine models of vaginal candidiasis were established in estrogen-treated mice. Non-estrogen-treated mice were used as controls (group C). The level of IL-23 p19 mRNA in murine vaginal tissue was determined by RT-PCR. Significantly increased levels of IL-23p19mRNA were observed on the 4th, the 7th and 14th day after inoculation in immuno-competent group when compared with that in control group (P<0.01, P<0.05). However, significant increase of IL-23 p19mRNA were only observed on the 7th day and the 14th day after inoculatuon in immuno-suppressed groups (P<0.05). On the 4th and 7th day, the levels of IL-23 p19mRNA were significantly increased in immuno-competent group than those in immuno-suppressed group (P <0.05). Local IL-23 may play a role in the pathogenesis of murine vaginal candidiasis and has a protective function during infection. Low vaginal IL-23 level may correlate with the increased susceptibility to Candida albicans in immuno-suppressed group.

Development of Ultra-Super Sensitive Immunohistochemistry and Its Application to the Etiological Study of Adult T-Cell Leukemia/Lymphoma

PubMed Central

Hasui, Kazuhisa; Wang, Jia; Tanaka, Yuetsu; Izumo, Shuji; Eizuru, Yoshito; Matsuyama, Takami

2012-01-01

Antigen retrieval (AR) and ultra-super sensitive immunohistochemistry (ultra-IHC) have been established for application to archival human pathology specimens. The original ultra-IHC was the ImmunoMax method or the catalyzed signal amplification system (ImmunoMax/CSA method), comprising the streptavidin-biotin complex (sABC) method and catalyzed reporter deposition (CARD) reaction with visualization of its deposition. By introducing procedures to diminish non-specific staining in the original ultra-IHC method, we developed the modified ImmunoMax/CSA method with AR heating sections in an AR solution (heating-AR). The heating-AR and modified ImmunoMax/CSA method visualized expression of the predominantly simple present form of HTLV-1 proviral DNA pX region p40Tax protein (Tax) in adult T-cell leukemia/lymphoma (ATLL) cells in archival pathology specimens in approximately 75% of cases. The simple present form of Tax detected exhibited a close relation with ATLL cell proliferation. We also established a new simplified CSA (nsCSA) system by replacing the sABC method with the secondary antibody- and horse radish peroxidase-labeled polymer reagent method, introducing the pretreatments blocking non-specific binding of secondary antibody reagent, and diminishing the diffusion of deposition in the CARD reaction. Combined with AR treating sections with proteinase K solution (enzymatic-AR), the nsCSA system visualized granular immunostaining of the complex present form of Tax in a small number of ATLL cells in most cases, presenting the possibility of etiological pathological diagnosis of ATLL and suggesting that the complex present form of Tax-positive ATLL cells were young cells derived from ATLL stem cells. The heating-AR and ultra-IHC detected physiological expression of the p53 protein and its probable phosphorylation by Tax in peripheral blood mononuclear cells of peripheral blood tissue specimens from HTLV-1 carriers, as well as physiological and pathological expression of the molecules involved with G1 phase progression and G1–S phase transition (E2F-1, E2F-4, DP-1, and cyclin E) in ATLL and peripheral T-cell lymphoma cells. The ultra-IHC with AR is useful for etiological pathological diagnosis of ATLL since HTLV-1 pathogenicity depends on that of Tax, and can be a useful tool for studies translating advanced molecular biology and pathology to human pathology. PMID:22685351

Fallen Angels or Risen Apes? A Tale of the Intricate Complexities of Imbalanced Immune Responses in the Pathogenesis and Progression of Immune-Mediated and Viral Cancers

PubMed Central

Ondondo, Beatrice Omusiro

2014-01-01

Excessive immune responses directed against foreign pathogens, self-antigens, or commensal microflora can cause cancer establishment and progression if the execution of tight immuno-regulatory mechanisms fails. On the other hand, induction of potent tumor antigen-specific immune responses together with stimulation of the innate immune system is a pre-requisite for effective anti-tumor immunity, and if suppressed by the strong immuno-regulatory mechanisms can lead to cancer progression. Therefore, it is crucial that the inevitable co-existence of these fundamental, yet conflicting roles of immune-regulatory cells is carefully streamlined as imbalances can be detrimental to the host. Infection with chronic persistent viruses is characterized by severe immune dysfunction resulting in T cell exhaustion and sometimes deletion of antigen-specific T cells. More often, this is due to increased immuno-regulatory processes, which are triggered to down-regulate immune responses and limit immunopathology. However, such heightened levels of immune disruption cause a concomitant loss of tumor immune-surveillance and create a permissive microenvironment for cancer establishment and progression, as demonstrated by increased incidences of cancer in immunosuppressed hosts. Paradoxically, while some cancers arise as a consequence of increased immuno-regulatory mechanisms that inhibit protective immune responses and impinge on tumor surveillance, other cancers arise due to impaired immuno-regulatory mechanisms and failure to limit pathogenic inflammatory responses. This intricate complexity, where immuno-regulatory cells can be beneficial in certain immune settings but detrimental in other settings underscores the need for carefully formulated interventions to equilibrate the balance between immuno-stimulatory and immuno-regulatory processes. PMID:24639678

Towards Translational ImmunoPET/MR Imaging of Invasive Pulmonary Aspergillosis: The Humanised Monoclonal Antibody JF5 Detects Aspergillus Lung Infections In Vivo

PubMed Central

Davies, Genna; Rolle, Anna-Maria; Maurer, Andreas; Spycher, Philipp R.; Schillinger, Claudia; Solouk-Saran, Djamschid; Hasenberg, Mike; Weski, Juliane; Fonslet, Jesper; Dubois, Adrien; Boschetti, Frederic; Denat, Franck; Gunzer, Matthias; Eichner, Martin; Ryder, Lauren S; Jensen, Mikael; Schibli, Roger; Pichler, Bernd J.; Wiehr, Stefan; Thornton, Christopher R.

2017-01-01

Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease of hematological malignancy or bone marrow transplant patients caused by the ubiquitous environmental fungus Aspergillus fumigatus. Current diagnostic tests for the disease lack sensitivity as well as specificity, and culture of the fungus from invasive lung biopsy, considered the gold standard for IPA detection, is slow and often not possible in critically ill patients. In a previous study, we reported the development of a novel non-invasive procedure for IPA diagnosis based on antibody-guided positron emission tomography and magnetic resonance imaging (immunoPET/MRI) using a [64Cu]DOTA-labeled mouse monoclonal antibody (mAb), mJF5, specific to Aspergillus. To enable translation of the tracer to the clinical setting, we report here the development of a humanised version of the antibody (hJF5), and pre-clinical imaging of lung infection using a [64Cu]NODAGA-hJF5 tracer. The humanised antibody tracer shows a significant increase in in vivo biodistribution in A. fumigatus infected lungs compared to its radiolabeled murine counterpart [64Cu]NODAGA-mJF5. Using reverse genetics of the pathogen, we show that the antibody binds to the antigenic determinant β1,5-galactofuranose (Galf) present in a diagnostic mannoprotein antigen released by the pathogen during invasive growth in the lung. The absence of the epitope Galf in mammalian carbohydrates, coupled with the enhanced imaging capabilities of the hJF5 antibody, means that the [64Cu]NODAGA-hJF5 tracer developed here represents an ideal candidate for the diagnosis of IPA and translation to the clinical setting. PMID:28912884

Microarray-based IgE detection in tears of patients with vernal keratoconjunctivitis.

PubMed

Leonardi, Andrea; Borghesan, Franco; Faggian, Diego; Plebani, Mario

2015-11-01

A specific allergen sensitization can be demonstrated in approximately half of the vernal keratoconjunctivitis (VKC) patients by conventional allergic tests. The measurement of specific IgE in tears using a multiplex allergen microarray may offer advantages to identify local sensitization to a specific allergen. In spring-summer 2011, serum and tears samples were collected from 10 active VKC patients (three females, seven males) and 10 age-matched normal subjects. Skin prick test, symptoms score and full ophthalmological examination were performed. Specific serum and tear IgE were assayed using ImmunoCAP ISAC, a microarray containing 103 components derived from 47 allergens. Normal subjects resulted negative for the presence of specific IgE both in serum and in tears. Of the 10 VKC patients, six resulted positive to specific IgE in serum and/or tears. In three of these six patients, specific IgE was found positive only in tears. Cross-reactivity between specific markers was found in three patients. Grass, tree, mites, animal but also food allergen-specific IgE were found in tears. Conjunctival provocation test performed out of season confirmed the specific local conjunctival reactivity. Multiple specific IgE measurements with single protein allergens using a microarray technique in tear samples are a useful, simple and non-invasive diagnostic tool. ImmunoCAP ISAC detects allergen sensitization at component level and adds important information by defining both cross- and co-sensitization to a large variety of allergen molecules. The presence of specific IgE only in tears of VKC patients reinforces the concept of possible local sensitization. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Carbon nanotubes-based chemiresistive immunosensor for small molecules: detection of nitroaromatic explosives.

PubMed

Park, Miso; Cella, Lakshmi N; Chen, Wilfred; Myung, Nosang V; Mulchandani, Ashok

2010-12-15

In recent years, there has been a growing focus on use of one-dimensional (1-D) nanostructures, such as carbon nanotubes and nanowires, as transducer elements for label-free chemiresistive/field-effect transistor biosensors as they provide label-free and high sensitivity detection. While research to-date has elucidated the power of carbon nanotubes- and other 1-D nanostructure-based field effect transistors immunosensors for large charged macromolecules such as proteins and viruses, their application to small uncharged or charged molecules has not been demonstrated. In this paper we report a single-walled carbon nanotubes (SWNTs)-based chemiresistive immunosensor for label-free, rapid, sensitive and selective detection of 2,4,6-trinitrotoluene (TNT), a small molecule. The newly developed immunosensor employed a displacement mode/format in which SWNTs network forming conduction channel of the sensor was first modified with trinitrophenyl (TNP), an analog of TNT, and then ligated with the anti-TNP single chain antibody. Upon exposure to TNT or its derivatives the bound antibodies were displaced producing a large change, several folds higher than the noise, in the resistance/conductance of SWNTs giving excellent limit of detection, sensitivity and selectivity. The sensor detected between 0.5 ppb and 5000 ppb TNT with good selectivity to other nitroaromatic explosives and demonstrated good accuracy for monitoring TNT in untreated environmental water matrix. We believe this new displacement format can be easily generalized to other one-dimensional nanostructure-based chemiresistive immuno/affinity-sensors for detecting small and/or uncharged molecules of interest in environmental monitoring and health care. Copyright © 2010 Elsevier B.V. All rights reserved.

An Embedded Microretroreflector-Based Microfluidic Immunoassay Platform

PubMed Central

Raja, Balakrishnan; Pascente, Carmen; Knoop, Jennifer; Shakarisaz, David; Sherlock, Tim; Kemper, Steven; Kourentzi, Katerina; Renzi, Ronald F.; Hatch, Anson V.; Olano, Juan; Peng, Bi-Hung; Ruchhoeft, Paul; Willson, Richard

2017-01-01

We present a microfluidic immunoassay platform based on the use of linear microretroreflectors embedded in a transparent polymer layer as an optical sensing surface, and micron-sized magnetic particles as light-blocking labels. Retroreflectors return light directly to its source and are highly detectable using inexpensive optics. The analyte is immuno-magnetically pre-concentrated from a sample and then captured on an antibody-modified microfluidic substrate comprised of embedded microretroreflectors, thereby blocking reflected light. Fluidic force discrimination is used to increase specificity of the assay, following which a difference imaging algorithm that can see single 3 μm magnetic particles without optical calibration is used to detect and quantify signal intensity from each sub-array of retroreflectors. We demonstrate the utility of embedded microretroreflectors as a new sensing modality through a proof-of-concept immunoassay for a small, obligate intracellular bacterial pathogen, Rickettsia conorii, the causative agent of Mediterranean Spotted Fever. The combination of large sensing area, optimized surface chemistry and microfluidic protocols, automated image capture and analysis, and high sensitivity of the difference imaging results in a sensitive immunoassay with a limit of detection of roughly 4000 R. conorii per mL. PMID:27025227

Christmas-tree Derived Amplification Immuno-strategy for Sensitive Visual Detection of Vibrio parahaemolyticus Based on Gold Label Silver Stain Technology.

PubMed

Song, Xinxin; Wu, Yanjie; Wu, Lin; Hu, Yufang; Li, Wenrou; Guo, Zhiyong; Su, Xiurong; Jiang, Xiaohua

2017-01-01

A developed Christmas-tree derived immunosensor based on a gold label silver stain (GLSS) technique was fabricated for a highly sensitive analysis of Vibrio parahaemolyticu (VP). In this strategy, captured VP antibody (cAb) was immobilized on a solid substrate; then, the VPs were sequentially tagged with a signal probe by incubating the assay with a detection VP antibody (dAb) conjugated gold nanoparticles (AuNPs)-labeled graphite-like carbon nitride (g-C 3 N 4 ). Finally, the attached signal probe could harvest a visible signal by the silver meal deposition, and then followed by homebrew Matlab 6.0 as a grey value acquisition. In addition, the overall design of the biosensor was established in abundant AuNPs and g-C 3 N 4 with a two-dimensional structure, affording a bulb-decorated Christmas-tree model. Moreover, with the optimized conditions, the detection limit of the as-proposed biosensor is as low as 10 2 CFU (Colony-Forming Units) mL -1 , exhibiting an increase of two orders of magnitude compared with the traditional immune-gold method. Additionally, the developed visible immunosensor was also successfully applied to the analysis of complicated samples.

Schimke immuno-osseous dysplasia: case report and review of 25 patients

PubMed Central

Saraiva, J.; Dinis, A.; Resende, C.; Faria, E.; Gomes, C.; Correia, A; Gil, J.; da Fonseca, N.

1999-01-01

Immuno-osseous dysplasia is characterised by spondyloepiphyseal dysplasia, lymphopenia with defective cellular immunity, and progressive renal disease. We describe a patient with a severe form of the disease, review the features of another 24 patients, and discuss the previous classification. The differences between the two groups are not striking, and although similarities are greater between affected sibs, the same diagnosis of Schimke immuno-osseous dysplasia should apply to them all. The aetiology and physiopathology of this rare osteochondrodysplasia of presumed autosomal recessive inheritance remain unknown.


Keywords: osteochondrodysplasia; immuno-osseous dysplasia; spondyloepiphyseal dysplasia; defective cellular immunity PMID:10528861

[Heart fatty-acid binding protein (h-FABP): a new cardiac marker].

PubMed

Servonnet, A; Delacour, H; Dehan, C; Gardet, V

2006-01-01

Heart Fatty-Acid Binding Protein (h-FABP) is a small cytosolic protein that is abundant in the heart and found at lower concentrations in muscle or in the brain. h-FABP is released into the circulation shortly after the onset of ischemia. Several studies indicate its usefulness in cardiology: exclusion of acute myocardial infarction, detection of reperfusion, prognostic value... A rapid immuno-chromatographic assay (Cardiodetect) was recently commercialized in France with a result obtainable within 15 minutes. We review the strengths and weakness of h-FABP for detecting myocardial injury.

Advances in the design of nanomaterial-based electrochemical affinity and enzymatic biosensors for metabolic biomarkers: A review.

PubMed

Farzin, Leila; Shamsipur, Mojtaba; Samandari, Leila; Sheibani, Shahab

2018-05-02

This review (with 340 refs) focuses on methods for specific and sensitive detection of metabolites for diagnostic purposes, with particular emphasis on electrochemical nanomaterial-based sensors. It also covers novel candidate metabolites as potential biomarkers for diseases such as neurodegenerative diseases, autism spectrum disorder and hepatitis. Following an introduction into the field of metabolic biomarkers, a first major section classifies electrochemical biosensors according to the bioreceptor type (enzymatic, immuno, apta and peptide based sensors). A next section covers applications of nanomaterials in electrochemical biosensing (with subsections on the classification of nanomaterials, electrochemical approaches for signal generation and amplification using nanomaterials, and on nanomaterials as tags). A next large sections treats candidate metabolic biomarkers for diagnosis of diseases (in the context with metabolomics), with subsections on biomarkers for neurodegenerative diseases, autism spectrum disorder and hepatitis. The Conclusion addresses current challenges and future perspectives. Graphical abstract This review focuses on the recent developments in electrochemical biosensors based on the use of nanomaterials for the detection of metabolic biomarkers. It covers the critical metabolites for some diseases such as neurodegenerative diseases, autism spectrum disorder and hepatitis.

[Comparative research into sensitivity and specificity of immune-enzyme analysis with chemiluminescence and colorimetric detection for detecting antigens and antibodies to avian influenza viruses and newcastle disease].

PubMed

Vitkova, O N; Kapustina, T P; Mikhailova, V V; Safonov, G A; Vlasova, N N; Belousova, R V

2015-01-01

The goal of this work was to demonstrate the results of the development of the enzyme-linked immunosorbent tests with chemiluminescence detection and colorimetric detection of specific viral antigens and antibodies for identifying the avian influenza and the Newcastle disease viruses: high sensitivity and specificity of the immuno- chemiluminescence assay, which are 10-50 times higher than those of the ELISA colorimetric method. The high effectiveness of the results and the automation of the process of laboratory testing (using a luminometer) allow these methods to be recommended for including in primary screening tests for these infectious diseases.

Review: Microbial Analysis in Dielectrophoretic Microfluidic Systems

PubMed Central

Fernandez, Renny E.; Rohani, Ali; Farmehini, Vahid; Swami, Nathan S.

2017-01-01

Infections caused by various known and emerging pathogenic microorganisms, including antibiotic-resistant strains, are a major threat to global health and well-being. This highlights the urgent need for detection systems for microbial identification, quantification and characterization towards assessing infections, prescribing therapies and understanding the dynamic cellular modifications. Current state-of-the-art microbial detection systems exhibit a trade-off between sensitivity and assay time, which could be alleviated by selective and label-free microbial capture onto the sensor surface from dilute samples. AC electrokinetic methods, such as dielectrophoresis, enable frequency-selective capture of viable microbial cells and spores due to polarization based on their distinguishing size, shape and sub-cellular compositional characteristics, for downstream coupling to various detection modalities. Following elucidation of the polarization mechanisms that distinguish bacterial cells from each other, as well as from mammalian cells, this review compares the microfluidic platforms for dielectrophoretic manipulation of microbials and their coupling to various detection modalities, including immuno-capture, impedance measurement, Raman spectroscopy and nucleic acid amplification methods, as well as for phenotypic assessment of microbial viability and antibiotic susceptibility. Based on the urgent need within point-of-care diagnostics towards reducing assay times and enhancing capture of the target organism, as well as the emerging interest in isolating intact microbials based on their phenotype and subcellular features, we envision widespread adoption of these label-free and selective electrokinetic techniques. PMID:28372723

An isothermal based recombinase polymerase amplification assay for rapid, sensitive and robust indexing of citrus yellow mosaic virus.

PubMed

Kumar, P V; Sharma, S K; Rishi, N; Ghosh, D K; Baranwal, V K

Management of viral diseases relies on definite and sensitive detection methods. Citrus yellow mosaic virus (CYMV), a double stranded DNA virus of the genus Badnavirus, causes yellow mosaic disease in citrus plants. CYMV is transmitted through budwood and requires a robust and simplified indexing protocol for budwood certification programme. The present study reports development and standardization of an isothermal based recombinase polymerase amplification (RPA) assay for a sensitive, rapid, easy, and cost-effective method for detection and diagnosis of CYMV. Two different oligonucleotide primer sets were designed from ORF III (coding for polyprotein) and ORF II (coding for virion associated protein) regions of CYMV to perform amplification assays. Comparative evaluation of RPA, PCR and immuno-capture recombinase polymerase amplification (IC-RPA) based assays were done using purified DNA and plant crude sap. CYMV infection was efficiently detected from the crude sap in RPA and IC-RPA assays. The primer set used in RPA was specific and did not show any cross-amplification with banana streak MY virus (BSMYV), another Badnavirus species. The results from the present study indicated that RPA assay can be used easily in routine indexing of citrus planting material. To the best of our knowledge, this is the first report on development of a rapid and simplified isothermal detection assay for CYMV and can be utilized as an effective technique in quarantine and budwood certification process.

Real-Time Tau Protein Detection by Sandwich-Based Piezoelectric Biosensing: Exploring Tubulin as a Mass Enhancer.

PubMed

Li, Dujuan; Scarano, Simona; Lisi, Samuele; Palladino, Pasquale; Minunni, Maria

2018-03-22

Human tau protein is one of the most advanced and accepted biomarkers for AD and tauopathies diagnosis in general. In this work, a quartz crystal balance (QCM) immunosensor was developed for the detection of human tau protein in buffer and artificial cerebrospinal fluid (aCSF), through both direct and sandwich assays. Starting from a conventional immuno-based sandwich strategy, two monoclonal antibodies recognizing different epitopes of tau protein were used, achieving a detection limit for the direct assay in nanomolar range both in HBES-EP and aCSF. Afterward, for exploring alternative specific receptors as secondary recognition elements for tau protein biosensing, we tested tubulin and compared its behavior to a conventional secondary antibody in the sandwich assay. Tau-tubulin binding has shown an extended working range coupled to a signal improvement in comparison with the conventional secondary antibody-based approach, showing a dose-response trend at lower tau concentration than is usually investigated and closer to the physiological levels in the reference matrix for protein tau biomarker. Our results open up new and encouraging perspectives for the use of tubulin as an alternative receptor for tau protein with interesting features due to the possibility of taking advantage of its polymerization and reversible binding to this key hallmark of Alzheimer's disease.

False-positive cryptococcal antigen latex agglutination caused by disinfectants and soaps.

PubMed Central

Blevins, L B; Fenn, J; Segal, H; Newcomb-Gayman, P; Carroll, K C

1995-01-01

Five disinfectants or soaps were tested to determine if any could be responsible for false-positive results obtained with the Latex-Crypto Antigen Detection System kit (Immuno-Mycologics, Inc., Norman, Okla.). Three disinfectants or soaps (Derma soap, 7X, and Bacdown) produced false-positive agglutination after repeated washing of ring slides during testing of a known negative cerebrospinal fluid specimen. PMID:7650214

3D biomaterial matrix to support long term, full thickness, immuno-competent human skin equivalents with nervous system components.

PubMed

Vidal, Sarah E Lightfoot; Tamamoto, Kasey A; Nguyen, Hanh; Abbott, Rosalyn D; Cairns, Dana M; Kaplan, David L

2018-04-24

Current commercially available human skin equivalents (HSEs) are used for relatively short term studies (∼1 week) due in part to the time-dependent contraction of the collagen gel-based matrix and the limited cell types and skin tissue components utilized. In contrast, here we describe a new matrix consisting of a silk-collagen composite system that provides long term, stable cultivation with reduced contraction and degradation over time. This matrix supports full thickness skin equivalents which include nerves. The unique silk-collagen composite system preserves cell-binding domains of collagen while maintaining the stability and mechanics of the skin system for long-term culture with silk. The utility of this new composite protein-based biomaterial was demonstrated by bioengineering full thickness human skin systems using primary cells, including nerves and immune cells to establish an HSE with a neuro-immuno-cutaneous system. The HSEs with neurons and hypodermis, compared to in vitro skin-only HSEs controls, demonstrated higher secretion of pro-inflammatory cytokines. Proteomics analysis confirmed the presence of several proteins associated with inflammation across all sample groups, but HSEs with neurons had the highest amount of detected protein due to the complexity of the model. This improved, in vitro full thickness HSE model system utilizes cross-linked silk-collagen as the biomaterial and allows reduced reliance on animal models and provides a new in vitro tissue system for the assessment of chronic responses related to skin diseases and drug discovery. Copyright © 2018 Elsevier Ltd. All rights reserved.

Design and Fabrication of a PDMS Microchip Based Immunoassay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shao, Guocheng; Wang, Wanjun; Wang, Jun

2010-07-01

In this paper, we describe the design and fabrication process of a polydimethylsiloxane (PDMS) microchip for on-chip multiplex immunoassay application. The microchip consists of a PDMS microfluidic channel layer and a micro pneumatic valve control layer. By selectively pressurizing the pneumatic microvalves, immuno reagents were controlled to flow and react in certain fluidic channel sites. Cross contamination was prevented by tightly closed valves. Our design was proposed to utilize PDMS micro channel surface as the solid phase immunoassay substrate and simultaneously detect four targets antigens on chip. Experiment result shows that 20psi valve pressure is sufficient to tightly close amore » 200µm wide micro channel with flow rate up to 20µl/min.« less

Diagnostic accuracy of three monoclonal stool tests in a large series of untreated Helicobacter pylori infected patients.

PubMed

Lario, Sergio; Ramírez-Lázaro, María José; Montserrat, Antònia; Quílez, María Elisa; Junquera, Félix; Martínez-Bauer, Eva; Sanfeliu, Isabel; Brullet, Enric; Campo, Rafael; Segura, Ferran; Calvet, Xavier

2016-06-01

Immunochromatographic tests need to be improved in order to enhance their reliability. Recently, several new kits have appeared on the market. The objective was to evaluate the diagnostic accuracy of three monoclonal rapid stool tests - the new Uni-Gold™ H.pylori Antigen (Trinity Biotech, Ireland), the RAPID Hp StAR (Oxoid Ltd., UK) and the ImmunoCard STAT! HpSA (Meridian Diagnostics, USA) - for detecting H. pylori infection prior to eradication treatment. Diagnostic accuracy (sensitivity and specificity) and reliability (concordance between observers) were evaluated in 250 untreated consecutive dyspeptic patients. The gold standard for diagnosing H. pylori infection was defined as the concordance of two or more of rapid urease test (RUT), histopathology and urease breath test (UBT) or positive culture in isolation. Readings of immunochromatographic tests were performed by two different observers. Sensitivity, specificity, positive and negative predictive values and 95% confidence intervals were calculated. Sensitivity and specificity were compared using the McNemar test. The three tests showed a good correlation, with Kappa values>0.9. RAPID Hp StAR had a sensitivity of 91%-92% and a specificity ranging from 77% to 85%. Its sensitivity was higher than that of Uni-Gold™ H.pylori Antigen and ImmunoCard STAT! HpSA (p<0.01). Uni-Gold™ H.pylori Antigen kit showed a sensitivity of 83%, similar to ImmunoCard STAT! HpSA. Specificity of Uni-Gold™ H.pylori Antigen approached 90% (87-89%) and was superior to that of RAPID Hp StAR (p<0.01). Uni-Gold™ H.pylori Antigen and ImmunoCard STAT! HpSA present similar levels of diagnostic accuracy. RAPID Hp StAR was the most sensitive but less reliable of the three immunochromatographic stool tests. None are as accurate and reliable as UBT, RUT and histology. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

The current preference for the immuno-analytical ELISA method for quantitation of steroid hormones (endocrine disruptor compounds) in wastewater in South Africa.

PubMed

Manickum, Thavrin; John, Wilson

2015-07-01

The availability of national test centers to offer a routine service for analysis and quantitation of some selected steroid hormones [natural estrogens (17-β-estradiol, E2; estrone, E1; estriol, E3), synthetic estrogen (17-α-ethinylestradiol, EE2), androgen (testosterone), and progestogen (progesterone)] in wastewater matrix was investigated; corresponding internationally used chemical- and immuno-analytical test methods were reviewed. The enzyme-linked immunosorbent assay (ELISA) (immuno-analytical technique) was also assessed for its suitability as a routine test method to quantitate the levels of these hormones at a sewage/wastewater treatment plant (WTP) (Darvill, Pietermaritzburg, South Africa), over a 2-year period. The method performance and other relevant characteristics of the immuno-analytical ELISA method were compared to the conventional chemical-analytical methodology, like gas/liquid chromatography-mass spectrometry (GC/LC-MS), and GC-LC/tandem mass spectrometry (MSMS), for quantitation of the steroid hormones in wastewater and environmental waters. The national immuno-analytical ELISA technique was found to be sensitive (LOQ 5 ng/L, LOD 0.2-5 ng/L), accurate (mean recovery 96%), precise (RSD 7-10%), and cost-effective for screening and quantitation of these steroid hormones in wastewater and environmental water matrix. A survey of the most current international literature indicates a fairly equal use of the LC-MS/MS, GC-MS/MS (chemical-analytical), and ELISA (immuno-analytical) test methods for screening and quantitation of the target steroid hormones in both water and wastewater matrix. Internationally, the observed sensitivity, based on LOQ (ng/L), for the steroid estrogens E1, E2, EE2, is, in decreasing order: LC-MSMS (0.08-9.54) > GC-MS (1) > ELISA (5) (chemical-analytical > immuno-analytical). At the national level, the routine, unoptimized chemical-analytical LC-MSMS method was found to lack the required sensitivity for meeting environmental requirements for steroid hormone quantitation. Further optimization of the sensitivity of the chemical-analytical LC-tandem mass spectrometry methods, especially for wastewater screening, in South Africa is required. Risk assessment studies showed that it was not practical to propose standards or allowable limits for the steroid estrogens E1, E2, EE2, and E3; the use of predicted-no-effect concentration values of the steroid estrogens appears to be appropriate for use in their risk assessment in relation to aquatic organisms. For raw water sources, drinking water, raw and treated wastewater, the use of bioassays, with trigger values, is a useful screening tool option to decide whether further examination of specific endocrine activity may be warranted, or whether concentrations of such activity are of low priority, with respect to health concerns in the human population. The achievement of improved quantitation limits for immuno-analytical methods, like ELISA, used for compound quantitation, and standardization of the method for measuring E2 equivalents (EEQs) used for biological activity (endocrine: e.g., estrogenic) are some areas for future EDC research.

Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides.

PubMed

Mishima, Eikan; Jinno, Daisuke; Akiyama, Yasutoshi; Itoh, Kunihiko; Nankumo, Shinnosuke; Shima, Hisato; Kikuchi, Koichi; Takeuchi, Yoichi; Elkordy, Alaa; Suzuki, Takehiro; Niizuma, Kuniyasu; Ito, Sadayoshi; Tomioka, Yoshihisa; Abe, Takaaki

2015-01-01

The biological roles of RNA modifications are still largely not understood. Thus, developing a method for detecting RNA modifications is important for further clarification. We developed a method for detecting RNA modifications called immuno-northern blotting (INB) analysis and herein introduce its various capabilities. This method involves the separation of RNAs using either polyacrylamide or agarose gel electrophoresis, followed by transfer onto a nylon membrane and subsequent immunoblotting using antibodies against modified nucleosides for the detection of specific modifications. We confirmed that INB with the antibodies for 1-methyladenosine (m1A), N6-methyladenosine (m6A), pseudouridine, and 5-methylcytidine (m5C) showed different modifications in a variety of RNAs from various species and organelles. INB with the anti-m5C antibody revealed that the antibody cross-reacted with another modification on DNA, suggesting the application of this method for characterization of the antibody for modified nucleosides. Additionally, using INB with the antibody for m1A, which is a highly specific modification in eukaryotic tRNA, we detected tRNA-derived fragments known as tiRNAs under the cellular stress response, suggesting the application for tracking target RNA containing specific modifications. INB with the anti-m6A antibody confirmed the demethylation of m6A by the specific demethylases fat mass and obesity-associated protein (FTO) and ALKBH5, suggesting its application for quantifying target modifications in separated RNAs. Furthermore, INB demonstrated that the knockdown of FTO and ALKBH5 increased the m6A modification in small RNAs as well as in mRNA. The INB method has high specificity, sensitivity, and quantitative capability, and it can be employed with conventional experimental apparatus. Therefore, this method would be useful for research on RNA modifications and metabolism.

Immuno-oncology Clinical Trial Design: Limitations, Challenges, and Opportunities

PubMed Central

Baik, Christina S.; Rubin, Eric H.; Forde, Patrick M.; Mehnert, Janice M.; Collyar, Deborah; Butler, Marcus O.; Dixon, Erica L.; Chow, Laura Q.M.

2017-01-01

Recent advances in immuno-oncology and regulatory approvals have been rapid and paradigm shifting in many difficult-to-treat malignancies. Despite immune checkpoint inhibitor therapy becoming the standard of care across multiple tumor types, there are many unanswered questions that need to be addressed before this therapeutic modality can be fully harnessed. Areas of limitations include treatment of patients not sufficiently represented in clinical trials, uncertainty of the optimal treatment dosing and duration, and lack of understanding regarding long-term immune related toxicities and atypical tumor responses. Patients such as those with autoimmune disease, chronic viral infections, limited performance status, and brain metastases were often excluded from initial trials due to concerns of safety. However, limited data suggest that some of these patients can benefit from therapy with manageable toxicities; thus, future studies should incorporate these patients to clearly define safety and efficacy. There are still controversies regarding the optimal dosing strategy that can vary from weight-based to flat dosing, with undefined treatment duration. Further elucidation of the optimal dosing approach and evaluation of predictive biomarkers should be incorporated in the design of future trials. Finally, there are long-term immune-mediated toxicities, atypical tumor responses such as pseudoprogression and endpoints unique to immuno-oncology that are not adequately captured by traditional trial designs; thus, novel study designs are needed. In this article, we discuss in detail the above challenges and propose needed areas of research for exploration and incorporation in the next generation of immuno-oncology clinical trials. PMID:28864727

Search for Allergens from the Pollen Proteome of Sunflower (Helianthus annuus L.): A Major Sensitizer for Respiratory Allergy Patients.

PubMed

Ghosh, Nandini; Sircar, Gaurab; Saha, Bodhisattwa; Pandey, Naren; Gupta Bhattacharya, Swati

2015-01-01

Respiratory allergy triggered by pollen allergens is increasing at an alarming rate worldwide. Sunflower pollen is thought to be an important source of inhalant allergens. Present study aims to identify the prevalence of sunflower pollinosis among the Indian allergic population and characterizes the pollen allergens using immuno-proteomic tools. Clinico-immunological tests were performed to understand the prevalence of sensitivity towards sunflower pollen among the atopic population. Sera from selected sunflower positive patients were used as probe to detect the IgE-reactive proteins from the one and two dimensional electrophoretic separated proteome of sunflower pollen. The antigenic nature of the sugar moiety of the glycoallergens was studied by meta-periodate modification of IgE-immunoblot. Finally, these allergens were identified by mass-spectrometry. Prevalence of sunflower pollen sensitization was observed among 21% of the pollen allergic population and associated with elevated level of specific IgE and histamine in the sera of these patients. Immunoscreening of sunflower pollen proteome with patient sera detected seven IgE-reactive proteins with varying molecular weight and pI. Hierarchical clustering of 2D-immunoblot data highlighted three allergens characterized by a more frequent immuno-reactivity and increased levels of IgE antibodies in the sera of susceptible patients. These allergens were considered as the major allergens of sunflower pollen and were found to have their glycan moiety critical for inducing IgE response. Homology driven search of MS/MS data of these IgE-reactive proteins identified seven previously unreported allergens from sunflower pollen. Three major allergenic proteins were identified as two pectate lyases and a cysteine protease. Novelty of the present report is the identification of a panel of seven sunflower pollen allergens for the first time at immuno-biochemical and proteomic level, which substantiated the clinical evidence of sunflower allergy. Further purification and recombinant expression of these allergens will improve component-resolved diagnosis and therapy of pollen allergy.

Evaluation of different immuno-magnetic beads and selective plating media for the confirmation of Shiga toxin-producing Escherichia coli detected by molecular methods in beef trim enrichment broths

USDA-ARS?s Scientific Manuscript database

Introduction: Shiga toxin-producing E. coli of serogroups O26, O45, O103, O111, O121 and O145 are a testing concern for the beef industry and regulators. Numerous tests are available that attempt to predict the presence of these pathogens. However, potential positive results require culture confir...

2005 6th Annual Science and Engineering Technology Conference

DTIC Science & Technology

2005-04-21

BioFAC VBAIDS Hybrid: PCR/Immuno Fast PCR Fast Immunoassay Mass Spec (Pyrolysis) SIBS UV -LIF IR Fluorochrome Charge Detect. BioCADS Trigger Advanced...Weights Beam forming Signal Processing mapped to GPU architecture Vector Processor STAP (STAP-BOY) GaN High Frequency Transistor (WBG-RF) UV Laser...Service anti- counterfeiting • Embedded security strips Technology Limitations and Barriers • Training and cost (training intensive) Land Borders North Land

Direct Detection of T- and B-Memory Lymphocytes by ImmunoSpot® Assays Reveals HCMV Exposure that Serum Antibodies Fail to Identify.

PubMed

Terlutter, Fredrik; Caspell, Richard; Nowacki, Tobias M; Lehmann, Alexander; Li, Ruliang; Zhang, Ting; Przybyla, Anna; Kuerten, Stefanie; Lehmann, Paul V

2018-05-19

It is essential to identify donors who have not been infected with human cytomegalovirus (HCMV) in order to avoid transmission of HCMV to recipients of blood transfusions or organ transplants. In the present study, we tested the reliability of seronegativity as an indicator for the lack of HCMV exposure in healthy human blood donors. Eighty-two HCMV seronegative individuals were identified, and their peripheral blood mononuclear cells (PBMC) were tested in ImmunoSpot® assays for the presence of HCMV-specific T- and B-memory lymphocytes. Eighty-two percent (67 of 82) of these HCMV seronegative individuals featured at least one memory cell that was lineage specific for HCMV, with the majority of these subjects possessing CD4+ and CD8+ T cells, as well as B cells, providing three independent lines of evidence for having developed immunity to HCMV. Only 15 of these 82 donors (18%) showed neither T- nor B-cell memory to HCMV, consistent with immunological naïveté to the virus. The data suggest that measurements of serum antibodies frequently fail to reveal HCMV exposure in humans, which may be better identified by direct detection of HCMV-specific memory lymphocytes.

Use of hydrophilic extra-viral domain of canine distemper virus H protein for enzyme-linked immunosorbent assay development.

PubMed

Cho, Ki-hyun; Kim, Jeongmi; Yoo, Hyun-ah; Kim, Dae-hee; Park, Seung-yong; Song, Chang-seon; Choi, In-soo; Lee, Joong-bok

2014-12-01

Simple methods for measuring the levels of serum antibody against canine distemper virus (CDV) would assist in the effective vaccination of dogs. To develop an enzyme-linked immunosorbent assay (ELISA) specific for CDV, we expressed hydrophilic extra-viral domain (HEVD) protein of the A75/17-CDV H gene in a pET 28a plasmid-based Escherichia (E.) coli vector system. Expression was confirmed by dot and Western blotting. We proposed that detection of E. coli-expressed H protein might be conformation- dependent because intensities of the reactions observed with these two methods varied. The H gene HEVD protein was further purified and used as an antigen for an ELISA. Samples from dogs with undetectable to high anti-CDV antibody titers were analyzed using this HEVD-specific ELISA and a commercial CDV antibody detection kit (ImmunoComb). Levels of HEVD antigenicity measured with the assays and immunochromatography correlated. These data indicated that the HEDV protein may be used as antigen to develop techniques for detecting antibodies against CDV.

Nanocolloidal gold-based immuno-dip strip assay for rapid detection of Sudan red I in food samples.

PubMed

Wang, Jia; Wang, Zhanhui; Liu, Jing; Li, Hao; Li, Qing X; Li, Ji; Xu, Ting

2013-02-15

A semiquantitative dip strip assay was developed using nanocolloidal gold-labelled monoclonal antibody (Mab) 8A10 for the rapid detection of Sudan red I in food samples. A protein-Sudan red I conjugate was coated on a nitro cellulose membrane strip in a defined test line. In flow of the complex of nanocolloidal gold labelled-Mab and Sudan red I along the strip, intensive red colour that was formed in the test line reflected the Sudan red I concentration. The test required 10 min and had a visual limit of detection of 10 ng/g Sudan red I in tomato sauce and chilli powder samples. The results of the strip assay agreed well with those of a high performance liquid chromatography method for both spiked and real commercial samples. The strip was stable for at least 2 months at 4°C. The strip assay offers the potential as a useful rapid and simple method for screening of Sudan red I in food samples. Copyright © 2012 Elsevier Ltd. All rights reserved.

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

PubMed Central

Yamada, Norishige; Ogawa, Akiyo; Ogawa, Yuya

2014-01-01

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell level to detect the spatial dynamics of RNA localization with simultaneous insight into the localization of proteins, epigenetic modifications and other details which can be highlighted by immunofluorescence. X-chromosome inactivation is a paradigm for long non-coding RNA (lncRNA)-mediated gene silencing. X-inactive specific transcript (Xist) lncRNA accumulation (called an Xist cloud) on one of the two X-chromosomes in mammalian females is a critical step to initiate X-chromosome inactivation. Xist RNA directly or indirectly interacts with various chromatin-modifying enzymes and introduces distinct epigenetic landscapes to the inactive X-chromosome (Xi). One known epigenetic hallmark of the Xi is the Histone H3 trimethyl-lysine 27 (H3K27me3) modification. Here, we describe a simple and quick immuno-FISH protocol for detecting Xist RNA using RNA FISH with multiple oligonucleotide probes coupled with immunofluorescence of H3K27me3 to examine the localization of Xist RNA and associated epigenetic modifications. Using oligonucleotide probes results in a shorter incubation time and more sensitive detection of Xist RNA compared to in vitro transcribed RNA probes (riboprobes). This protocol provides a powerful tool for understanding the dynamics of lncRNAs and its associated epigenetic modification, chromatin structure, nuclear organization and transcriptional regulation. PMID:25489864

Immuno capture PCR for rapid and sensitive identification of pathogenic Bacillus anthracis.

PubMed

Makam, Shivakiran S; Majumder, Saugata; Kingston, Joseph J; Urs, Radhika M; Tuteja, Urmil; Sripathi, Murali H; Batra, Harsh V

2013-12-01

Immuno capture PCR (IPCR) is a technique capable of detecting the pathogens with high specificity and sensitivity. Rapid and accurate detection of Bacillus anthracis was achieved using anti-EA1 antibodies to capture the cells and two primer sets targeting the virulence factors of the pathogen i.e., protective antigen (pag) and capsule (cap) in an IPCR format. Monoclonal antibodies specific to B. anthracis were generated against extractable antigen 1 protein and used as capture antibody onto 96 well polystyrene plates. Following the binding of the pathogen, the DNA extraction was carried out in the well itself and further processed for PCR assay. We compared IPCR described here with conventional duplex PCR using the same primers and sandwich ELISA using the monoclonal antibodies developed in the present study. IPCR was capable of detecting as few as 10 and 100 cfu ml⁻¹ of bacterial cells and spores, respectively. IPCR was found to be 2-3 logs more sensitive than conventional duplex PCR and the sandwich ELISA. The effect of other bacteria and any organic materials on IPCR was also analyzed and found that this method was robust with little change in the sensitivity in the presence of interfering agents. Moreover, we could demonstrate a simple process of microwave treatment for spore disruption which otherwise are resistant to chemical treatments. Also, the IPCR could clearly distinguish the pathogenic and nonpathogenic strains of B. anthracis in the same assay. This can help in saving resources on unnecessary decontamination procedures during false alarms.

Immuno and Affinity Cytochemical Analysis of Cell Wall Composition in the Moss Physcomitrella patens.

PubMed

Berry, Elizabeth A; Tran, Mai L; Dimos, Christos S; Budziszek, Michael J; Scavuzzo-Duggan, Tess R; Roberts, Alison W

2016-01-01

In contrast to homeohydric vascular plants, mosses employ a poikilohydric strategy for surviving in the dry aerial environment. A detailed understanding of the structure, composition, and development of moss cell walls can contribute to our understanding of not only the evolution of overall cell wall complexity, but also the differences that have evolved in response to selection for different survival strategies. The model moss species Physcomitrella patens has a predominantly haploid lifecycle consisting of protonemal filaments that regenerate from protoplasts and enlarge by tip growth, and leafy gametophores composed of cells that enlarge by diffuse growth and differentiate into several different types. Advantages for genetic studies include methods for efficient targeted gene modification and extensive genomic resources. Immuno and affinity cytochemical labeling were used to examine the distribution of polysaccharides and proteins in regenerated protoplasts, protonemal filaments, rhizoids, and sectioned gametophores of P. patens. The cell wall composition of regenerated protoplasts was also characterized by flow cytometry. Crystalline cellulose was abundant in the cell walls of regenerating protoplasts and protonemal cells that developed on media of high osmolarity, whereas homogalactuonan was detected in the walls of protonemal cells that developed on low osmolarity media and not in regenerating protoplasts. Mannan was the major hemicellulose detected in all tissues tested. Arabinogalactan proteins were detected in different cell types by different probes, consistent with structural heterogneity. The results reveal developmental and cell type specific differences in cell wall composition and provide a basis for analyzing cell wall phenotypes in knockout mutants.

Nano-biosensors to detect beta-amyloid for Alzheimer's disease management.

PubMed

Kaushik, Ajeet; Jayant, Rahul Dev; Tiwari, Sneham; Vashist, Arti; Nair, Madhavan

2016-06-15

Beta-amyloid (β-A) peptides are potential biomarkers to monitor Alzheimer's diseases (AD) for diagnostic purposes. Increased β-A level is neurotoxic and induces oxidative stress in brain resulting in neurodegeneration and causes dementia. As of now, no sensitive and inexpensive method is available for β-A detection under physiological and pathological conditions. Although, available methods such as neuroimaging, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) detect β-A, but they are not yet extended at point-of-care (POC) due to sophisticated equipments, need of high expertize, complicated operations, and challenge of low detection limit. Recently, β-A antibody based electrochemical immuno-sensing approach has been explored to detect β-A at pM levels within 30-40 min compared to 6-8h of ELISA test. The introduction of nano-enabling electrochemical sensing technology could enable rapid detection of β-A at POC and may facilitate fast personalized health care delivery. This review explores recent advancements in nano-enabling electrochemical β-A sensing technologies towards POC application to AD management. These analytical tools can serve as an analytical tool for AD management program to obtain bio-informatics needed to optimize therapeutics for neurodegenerative diseases diagnosis management. Copyright © 2016 Elsevier B.V. All rights reserved.

Rapid electrochemical detection of polyaniline-labeled Escherichia coli O157:H7.

PubMed

Setterington, Emma B; Alocilja, Evangelyn C

2011-01-15

There is a high demand for rapid, sensitive, and field-ready detection methods for Escherichia coli O157:H7, a highly infectious and potentially fatal food and water borne pathogen. In this study, E. coli O157:H7 cells are isolated via immunomagnetic separation (IMS) and labeled with biofunctionalized electroactive polyaniline (immuno-PANI). Labeled cell complexes are deposited onto a disposable screen-printed carbon electrode (SPCE) sensor and pulled to the electrode surface by an external magnetic field, to amplify the electrochemical signal generated by the polyaniline. Cyclic voltammetry is used to detect polyaniline and signal magnitude indicates the presence or absence of E. coli O157:H7. As few as 7CFU of E. coli O157:H7 (corresponding to an original concentration of 70 CFU/ml) were successfully detected on the SPCE sensor. The assay requires 70 min from sampling to detection, giving it a major advantage over standard culture methods in applications requiring high-throughput screening of samples and rapid results. The method can be performed with portable, handheld instrumentation and no biological modification of the sensor surface is required. Potential applications include field-based pathogen detection for food and water safety, environmental monitoring, healthcare, and biodefense. Copyright © 2010 Elsevier B.V. All rights reserved.

Protein detection by Simple Western™ analysis.

PubMed

Harris, Valerie M

2015-01-01

Protein Simple© has taken a well-known protein detection method, the western blot, and revolutionized it. The Simple Western™ system uses capillary electrophoresis to identify and quantitate a protein of interest. Protein Simple© provides multiple detection apparatuses (Wes, Sally Sue, or Peggy Sue) that are suggested to save scientists valuable time by allowing the researcher to prepare the protein sample, load it along with necessary antibodies and substrates, and walk away. Within 3-5 h the protein will be separated by size, or charge, immuno-detection of target protein will be accurately quantitated, and results will be immediately made available. Using the Peggy Sue instrument, one study recently examined changes in MAPK signaling proteins in the sex-determining stage of gonadal development. Here the methodology is described.

What are the reasons for low use of graphene quantum dots in immunosensing of cancer biomarkers?

PubMed

Hasanzadeh, Mohammad; Shadjou, Nasrin

2017-02-01

Graphene quantum dots-based immunosensors have recently gained importance for detecting antigens and biomarkers responsible for cancer diagnosis. This paper reports a literature survey of the applications of graphene quantum dots for sensing cancer biomarkers. The survey sought to explore three questions: (1) Do graphene quantum dots improve immunosensing technology? (2) If so, can graphene quantum dots have a critical, positive impact on construction of immuno-devices? And (3) What is the reason for some troubles in the application of this technology? The number of published papers in the field seems positively answer the first two questions. However additional efforts must be made to move from the bench to the real diagnosis. Some approaches to improve the analytical performance of graphene quantum dots-based immunosensors through their figures of merit have been also discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

Age-Related Differences in Influenza B Infection by Lineage in a Community-Based Sentinel System, 2010-2011 to 2015-2016, Canada.

PubMed

Skowronski, Danuta M; Chambers, Catharine; De Serres, Gaston; Sabaiduc, Suzana; Winter, Anne-Luise; Dickinson, James A; Gubbay, Jonathan B; Fonseca, Kevin; Drews, Steven J; Charest, Hugues; Martineau, Christine; Krajden, Mel; Petric, Martin; Bastien, Nathalie; Li, Yan

2017-09-15

Age-related differences in influenza B lineage detection were explored in the community-based Canadian Sentinel Practitioner Surveillance Network (SPSN) from 2010-2011 to 2015-2016. Whereas >80% of B(Victoria) cases were <40 years old, B(Yamagata) cases showed a bimodal age distribution with 27% who were <20 years old and 61% who were 30-64 years old, but with a notable gap in cases between 20 and 29 years old (4%). Overall, the median age was 20 years lower for B(Victoria) vs B(Yamagata) cases (20 vs 40 years; P < .01). Additional phylodynamic and immuno-epidemiological research is needed to understand age-related variation in influenza B risk by lineage, with potential implications for prevention and control across the lifespan. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

A study on real-time low-quality content detection on Twitter from the users' perspective.

PubMed

Chen, Weiling; Yeo, Chai Kiat; Lau, Chiew Tong; Lee, Bu Sung

2017-01-01

Detection techniques of malicious content such as spam and phishing on Online Social Networks (OSN) are common with little attention paid to other types of low-quality content which actually impacts users' content browsing experience most. The aim of our work is to detect low-quality content from the users' perspective in real time. To define low-quality content comprehensibly, Expectation Maximization (EM) algorithm is first used to coarsely classify low-quality tweets into four categories. Based on this preliminary study, a survey is carefully designed to gather users' opinions on different categories of low-quality content. Both direct and indirect features including newly proposed features are identified to characterize all types of low-quality content. We then further combine word level analysis with the identified features and build a keyword blacklist dictionary to improve the detection performance. We manually label an extensive Twitter dataset of 100,000 tweets and perform low-quality content detection in real time based on the characterized significant features and word level analysis. The results of our research show that our method has a high accuracy of 0.9711 and a good F1 of 0.8379 based on a random forest classifier with real time performance in the detection of low-quality content in tweets. Our work therefore achieves a positive impact in improving user experience in browsing social media content.

A study on real-time low-quality content detection on Twitter from the users’ perspective

PubMed Central

Yeo, Chai Kiat; Lau, Chiew Tong; Lee, Bu Sung

2017-01-01

Detection techniques of malicious content such as spam and phishing on Online Social Networks (OSN) are common with little attention paid to other types of low-quality content which actually impacts users’ content browsing experience most. The aim of our work is to detect low-quality content from the users’ perspective in real time. To define low-quality content comprehensibly, Expectation Maximization (EM) algorithm is first used to coarsely classify low-quality tweets into four categories. Based on this preliminary study, a survey is carefully designed to gather users’ opinions on different categories of low-quality content. Both direct and indirect features including newly proposed features are identified to characterize all types of low-quality content. We then further combine word level analysis with the identified features and build a keyword blacklist dictionary to improve the detection performance. We manually label an extensive Twitter dataset of 100,000 tweets and perform low-quality content detection in real time based on the characterized significant features and word level analysis. The results of our research show that our method has a high accuracy of 0.9711 and a good F1 of 0.8379 based on a random forest classifier with real time performance in the detection of low-quality content in tweets. Our work therefore achieves a positive impact in improving user experience in browsing social media content. PMID:28793347

Radiofrequency ablation of liver tumors in combination with local OK-432 injection prolongs survival and suppresses distant tumor growth in the rabbit model with intra- and extrahepatic VX2 tumors.

PubMed

Kageyama, Ken; Yamamoto, Akira; Okuma, Tomohisa; Hamamoto, Shinichi; Takeshita, Toru; Sakai, Yukimasa; Nishida, Norifumi; Matsuoka, Toshiyuki; Miki, Yukio

2013-10-01

To evaluate survival and distant tumor growth after radiofrequency ablation (RFA) and local OK-432 injection at a single tumor site in a rabbit model with intra- and extrahepatic VX2 tumors and to examine the effect of this combination therapy, which we termed immuno-radiofrequency ablation (immunoRFA), on systemic antitumor immunity in a rechallenge test. Our institutional animal care committee approved all experiments. VX2 tumors were implanted to three sites: two in the liver and one in the left ear. Rabbits were randomized into four groups of seven to receive control, RFA alone, OK-432 alone, and immunoRFA treatments at a single liver tumor at 1 week after implantation. Untreated liver and ear tumor volumes were measured after the treatment. As the rechallenge test, tumors were reimplanted into the right ear of rabbits, which survived the 35 weeks and were followed up without additional treatment. Statistical significance was examined by log-rank test for survival and Student's t test for tumor volume. Survival was significantly prolonged in the immunoRFA group compared to the other three groups (P < 0.05). Untreated liver and ear tumor sizes became significantly smaller after immunoRFA compared to controls (P < 0.05). In the rechallenge test, the reimplanted tumors regressed without further therapy compared to the ear tumors of the control group (P < 0.05). ImmunoRFA led to improved survival and suppression of distant untreated tumor growth. Decreases in size of the distant untreated tumors and reimplanted tumors suggested that systemic antitumor immunity was enhanced by immunoRFA.

Radiofrequency Ablation of Liver Tumors in Combination with Local OK-432 Injection Prolongs Survival and Suppresses Distant Tumor Growth in the Rabbit Model with Intra- and Extrahepatic VX2 Tumors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kageyama, Ken, E-mail: kageyamaken0112@gmail.com; Yamamoto, Akira, E-mail: loveakirayamamoto@gmail.com; Okuma, Tomohisa, E-mail: o-kuma@msic.med.osaka-cu.ac.jp

Purpose: To evaluate survival and distant tumor growth after radiofrequency ablation (RFA) and local OK-432 injection at a single tumor site in a rabbit model with intra- and extrahepatic VX2 tumors and to examine the effect of this combination therapy, which we termed immuno-radiofrequency ablation (immunoRFA), on systemic antitumor immunity in a rechallenge test. Methods: Our institutional animal care committee approved all experiments. VX2 tumors were implanted to three sites: two in the liver and one in the left ear. Rabbits were randomized into four groups of seven to receive control, RFA alone, OK-432 alone, and immunoRFA treatments at amore » single liver tumor at 1 week after implantation. Untreated liver and ear tumor volumes were measured after the treatment. As the rechallenge test, tumors were reimplanted into the right ear of rabbits, which survived the 35 weeks and were followed up without additional treatment. Statistical significance was examined by log-rank test for survival and Student's t test for tumor volume. Results: Survival was significantly prolonged in the immunoRFA group compared to the other three groups (P < 0.05). Untreated liver and ear tumor sizes became significantly smaller after immunoRFA compared to controls (P < 0.05). In the rechallenge test, the reimplanted tumors regressed without further therapy compared to the ear tumors of the control group (P < 0.05). Conclusion: ImmunoRFA led to improved survival and suppression of distant untreated tumor growth. Decreases in size of the distant untreated tumors and reimplanted tumors suggested that systemic antitumor immunity was enhanced by immunoRFA.« less

Immunoconjugates and long circulating systems: Origins, current state of the art and future directions☆

PubMed Central

Koshkaryev, Alexander; Sawant, Rupa; Deshpande, Madhura; Torchilin, Vladimir

2012-01-01

Significant progress has been made recently in the area of immunoconjugated drugs and drug delivery systems (DDS). The immuno-modification of either the drug or DDS has proven to be a very promising approach that has significantly improved the targeted accumulation in pathological sites while decreasing its undesirable side effects in healthy tissues. The arrangement for both prolonged life in the circulation and specific target recognition represents another potent strategy in the development of immuno-targeted systems. The longevity of immuno-targeted DDS such as immunoliposomes and immunomicelles improves their targetability even in the presence of the additional passive accumulation in areas with a compromised vasculature. The added use of the immuno-targeted systems takes advantage of the specific microenvironment of pathological sites including lowered pH, increased temperature, and variation in the enzymatic activity. “Smart” stimulus-responsive systems combine different valuable functionalities including PEG-protection, targeting antibody, cell-penetration, and stimulus-sensitive functions. In this review we examined the evolution, current status and future directions in the area of therapeutical immunoconjugates and long-circulating immuno-targeted DDS. PMID:22964425

Selection of specific inhibitor peptides in enzyme-linked immunosorbent assay (ELISA) of cardiac troponin I using immuno-dominant epitopes as competitor.

PubMed

Rezaee, Majid Asiabanha; Rasaee, Mohammad Javad; Mohammadnejad, Javad

2017-01-01

Human cardiac troponin I (cTni) is the gold marker for early diagnosis of myocardial infarction. In this regard, four immune-dominant epitopes of cTni were predicted and their 3D structures were determined. Thereafter, the competitive performance of the peptides was monitored with the developed polyclonal antibody-based indirect competitive ELISA; a half-maximal inhibitory concentration (IC50) of 0.49 (µg/mL) and detection limit of 0.037 (µg/mL) were achieved for recombinant cTni. The competitive ELISA determined sensitivity levels of 0.306, 0.141, 0.960, and 0.155 (µg/mL), respectively, for each peptide as competitor. We indicated that two of the selected epitopes have significant sensitivity scales and inhibition ability.

Robust video copy detection approach based on local tangent space alignment

NASA Astrophysics Data System (ADS)

Nie, Xiushan; Qiao, Qianping

2012-04-01

We propose a robust content-based video copy detection approach based on local tangent space alignment (LTSA), which is an efficient dimensionality reduction algorithm. The idea is motivated by the fact that the content of video becomes richer and the dimension of content becomes higher. It does not give natural tools for video analysis and understanding because of the high dimensionality. The proposed approach reduces the dimensionality of video content using LTSA, and then generates video fingerprints in low dimensional space for video copy detection. Furthermore, a dynamic sliding window is applied to fingerprint matching. Experimental results show that the video copy detection approach has good robustness and discrimination.

Multiplexing T- and B-Cell FLUOROSPOT Assays: Experimental Validation of the Multi-Color ImmunoSpot® Software Based on Center of Mass Distance Algorithm.

PubMed

Karulin, Alexey Y; Megyesi, Zoltán; Caspell, Richard; Hanson, Jodi; Lehmann, Paul V

2018-01-01

Over the past decade, ELISPOT has become a highly implemented mainstream assay in immunological research, immune monitoring, and vaccine development. Unique single cell resolution along with high throughput potential sets ELISPOT apart from flow cytometry, ELISA, microarray- and bead-based multiplex assays. The necessity to unambiguously identify individual T and B cells that do, or do not co-express certain analytes, including polyfunctional cytokine producing T cells has stimulated the development of multi-color ELISPOT assays. The success of these assays has also been driven by limited sample/cell availability and resource constraints with reagents and labor. There are few commercially available test kits and instruments available at present for multi-color FLUOROSPOT. Beyond commercial descriptions of competing systems, little is known about their accuracy in experimental settings detecting individual cells that secrete multiple analytes vs. random overlays of spots. Here, we present a theoretical and experimental validation study for three and four color T- and B-cell FLUOROSPOT data analysis. The ImmunoSpot ® Fluoro-X™ analysis system we used includes an automatic image acquisition unit that generates individual color images free of spectral overlaps and multi-color spot counting software based on the maximal allowed distance between centers of spots of different colors or Center of Mass Distance (COMD). Using four color B-cell FLUOROSPOT for IgM, IgA, IgG1, IgG3; and three/four color T-cell FLUOROSPOT for IL-2, IFN-γ, TNF-α, and GzB, in serial dilution experiments, we demonstrate the validity and accuracy of Fluoro-X™ multi-color spot counting algorithms. Statistical predictions based on the Poisson spatial distribution, coupled with scrambled image counting, permit objective correction of true multi-color spot counts to exclude randomly overlaid spots.

Epithelium percentage estimation facilitates epithelial quantitative protein measurement in tissue specimens.

PubMed

Chen, Jing; Toghi Eshghi, Shadi; Bova, George Steven; Li, Qing Kay; Li, Xingde; Zhang, Hui

2013-12-01

The rapid advancement of high-throughput tools for quantitative measurement of proteins has demonstrated the potential for the identification of proteins associated with cancer. However, the quantitative results on cancer tissue specimens are usually confounded by tissue heterogeneity, e.g. regions with cancer usually have significantly higher epithelium content yet lower stromal content. It is therefore necessary to develop a tool to facilitate the interpretation of the results of protein measurements in tissue specimens. Epithelial cell adhesion molecule (EpCAM) and cathepsin L (CTSL) are two epithelial proteins whose expressions in normal and tumorous prostate tissues were confirmed by measuring staining intensity with immunohistochemical staining (IHC). The expressions of these proteins were measured by ELISA in protein extracts from OCT embedded frozen prostate tissues. To eliminate the influence of tissue heterogeneity on epithelial protein quantification measured by ELISA, a color-based segmentation method was developed in-house for estimation of epithelium content using H&E histology slides from the same prostate tissues and the estimated epithelium percentage was used to normalize the ELISA results. The epithelium contents of the same slides were also estimated by a pathologist and used to normalize the ELISA results. The computer based results were compared with the pathologist's reading. We found that both EpCAM and CTSL levels, measured by ELISA assays itself, were greatly affected by epithelium content in the tissue specimens. Without adjusting for epithelium percentage, both EpCAM and CTSL levels appeared significantly higher in tumor tissues than normal tissues with a p value less than 0.001. However, after normalization by the epithelium percentage, ELISA measurements of both EpCAM and CTSL were in agreement with IHC staining results, showing a significant increase only in EpCAM with no difference in CTSL expression in cancer tissues. These results were obtained with normalization by both the computer estimated and pathologist estimated epithelium percentage. Our results show that estimation of tissue epithelium percentage using our color-based segmentation method correlates well with pathologists' estimation of tissue epithelium percentages. The epithelium contents estimated by color-based segmentation may be useful in immuno-based analysis or clinical proteomic analysis of tumor proteins. The codes used for epithelium estimation as well as the micrographs with estimated epithelium content are available online.

Direct Detection of Soil-Bound Prions

PubMed Central

Genovesi, Sacha; Leita, Liviana; Sequi, Paolo; Andrighetto, Igino; Sorgato, M. Catia; Bertoli, Alessandro

2007-01-01

Scrapie and chronic wasting disease are contagious prion diseases affecting sheep and cervids, respectively. Studies have indicated that horizontal transmission is important in sustaining these epidemics, and that environmental contamination plays an important role in this. In the perspective of detecting prions in soil samples from the field by more direct methods than animal-based bioassays, we have developed a novel immuno-based approach that visualises in situ the major component (PrPSc) of prions sorbed onto agricultural soil particles. Importantly, the protocol needs no extraction of the protein from soil. Using a cell-based assay of infectivity, we also report that samples of agricultural soil, or quartz sand, acquire prion infectivity after exposure to whole brain homogenates from prion-infected mice. Our data provide further support to the notion that prion-exposed soils retain infectivity, as recently determined in Syrian hamsters intracerebrally or orally challanged with contaminated soils. The cell approach of the potential infectivity of contaminated soil is faster and cheaper than classical animal-based bioassays. Although it suffers from limitations, e.g. it can currently test only a few mouse prion strains, the cell model can nevertheless be applied in its present form to understand how soil composition influences infectivity, and to test prion-inactivating procedures. PMID:17957252

In vivo detection of free radicals using molecular MRI and immuno-spin trapping in a mouse model for amyotrophic lateral sclerosis.

PubMed

Towner, Rheal A; Smith, Nataliya; Saunders, Debra; Lupu, Florea; Silasi-Mansat, Robert; West, Melinda; Ramirez, Dario C; Gomez-Mejiba, Sandra E; Bonini, Marcelo G; Mason, Ronald P; Ehrenshaft, Marilyn; Hensley, Kenneth

2013-10-01

Free radicals associated with oxidative stress play a major role in amyotrophic lateral sclerosis (ALS). By combining immuno-spin trapping and molecular magnetic resonance imaging, in vivo trapped radical adducts were detected in the spinal cords of SOD1(G93A)-transgenic (Tg) mice, a model for ALS. For this study, the nitrone spin trap DMPO (5,5-dimethyl-1-pyrroline N-oxide) was administered (ip) over 5 days before administration (iv) of an anti-DMPO probe (anti-DMPO antibody covalently bound to an albumin-gadolinium-diethylenetriamine pentaacetic acid-biotin MRI contrast agent) to trap free radicals. MRI was used to detect the presence of the anti-DMPO radical adducts by a significant sustained increase in MR signal intensities (p < 0.05) or anti-DMPO probe concentrations measured from T₁ relaxations (p < 0.01). The biotin moiety of the anti-DMPO probe was targeted with fluorescence-labeled streptavidin to locate the probe in excised tissues. Negative controls included either Tg ALS mice initially administered saline rather than DMPO followed by the anti-DMPO probe or non-Tg mice initially administered DMPO and then the anti-DMPO probe. The anti-DMPO probe was found to bind to neurons via colocalization fluorescence microscopy. DMPO adducts were also confirmed in diseased/nondiseased tissues from animals administered DMPO. Apparent diffusion coefficients from diffusion-weighted images of spinal cords from Tg mice were significantly elevated (p < 0.001) compared to wild-type controls. This is the first report regarding the detection of in vivo trapped radical adducts in an ALS model. This novel, noninvasive, in vivo diagnostic method can be applied to investigate the involvement of free radical mechanisms in ALS rodent models. Copyright © 2013 Elsevier Inc. All rights reserved.

Development and evaluation of IgY ImmunoCapture PCR ELISA for detection of Staphylococcus aureus enterotoxin A devoid of protein A interference.

PubMed

Reddy, Prakash; Ramlal, Shylaja; Sripathy, Murali Harishchandra; Batra, Harsh Vardhan

2014-06-01

In the present study, a sensitive and specific IgY mediated ImmunoCapture-PCR-ELISA (IC-PCR-ELISA) was developed for the detection of staphylococcal enterotoxin A (SEA) from culture supernatants and suspected contaminated samples. Due to the virtue of avian immunoglobulins (IgY) to have the least affinity towards staphylococcal protein A (SpA) responsible for false positives, we employed anti-SEA IgY for capture of SEA toxin and revealed with SEA specific rabbit antibodies conjugated to a 524bp DNA marker. Biotin-11-dUTP was incorporated during PCR amplification and post PCR analysis was performed by PCR-ELISA. Unlike IgG immunocapture, IgY mediated immunocapture of SEA was free from false positives due to protein A. The developed assay was specific to SEA except for minor cross reactivity with staphylococcal enterotoxin E (SEE). Several raw milk samples were evaluated for the presence of SEA with and without enrichment. Three samples were found to be positive for SEA after enrichment for 8h. Though IC-PCR-ELISA for SEA showed 100% correlation with PCR analysis for sea gene, the assay was unique in terms of sensitivity of detecting ~10pg/ml of SEA toxin from spiked milk samples. Result of IC-PCR-ELISA was further confirmed by conventional methods of isolation and characterization. The presented method can be very useful for rapid analysis of milk samples for SEA contamination and can be further extended for detection of multiple SE's in different wells of same PCR plate using common DNA substrate. Copyright © 2014 Elsevier B.V. All rights reserved.

Production of monoclonal antibodies and development of a quantitative immuno-polymerase chain reaction assay to detect and quantify recombinant Glutathione S-transferase.

PubMed

Abud, J E; Luque, E H; Ramos, J G; Rodriguez, H A

2017-07-01

GST-tagged proteins are important tools for the production of recombinant proteins. Removal of GST tag from its fusion protein, frequently by harsh chemical treatments or proteolytic methods, is often required. Thus, the monitoring of the proteins in tag-free form requires a significant effort to determine the remnants of GST during purification process. In the present study, we developed both a conventional enzyme-linked immunosorbent assay (ELISA) and an immuno-polymerase chain reaction (IPCR) assay, both specific for detection of recombinant GST (rGST). rGST was expressed in Escherichia coli JM109, using a pGEX4T-3 vector, and several anti-rGST monoclonal antibodies were generated using hybridoma technology. Two of these were rationally selected as capture and detection antibodies, allowing the development of a sandwich ELISA with a limit of detection (LOD) of 0.01 μg/ml. To develop the rGST-IPCR assay, we selected "Universal-IPCR" format, comprising the biotin-avidin binding as the coupling system. In addition, the rGST-IPCR was developed in standard PCR tubes, and the surface adsorption of antibodies on PCR tubes, the optimal neutravidin concentrations, the generation of a reporter DNA and the concentration effect were studied and determined. Under optimized assay conditions, the rGST-IPCR assay provided a 100-fold increase in the LOD as well as an expanded working range, in comparison with rGST-ELISA. The proposed method exhibited great potentiality for application in several fields in which measurement of very low levels of GST is necessary, and might provide a model for other IPCR assays. Copyright © 2017 Elsevier Inc. All rights reserved.

Concentration and detection of bacteria in virtual environmental samples based on non-immunomagnetic separation and quantum dots by using a laboratory-made system

NASA Astrophysics Data System (ADS)

Cheng, Zhi; Wu, Taihu; Chen, Feng; Du, Yaohua; Gu, Biao; Li, Chao; Yang, Zijian

2012-03-01

This study investigated a method that simultaneously detects three bacteria, Salmonella typhimurium, Escherichia coli, and Staphylococcus aureus via an approach that combines un-immunized magnetic nanoparticles for the enrichment and antibody-conjugated quantum dots (QDs) as fluorescence markers, by using a laboratory-made system. In the enrichment procedure, the un-immunized superparamagnetic polymer nanoparticles and the three bacteria formed "beadcell" complex. Magnetic nanoparticles with different size were used and some interferents were added into the bacteria suspension respectively to check the influence on concentration efficiency. In the immuno-fluorescence labeling procedure, QDs with different emission wavelenghs were immobilized with antibody. Antibody conjugated QDs capture the bacteria selectively and specifically so that "sandwich" complex were formed. The suspension of the labeled bacteria was trickled onto a microporous membrane. A 450nm semiconductor laser was used as a part of the laboratory-made system to excite the QDs. Three PMT detectors were utilized to detect the fluorescence intensity. These un-immunized magnetic nanoparticles can be applied in nonspecific separation and enrichment of bacteria from environmental samples, and this method, of which the detection procedures are completed within 2 h, can be applied to the cost-effective and rapid detecting of bacterial contamination.

Combined time-lapse cinematography and immuno-electron microscopy.

PubMed

Balfour, B M; Goscicka, T; MacKenzie, J L; Gautam, A; Tate, M; Clark, J

1990-04-01

A method was developed to record interactions between mobile non-adherent immunocytes by time-lapse cinematography and then to study the same cells by immuno-electron microscopy, using monoclonal antibodies against surface components. For this purpose a modified stage was designed to fit an inverted microscope. The attachment included a device to cool the culture chamber with N2 gas, a micro-injector for monoclonal antibody and immuno-gold treatment, and two pairs of washing needles to change the medium without disturbance. The technique was first employed to study the formation of aggregates around the antigen-presenting cells in cultures containing cells from hyper-immunized animals. Recently peripheral blood cells from normal subjects and patients with immune deficiency syndromes were stimulated with pokeweed mitogen, cluster formation was recorded, and the cells were processed for immuno-electron microscopy.

Mass-tag enhanced immuno-laser desorption/ionization mass spectrometry for sensitive detection of intact protein antigens.

PubMed

Lorey, Martina; Adler, Belinda; Yan, Hong; Soliymani, Rabah; Ekström, Simon; Yli-Kauhaluoma, Jari; Laurell, Thomas; Baumann, Marc

2015-05-19

A new read-out method for antibody arrays using laser desorption/ionization-mass spectrometry (LDI-MS) is presented. Small, photocleavable reporter molecules with a defined mass called "mass-tags" are used for detection of immunocaptured proteins from human plasma. Using prostate specific antigen (PSA), a biomarker for prostate cancer, as a model antigen, a high sensitivity generic detection methodology based immunocapture with a primary antibody and with a biotin labeled secondary antibody coupled to mass-tagged avidin is demonstrated. As each secondary antibody can bind several avidin molecules, each having a large number of mass-tags, signal amplification can be achieved. The developed PSA sandwich mass-tag analysis method provided a limit of detection below 200 pg/mL (6 pM) for a 10 μL plasma sample, well below the clinically relevant cutoff value of 3-4 ng/mL. This brings the limit of detection (LOD) for detection of intact antigens with matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) down to levels comparable to capture by anti-peptide antibodies selected reaction monitoring (SISCAPA SRM) and enzyme linked immunosorbent assay (ELISA), as 6 pM corresponds to a maximal amount of 60 amol PSA captured on-spot. We propose the potential use of LDI (laser desorption/ionization) with mass-tag read-out implemented in a sandwich assay format for low abundant and/or early disease biomarker detection.

Immuno-Oncology-The Translational Runway for Gene Therapy: Gene Therapeutics to Address Multiple Immune Targets.

PubMed

Weß, Ludger; Schnieders, Frank

2017-12-01

Cancer therapy is once again experiencing a paradigm shift. This shift is based on extensive clinical experience demonstrating that cancer cannot be successfully fought by addressing only single targets or pathways. Even the combination of several neo-antigens in cancer vaccines is not sufficient for successful, lasting tumor eradication. The focus has therefore shifted to the immune system's role in cancer and the striking abilities of cancer cells to manipulate and/or deactivate the immune system. Researchers and pharma companies have started to target the processes and cells known to support immune surveillance and the elimination of tumor cells. Immune processes, however, require novel concepts beyond the traditional "single-target-single drug" paradigm and need parallel targeting of diverse cells and mechanisms. This review gives a perspective on the role of gene therapy technologies in the evolving immuno-oncology space and identifies gene therapy as a major driver in the development and regulation of effective cancer immunotherapy. Present challenges and breakthroughs ranging from chimeric antigen receptor T-cell therapy, gene-modified oncolytic viruses, combination cancer vaccines, to RNA therapeutics are spotlighted. Gene therapy is recognized as the most prominent technology enabling effective immuno-oncology strategies.

Cost-effective and label-free holographic biosensor for detection of herpes simplex virus (Conference Presentation)

NASA Astrophysics Data System (ADS)

Ray, Aniruddha; Ho, Ha; Daloglu, Mustafa; Torres, Avee; McLeod, Euan; Ozcan, Aydogan

2017-03-01

Herpes is one of the most widespread sexually transmitted viral diseases. Timely detection of Herpes Simplex Virus (HSV) can help prevent the rampant spreading of the virus. Current detection techniques such as viral culture, immuno-assays or Polymerase-Chain-Reaction, are time extensive and require expert handling. Here we present a field-portable, easy-to-use, and cost-effective biosensor for the detection of HSV based on holographic imaging. The virus is first captured from a target solution onto specifically developed substrates, prepared by coating glass coverslips with HSV-specific antibodies, and imaged using a lensfree holographic microscope. Several light-emitting-diodes (LEDs), coupled to multi-mode optical-fibers, are used to illuminate the sample containing the viruses. A micro-controller is used to activate the LEDs one at a time and in-line holograms are recorded using a CMOS imager placed immediately above the substrate. These sub-pixel shifted holograms are used to generate a super-resolved hologram, which is reconstructed to obtain the phase and amplitude images of the viruses. The signal of the viruses is enhanced using self-assembled PEG-based nanolenses, formed around the viral particles. Based on the phase information of the reconstructed images we can estimate the size of the viral particles, with an accuracy of +/- 11 nm, as well as quantify the viral load. The limit-of-detection of this system is estimated to be <500 viral copies per 100 μL sample volume that is imaged over 30 mm^2 field-of-view. This holographic microscopy based biosensor is label-free, cost-effective and field-portable, providing results in 2 hours, including sample preparation and imaging time.

Prostate cancer marker panel with single cell sensitivity in urine.

PubMed

Nickens, Kristen P; Ali, Amina; Scoggin, Tatiana; Tan, Shyh-Han; Ravindranath, Lakshmi; McLeod, David G; Dobi, Albert; Tacha, David; Sesterhenn, Isabell A; Srivastava, Shiv; Petrovics, Gyorgy

2015-06-15

Over one million men undergo prostate biopsies annually in the United States, a majority of whom due to elevated serum PSA. More than half of the biopsies turn out to be negative for prostate cancer (CaP). The limitations of both the PSA test and the biopsy procedure have led to the development for more precise CaP detection assays in urine (e.g., PCA3, TMPRSS2-ERG) or blood (e.g., PHI, 4K). Here, we describe the development and evaluation of the Urine CaP Marker Panel (UCMP) assay for sensitive and reproducible detection of CaP cells in post-digital rectal examination (post-DRE) urine. The cellular content of the post-DRE urine was captured on a translucent filter membrane, which is placed on Cytoclear slides for direct evaluation by microscopy and immuno-cytochemistry (ICC). Cells captured on the membrane were assayed for PSA and Prostein expression to identify prostate epithelial cells, and for ERG and AMACR to identify prostate tumor cells. Immunostained cells were analyzed for quantitative and qualitative features and correlated with biopsy positive and negative status for malignancy. The assay was optimized for single cell capture sensitivity and downstream evaluations by spiking a known number of cells from established CaP cell lines, LNCaP and VCaP, into pre-cleared control urine. The cells captured from the post-DRE urine of subjects, obtained prior to biopsy procedure, were co-stained for ERG, AMACR (CaP specific), and Prostein or PSA (prostate epithelium specific) rendering a whole cell based analysis and characterization. A feasibility cohort of 63 post-DRE urine specimens was assessed. Comparison of the UCMP results with blinded biopsy results showed an assay sensitivity of 64% (16 of 25) and a specificity of 68.8% (22 of 32) for CaP detection by biopsy. This pilot study assessing a minimally invasive CaP detection assay with single cell sensitivity cell-capture and characterization from the post-DRE urine holds promise for further development of this novel assay platform. Prostate 75: 969-975, 2015. © 2015 The Authors. The Prostate, published by Wiley Periodicals, Inc. © 2015 The Authors. The Prostate, published by Wiley Periodicals, Inc.

Ornamental comb colour predicts T-cell-mediated immunity in male red grouse Lagopus lagopus scoticus

NASA Astrophysics Data System (ADS)

Mougeot, Francois

2008-02-01

Sexual ornaments might reliably indicate the ability to cope with parasites and diseases, and a better ability to mount a primary inflammatory response to a novel challenge. Carotenoid-based ornaments are amongst the commonest sexual signals of birds and often influence mate choice. Because carotenoids are immuno-stimulants, signallers may trade-off allocating these to ornamental colouration or using them for immune responses, so carotenoid-based ornaments might be particularly useful as honest indicators of immuno-compentence. Tetraonid birds, such as the red grouse Lagopus lagopus scoticus, exhibit supra-orbital yellow red combs, a conspicuous ornament which functions in intra- and inter-sexual selection. The colour of combs is due to epidermal pigmentation by carotenoids, while their size is testosterone-dependent. In this study, I investigated whether comb characteristics, and in particular, comb colour, indicated immuno-competence in free-living male red grouse. I assessed T-cell-mediated immunity using a standardised challenge with phytohaemagglutinin. Red grouse combs reflect in the red and in the ultraviolet spectrum of light, which is not visible to humans but that grouse most likely see, so I measured comb colour across the whole bird visible spectrum (300 700 nm) using a reflectance spectrometer. I found that males with bigger and redder combs, but with less ultraviolet reflectance, had greater T-cell-mediated immune response. Comb colour predicted T-cell-mediated immune response better than comb size, indicating that the carotenoid-based colouration of this ornament might reliably signal this aspect of male quality.

Antibody-Coupled Magnetic Beads Can Be Reused in Immuno-MRM Assays To Reduce Cost and Extend Antibody Supply.

PubMed

Zhao, Lei; Whiteaker, Jeffrey R; Voytovich, Uliana J; Ivey, Richard G; Paulovich, Amanda G

2015-10-02

Immunoaffinity enrichment of peptides coupled to targeted, multiple reaction monitoring mass spectrometry (immuno-MRM) enables precise quantification of peptides. Affinity-purified polyclonal antibodies are routinely used as affinity reagents in immuno-MRM assays, but they are not renewable, limiting the number of experiments that can be performed. In this technical note, we describe a workflow to regenerate anti-peptide polyclonal antibodies coupled to magnetic beads for enrichments in multiplex immuno-MRM assays. A multiplexed panel of 44 antibodies (targeting 60 peptides) is used to show that peptide analytes can be effectively stripped off of antibodies using acid washing without compromising assay performance. The performance of the multiplexed panel (determined by correlation, agreement, and precision of reused assays) is reproducible (R(2) between 0.81 and 0.99) and consistent (median CVs 8-15%) for at least 10 times of washing and reuse. Application of this workflow to immuno-MRM studies greatly reduces per sample assay cost and increases the number of samples that can be interrogated with a limited supply of polyclonal antibody reagent. This allows more characterization for promising and desirable targets prior to committing funds and efforts to conversion to a renewable monoclonal antibody.

QTL analysis of citrus tristeza virus-citradia interaction.

PubMed

Asins, M J; Bernet, G P; Ruiz, C; Cambra, M; Guerri, J; Carbonell, E A

2004-02-01

Citrus tristeza virus (CTV) has caused the death of millions of trees grafted on sour orange ( Citrus aurantium). However, this rootstock is very well adapted to the Mediterranean, semi-arid conditions. The aim of the present research is to genetically analyze the accumulation of CTV in a progeny derived from the cross between C. aurantium and Poncirus trifoliata, both resistant to CTV isolate T-346. Graft propagation of 104 hybrids was done on healthy sweet orange as a rootstock. Three months later, each rootstock was graft inoculated with two patches of infected tissue (isolate T-346). One, 2, and sometimes, 3 and 4 years after inoculation, hybrids and infected patches were tested for CTV by tissue-blot immuno-assay. Additionally, CTV multiplication was evaluated every year as the optical density of double-antibody sandwich enzyme-linked immuno-sorbent assay reactions. Linkage maps for P. trifoliata based on 63 markers, and for C. aurantium based on 157 markers, were used. Most molecular markers were microsatellites and IRAP (inter-retrotransposon amplified polymorphisms). Some analogues of resistance and expressed sequences were also included for candidate gene analysis. Resistance against CTV was analyzed as a quantitative trait (CTV accumulation) by QTL (quantitative trait loci) analysis to avoid the assumption of monogenic control. Three major resistance QTLs were detected where the P. trifoliata resistance gene, Ctv-R, had been previously located in other progenies. Up to five minor QTLs were detected ( Ctv-A(1) to Ctv-A(5)). A significant epistatic interaction involving Ctv-R(1) and Ctv-A(1) was also found. An analogue of a resistance gene is a candidate for Ctv-A(3), and two expressed sequences are candidates for Ctv-A(1) and Ctv-A(5). Single-strand conformational polymorphism analysis of CTV genes QTL P20 and P25 (coat protein) in susceptible hybrids, was carried out to test whether or not any QTL accumulation was a defeated resistance gene. Since the same haplotype of the virus was visualized independently on the CTV titer, differences in the amount of virions are not explained through the selection of CTV genotypes by the host, but through differences among citradias in CTV replication and/or movement.

Immuno and Affinity Cytochemical Analysis of Cell Wall Composition in the Moss Physcomitrella patens

DOE PAGES

Berry, Elizabeth A.; Tran, Mai L.; Dimos, Christos S.; ...

2016-03-08

In contrast to homeohydric vascular plants, mosses employ a poikilohydric strategy for surviving in the dry aerial environment. A detailed understanding of the structure, composition, and development of moss cell walls can contribute to our understanding of not only the evolution of overall cell wall complexity, but also the differences that have evolved in response to selection for different survival strategies. The model moss species Physcomitrella patens has a predominantly haploid lifecycle consisting of protonemal filaments that regenerate from protoplasts and enlarge by tip growth, and leafy gametophores composed of cells that enlarge by diffuse growth and differentiate into severalmore » different types. Advantages for genetic studies include methods for efficient targeted gene modification and extensive genomic resources. Immuno and affinity cytochemical labeling were used to examine the distribution of polysaccharides and proteins in regenerated protoplasts, protonemal filaments, rhizoids, and sectioned gametophores of P. patens. The cell wall composition of regenerated protoplasts was also characterized by flow cytometry. Crystalline cellulose was abundant in the cell walls of regenerating protoplasts and protonemal cells that developed on media of high osmolarity, whereas homogalactuonan was detected in the walls of protonemal cells that developed on low osmolarity media and not in regenerating protoplasts. Mannan was the major hemicellulose detected in all tissues tested. Arabinogalactan proteins were detected in different cell types by different probes, consistent with structural heterogneity. The results reveal developmental and cell type specific differences in cell wall composition and provide a basis for analyzing cell wall phenotypes in knockout mutants.« less

Immuno and Affinity Cytochemical Analysis of Cell Wall Composition in the Moss Physcomitrella patens

PubMed Central

Berry, Elizabeth A.; Tran, Mai L.; Dimos, Christos S.; Budziszek, Michael J.; Scavuzzo-Duggan, Tess R.; Roberts, Alison W.

2016-01-01

In contrast to homeohydric vascular plants, mosses employ a poikilohydric strategy for surviving in the dry aerial environment. A detailed understanding of the structure, composition, and development of moss cell walls can contribute to our understanding of not only the evolution of overall cell wall complexity, but also the differences that have evolved in response to selection for different survival strategies. The model moss species Physcomitrella patens has a predominantly haploid lifecycle consisting of protonemal filaments that regenerate from protoplasts and enlarge by tip growth, and leafy gametophores composed of cells that enlarge by diffuse growth and differentiate into several different types. Advantages for genetic studies include methods for efficient targeted gene modification and extensive genomic resources. Immuno and affinity cytochemical labeling were used to examine the distribution of polysaccharides and proteins in regenerated protoplasts, protonemal filaments, rhizoids, and sectioned gametophores of P. patens. The cell wall composition of regenerated protoplasts was also characterized by flow cytometry. Crystalline cellulose was abundant in the cell walls of regenerating protoplasts and protonemal cells that developed on media of high osmolarity, whereas homogalactuonan was detected in the walls of protonemal cells that developed on low osmolarity media and not in regenerating protoplasts. Mannan was the major hemicellulose detected in all tissues tested. Arabinogalactan proteins were detected in different cell types by different probes, consistent with structural heterogneity. The results reveal developmental and cell type specific differences in cell wall composition and provide a basis for analyzing cell wall phenotypes in knockout mutants. PMID:27014284

Evaluation of a QuECHERS-like extraction approach for the determination of PBDEs in mussels by immuno-assay-based screening methods

USDA-ARS?s Scientific Manuscript database

A sample preparation method was evaluated for the determination of polybrominated diphenyl ethers (PBDEs) in mussel samples, by using colorimetric and electrochemical immunoassay-based screening methods. A simple sample preparation in conjunction with a rapid screening method possesses the desired c...

Technology assessment and strategy for development of a Rapid Field Water Microbiology Test Kit. Technical report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Preston, D.R.; Schaub, S.A.

1991-09-01

A literature and market search of existing technology for the detection, identification, and quantification of microorganisms in water was conducted. Based upon the availability of technologies and their configurations, an assessment of the appropriate strategies to pursue for the near and long term development plans in development of the Rapid Field Bacteriology Test Kit was performed. Near term technologies to improve the Army's capability to detect microorganisms would appear to be essentially improvements in versatility and measurement of coliform indicator organisms. New chromogenic and fluorogenic indicator substances associated with new substrates appear to be best suited for test kit developmentmore » either for quantitative membrane filter tests or presence/absence and multiple fermentation tests. Test times, incubator requirements, and operator involvement appear to be similar to older technologies. Long term development would appear to favor such technologies as genetic probes with amplification of the hydridized nucleic acid materials of positive samples, and some immunological based systems such as enzyme linked, immuno-sorbent assays. In both cases, the major problems would appear to be sample preparation and development of signal strengths from the reactions which would allow the user to see results in 1 hour.« less

Highly sensitive dendrimer-based nanoplasmonic biosensor for drug allergy diagnosis.

PubMed

Soler, Maria; Mesa-Antunez, Pablo; Estevez, M-Carmen; Ruiz-Sanchez, Antonio Jesus; Otte, Marinus A; Sepulveda, Borja; Collado, Daniel; Mayorga, Cristobalina; Torres, Maria Jose; Perez-Inestrosa, Ezequiel; Lechuga, Laura M

2015-04-15

A label-free biosensing strategy for amoxicillin (AX) allergy diagnosis based on the combination of novel dendrimer-based conjugates and a recently developed nanoplasmonic sensor technology is reported. Gold nanodisks were functionalized with a custom-designed thiol-ending-polyamido-based dendron (d-BAPAD) peripherally decorated with amoxicilloyl (AXO) groups (d-BAPAD-AXO) in order to detect specific IgE generated in patient's serum against this antibiotic during an allergy outbreak. This innovative strategy, which follows a simple one-step immobilization procedure, shows exceptional results in terms of sensitivity and robustness, leading to a highly-reproducible and long-term stable surface which allows achieving extremely low limits of detection. Moreover, the viability of this biosensor approach to analyze human biological samples has been demonstrated by directly analyzing and quantifying specific anti-AX antibodies in patient's serum without any sample pretreatment. An excellent limit of detection (LoD) of 0.6ng/mL (i.e. 0.25kU/L) has been achieved in the evaluation of clinical samples evidencing the potential of our nanoplasmonic biosensor as an advanced diagnostic tool to quickly identify allergic patients. The results have been compared and validated with a conventional clinical immunofluorescence assay (ImmunoCAP test), confirming an excellent correlation between both techniques. The combination of a novel compact nanoplasmonic platform and a dendrimer-based strategy provides a highly sensitive label free biosensor approach with over two times better detectability than conventional SPR. Both the biosensor device and the carrier structure hold great potential in clinical diagnosis for biomarker analysis in whole serum samples and other human biological samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Strategies for the Identification and Tracking of Cronobacter Species: An Opportunistic Pathogen of Concern to Neonatal Health

PubMed Central

Yan, Qiongqiong; Fanning, Séamus

2015-01-01

Cronobacter species are emerging opportunistic food-borne pathogens, which consists of seven species, including C. sakazakii, C. malonaticus, C. muytjensii, C. turicensis, C. dublinensis, C. universalis, and C. condimenti. The organism can cause severe clinical infections, including necrotizing enterocolitis, septicemia, and meningitis, predominately among neonates <4 weeks of age. Cronobacter species can be isolated from various foods and their surrounding environments; however, powdered infant formula (PIF) is the most frequently implicated food source linked with Cronobacter infection. This review aims to provide a summary of laboratory-based strategies that can be used to identify and trace Cronobacter species. The identification of Cronobacter species using conventional culture method and immuno-based detection protocols were first presented. The molecular detection and identification at genus-, and species-level along with molecular-based serogroup approaches are also described, followed by the molecular sub-typing methods, in particular pulsed-field gel electrophoresis and multi-locus sequence typing. Next generation sequence approaches, including whole genome sequencing, DNA microarray, and high-throughput whole-transcriptome sequencing, are also highlighted. Appropriate application of these strategies would contribute to reduce the risk of Cronobacter contamination in PIF and production environments, thereby improving food safety and protecting public health. PMID:26000266

Quantitative targeted proteomic analysis of potential markers of tyrosine kinase inhibitor (TKI) sensitivity in EGFR mutated lung adenocarcinoma.

PubMed

Awasthi, Shivangi; Maity, Tapan; Oyler, Benjamin L; Qi, Yue; Zhang, Xu; Goodlett, David R; Guha, Udayan

2018-04-13

Lung cancer causes the highest mortality among all cancers. Patients harboring kinase domain mutations in the epidermal growth factor receptor (EGFR) respond to EGFR tyrosine kinase inhibitors (TKIs), however, acquired resistance always develops. Moreover, 30-40% of patients with EGFR mutations exhibit primary resistance. Hence, there is an unmet need for additional biomarkers of TKI sensitivity that complement EGFR mutation testing and predict treatment response. We previously identified phosphopeptides whose phosphorylation is inhibited upon treatment with EGFR TKIs, erlotinib and afatinib in TKI sensitive cells, but not in resistant cells. These phosphosites are potential biomarkers of TKI sensitivity. Here, we sought to develop modified immuno-multiple reaction monitoring (immuno-MRM)-based quantitation assays for select phosphosites including EGFR-pY1197, pY1172, pY998, AHNAK-pY160, pY715, DAPP1-pY139, CAV1-pY14, INPPL1-pY1135, NEDD9-pY164, NF1-pY2579, and STAT5A-pY694. These sites were significantly hypophosphorylated by erlotinib and a 3rd generation EGFR TKI, osimertinib, in TKI-sensitive H3255 cells, which harbor the TKI-sensitizing EGFR L858R mutation. However, in H1975 cells, which harbor the TKI-resistant EGFR L858R/T790M mutant, osimertinib, but not erlotinib, could significantly inhibit phosphorylation of EGFR-pY-1197, STAT5A-pY694 and CAV1-pY14, suggesting these sites also predict response in TKI-resistant cells. We could further validate EGFR-pY-1197 as a biomarker of TKI sensitivity by developing a calibration curve-based modified immuno-MRM assay. In this report, we have shown the development and optimization of MRM assays coupled with global phosphotyrosine enrichment (modified immuno-MRM) for a list of 11 phosphotyrosine peptides. Our optimized assays identified the targets reproducibly in biological samples with good selectivity. We also developed and characterized quantitation methods to determine endogenous abundance of these targets and correlated the results of the relative quantification with amounts estimated from the calibration curves. This approach represents a way to validate and verify biomarker candidates discovered from large-scale global phospho-proteomics analysis. The application of these modified immuno-MRM assays in lung adenocarcinoma cells provides proof-of concept for the feasibility of clinical applications. These assays may be used in prospective clinical studies of EGFR TKI treatment of EGFR mutant lung cancer to correlate treatment response and other clinical endpoints. Copyright © 2018. Published by Elsevier B.V.

A novel mast cell co-culture microfluidic chip for the electrochemical evaluation of food allergen.

PubMed

Jiang, Hui; Jiang, Donglei; Zhu, Pei; Pi, Fuwei; Ji, Jian; Sun, Chao; Sun, Jiadi; Sun, Xiulan

2016-09-15

In this study a novel cell-to-cell electrochemical microfluidic chip was developed for qualitative and quantitative analysis of food allergen. Microfluidic cell culture, food allergen-induced cell morphological changes, and cell metabolism measurements were performed simultaneously using the aforementioned device. RBL-2H3 mast cells and ANA-1 macrophages have been used within a cell co-culture model to observe their allergic response when they are introduced to the antigen stimulus. Two cell cultivation microfluidic channels are located in the microfluidic chip, which is fabricated with four groups of gold electrodes, with an additional "capillary". In order to detect the allergic response, the cells were stimulated with dinitrophenylated bovine serum albumin (DNP-BSA) without anti-DNP IgE incubation. When exocytosis occurs, the cell-secreted inflammatory cytokines were measured by enzyme-linked immuno sorbent assay (ELISA) and cell impedance changes were detected using cell-based electrochemical assay. Results indicate that the real-time cell allergic response are accurately monitored by this electrochemical microfluidic chip, which provides a general example of rapidly prototyped low-cost biosensor technology for applications in both food allergen detection and investigation. Copyright © 2016 Elsevier B.V. All rights reserved.

Early Detection of Breast Cancer Using Posttranslationally Modified Biomarkers

DTIC Science & Technology

2013-09-01

goat antimouse IgG in PBS (Jackson Immuno Research Laboratory Inc., West Grove, PA) was added to each well. The slides were incubated for another 40...a concentration of 10 μg/mL; this was followed by incubation for 1 h at 37 °C. The secondary antibody, FITC-conjugated goat antimouse, was incubated...mIgG) was used as the negative control, and glycolipid-specific antibody anti-Globo-H (MBr1)14−16 (Enzo Life Sciences, Inc., Farm - ingdale, NY) was used

Fabrication of a Novel Highly Sensitive and Selective Immunosensor for Botulinum Neurotoxin Serotype A Based on an Effective Platform of Electrosynthesized Gold Nanodendrites/Chitosan Nanoparticles.

PubMed

Sorouri, Rahim; Bagheri, Hasan; Afkhami, Abbas; Salimian, Jafar

2017-05-09

In this work, a novel nanocomposite consisting of electrosynthesized gold nanodendrites and chitosan nanoparticles (AuNDs/CSNPs) has been prepared to fabricate an impedimetric immunosensor based on a screen printed carbon electrode (SPCE) for the rapid and sensitive immunoassay of botulinum neurotoxin A (BoNT/A). BoNT/A polyclonal antibody was immobilized on the nanocomposite-modified SPCE for the signal amplification. The structure of the prepared nanocomposite was investigated by transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). The charge transfer resistance (R CT ) changes were used to detect BoNT/A as the specific immuno-interactions at the immunosensor surface that efficiently limited the electron transfer of Fe(CN)₆ 3-/4- as a redox probe at pH = 7.4. A linear relationship was observed between the %∆ R CT and the concentration logarithm of BoNT/A within the range of 0.2 to 230 pg·mL -1 with a detection limit (S/N = 3) of 0.15 pg·mL -1 . The practical applicability of the proposed sensor was examined by evaluating the detection of BoNT/A in milk and serum samples with satisfactory recoveries. Therefore, the prepared immunosensor holds great promise for the fast, simple and sensitive detection of BoNT/A in various real samples.

Fabrication of a Novel Highly Sensitive and Selective Immunosensor for Botulinum Neurotoxin Serotype A Based on an Effective Platform of Electrosynthesized Gold Nanodendrites/Chitosan Nanoparticles

PubMed Central

Sorouri, Rahim; Bagheri, Hasan; Afkhami, Abbas; Salimian, Jafar

2017-01-01

In this work, a novel nanocomposite consisting of electrosynthesized gold nanodendrites and chitosan nanoparticles (AuNDs/CSNPs) has been prepared to fabricate an impedimetric immunosensor based on a screen printed carbon electrode (SPCE) for the rapid and sensitive immunoassay of botulinum neurotoxin A (BoNT/A). BoNT/A polyclonal antibody was immobilized on the nanocomposite-modified SPCE for the signal amplification. The structure of the prepared nanocomposite was investigated by transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). The charge transfer resistance (RCT) changes were used to detect BoNT/A as the specific immuno-interactions at the immunosensor surface that efficiently limited the electron transfer of Fe(CN)63−/4− as a redox probe at pH = 7.4. A linear relationship was observed between the %∆RCT and the concentration logarithm of BoNT/A within the range of 0.2 to 230 pg·mL−1 with a detection limit (S/N = 3) of 0.15 pg·mL−1. The practical applicability of the proposed sensor was examined by evaluating the detection of BoNT/A in milk and serum samples with satisfactory recoveries. Therefore, the prepared immunosensor holds great promise for the fast, simple and sensitive detection of BoNT/A in various real samples. PMID:28486408

The feasibility study based on e-commerce instructions-focuses on detection and deletion of illegal content

NASA Astrophysics Data System (ADS)

Guo, Tianze; Bi, Siyu; Liu, Jiaming

2018-04-01

This essay legally restrains the illegal content based on the e-commerce directive and introduces that the European countries detect and notify illegal content through the instructions of competent authorities, notification of credible flaggers, user reports and technical tools. The illegal content should be deleted through the service terms and transparency report basing on prevent excessive deletions system. At the same time, use filters to detect and filter to against the recurrence of illegal content. By analyzing the advantages of China under the environment of cracking down on illegal content, this essay concludes that the success of China in cracking down on illegal content lies in all-round collaborative management model of countries, governments, enterprises and individuals. At the end of the essay, one is to build a training corpus that can automatically update the ability to identify the illegal content. And it proposes an optimization scheme that establish a complete set of address resolution procedures and classify IP address data according to big data analysis and DNS protection module to prevent hackers from spreading illegal content by tampering with DNS segments.

Recent Developments in Enzyme, DNA and Immuno-Based Biosensors.

PubMed

Asal, Melis; Özen, Özlem; Şahinler, Mert; Polatoğlu, İlker

2018-06-13

Novel sensitive, rapid and economical biosensors are being developed in a wide range of medical environmental and food applications. In this paper, we review some of the main advances in the field over the past few years by discussing recent studies from literature. A biosensor, which is defined as an analytical device consisting of a biomolecule, a transducer and an output system, can be categorized according to the type of the incorporated biomolecule. The biomolecules can be enzymes, antibodies, ssDNA, organelles, cells etc. The main biosensor categories classified according to the biomolecules are enzymatic biosensors, immunosensors and DNA-based biosensors. These sensors can measure analytes produced or reduced during reactions at lower costs compared to the conventional detection techniques. Numerous types of biosensor studies conducted over the last decade have been explored here to reveal their key applications in medical, environmental and food industries which provide comprehensive perspective to the readers. Overviews of the working principles and applications of the reviewed sensors are also summarized.

Quantitative assessment of serum-specific IgE in the diagnosis of human cystic echinococcosis.

PubMed

Marinova, I; Nikolov, G; Michova, A; Kurdova, R; Petrunov, B

2011-07-01

Anti-Echinococcus serum immunoglobulin (Ig)E was assessed by the ImmunoCAP system and compared with anti-Echinococcus serum IgG assessed by enzyme-linked immunosorbent assay (ELISA) and Western blot. The ImmunoCAP system revealed very high specificity (one false positive of 110 healthy individuals), low cross-reactivity (one false positive of 58 patients with other diseases) and decreased sensitivity (73.55%). Receiver operating characteristic analysis displayed a beneficial diagnostic value with high accuracy. Comparison of the ImmunoCAP system with ELISA and Western blot showed significantly higher specificity and significantly lower cross-reactivity compared with the ELISA. Examination of sera from 155 patients with cystic echinococcosis (CE) showed varying levels of anti-Echinococcus IgE (range, 0.01-118.33 kUA/L). However, most samples had moderately elevated IgE levels. Analysis of serum-specific IgE revealed significantly higher sensitivity of the ImmunoCAP system and significantly higher antibody levels in hepatic CE compared with pulmonary CE. © 2011 Blackwell Publishing Ltd.

Determination of total sulfur content via sulfur-specific chemiluminescence detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kubala, S.W.; Campbell, D.N.; DiSanzo, F.P.

A specially designed system, based upon sulfur-specific chemiluminescence detection (SSCD), was developed to permit the determination of total sulfur content in a variety of samples. This type of detection system possesses several advantages such as excellent linearity and selectivity, low minimum detectable levels, and an equimolar response to various sulfur compounds. This paper will focus on the design and application of a sulfur-specific chemiluminescence detection system for use in determining total sulfur content in gasoline.

In Vivo and In Situ Detection of Macromolecular Free Radicals Using Immuno-Spin Trapping and Molecular Magnetic Resonance Imaging.

PubMed

Towner, Rheal A; Smith, Nataliya

2018-05-20

In vivo free radical imaging in preclinical models of disease has become a reality. Free radicals have traditionally been characterized by electron spin resonance (ESR) or electron paramagnetic resonance (EPR) spectroscopy coupled with spin trapping. The disadvantage of the ESR/EPR approach is that spin adducts are short-lived due to biological reductive and/or oxidative processes. Immuno-spin trapping (IST) involves the use of an antibody that recognizes macromolecular 5,5-dimethyl-pyrroline-N-oxide (DMPO) spin adducts (anti-DMPO antibody), regardless of the oxidative/reductive state of trapped radical adducts. Recent Advances: The IST approach has been extended to an in vivo application that combines IST with molecular magnetic resonance imaging (mMRI). This combined IST-mMRI approach involves the use of a spin-trapping agent, DMPO, to trap free radicals in disease models, and administration of an mMRI probe, an anti-DMPO probe, which combines an antibody against DMPO-radical adducts and an MRI contrast agent, resulting in targeted free radical adduct detection. The combined IST-mMRI approach has been used in several rodent disease models, including diabetes, amyotrophic lateral sclerosis (ALS), gliomas, and septic encephalopathy. The advantage of this approach is that heterogeneous levels of trapped free radicals can be detected directly in vivo and in situ to pin point where free radicals are formed in different tissues. The approach can also be used to assess therapeutic agents that are either free radical scavengers or generate free radicals. Smaller probe constructs and radical identification approaches are being considered. The focus of this review is on the different applications that have been studied, advantages and limitations, and future directions. Antioxid. Redox Signal. 28, 1404-1415.

ImmunoScenarios: A Game for the Immune System.

ERIC Educational Resources Information Center

Taylor, Mark F.; Jackson, Sally W.

1996-01-01

Describes a board game, ImmunoScenarios, which was developed to reinforce the ideas about the immune system discussed in lecture classes. Emphasizes important characteristics of the body's specific defense system including specificity, cooperation among various cells, and memory. Includes directions for playing, student handouts, and scenarios.…

21 CFR 866.3460 - Rabiesvirus immuno-fluorescent reagents.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Rabiesvirus immuno-fluorescent reagents. 866.3460 Section 866.3460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3460 Rabiesvirus...

21 CFR 866.3460 - Rabiesvirus immuno-fluorescent reagents.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Rabiesvirus immuno-fluorescent reagents. 866.3460 Section 866.3460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3460 Rabiesvirus...

21 CFR 866.3460 - Rabiesvirus immuno-fluorescent reagents.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Rabiesvirus immuno-fluorescent reagents. 866.3460 Section 866.3460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3460 Rabiesvirus...

21 CFR 866.3460 - Rabiesvirus immuno-fluorescent reagents.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Rabiesvirus immuno-fluorescent reagents. 866.3460 Section 866.3460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3460 Rabiesvirus...

21 CFR 866.3460 - Rabiesvirus immuno-fluorescent reagents.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Rabiesvirus immuno-fluorescent reagents. 866.3460 Section 866.3460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3460 Rabiesvirus...

High-Affinity Recombinant Antibody Fragments (Fabs) Can Be Applied in Peptide Enrichment Immuno-MRM Assays

PubMed Central

2015-01-01

High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an automated laboratory and thus may offer advantages over conventional monoclonal antibodies. However, recombinant antibodies are typically obtained as fragments (Fab or scFv) expressed from E. coli, and it is not known whether these antibody formats are compatible with the established protocols and whether the affinities necessary for immunocapture of small linear peptides can be achieved with this technology. Hence, we performed a feasibility study to ask: (a) whether it is feasible to isolate high-affinity Fabs to small linear antigens and (b) whether it is feasible to incorporate antibody fragments into robust, quantitative immuno-MRM assays. We describe successful isolation of high-affinity Fab fragments against short (tryptic) peptides from a human combinatorial Fab library. We analytically characterize three immuno-MRM assays using recombinant Fabs, full-length IgGs constructed from these Fabs, or traditional monoclonals. We show that the antibody fragments show similar performance compared with traditional mouse- or rabbit-derived monoclonal antibodies. The data establish feasibility of isolating and incorporating high-affinity Fabs into peptide immuno-MRM assays. PMID:24568200

High-affinity recombinant antibody fragments (Fabs) can be applied in peptide enrichment immuno-MRM assays.

PubMed

Whiteaker, Jeffrey R; Zhao, Lei; Frisch, Christian; Ylera, Francisco; Harth, Stefan; Knappik, Achim; Paulovich, Amanda G

2014-04-04

High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an automated laboratory and thus may offer advantages over conventional monoclonal antibodies. However, recombinant antibodies are typically obtained as fragments (Fab or scFv) expressed from E. coli, and it is not known whether these antibody formats are compatible with the established protocols and whether the affinities necessary for immunocapture of small linear peptides can be achieved with this technology. Hence, we performed a feasibility study to ask: (a) whether it is feasible to isolate high-affinity Fabs to small linear antigens and (b) whether it is feasible to incorporate antibody fragments into robust, quantitative immuno-MRM assays. We describe successful isolation of high-affinity Fab fragments against short (tryptic) peptides from a human combinatorial Fab library. We analytically characterize three immuno-MRM assays using recombinant Fabs, full-length IgGs constructed from these Fabs, or traditional monoclonals. We show that the antibody fragments show similar performance compared with traditional mouse- or rabbit-derived monoclonal antibodies. The data establish feasibility of isolating and incorporating high-affinity Fabs into peptide immuno-MRM assays.

Application of ImmunoScore Model for the Differentiation between Active Tuberculosis and Latent Tuberculosis Infection as Well as Monitoring Anti-tuberculosis Therapy.

PubMed

Zhou, Yu; Du, Juan; Hou, Hong-Yan; Lu, Yan-Fang; Yu, Jing; Mao, Li-Yan; Wang, Feng; Sun, Zi-Yong

2017-01-01

Tuberculosis (TB) is a leading global public health problem. To achieve the end TB strategy, non-invasive markers for diagnosis and treatment monitoring of TB disease are urgently needed, especially in high-endemic countries such as China. Interferon-gamma release assays (IGRAs) and tuberculin skin test (TST), frequently used immunological methods for TB detection, are intrinsically unable to discriminate active tuberculosis (ATB) from latent tuberculosis infection (LTBI). Thus, the specificity of these methods in the diagnosis of ATB is dependent upon the local prevalence of LTBI. The pathogen-detecting methods such as acid-fast staining and culture, all have limitations in clinical application. ImmunoScore (IS) is a new promising prognostic tool which was commonly used in tumor. However, the importance of host immunity has also been demonstrated in TB pathogenesis, which implies the possibility of using IS model for ATB diagnosis and therapy monitoring. In the present study, we focused on the performance of IS model in the differentiation between ATB and LTBI and in treatment monitoring of TB disease. We have totally screened five immunological markers (four non-specific markers and one TB-specific marker) and successfully established IS model by using Lasso logistic regression analysis. As expected, the IS model can effectively distinguish ATB from LTBI (with a sensitivity of 95.7% and a specificity of 92.1%) and also has potential value in the treatment monitoring of TB disease.

Application of circular consensus sequencing and network analysis to characterize the bovine IgG repertoire

USDA-ARS?s Scientific Manuscript database

Background: Vertebrate immune systems generate diverse repertoires of antibodies capable of mediating response to a variety of antigens. Next generation sequencing methods provide unique approaches to a number of immuno-based research areas including antibody discovery and engineering, disease surve...

Optimizing the Immuno-Surface Characteristics for Bio-Sensors and Filters Through Modeling and Experiments

DTIC Science & Technology

2005-06-01

immobilization of antibodies o Adsorbed, aminophase, heterobifunctional crosslinkers (GMBS, BMPS, EMCS) o GMBS attaches the most antibodies o ProteinA ...play a role in getting the antigen close enough to the immuno-surface to potentially interact as well as the short range molecular forces that

21 CFR 866.5860 - Total spinal fluid immuno-logical test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... diagnosis of multiple sclerosis and other diseases of the nervous system. (b) Classification. Class I... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Total spinal fluid immuno-logical test system. 866... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866...

21 CFR 866.5860 - Total spinal fluid immuno-logical test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... diagnosis of multiple sclerosis and other diseases of the nervous system. (b) Classification. Class I... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Total spinal fluid immuno-logical test system. 866... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866...

21 CFR 866.5860 - Total spinal fluid immuno-logical test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... diagnosis of multiple sclerosis and other diseases of the nervous system. (b) Classification. Class I... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Total spinal fluid immuno-logical test system. 866... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866...

21 CFR 866.5860 - Total spinal fluid immuno-logical test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... diagnosis of multiple sclerosis and other diseases of the nervous system. (b) Classification. Class I... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Total spinal fluid immuno-logical test system. 866... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866...

21 CFR 866.5860 - Total spinal fluid immuno-logical test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... diagnosis of multiple sclerosis and other diseases of the nervous system. (b) Classification. Class I... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Total spinal fluid immuno-logical test system. 866... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866...

77 FR 12240 - Application(s) for Duty-Free Entry of Scientific Instruments

Federal Register 2010, 2011, 2012, 2013, 2014

2012-02-29

... Institute, 5353 Parkside Dr MC 19-RE, Jupiter, FL 33458. Instrument: Freeze Fracture/Freeze Etch device... localization of membrane proteins using freeze fracture replica immuno- gold labeling, including all kinds of receptors and channels. Because freeze-fracture replica immuno-gold labeling has a high sensitivity for the...

Progress in Life Marker Chip Technology for Detection of Life on Mars

NASA Astrophysics Data System (ADS)

Sims, M. R.; Cullen, D. C.; Laan, E.; Borst, G.; Prak, A.; Richter, L.; Gaubert, F.; Steele, A.; Parnell, J.; Sephton, M.

2007-12-01

Detection of Life on Mars will rely on detection of biomarkers, physical or chemical structures that can be associated with Life. As a possible payload for the ESA ExoMars rover mission planned in 2013 and other future missions a Life Marker Chip instrument is being developed. This instrument uses immuno-assay techniques to detect the relevant biomarkers. This paper describes the typical targets it will search for, its operating principle and the status of development. 63 biomarker targets have been identified and assays have been developed for a limited subset. Assay development includes use of recombinant DNA techniques to generate the molecular receptors (antibodies). This type of instrument has applications in terrestrial research e.g. sub-glacial lakes as well as planetary exploration. Breadboard demonstrators have been built of the assay system and key components of the micro-fluidics. Results from these breadboards will be presented, along with plans for future development.

Label-free impedimetric immunosensor for sensitive detection of ochratoxin A.

PubMed

Radi, Abd-Elgawad; Muñoz-Berbel, Xavier; Lates, Vasilica; Marty, Jean-Louis

2009-03-15

A novel label-free electrochemical impedimetric immunosensor for sensitive detection of ochratoxin A (OTA) was reported. A two-step reaction protocol was elaborated to modify the gold electrode. The electrode was first derivatized by electrochemical reduction of in situ generated 4-carboxyphenyl diazonium salt (4-CPDS) in acidic aqueous solution yielded stable 4-carboxyphenyl (4-CP) monolayer. The ochratoxin A antibody was then immobilized making use of the carbodiimide chemistry. The steps of the immunosensor elaboration and the immunochemical reaction between ochratoxin A and the surface-bound antibody were interrogated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The impedance change, due to the specific immuno-interaction at the immunosensor surface was utilized to detect ochratoxin A. The increase in electron-transfer resistance (DeltaR(et)) values was linearly proportional to the concentration of OTA in the range of 1-20ngmL(-1), with a detection limit of 0.5ngmL(-1).

On-Orbit, Immuno-Based, Label-Free White Blood Cell Counting System with Microelectromechanical Sensor Technology (OILWBCS-MEMS)

NASA Technical Reports Server (NTRS)

Edmonds, Jessica

2015-01-01

Aurora Flight Sciences, in partnership with Draper Laboratory, has developed a miniaturized system to count white blood cells in microgravity environments. The system uses MEMS technology to simultaneously count total white blood cells, the five white blood cell differential subgroups, and various lymphocyte subtypes. The OILWBCS-MEMS detection technology works by immobilizing an array of white blood cell-specific antibodies on small, gold-coated membranes. When blood flows across the membranes, specific cells' surface protein antigens bind to their corresponding antibodies. This binding can be measured and correlated to cell counts. In Phase I, the partners demonstrated surface chemistry sensitivity and specificity for total white blood cells and two lymphocyte subtypes. In Phase II, a functional prototype demonstrated end-to-end operation. This rugged, miniaturized device requires minimal blood sample preparation and will be useful for both space flight and terrestrial applications.

Combined molecular MRI and immuno-spin-trapping for in vivo detection of free radicals in orthotopic mouse GL261 gliomas.

PubMed

Towner, Rheal A; Smith, Nataliya; Saunders, Debra; De Souza, Patricia Coutinho; Henry, Leah; Lupu, Florea; Silasi-Mansat, Robert; Ehrenshaft, Marilyn; Mason, Ronald P; Gomez-Mejiba, Sandra E; Ramirez, Dario C

2013-12-01

Free radicals play a major role in gliomas. By combining immuno-spin-trapping (IST) and molecular magnetic resonance imaging (mMRI), in vivo levels of free radicals were detected within mice bearing orthotopic GL261 gliomas. The nitrone spin trap DMPO (5,5-dimethyl pyrroline N-oxide) was administered prior to injection of an anti-DMPO probe (anti-DMPO antibody covalently bound to a bovine serum albumin (BSA)-Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)-biotin MRI contrast agent) to trap tumor-associated free radicals. mMRI detected the presence of anti-DMPO adducts by either a significant sustained increase (p<0.001) in MR signal intensity or a significant decrease (p<0.001) in T1 relaxation, measured as %T1 change. In vitro assessment of the anti-DMPO probe indicated a significant decrease (p<0.0001) in T1 relaxation in GL261 cells that were oxidatively stressed with hydrogen peroxide, compared to controls. The biotin moiety of the anti-DMPO probe was targeted with fluorescently-labeled streptavidin to locate the anti-DMPO probe in excised brain tissues. As a negative control a non-specific IgG antibody covalently bound to the albumin-Gd-DTPA-biotin construct was used. DMPO adducts were also confirmed in tumor tissue from animals administered DMPO, compared to non-tumor brain tissue. GL261 gliomas were found to have significantly increased malondialdehyde (MDA) protein adducts (p<0.001) and 3-nitrotyrosine (3-NT) (p<0.05) compared to normal mouse brain tissue, indicating increased oxidized lipids and proteins, respectively. Co-localization of the anti-DMPO probe with either 3-NT or 4-hydroxynonenal was also observed. This is the first report regarding the detection of in vivo levels of free radicals from a glioma model. © 2013.

Single-cell mRNA profiling reveals transcriptional heterogeneity among pancreatic circulating tumour cells.

PubMed

Lapin, Morten; Tjensvoll, Kjersti; Oltedal, Satu; Javle, Milind; Smaaland, Rune; Gilje, Bjørnar; Nordgård, Oddmund

2017-05-31

Single-cell mRNA profiling of circulating tumour cells may contribute to a better understanding of the biology of these cells and their role in the metastatic process. In addition, such analyses may reveal new knowledge about the mechanisms underlying chemotherapy resistance and tumour progression in patients with cancer. Single circulating tumour cells were isolated from patients with locally advanced or metastatic pancreatic cancer with immuno-magnetic depletion and immuno-fluorescence microscopy. mRNA expression was analysed with single-cell multiplex RT-qPCR. Hierarchical clustering and principal component analysis were performed to identify expression patterns. Circulating tumour cells were detected in 33 of 56 (59%) examined blood samples. Single-cell mRNA profiling of intact isolated circulating tumour cells revealed both epithelial-like and mesenchymal-like subpopulations, which were distinct from leucocytes. The profiled circulating tumour cells also expressed elevated levels of stem cell markers, and the extracellular matrix protein, SPARC. The expression of SPARC might correspond to an epithelial-mesenchymal transition in pancreatic circulating tumour cells. The analysis of single pancreatic circulating tumour cells identified distinct subpopulations and revealed elevated expression of transcripts relevant to the dissemination of circulating tumour cells to distant organ sites.

Unusual immuno-modulatory triterpene-caffeates in the skins of russeted varieties of apples and pears.

PubMed

Andre, Christelle M; Larsen, Lesley; Burgess, Elaine J; Jensen, Dwayne J; Cooney, Janine M; Evers, Danièle; Zhang, Jingli; Perry, Nigel B; Laing, William A

2013-03-20

Three triterpene-caffeates have been isolated from skins of a russeted apple cultivar "Merton Russet" and identified by LC-MS and NMR as betulinic acid-3-cis-caffeate, betulinic acid-3-trans-caffeate, and oleanolic acid-3-trans-caffeate. Betulinic acid-3-trans-caffeate and oleanolic acid-3-trans-caffeate were also found in russeted pear skins. These compounds have not been previously reported in apples or pears, or in any other foods. Their presence was related to suberized tissue as they were only found in russet portions of the partially russeted apple cultivar "Cox's Orange Pippin" and were not detected in the waxy apple cultivar "Royal Gala". High concentrations of betulinic acid-3-trans-caffeate were found in the bark of both "Merton Russet" and "Royal Gala" trees. The three triterpene-caffeates showed anti-inflammatory activity in vitro, inhibiting NF-κB activation with IC50's of 6-9 μM. Betulinic acid-3-trans-caffeate, the predominant compound in the apples, was immuno-modulatory at around 10 μM in the in vitro and ex vivo bioassays, boosting production of the pro-inflammatory cytokine TNFα in cells stimulated with bacterial lipopolysaccharides.

Selenium-regulated hierarchy of human selenoproteome in cancerous and immortalized cells lines.

PubMed

Touat-Hamici, Zahia; Bulteau, Anne-Laure; Bianga, Juliusz; Jean-Jacques, Hélène; Szpunar, Joanna; Lobinski, Ryszard; Chavatte, Laurent

2018-04-13

Selenoproteins (25 genes in human) co-translationally incorporate selenocysteine using a UGA codon, normally used as a stop signal. The human selenoproteome is primarily regulated by selenium bioavailability with a tissue-specific hierarchy. We investigated the hierarchy of selenoprotein expression in response to selenium concentration variation in four cell lines originating from kidney (HEK293, immortalized), prostate (LNCaP, cancer), skin (HaCaT, immortalized) and liver (HepG2, cancer), using complementary analytical methods. We performed (i) enzymatic activity, (ii) RT-qPCR, (iii) immuno-detection, (iv) selenium-specific mass spectrometric detection after non-radioactive 76 Se labeling of selenoproteins, and (v) luciferase-based reporter constructs in various cell extracts. We characterized cell-line specific alterations of the selenoproteome in response to selenium variation that, in most of the cases, resulted from a translational control of gene expression. We established that UGA-selenocysteine recoding efficiency, which depends on the nature of the SECIS element, dictates the response to selenium variation. We characterized that selenoprotein hierarchy is cell-line specific with conserved features. This analysis should be done prior to any experiments in a novel cell line. We reported a strategy based on complementary methods to evaluate selenoproteome regulation in human cells in culture. Copyright © 2018 Elsevier B.V. All rights reserved.

Alpha-Tocopherol alters transcription activities that modulate tumor necrosis factor alpha (TNF-¿)-induced inflammatory response in bovine cells

USDA-ARS?s Scientific Manuscript database

To further investigate the potential role of '-tocopherol in maintaining immuno-homeostasis in bovine cells (Madin-Darby bovine kidney epithelial cell line), we undertook in vitro experiments using recombinant TNF-a as an immuno-stimulant to simulate inflammation response in cells with and without '...

21 CFR 866.5400 - Alpha-globulin immuno-logical test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... alpha-globulin (a serum protein) in serum and other body fluids. Measurement of alpha-globulin may aid... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Alpha-globulin immuno-logical test system. 866.5400 Section 866.5400 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN...

21 CFR 866.5350 - Fibrinopeptide A immuno-logical test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... fibrinopeptide A (a blood-clotting factor) in plasma and other body fluids. Measurement of fibrinopeptide A may... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Fibrinopeptide A immuno-logical test system. 866.5350 Section 866.5350 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN...

21 CFR 866.5350 - Fibrinopeptide A immuno-logical test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... fibrinopeptide A (a blood-clotting factor) in plasma and other body fluids. Measurement of fibrinopeptide A may... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Fibrinopeptide A immuno-logical test system. 866.5350 Section 866.5350 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN...

21 CFR 866.5350 - Fibrinopeptide A immuno-logical test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... fibrinopeptide A (a blood-clotting factor) in plasma and other body fluids. Measurement of fibrinopeptide A may... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Fibrinopeptide A immuno-logical test system. 866.5350 Section 866.5350 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN...

21 CFR 866.5400 - Alpha-globulin immuno-logical test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... alpha-globulin (a serum protein) in serum and other body fluids. Measurement of alpha-globulin may aid... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Alpha-globulin immuno-logical test system. 866.5400 Section 866.5400 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN...

21 CFR 866.5350 - Fibrinopeptide A immuno-logical test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... fibrinopeptide A (a blood-clotting factor) in plasma and other body fluids. Measurement of fibrinopeptide A may... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Fibrinopeptide A immuno-logical test system. 866.5350 Section 866.5350 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN...

21 CFR 866.5400 - Alpha-globulin immuno-logical test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... alpha-globulin (a serum protein) in serum and other body fluids. Measurement of alpha-globulin may aid... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Alpha-globulin immuno-logical test system. 866.5400 Section 866.5400 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN...

21 CFR 866.5400 - Alpha-globulin immuno-logical test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... alpha-globulin (a serum protein) in serum and other body fluids. Measurement of alpha-globulin may aid... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Alpha-globulin immuno-logical test system. 866.5400 Section 866.5400 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN...

A dual challenge of corticotropin releasing hormone and vasopressin alters immune cell profiles in beef heifers

USDA-ARS?s Scientific Manuscript database

The duration and magnitude of cortisol release can have different effects on the immune response. Over the last decade, studies have suggested that acute stress, when cortisol is elevated for a short duration of time, can be immuno-stimulatory rather than immuno-suppressive. This study was designed ...

Cancer immunotherapy without frontiers: 2nd Annual Immuno-Oncology Meeting of the Centro de Investigación de Cancer en Sonora (CICS), Ciudad Obregón, Sonora México, Dec 2-4, 2016.

PubMed

Gallardo-Rincón, Dolores; Marquez, Juan Pablo; Celis, Esteban

2017-09-01

This meeting in immuno-oncology brought together clinicians and scientists from United States, Canada, and México with the goal of breaking down international walls and establishing new collaborations.

MiRNAs: dynamic regulators of immune cell functions in inflammation and cancer.

PubMed

Hirschberger, Simon; Hinske, Ludwig Christian; Kreth, Simone

2018-09-01

MicroRNAs (miRNAs), small noncoding RNA molecules, have emerged as important regulators of almost all cellular processes. By binding to specific sequence motifs within the 3'- untranslated region of their target mRNAs, they induce either mRNA degradation or translational repression. In the human immune system, potent miRNAs and miRNA-clusters have been discovered, that exert pivotal roles in the regulation of gene expression. By targeting cellular signaling hubs, these so-called immuno-miRs have fundamental regulative impact on both innate and adaptive immune cells in health and disease. Importantly, they also act as mediators of tumor immune escape. Secreted by cancer cells and consecutively taken up by immune cells, immuno-miRs are capable to influence immune functions towards a blunted anti-tumor response, thus shaping a permissive tumor environment. This review provides an overview of immuno-miRs and their functional impact on individual immune cell entities. Further, implications of immuno-miRs in the amelioration of tumor surveillance are discussed. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Regulatory T Lymphocytes in Periodontitis: A Translational View

PubMed Central

2018-01-01

Periodontitis is a chronic immuno-inflammatory disease in which the disruption of the balance between host and microbiota interactions is key to the onset and progression of the disease. The immune homeostasis associated with periodontal health requires a regulated immuno-inflammatory response, during which the presence of regulatory T cells (Tregs) is essential to ensure a controlled response that minimizes collateral tissue damage. Since Tregs modulate both innate and adaptive immunity, pathological conditions that may resolve by the acquisition of immuno-tolerance, such as periodontitis, may benefit by the use of Treg immunotherapy. In recent years, many strategies have been proposed to take advantage of the immuno-suppressive capabilities of Tregs as immunotherapy, including the ex vivo and in vivo manipulation of the Treg compartment. Ongoing research in both basic and translational studies let us gain a better understanding of the diversity of Treg subsets, their phenotypic plasticity, and suppressive functions, which can be used as a substrate for new immunotherapies. Certainly, as our knowledge of Treg biology increases, we will be capable to develop new therapies designed to enhance the stability and function of Tregs during periodontitis.

Multiplexed fluorescent microarray for human salivary protein analysis using polymer microspheres and fiber-optic bundles.

PubMed

Nie, Shuai; Benito-Peña, Elena; Zhang, Huaibin; Wu, Yue; Walt, David R

2013-10-10

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 μm polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 μm microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.

Assessment of radionuclide contents in food in Hong Kong

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, K.N.; Mao, S.Y.

1999-12-01

Baseline values of concentrations of the natural radionuclides ({sup 238}U, {sup 226}Ra, {sup 228}Ra/{sup 232}Th, {sup 210}Pb) and artificial radionuclides ({sup 137}Cs, {sup 60}Co) in food and drinks (tap water, milk, and water-based drinks) were determined by gamma spectroscopy. All food and drinks were found to contain detectable {sup 40}K contents: 0.1 to 160 Bq Kg{sup {minus}1} for food and 0.006 to 61 Bq L{sup {minus}1} for drinks. Most of the other natural radionuclides in solid food were found to have contents below the minimum detectable activities (MDA). More samples in the leafy vegetable, tomato, carrot and potato categories containedmore » detectable amounts of {sup 228}Ra than the meat, cereal, and fish categories, with concentrations up to 1.2 Bq kg{sup {minus}1} for the former categories and 0.35 Bq kg{sup {minus}1} for the latter categories. The {sup 238}U and {sup 226}Ra radionuclides were detectable in most of the water-based drink samples, and the {sup 228}Ra and {sup 210}Pb radionuclides were detectable in fewer water-based drink samples. The {sup 137}Cs contents in solid food were detectable in most of the solid food samples (reaching 0.59 Bq kg{sup {minus}1}), but in drinks the {sup 137}Cs contents were very low and normally lower than the MDA values. Nearly all the {sup 60}Co contents in food and drinks were below the MDA values and their contents were below those of {sup 137}Cs.« less

Noninvasive Interrogation of DLL3 Expression in Metastatic Small Cell Lung Cancer.

PubMed

Sharma, Sai Kiran; Pourat, Jacob; Abdel-Atti, Dalya; Carlin, Sean D; Piersigilli, Alessandra; Bankovich, Alexander J; Gardner, Eric E; Hamdy, Omar; Isse, Kumiko; Bheddah, Sheila; Sandoval, Joseph; Cunanan, Kristen M; Johansen, Eric B; Allaj, Viola; Sisodiya, Vikram; Liu, David; Zeglis, Brian M; Rudin, Charles M; Dylla, Scott J; Poirier, John T; Lewis, Jason S

2017-07-15

The Notch ligand DLL3 has emerged as a novel therapeutic target expressed in small cell lung cancer (SCLC) and high-grade neuroendocrine carcinomas. Rovalpituzumab teserine (Rova-T; SC16LD6.5) is a first-in-class DLL3-targeted antibody-drug conjugate with encouraging initial safety and efficacy profiles in SCLC in the clinic. Here we demonstrate that tumor expression of DLL3, although orders of magnitude lower in surface protein expression than typical oncology targets of immunoPET, can serve as an imaging biomarker for SCLC. We developed 89 Zr-labeled SC16 antibody as a companion diagnostic agent to facilitate selection of patients for treatment with Rova-T based on a noninvasive interrogation of the in vivo status of DLL3 expression using PET imaging. Despite low cell-surface abundance of DLL3, immunoPET imaging with 89 Zr-labeled SC16 antibody enabled delineation of subcutaneous and orthotopic SCLC tumor xenografts as well as distant organ metastases with high sensitivity. Uptake of the radiotracer in tumors was concordant with levels of DLL3 expression and, most notably, DLL3 immunoPET yielded rank-order correlation for response to SC16LD6.5 therapy in SCLC patient-derived xenograft models. Cancer Res; 77(14); 3931-41. ©2017 AACR . ©2017 American Association for Cancer Research.

Establishing a sample-to cut-off ratio for lab-diagnosis of hepatitis C virus in Indian context.

PubMed

Tiwari, Aseem K; Pandey, Prashant K; Negi, Avinash; Bagga, Ruchika; Shanker, Ajay; Baveja, Usha; Vimarsh, Raina; Bhargava, Richa; Dara, Ravi C; Rawat, Ganesh

2015-01-01

Lab-diagnosis of hepatitis C virus (HCV) is based on detecting specific antibodies by enzyme immuno-assay (EIA) or chemiluminescence immuno-assay (CIA). Center for Disease Control reported that signal-to-cut-off (s/co) ratios in anti-HCV antibody tests like EIA/CIA can be used to predict the probable result of supplemental test; above a certain s/co value it is most likely to be true-HCV positive result and below that certain s/co it is most likely to be false-positive result. A prospective study was undertaken in patients in tertiary care setting for establishing this "certain" s/co value. The study was carried out in consecutive patients requiring HCV testing for screening/diagnosis and medical management. These samples were tested for anti-HCV on CIA (VITROS(®) Anti-HCV assay, Ortho-Clinical Diagnostics, New Jersey) for calculating s/co value. The supplemental nucleic acid test used was polymerase chain reaction (PCR) (Abbott). PCR test results were used to define true negatives, false negatives, true positives, and false positives. Performance of different putative s/co ratios versus PCR was measured using sensitivity, specificity, positive predictive value and negative predictive value and most appropriate s/co was considered on basis of highest specificity at sensitivity of at least 95%. An s/co ratio of ≥6 worked out to be over 95% sensitive and almost 92% specific in 438 consecutive patient samples tested. The s/co ratio of six can be used for lab-diagnosis of HCV infection; those with s/co higher than six can be diagnosed to have HCV infection without any need for supplemental assays.

Non-immunogenic dextran-coated superparamagnetic iron oxide nanoparticles: a biocompatible, size-tunable contrast agent for magnetic resonance imaging.

PubMed

Unterweger, Harald; Janko, Christina; Schwarz, Marc; Dézsi, László; Urbanics, Rudolf; Matuszak, Jasmin; Őrfi, Erik; Fülöp, Tamás; Bäuerle, Tobias; Szebeni, János; Journé, Clément; Boccaccini, Aldo R; Alexiou, Christoph; Lyer, Stefan; Cicha, Iwona

2017-01-01

Iron oxide-based contrast agents have been in clinical use for magnetic resonance imaging (MRI) of lymph nodes, liver, intestines, and the cardiovascular system. Superparamagnetic iron oxide nanoparticles (SPIONs) have high potential as a contrast agent for MRI, but no intravenous iron oxide-containing agents are currently approved for clinical imaging. The aim of our work was to analyze the hemocompatibility and immuno-safety of a new type of dextran-coated SPIONs (SPIONdex) and to characterize these nanoparticles with ultra-high-field MRI. Key parameters related to nanoparticle hemocompatibility and immuno-safety were investigated in vitro and ex vivo. To address concerns associated with hypersensitivity reactions to injectable nanoparticulate agents, we analyzed complement activation-related pseudoallergy (CARPA) upon intravenous administration of SPIONdex in a pig model. Furthermore, the size-tunability of SPIONdex and the effects of size reduction on their biocompatibility were investigated. In vitro, SPIONdex did not induce hemolysis, complement or platelet activation, plasma coagulation, or leukocyte procoagulant activity, and had no relevant effect on endothelial cell viability or endothelial-monocytic cell interactions. Furthermore, SPIONdex did not induce CARPA even upon intravenous administration of 5 mg Fe/kg in pigs. Upon SPIONdex administration in mice, decreased liver signal intensity was observed after 15 minutes and was still detectable 24 h later. In addition, by changing synthesis parameters, a reduction in particle size <30 nm was achieved, without affecting their hemo- and biocompatibility. Our findings suggest that due to their excellent biocompatibility, safety upon intravenous administration and size-tunability, SPIONdex particles may represent a suitable candidate for a new-generation MRI contrast agent.

Allergen content of grass pollen preparations for skin prick testing and sublingual immunotherapy.

PubMed

Sander, I; Fleischer, C; Meurer, U; Brüning, T; Raulf-Heimsoth, M

2009-10-01

The allergen content of diagnostics and immunotherapeutics is crucial for effective diagnosis and treatment. The aim of this study was to quantify and compare the allergen content of different grass pollen preparations for skin prick testing and sublingual immunotherapy (SLIT). Five skin prick test (SPT) solutions and 10 sublingual immunotherapeutics were analysed for protein and allergen concentration by Bradford assay, inhibition of IgE-binding to Phleum pratense ImmunoCAPs and content of the main allergen Phl p 5 by two-site enzyme immunoassay. In addition, the grass pollen preparations were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analyses. Protein concentrations of SPT solutions ranged from 15 to 427 microg/ml, and Phl p 5 concentrations ranged from 0.15 to 18.3 microg/ml. The ranking of SPT solutions concerning Phl p 5 content and IgE inhibition capacity was the same, and the ranking of protein and allergen content was closely correlated (r = 0.9). Protein content of the maintenance doses of the immunotheurapeutics ranged from 5 to 153 microg, Phl p 5 content ranged from 0.2 to 21.6 microg. IgE inhibition capacity of the maintenance doses was closely correlated to their Phl p 5 and protein content. SDS-PAGE and immunoblots confirmed the differences in protein and allergen content. Grass pollen preparations for SPT and SLIT varied greatly concerning protein and allergen content. Whereas this result corresponds to previous analyses results of SPT solutions, it was the first comparison of grass pollen immunotherapeutics. For diagnosis and therapy, these differences should be taken into account.

Epitope mapping of salmonella flagellar hook-associated protein, FlgK, with mass spectrometry-based immuno-capture proteomics using chicken (gallus gallus domesticus] sera

USDA-ARS?s Scientific Manuscript database

Salmonella, a Gram-negative rod, is the leading foodborne pathogen associated with human acute bacterial gastroenteritis worldwide. The Salmonella flagellum is responsible for bacterial movement, colonization and invasion in the host gastrointestinal tract. The flagellum has a complex structure, c...

Bioactive components on immuno-enhancement effects in the traditional Chinese medicine Shenqi Fuzheng Injection based on relevance analysis between chemical HPLC fingerprints and in vivo biological effects.

PubMed

Wang, Jinxu; Tong, Xin; Li, Peibo; Liu, Menghua; Peng, Wei; Cao, Hui; Su, Weiwei

2014-08-08

Shenqi Fuzheng Injection (SFI) is an injectable traditional Chinese herbal formula comprised of two Chinese herbs, Radix codonopsis and Radix astragali, which were commonly used to improve immune functions against chronic diseases in an integrative and holistic way in China and other East Asian countries for thousands of years. This present study was designed to explore the bioactive components on immuno-enhancement effects in SFI using the relevance analysis between chemical fingerprints and biological effects in vivo. According to a four-factor, nine-level uniform design, SFI samples were prepared with different proportions of the four portions separated from SFI via high speed counter current chromatography (HSCCC). SFI samples were assessed with high performance liquid chromatography (HPLC) for 23 identified components. For the immunosuppressed murine experiments, biological effects in vivo were evaluated on spleen index (E1), peripheral white blood cell counts (E2), bone marrow cell counts (E3), splenic lymphocyte proliferation (E4), splenic natural killer cell activity (E5), peritoneal macrophage phagocytosis (E6) and the amount of interleukin-2 (E7). Based on the hypothesis that biological effects in vivo varied with differences in components, multivariate relevance analysis, including gray relational analysis (GRA), multi-linear regression analysis (MLRA) and principal component analysis (PCA), were performed to evaluate the contribution of each identified component. The results indicated that the bioactive components of SFI on immuno-enhancement activities were calycosin-7-O-β-d-glucopyranoside (P9), isomucronulatol-7,2'-di-O-glucoside (P11), biochanin-7-glucoside (P12), 9,10-dimethoxypterocarpan-3-O-xylosylglucoside (P15) and astragaloside IV (P20), which might have positive effects on spleen index (E1), splenic lymphocyte proliferation (E4), splenic natural killer cell activity (E5), peritoneal macrophage phagocytosis (E6) and the amount of interleukin-2 (E7), while 5-hydroxymethyl-furaldehyde (P5) and lobetyolin (P13) might have negative effects on E1, E4, E5, E6 and E7. Finally, the bioactive HPLC fingerprint of SFI based on its bioactive components on immuno-enhancement effects was established for quality control of SFI. In summary, this study provided a perspective to explore the bioactive components in a traditional Chinese herbal formula with a series of HPLC and animal experiments, which would be helpful to improve quality control and inspire further clinical studies of traditional Chinese medicines. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

⁸⁹Zr-Labeled Versus ¹²⁴I-Labeled αHER2 Fab with Optimized Plasma Half-Life for High-Contrast Tumor Imaging In Vivo.

PubMed

Mendler, Claudia T; Gehring, Torben; Wester, Hans-Jürgen; Schwaiger, Markus; Skerra, Arne

2015-07-01

Immuno-PET imaging of the tumor antigen HER2/neu allows for the noninvasive detection and monitoring of oncogene expression; such detection and monitoring are of prognostic value in patients with breast cancer. Compared with the full-size antibody trastuzumab, smaller protein tracers with more rapid blood clearance permit higher imaging contrast at earlier time points. Antigen-binding fragments (Fabs) of antibodies with moderately prolonged circulation achieved through the genetic fusion with a long, conformationally disordered chain of the natural amino acids Pro, Ala, and Ser (PASylation)-a biologic alternative to chemical conjugation with polyethylene glycol, PEG-offer a promising tracer format with improved pharmacokinetics for in vivo imaging. Recently, the transition metal radionuclide (89)Zr has attracted increasing interest for immuno-PET studies, complementing the conventional halogen radionuclide (124)I. To allow direct comparison of these 2 radioactive labels for the same protein tracer, the recombinant αHER2 Fab fused with 200 Pro, Ala, and Ser (PAS200) residues was either conjugated with (124)I via an iodination reagent or coupled with deferoxamine (Df) and complexed with (89)Zr. After confirmation of the stability of both radioconjugates and quality control in vitro, immuno-PET and biodistribution studies were performed with CD1-Foxn1(nu) mice bearing HER2-positive human tumor xenografts. (89)Zr⋅Df-Fab-PAS200 and (124)I-Fab-PAS200 showed specific tumor uptake of 11 and 2.3 percentage injected dose per gram 24 h after injection, respectively; both led to high tumor-to-blood (3.6 and 4.4, respectively) and tumor-to-muscle (20 and 43, respectively) ratios. With regard to off-target accumulation, overt (124)I activity was seen in the thyroid, as expected, whereas high kidney uptake was evident for (89)Zr; the latter was probably due to glomerular filtration and reabsorption of the protein tracer in proximal tubular cells. Both (89)Zr- and (124)I-labeled versions of αHER2 Fab-PAS200 allowed PET tumor imaging with high contrast. With its residualizing radiometal, the tracer (89)Zr⋅Df-Fab-PAS200 showed better in vivo stability and higher tumor uptake. © 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Optimized small molecule antibody labeling efficiency through continuous flow centrifugal diafiltration.

PubMed

Cappione, Amedeo; Mabuchi, Masaharu; Briggs, David; Nadler, Timothy

2015-04-01

Protein immuno-detection encompasses a broad range of analytical methodologies, including western blotting, flow cytometry, and microscope-based applications. These assays which detect, quantify, and/or localize expression for one or more proteins in complex biological samples, are reliant upon fluorescent or enzyme-tagged target-specific antibodies. While small molecule labeling kits are available with a range of detection moieties, the workflow is hampered by a requirement for multiple dialysis-based buffer exchange steps that are both time-consuming and subject to sample loss. In a previous study, we briefly described an alternative method for small-scale protein labeling with small molecule dyes whereby all phases of the conjugation workflow could be performed in a single centrifugal diafiltration device. Here, we expand on this foundational work addressing functionality of the device at each step in the workflow (sample cleanup, labeling, unbound dye removal, and buffer exchange/concentration) and the implications for optimizing labeling efficiency. When compared to other common buffer exchange methodologies, centrifugal diafiltration offered superior performance as measured by four key parameters (process time, desalting capacity, protein recovery, retain functional integrity). Originally designed for resin-based affinity purification, the device also provides a platform for up-front antibody purification or albumin carrier removal. Most significantly, by exploiting the rapid kinetics of NHS-based labeling reactions, the process of continuous diafiltration minimizes reaction time and long exposure to excess dye, guaranteeing maximal target labeling while limiting the risks associated with over-labeling. Overall, the device offers a simplified workflow with reduced processing time and hands-on requirements, without sacrificing labeling efficiency, final yield, or conjugate performance. Copyright © 2015 Elsevier B.V. All rights reserved.

Technology evaluation: C242-DM1, ImmunoGen Inc.

PubMed

Smith, S

2001-04-01

C242-DM1 is a tumor-activated immunotoxin under development by GlaxoSmithKline plc (formerly SmithKline Beecham plc), under licence from ImmunoGen Inc, as a potential treatment for colon tumor. It consists of a colon cancer-specific humanized antibody, C242, conjugated to the maytansine derivative DM1. In preclinical studies, C242-DM1 caused complete tumor regression in animal models of both human pancreatic and non-small cell lung cancer (NSCLC) at non-toxic doses. C242-DM1 has also been evaluated in an immunoconjugate combination with J-591 (Cornell University). The J591-DM1 immunoconjugate demonstrated effective, antigen-specific delivery of a highly cytotoxic drug to PSMA-positive Pca cells in vitro and in vivo with low systemic toxicity. Results from studies in monkeys showed that C242-DM1 had no significant toxicity or side effects, when administered at doses higher than those that were previously shown to completely eradicate human colon tumors in mice [271420]. ImmunoGen acquired the right to evaluate, and an option to license, technology related to maytansines from Takeda. In February 1999, ImmunoGen and SmithKline Beecham signed a US $45 million development and commercialization agreement for C242-DM1 [313493]. In August 1997, Immunogen received an SBIR grant to advance development of huC242-DM1 [258356]. EP-00425235, held by ImmunoGen, covers conjugated forms of ansamitocin (maytansine) derivatives. Takeda holds several patents for the production of ansamitocin and its analogs, the first one being JP-53124692.

[Cutaneous chondroid syringoma].

PubMed

Aoun, Agathe; Dufrenot-Petitjean-Roget, Leila; Amazan, Emmanuelle; Derancourt, Christian; Alexandre, Marina; Quist, Danièle; Grossin, Maggy; Molinié, Vincent

2015-08-01

Chondroid syringoma (CS) is a rare cutaneous tumor characterized by mixte epithelial and mesenchymal component. The confident histological diagnosis can be obtained by immuno-histochemistry study. Here we present 10 new cases with their clinico-hystological characteristics. The 10 cases were observed between January 2000 and august 2013, in Fort-de-France and Louis-Mourier universitary hospitals. For all the cases a controlled histological study was performed by a dermatopathologist expert and immuno-histochemistry was added. Clinical and immuno-histological data were analyzed. The lesions were almost localized on the face (3/10) and the extremities (3/10). The size was about 1.2 to 5.2cm. Every case was treated by surgery, no malignant case was diagnosed. Histologically, all the 10 cases presented as a well-limited dermic tumor with a mixte epithelial and mesenchymal component. The stroma was myxo-chondroid, and the epithelial component consisted in epithelial cavities lined by one or two cell layers with eccrine (4/10) or apocrine (5/10) features. Immuno-chemistry study reveals positivity for EMA, ACE and CK7 for the internal cells, and positivity for S100 protein and vimentin of the extern cell layer. Chondroid syringoma is characterized by a mixte epithelial with eccrine and apocrine cells and a myxo-chondroid stroma. Our study has some clinical and histological particularities (lesions on the extremities, epidermic connecting…). The main differentials diagnoses are the other annexial tumors. The treatment is surgical. The histological diagnosis of CS is quite easy, but in case of doubt, immuno-chemistry will help, showing a double mesenchymal and epithelial differentiation. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Impairment in immuno-modulatory function of Flk1(+)CD31(-)CD34(-) MSCs from MDS-RA patients.

PubMed

Han, Qin; Sun, Zhao; Liu, Lihui; Chen, Bin; Cao, Ying; Li, Kanghua; Zhao, Robert Chunhua

2007-11-01

Myelodysplastic syndromes are a group of hematopoietic disorders characterized by hematopoietic stem cell dysregulation and abnormalities in the immune system. Mesenchymal stem cells (MSCs) and their derived stromal cells constitute a bone marrow microenvironment, which is the niche for hematopoiesis and a key compartment for immune development and regulation. Existing evidence has shown that MSCs from MDS patients have impaired capacity in supporting hematopoiesis. Here, we conducted an investigation to determine whether the immuno-modulatory function of MSCs is also impaired in MDS-RA (refractory anemia) patients. Flk1(+)CD31(-)CD34(-) MSCs were isolated from 15 MDS-RA patients and cultured for testing biological and immunological characteristics. MDS-RA patient-derived Flk1(+)CD31(-)CD34(-) MSCs showed normal morphology, phenotype and karyotype but appeared impaired in immuno-modulatory function. The capacity of patient Flk1(+)CD31(-)CD34(-) MSCs to inhibit T lymphocyte activation and proliferation was impaired in vitro. In conclusion, MDS-RA patient-derived MSCs have impaired immuno-modulatory functions, suggesting that the dysregulation of hematopoiesis and immune response may originate from MSCs rather than HSCs. MSCs might be a potential target for developing efficacious cures for MDS.

CARTILAGE OLIGOMERIC MATRIX PROTEIN ENHANCES MATRIX ASSEMBLY DURING CHONDROGENESIS OF HUMAN MESENCHYMAL STEM CELLS

PubMed Central

Haleem-Smith, Hana; Calderon, Raul; Song, Yingjie; Tuan, Rocky S.; Chen, Faye H.

2011-01-01

Cartilage oligomeric matrix protein/thrombospondin-5 (COMP/TSP5) is an abundant cartilage extracellular matrix (ECM) protein that interacts with major cartilage ECM components, including aggrecan and collagens. To test our hypothesis that COMP/TSP5 functions in the assembly of the ECM during cartilage morphogenesis, we have employed mesenchymal stem cell (MSC) chondrogenesis in vitro as a model to examine the effects of COMP over-expression on neo-cartilage formation. Human bone marrow-derived MSCs were transfected with either full-length COMP cDNA or control plasmid, followed by chondrogenic induction in three-dimensional pellet or alginate-hydrogel culture. MSC chondrogenesis and ECM production was estimated based on quantitation of sulfated glycosaminoglycan (sGAG) accumulation, immunohistochemistry of the presence and distribution of cartilage ECM proteins, and real-time RT-PCR analyis of mRNA expression of cartilage markers. Our results showed that COMP over-expression resulted in increased total sGAG content during the early phase of MSC chondrogenesis, and increased immuno-detectable levels of aggrecan and collagen type II in the ECM of COMP-transfected pellet and alginate cultures, indicating more abundant cartilaginous matrix. COMP transfection did not significantly increase the transcript levels of the early chondrogenic marker, Sox9, or aggrecan, suggesting that enhancement of MSC cartilage ECM was effected at post-transcriptional levels. These findings strongly suggest that COMP functions in mesenchymal chondrogenesis by enhancing cartilage ECM organization and assembly. The action of COMP is most likely mediated not via direct changes in cartilage matrix gene expression but via interactions of COMP with other cartilage ECM proteins, such as aggrecan and collagens, that result in enhanced assembly and retention. PMID:22095699

[Survey of aflatoxins contamination of foodstuffs and edible oil in Shenzhen].

PubMed

Li, Ke; Qiu, Fen; Yang, Mei; Liang, Zhaohai; Zhou, Haitao

2013-07-01

To identify the aflatoxins contamination of foodstuffs and edible oil sold in Shenzhen. As research subjects stratified random sampling of 238 foodstuffs and edible oil, and applied with immuno-affinity column clean-up plus UPLC to determine the content of aflatoxin B1, B2, G1, and G2. Positive ratio of aflatoxin in rice, rice products, wheat flour, corn flour, edible oil were 35.3%, 33.8%, 13.9%, 46.7% and 24.5%,respectively. There were statistical differences between the positive ratio of aflatoxin in stereotypes packaged rice (26.5%) and bulk rice (56.3%) (chi2 = 11.6, P < 0.05). There were statistical differences between the positive ratio of aflatoxin in the rice produced in the area north of the Yangtze River (27.3%) and in the rice (41.4%) produced in the area south of the Yangtze River (chi2 = 7.257, P < 0.05). Aflatoxin B1 and B2 were detected in rice products, wheat flour, corn flour. Positive ratio of aflatoxin B1, B2, G1, and G2 were 24.5%, 24.5%, 11.3% and 3.8% in the edible oil,respectively. The over standard rate of aflatoxin B1 was 5.66%, excessive samples were producted bulk and self-pressed peanut oil from unlicensed workshop. All the four kinds of aflatoxin were detected, while subtype B1 and B2 dominated aflatoxin contamination in the rice and edible oil samples. There are differences between in the northern and southern rice, and the same as in the stereotypes packaged and bulk rice sold at Shenzhen.

[Novel therapy for malignant lymphoma: adoptive immuno-gene therapy using chimeric antigen receptor(CAR)-expressing T lymphocytes].

PubMed

Ozawa, Keiya

2014-03-01

Adoptive T-cell therapy using chimeric antigen receptor (CAR) technology is a novel approach to cancer immuno-gene therapy. CARs are hybrid proteins consisting of target-antigen-specific single-chain antibody fragment fused to intracellular T-cell activation domains (CD28 or CD137/CD3 zeta receptor). CAR-expressing engineered T lymphocytes can directly recognize and kill tumor cells in an HLA independent manner. In the United States, promising results have been obtained in the clinical trials of adoptive immuno-gene therapy using CD19-CAR-T lymphocytes for the treatment of refractory B-cell malignancies, including chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). In this review article, CD19-CAR-T gene therapy for refractory B-cell non-Hodgkin lymphoma is discussed.

Current perspectives of herpesviral retinitis and choroiditis.

PubMed

Madhavan, H N; Priya, K; Biswas, J

2004-10-01

Vision-threatening viral retinitis are primarily caused by members of the herpesvirus family. The biology and molecular characterization of herpesviruses, clinical presentations of retinopathies, pathology and pathogenesis including the host responses, epidemiology and the laboratory methods of aetiological diagnosis of these diseases are described. Clinical syndromes are acute retinal necrosis (ARN), progressive outer retinal necrosis (PORN), cytomegalovirus (CMV) retinitis, multifocal choroiditis and serpiginous choroiditis besides other viral retinopathies. Herpes simplex virus (HSV) retinitis is more common in immunocompetent persons while varicella zoster virus (VZV) affects both immunocompetent and immunosuppressed patients equally. CMV retinitis is most common among patients with AIDS. The currently employed laboratory methods of antigen detection, virus isolation and antibody detection by enzyme linked immuno-sorbent assay (ELISA) have low sensitivity. Polymerase chain reaction (PCR) has increased the value of diagnosis due to its high clinical sensitivity and absolute specificity in detection of herpesviruses in intraocular specimens.

A high-throughput AO/PI-based cell concentration and viability detection method using the Celigo image cytometry.

PubMed

Chan, Leo Li-Ying; Smith, Tim; Kumph, Kendra A; Kuksin, Dmitry; Kessel, Sarah; Déry, Olivier; Cribbes, Scott; Lai, Ning; Qiu, Jean

2016-10-01

To ensure cell-based assays are performed properly, both cell concentration and viability have to be determined so that the data can be normalized to generate meaningful and comparable results. Cell-based assays performed in immuno-oncology, toxicology, or bioprocessing research often require measuring of multiple samples and conditions, thus the current automated cell counter that uses single disposable counting slides is not practical for high-throughput screening assays. In the recent years, a plate-based image cytometry system has been developed for high-throughput biomolecular screening assays. In this work, we demonstrate a high-throughput AO/PI-based cell concentration and viability method using the Celigo image cytometer. First, we validate the method by comparing directly to Cellometer automated cell counter. Next, cell concentration dynamic range, viability dynamic range, and consistency are determined. The high-throughput AO/PI method described here allows for 96-well to 384-well plate samples to be analyzed in less than 7 min, which greatly reduces the time required for the single sample-based automated cell counter. In addition, this method can improve the efficiency for high-throughput screening assays, where multiple cell counts and viability measurements are needed prior to performing assays such as flow cytometry, ELISA, or simply plating cells for cell culture.

Detection of palytoxin-like compounds by a flow cytometry-based immunoassay supported by functional and analytical methods.

PubMed

Fraga, María; Vilariño, Natalia; Louzao, M Carmen; Fernández, Diego A; Poli, Mark; Botana, Luis M

2016-01-15

Palytoxin (PLTX) is a complex marine toxin produced by zoanthids (i.e. Palythoa), dinoflagellates (Ostreopsis) and cyanobacteria (Trichodesmium). PLTX outbreaks are usually associated with Indo-Pacific waters, however their recent repeated occurrence in Mediterranean-European Atlantic coasts demonstrate their current worldwide distribution. Human sickness and fatalities have been associated with toxic algal blooms and ingestion of seafood contaminated with PLTX-like molecules. These toxins represent a serious threat to human health. There is an immediate need to develop easy-to-use, rapid detection methods due to the lack of validated protocols for their detection and quantification. We have developed an immuno-detection method for PLTX-like molecules based on the use of microspheres coupled to flow-cytometry detection (Luminex 200™). The assay consisted of the competition between free PLTX-like compounds in solution and PLTX immobilized on the surface of microspheres for binding to a specific monoclonal anti-PLTX antibody. This method displays an IC50 of 1.83 ± 0.21 nM and a dynamic range of 0.47-6.54 nM for PLTX. An easy-to-perform extraction protocol, based on a mixture of methanol and acetate buffer, was applied to spiked mussel samples providing a recovery rate of 104 ± 8% and a range of detection from 374 ± 81 to 4430 ± 150 μg kg(-1) when assayed with this method. Extracts of Ostreopsis cf. siamensis and Palythoa tuberculosa were tested and yielded positive results for PLTX-like molecules. However, the data obtained for the coral sample suggested that this antibody did not detect 42-OH-PLTX efficiently. The same samples were further analyzed using a neuroblastoma cytotoxicity assay and UPLC-IT-TOF spectrometry, which also pointed to the presence of PLTX-like compounds. Therefore, this single detection method for PLTX provides a semi-quantitative tool useful for the screening of PLTX-like molecules in different matrixes. Copyright © 2015 Elsevier B.V. All rights reserved.

Immuno Nanosensor for the Ultrasensitive Naked Eye Detection of Tuberculosis.

PubMed

Mohd Bakhori, Noremylia; Yusof, Nor Azah; Abdullah, Jaafar; Wasoh, Helmi; Md Noor, Siti Suraiya; Ahmad Raston, Nurul Hanun; Mohammad, Faruq

2018-06-14

In the present study, a beneficial approach for the ultrasensitive and affordable naked eye detection and diagnosis of tuberculosis (TB) by utilizing plasmonic enzyme-linked immunosorbent assay (ELISA) via antibody-antigen interaction was studied. Here, the biocatalytic cycle of the intracellular enzymes links to the formation and successive growth of the gold nanoparticles (GNPs) for ultrasensitive detection. The formation of different colored solutions by the plasmonic nanoparticles in the presence of enzyme labels links directly to the existence or non-existence of the TB analytes in the sample solutions. For disease detection, the adapted protocol is based mainly on the conventional ELISA procedure that involves catalase-labeled antibodies, i.e., the enzymes consume hydrogen peroxide and further produce GNPs with the addition of gold (III) chloride. The amount of hydrogen peroxide remaining in the solution determines whether the GNPs solution is to be formed in the color blue or the color red, as it serves as a confirmation for the naked eye detection of TB analytes. However, the conventional ELISA method only shows tonal colors that need a high concentration of analyte to achieve high confidence levels for naked eye detection. Also, in this research, we proposed the incorporation of protein biomarker, Mycobacterium tuberculosis ESAT-6-like protein esxB (CFP-10), as a means of TB detection using plasmonic ELISA. With the use of this technique, the CFP-10 detection limit can be lowered to 0.01 µg/mL by the naked eye. Further, our developed technique was successfully tested and confirmed with sputum samples from patients diagnosed with positive TB, thereby providing enough evidence for the utilization of our technique in the early diagnosis of TB disease.

Comparison of antigen-capture ELISA to stool-culture methods for the detection of asymptomatic Entamoeba species infection in Kafer Daoud, Egypt.

PubMed

Abd-Alla, M D; Wahib, A A; Ravdin, J I

2000-05-01

We performed a prospective field study in the village of Kafer Daoud in Menofia, Egypt to compare the fecal culture method with enzyme linked immuno assay (ELISA) for detection of 170 kDa lectin antigen in feces for diagnosis of asymptomatic Entamoeba histolytica and Entamoeba dispar infection. All subjects with E. histolytica or E. dispar infection detected by culture also had positive ELISA for amebic antigen in their feces and an additional 57 Entameoba infections missed by culture were detected by ELISA (P < 0.001 compared to culture). The presence of fecal anti-lectin IgA antibodies and serum anti-LC3 (recombinant cysteine-rich lectin protein) IgG antibodies were positive predictors for E. histolytica infection (P < 0.03). Of interest, infection with Trichomonas hominis but not Blastocystis hominis was positively associated with E. histolytica infection (P < 0.05). In conclusion, ELISA for detection of fecal lectin antigen is a more sensitive method than fecal culture for detecting asymptomatic E. histolytica infection.

The changing face of clinical trials in the personalized medicine and immuno-oncology era: report from the international congress on clinical trials in Oncology & Hemato-Oncology (ICTO 2017).

PubMed

Golan, Talia; Milella, Michele; Ackerstein, Aliza; Berger, Ranaan

2017-12-28

In the past decade, the oncology community has witnessed major advances in the understanding of cancer biology and major breakthroughs in several different therapeutic areas, from solid tumors to hematological malignancies; moreover, the advent of effective immunotherapy approaches, such as immune-checkpoint blockade, is revolutionizing treatment algorithms in almost all oncology disease areas. As knowledge evolves and new weapons emerge in the "war against cancer", clinical and translational research need to adapt to a rapidly changing environment to effectively translate novel concepts into sustainable and accessible therapeutic options for cancer patients.With this in mind, translational cancer researchers, oncology professionals, treatment experts, CRO and industry leaders, as well as patient representatives gathered in London, 16-17 March 2017, for The International Congress on Clinical Trials in Oncology and Hemato-Oncology (ICTO2017), to discuss the changing face of oncology clinical trials in the new era of personalized medicine and immuno-oncology. A wide range of topics, including clinical trial design in immuno-oncology, biomarker-oriented drug development paths, statistical design and endpoint selection, challenges in the design and conduct of personalized medicine clinical trials, risk-based monitoring, financing and reimbursement, as well as best operational practices, were discussed in an open, highly interactive format, favoring networking among all relevant stakeholders. The most relevant data, approaches and issues emerged and discussed during the conference are summarized in this report.

Clinical and hematological data to group different chronic kidney disease patients: A practical approach to establish different groups of patients.

PubMed

Péterle, Vinícius B; Souza, Jéssica de O; Busato, Fernanda de O; Eutrópio, Frederico J; da Costa, Gisele de A P; Olivieri, David N; Tadokoro, Carlos E

2018-06-01

Chronic kidney disease (CKD) is the convergent point of several pathological processes, and its evolution is insidious and characterized by a progressive and irreversible loss of kidney function. This impaired function induces the accumulation of uremic toxins and individuals with terminal CKD often have altered physiological responses, including a persistent state of immuno-suppression and development of diseases. A better characterization and stratification of these patients with CKD in different immuno-compromised groups would contribute to more effective and personalized treatments. The focus of this study was to use two parameters to stratify patients with CKD into four separate groups that are representative of different immunological status. Patients with CKD were chosen randomly and stratified into four separate groups according to the period of time receiving dialysis treatment and leukocyte blood counts. The amount of apoptotic CD4 T cells were measured in each group of patients, and clinical/hematological parameters were correlated by multivariate analysis with each group. Observations reveal that one of the four groups of patients with CKD (group 3) had more apoptotic CD4 T cells than the other group; this group also had an increased malnutrition inflammation score (MIS), an elevated Kt/V, and a higher incidence of smoking. A simple two-parameter-based stratification strategy could be used to design effective immunological therapies that differentiate the degrees of immuno-suppression across groups of patients with CKD. © 2018 Wiley Periodicals, Inc.

Characterization of Anti-Salmonella enterica Serotype Typhi Antibody Responses in Bacteremic Bangladeshi Patients Using Immuno-affinity Proteomic-based Technology (IPT)

USDA-ARS?s Scientific Manuscript database

Salmonella enterica serotype Typhi (S. Typhi) is the cause of typhoid fever and a human-restricted pathogen. Currently available typhoid vaccines provide only 50-75% protection for 2-5 years, and available diagnostic assays to identify individuals with typhoid fever lack both sensitivity and specifi...

Characterization of Anti-Salmonella enterica Serotype Typhi Antibody Responses in Bacteremic Bangladeshi Patients by an Immuno-affinity Proteomic-based Technology (IPT)

USDA-ARS?s Scientific Manuscript database

Salmonella enterica serotype Typhi (S. Typhi) is the cause of typhoid fever and a human-restricted pathogen. Currently available typhoid vaccines provide only 50-75% protection for 2-5 years, and available diagnostic assays to identify individuals with typhoid fever lack both sensitivity and specif...

Microarray technology for major chemical contaminants analysis in food: current status and prospects.

PubMed

Zhang, Zhaowei; Li, Peiwu; Hu, Xiaofeng; Zhang, Qi; Ding, Xiaoxia; Zhang, Wen

2012-01-01

Chemical contaminants in food have caused serious health issues in both humans and animals. Microarray technology is an advanced technique suitable for the analysis of chemical contaminates. In particular, immuno-microarray approach is one of the most promising methods for chemical contaminants analysis. The use of microarrays for the analysis of chemical contaminants is the subject of this review. Fabrication strategies and detection methods for chemical contaminants are discussed in detail. Application to the analysis of mycotoxins, biotoxins, pesticide residues, and pharmaceutical residues is also described. Finally, future challenges and opportunities are discussed.

[Sandfly virus meningitis in a Danish traveller returning from Tuscany].

PubMed

Nissen, Nanna Bang; Jespersen, Sanne; Vinner, Lasse; Fomsgaard, Anders; Laursen, Alex

2011-10-03

We report the first case of Sandfly virus meningitis in a Danish traveller returning from Tuscany. A 52 year-old man was admitted with headache, fever and photophobia. Spinal fluid showed evidence of aseptic meningitis. Indirect immuno-fluorescence assays showed presence of immunoglobulin (Ig)M and IgG antibodies reactive against Toscana virus, and Phlebovirus RNA was detected in blood by real-time polymerase chain reaction. The patient recovered spontaneously. Since Sandfly virus is a very common cause of meningitis in the Mediterranean countries, it is important to be aware of this disease in travellers returning from these areas.

Effect of specimen storage, antibiotics, and feminine hygiene products on the detection of group B Streptococcus by culture and the STREP B OIA test.

PubMed

Ostroff, R M; Steaffens, J W

1995-07-01

Agar culture from vaginal swabs is the routine method for diagnosis of maternal Group B Streptococcus (GBS) colonization. Swab specimens are often transported to a clinical laboratory for processing. In these studies, specimen transport was simulated by inoculating swabs with GBS and storing them at selected temperatures and with or without transport medium. The recovery of viable GBS was assessed by agar culture. GBS antigen was detected immunologically with an Optical ImmunoAssay (OIA) method. Swabs that were stored with transport medium harbored viable but rapidly declining numbers of GBS. In contrast, a strong OIA signal was maintained. Recovery of viable GBS organisms declined more quickly when swabs were stored in the absence of transport medium, whereas detection of GBS antigen remained consistent. Both methods were tested for interference from either antibiotics or feminine hygiene products. These compounds inhibited the detection of GBS by culture but had no detrimental effect on the OIA result.

A Homogeneous Time-Resolved Fluorescence Immunoassay Method for the Measurement of Compound W

PubMed Central

Huang, Biao; Yu, Huixin; Bao, Jiandong; Zhang, Manda; Green, William L; Wu, Sing-Yung

2018-01-01

Objective: Using compound W (a 3,3′-diiodothyronine sulfate [T2S] immuno-crossreactive material)-specific polyclonal antibodies and homogeneous time-resolved fluorescence immunoassay assay techniques (AlphaLISA) to establish an indirect competitive compound W (ICW) quantitative detection method. Method: Photosensitive particles (donor beads) coated with compound W or T2S and rabbit anti-W antibody were incubated with biotinylated goat anti-rabbit antibody. This constitutes a detection system with streptavidin-coated acceptor particle. We have optimized the test conditions and evaluated the detection performance. Results: The sensitivity of the method was 5 pg/mL, and the detection range was 5 to 10 000 pg/mL. The intra-assay coefficient of variation averages <10% with stable reproducibility. Conclusions: The ICW-AlphaLISA shows good stability and high sensitivity and can measure a wide range of compound W levels in extracts of maternal serum samples. This may have clinical application to screen congenital hypothyroidism in utero. PMID:29449777

A Homogeneous Time-Resolved Fluorescence Immunoassay Method for the Measurement of Compound W.

PubMed

Huang, Biao; Yu, Huixin; Bao, Jiandong; Zhang, Manda; Green, William L; Wu, Sing-Yung

2018-01-01

Using compound W (a 3,3'-diiodothyronine sulfate [T 2 S] immuno-crossreactive material)-specific polyclonal antibodies and homogeneous time-resolved fluorescence immunoassay assay techniques (AlphaLISA) to establish an indirect competitive compound W (ICW) quantitative detection method. Photosensitive particles (donor beads) coated with compound W or T 2 S and rabbit anti-W antibody were incubated with biotinylated goat anti-rabbit antibody. This constitutes a detection system with streptavidin-coated acceptor particle. We have optimized the test conditions and evaluated the detection performance. The sensitivity of the method was 5 pg/mL, and the detection range was 5 to 10 000 pg/mL. The intra-assay coefficient of variation averages <10% with stable reproducibility. The ICW-AlphaLISA shows good stability and high sensitivity and can measure a wide range of compound W levels in extracts of maternal serum samples. This may have clinical application to screen congenital hypothyroidism in utero.

Autoantibodies in Autoimmune Hepatitis.

PubMed

Muratori, Luigi; Deleonardi, Gaia; Lalanne, Claudine; Barbato, Erica; Tovoli, Alessandra; Libra, Alessia; Lenzi, Marco; Cassani, Fabio; Muratori, Paolo

2015-01-01

The detection of diagnostic autoantibodies such as antinuclear antibodies (ANA), anti-smooth muscle antibodies (SMA), anti-liver/kidney microsomal type 1 (anti-LKM1), anti-liver cytosol type 1 (anti-LC1) and anti-soluble liver antigen (anti-SLA) is historically associated with the diagnosis of autoimmune hepatitis. When autoimmune hepatitis is suspected, the detection of one or any combination of diagnostic autoantibodies, by indirect immunofluorescence or immuno-enzymatic techniques with recombinant antigens, is a pivotal step to reach a diagnostic score of probable or definite autoimmune hepatitis. Diagnostic autoantibodies (ANA, SMA, anti-LKM1, anti-LC1, anti-SLA) are a cornerstone in the diagnosis of autoimmune hepatitis. Other ancillary autoantibodies, associated with peculiar clinical correlations, appear to be assay-dependent and institution-specific, and validation studies are needed. © 2015 S. Karger AG, Basel.

Highly sensitive immuno-assays for the determination of cotinine in serum and saliva. Comparison between RIA and an avidin-biotin ELISA.

PubMed

Benkirane, S; Nicolas, A; Galteau, M M; Siest, G

1991-06-01

Two immuno-assay methods (RIA and ELISA) have been developed for the accurate and sensitive measurement of cotinine in human body fluids (serum, saliva). RIA uses [3H]cotinine as antigen and charcoal/dextran for separating cotinine-bound antibodies from the free derivative. Another technique (ELISA) was developed to avoid the use of radio-labelled compounds and to determine cotinine in large populations, including passive or non-smokers who usually present very low concentrations. The two techniques were analytically validated. The detection limit was similar (0.1 micrograms/l) and the precision was better than 10% for both techniques. Non-smoker values ranged from 0.1 to 17 micrograms/l by ELISA and 0.1 to 27.5 micrograms/l by RIA, whereas smoker values ranged from 50 to 1000 micrograms/l (ELISA) and from 70 to 800 micrograms/l (RIA). The comparative analysis of cotinine in 96 human sera revealed a good correlation between the two methods (r = 0.97) and a reliable discrimination between the populations of non-smokers and smokers. As usual, the ELISA is more rapid (4 h 30 min) than the RIA (longer than 48 h). ELISA is proposed for use in the epidemiological investigation of the human tobacco risk.

Site-specific radical formation in DNA induced by Cu(II)-H2O2 oxidizing system, using ESR, Immuno-spin trapping, LC/MS and MS/MS

PubMed Central

Bhattacharjee, Suchandra; Deterding, Leesa J.; Chatterjee, Saurabh; Jiang, JinJie; Ehrenshaft, Marilyn; Lardinois, Olivier; Ramirez, Dario C.; Tomer, Kenneth B.; Mason, Ronald P.

2011-01-01

Oxidative stress-related damage to the DNA macromolecule produces a multitude of lesions that are implicated in mutagenesis, carcinogenesis, reproductive cell death and aging. Many of these lesions have been studied and characterized by various techniques. Of the techniques that are available, the comet assay, HPLC-EC, GC-MS, HPLC-MS and especially HPLC-MS/MS remain the most widely used and have provided invaluable information on these lesions. However, accurate measurement of DNA damage has been a matter of debate. In particular, there have been reports of artifactual oxidation leading to erroneously high damage estimates. Further, most of these techniques measure the end product of a sequence of events and thus provide only limited information on the initial radical mechanism. We report here a qualitative measurement of DNA damage induced by a Cu(II)-H2O2 oxidizing system using immuno spin-trapping (IST) with EPR, MS and MS/MS. The radical generated is trapped by DMPO immediately upon formation. The DMPO adduct formed is initially EPR active but subsequently is oxidized to the stable nitrone, which can then be detected by IST and further characterized by MS and MS/MS. PMID:21382477

Comparison of immunoglobulin E measurements on IMMULITE and ImmunoCAP in samples consisting of allergen-specific mouse-human chimeric monoclonal antibodies towards allergen extracts and four recombinant allergens.

PubMed

Szecsi, Pal B; Stender, Steen

2013-01-01

Specific immunoglobulin E (IgE) antibody in vitro tests are performed on enzyme immunoassay systems. Poor agreement among systems has been reported and comparisons have been made exclusively with allergen extracts - not with recombinant allergens. Here we compare the ImmunoCAP and the IMMULITE systems. Ten patient samples with positive IgE toward egg white, birch pollen or cat or dog dander were compared using allergen extracts or the recombinant allergens Gal d 1, Bet v 1, Fel d 1 and Can f 1 with the two assay systems. Comparisons were also performed using four monoclonal mouse-human chimeric IgE antibodies specific for the same allergenic components. IMMULITE estimated a higher allergen-specific IgE concentration in sera than ImmunoCAP when testing with allergen extracts as well as recombinant allergens. The chimeric antibodies gave an equivalent response in the total IgE and specific IgE (sIgE) with an average ratio of 1.08 (range 0.9-1.3) on ImmunoCAP. In contrast, IMMULITE exhibited sIgE signals that were substantially higher than the summed level of IgE for all four chimeric antibodies (average ratio 2.96 and range 1.7-4.3). Comparison using chimeric antibodies allowed the evaluation of the true performance of the systems. ImmunoCAP measured total IgE and sIgE equally, whereas IMMULITE displayed higher sIgE signals when compared to the summed level of total IgE for all four chimeric antibodies. Results obtained with the two assay systems are not interchangeable by means of mathematical conversion. Copyright © 2013 S. Karger AG, Basel.

Redox Regulation and the Autistic Spectrum: Role of Tryptophan Catabolites, Immuno-inflammation, Autoimmunity and the Amygdala

PubMed Central

Anderson, George; Maes, Michael

2014-01-01

The autistic spectrum disorders (ASD) form a set of multi-faceted disorders with significant genetic, epigenetic and environmental determinants. Oxidative and nitrosative stress (O&NS), immuno-inflammatory pathways, mitochondrial dysfunction and dysregulation of the tryptophan catabolite (TRYCATs) pathway play significant interactive roles in driving the early developmental etiology and course of ASD. O&NS interactions with immuno-inflammatory pathways mediate their effects centrally via the regulation of astrocyte and microglia responses, including regional variations in TRYCATs produced. Here we review the nature of these interactions and propose an early developmental model whereby different ASD genetic susceptibilities interact with environmental and epigenetic processes, resulting in glia biasing the patterning of central interarea interactions. A role for decreased local melatonin and N-acetylserotonin production by immune and glia cells may be a significant treatment target. PMID:24669209

Phosphorylation-specific status of RNAi triggers in pharmacokinetic and biodistribution analyses

PubMed Central

Trubetskoy, Vladimir S.; Griffin, Jacob B.; Nicholas, Anthony L.; Nord, Eric M.; Xu, Zhao; Peterson, Ryan M.; Wooddell, Christine I.; Rozema, David B.; Wakefield, Darren H.; Lewis, David L.

2017-01-01

Abstract The RNA interference (RNAi)-based therapeutic ARC-520 for chronic hepatitis B virus (HBV) infection consists of a melittin-derived peptide conjugated to N-acetylgalactosamine for hepatocyte targeting and endosomal escape, and cholesterol-conjugated RNAi triggers, which together result in HBV gene silencing. To characterize the kinetics of RNAi trigger delivery and 5΄-phosphorylation of guide strands correlating with gene knockdown, we employed a peptide-nucleic acid (PNA) hybridization assay. A fluorescent sense strand PNA probe binding to RNAi duplex guide strands was coupled with anion exchange high performance liquid chromatography to quantitate guide strands and metabolites. Compared to PCR- or ELISA-based methods, this assay enables separate quantitation of non-phosphorylated full-length guide strands from 5΄-phosphorylated forms that may associate with RNA-induced silencing complexes (RISC). Biodistribution studies in mice indicated that ARC-520 guide strands predominantly accumulated in liver. 5΄-phosphorylation of guide strands was observed within 5 min after ARC-520 injection, and was detected for at least 4 weeks corresponding to the duration of HBV mRNA silencing. Guide strands detected in RISC by AGO2 immuno-isolation represented 16% of total 5΄-phosphorylated guide strands in liver, correlating with a 2.7 log10 reduction of HBsAg. The PNA method enables pharmacokinetic analysis of RNAi triggers, elucidates potential metabolic processing events and defines pharmacokinetic-pharmacodynamic relationships. PMID:28180327

Lactic acid bacteria affect serum cholesterol levels, harmful fecal enzyme activity, and fecal water content.

PubMed

Lee, Do Kyung; Jang, Seok; Baek, Eun Hye; Kim, Mi Jin; Lee, Kyung Soon; Shin, Hea Soon; Chung, Myung Jun; Kim, Jin Eung; Lee, Kang Oh; Ha, Nam Joo

2009-06-11

Lactic acid bacteria (LAB) are beneficial probiotic organisms that contribute to improved nutrition, microbial balance, and immuno-enhancement of the intestinal tract, as well as lower cholesterol. Although present in many foods, most trials have been in spreads or dairy products. Here we tested whether Bifidobacteria isolates could lower cholesterol, inhibit harmful enzyme activities, and control fecal water content. In vitro culture experiments were performed to evaluate the ability of Bifidobacterium spp. isolated from healthy Koreans (20 approximately 30 years old) to reduce cholesterol-levels in MRS broth containing polyoxyethanylcholesterol sebacate. Animal experiments were performed to investigate the effects on lowering cholesterol, inhibiting harmful enzyme activities, and controlling fecal water content. For animal studies, 0.2 ml of the selected strain cultures (108 approximately 109 CFU/ml) were orally administered to SD rats (fed a high-cholesterol diet) every day for 2 weeks. B. longum SPM1207 reduced serum total cholesterol and LDL levels significantly (p < 0.05), and slightly increased serum HDL. B. longum SPM1207 also increased fecal LAB levels and fecal water content, and reduced body weight and harmful intestinal enzyme activities. Daily consumption of B. longum SPM1207 can help in managing mild to moderate hypercholesterolemia, with potential to improve human health by helping to prevent colon cancer and constipation.

Detecting Molecular Signatures of Life on Mars: the Life Marker Chip (lmc) Instrument

NASA Astrophysics Data System (ADS)

Derveni, Mariliza

In recent years, the rise of interest in planetary exploration and the emergence of Astrobiology as a promising field of research have lead to a number of programmes aiming to develop sensitive instruments for the detection of the molecular signatures of life in extreme environments. An antibody assay-based life detection instrument, the Life Marker Chip (LMC), is currently under development by a UK-lead international consortium for the European Space Agency's (ESA) ExoMars rover. This forms part of the joint ESA/NASA Mars exploration programme with the ExoMars Rover currently scheduled for launch in 2018. The organic molecules targeted for Life detection by the LMC are based on an assumption of "Earth-like" Life on Mars -extinct and/or extant. The molecular targets for the LMC have been chosen to represent markers of extinct Life, extant Life, abiotic chemistry (e.g. of meteoritic origin) and mission-borne Earth contamination. The LMC incorporates integrated liquid sample extraction and processing for dry Martian samples, which will be collected from up to 2m below the surface of Mars, where organic molecules, if present, are expected to be better preserved. The core technology of the LMC is a combination of optical evanescent waveguides, micro-fluidics, immuno-microarrays with fluorescent labels and CCD detector readout. Phage display recombinant antibody technology has been employed in order to acquire antibodies against a number of the LMC target molecules. The LMC hardware is currently in a breadboard phase of development. The recombinant antibody development for LMC targets is an on-going project, and testing of Earth-analogue Martian samples has been initiated

Lean management and medical laboratory: application in transfusionnal immuno-hematology.

PubMed

Thibert, Jean-Baptiste; Le Vacon, Françoise; Danic, Bruno

2017-10-01

Despite a common use in industrial applications, only a few studies describe the lean management methods in medical laboratory. These tools have been evaluated in analysis laboratory of blood donors, especially in immuno-hematology sector. The aim was to optimize the organization and maintain team cohesion and strong staff involvement in a restructuring context. The tools used and the results obtained are presented in this study.

Retinopathy following measles, mumps, and rubella vaccination in an immuno-incompetent girl.

PubMed

Schuil, J; van de Putte, E M; Zwaan, C M; Koole, F D; Meire, F M

1998-01-01

We describe a 4-year-old girl with subnormal visual acuity due to a bilateral retinopathy. The child had a history of encephalitis following MMR vaccination. Temporary retinopathy associated with measles, mumps, and rubella (MMR) vaccination has been described. Recently an idiopathic CD4+ T lymphocytopenia in the child was diagnosed. This cellular immunodeficiency supports our hypothesis of measles retinopathy after vaccination of an immuno-deficient child.

IMMUNO-MODULATORY PROPERTIES OF PREBIOTICS EXTRACTED FROM vernonia amygdalina.

PubMed

Im, Ezeonu; Ae, Asuquo; Bn, Ukwah; Po, Ukoha

2016-01-01

Vernonia amygdalina , commonly called bitter-leaf, is widely consumed in many parts of Africa, and Nigeria, in particular. The leaf extract has been reported to have antimicrobial, anti-plasmodial, anti-helminthic, as well as prebiotic properties, but its immuno-modulatory effects have not been well-studied, neither have the prebiotics been identified. This study evaluated the immuno-modulatory properties of the aqueous leaf extract and identified the prebiotic components. The immuno-modulatory potential was evaluated by monitoring the effects of oral administration of the extract on immunological, haematological and lipid profiles of Rattus norvegicus , while the prebiotic components were identified by thin layer chromatography (TLC), following liquid-liquid fractionation of the extract. Consumption of the extract caused significant increases in CD4+-, white blood cell-, total lymphocyte- and high density lipid (HDL) counts; decreases in low density lipid (LDL) and triglycerides and no significant effect on haemoglobin (Hb) and packed cell volume (PCV) in the blood of test animals. The water-soluble fraction of the extract contained most of the phyto-constituents of the extract and Thin Layer Chromatographic analysis of the fraction revealed the presence of fructo-oligosaccharide and galacto-oligosaccharide prebiotics. The results from this study have shown that the aqueous leaf extract of V. amygdalina has positive immune-modulatory and haematologic effects and contains some important prebiotic compounds.

High level expression and immunochemical characterization of botulinum neurotoxin type F light chain.

PubMed

Chauhan, Ritika; Chauhan, Vinita; Rao, Mula Kameshwar; Chaudhary, Dilip; Bhagyawant, Sameer; Dhaked, Ram Kumar

2018-06-01

Botulinum neurotoxins (BoNTs) are the most toxic biological substances known. Their potential use as biological warfare agent results in their classification as category A biowarfare agent by Centers for Disease Control and Prevention (CDC), USA. Presently, there are no approved detection system and pharmacological treatments for BoNT intoxication. Although a toxoid vaccine is available for immuno-prophylaxis, vaccines cannot reverse the effect of pre-translocated toxin. Direct handling of the live BoNTs for developing detection and therapeutics may pose fatal danger. This concern was addressed by purifying the recombinant catalytically active light chain of BoNT/F. BoNT/F-LC gene was amplified from the genomic DNA using specifically designed primers and expressed in Escherichia coli. Expression and purification profile were optimized under different conditions for biologically active light chain production. Specific polyclonal antibodies generated against type F illustrates in vivo neutralization in mice and rabbit. These antibodies play key role in conceiving the development of high throughput SPR based detection system which is a highly precise label free technique for protein interaction analysis. The presented work is first of its kind, signifying the production of highly stable and active rBoNT/F-LC and its immunochemical characterization. The study aids in paving the path towards developing a persistent detection system as well as in presenting comprehended scheme for in vitro small molecule therapeutics analysis. Copyright © 2018 Elsevier Inc. All rights reserved.

A novel method for detection of H9N2 influenza viruses by an aptamer-real time-PCR.

PubMed

Hmila, Issam; Wongphatcharachai, Manoosak; Laamiri, Nacira; Aouini, Rim; Marnissi, Boutheina; Arbi, Marwa; Sreevatsan, Srinand; Ghram, Abdeljelil

2017-05-01

H9N2 Influenza subtype has emerged in Tunisia causing epidemics in poultry and resulting in major economic losses. New mutations in their hemagglutinin and neuraminidase proteins were acquired, suggesting their potential to directly infect humans. Effective surveillance tools should be implemented to help prevent potential spillover of the virus across species. We have developed a highly sensitive real time immuno-polymerase chain reaction (RT-I-PCR) method for detecting H9N2 virus. The assay applies aptamers as ligands to capture and detect the virus. First, a panel of specific ssDNA aptamers was selected via a one step high stringency protocol. Next, the panel of selected aptamers was characterized for their affinities and their specificity to H9N2 virus. The aptamer showing the highest binding affinity to the virus was used as ligand to develop a highly sensitive sandwich Aptamer I-PCR. A 3-log increase in analytical sensitivity was achieved as compared to a routinely used ELISA antigen test, highlighting the potential of this approach to detect very low levels of virus particles. The test was validated using clinical samples and constitutes a rapid and a label-free platform, opening a new venue for the development of aptamer -based viability sensing for a variety of microorganisms of economic importance in Tunisia and surrounding regions. Copyright © 2017 Elsevier B.V. All rights reserved.

Time-gated luminescence microscopy allowing direct visual inspection of lanthanide-stained microorganisms in background-free condition.

PubMed

Jin, Dayong; Piper, James A

2011-03-15

Application of standard immuno-fluorescence microscopy techniques for detection of rare-event microorganisms in dirty samples is severely limited by autofluorescence of nontarget organisms or other debris. Time-gated detection using gateable array detectors in combination with microsecond-lifetime luminescent bioprobes (usually lanthanide-based) is highly effective in suppression of (nanosecond-lifetime) autofluorescence background; however, the complexity and cost of the instrumentation is a major barrier to application of these techniques to routine diagnostics. We report a practical, low-cost implementation of time-gated luminescence detection in a standard epifluorescence microscope which has been modified to include a high-power pulsed UV light-emitting diode (LED) illumination source and a standard fast chopper inserted in the focal plane behind a microscope eyepiece. Synchronization of the pulsed illumination/gated detection cycle is driven from the clock signal from the chopper. To achieve time-gated luminescence intensities sufficient for direct visual observation, we use high cycle rates, up to 2.5 kHz, taking advantage of the fast switching capabilities of the LED source. We have demonstrated real-time direct-visual inspection of europium-labeled Giardia lamblia cysts in dirty samples and Cryptosporidium parvum oocysts in fruit juice concentrate. The signal-to-background ratio has been enhanced by a factor of 18 in time-gated mode. The availability of low-cost, robust time-gated microscopes will aid development of long-lifetime luminescence bioprobes and accelerate their application in routine laboratory diagnostics.

F-18 Labeled Diabody-Luciferase Fusion Proteins for Optical-ImmunoPET

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Anna M.

2013-01-18

The goal of the proposed work is to develop novel dual-labeled molecular imaging probes for multimodality imaging. Based on small, engineered antibodies called diabodies, these probes will be radioactively tagged with Fluorine-18 for PET imaging, and fused to luciferases for optical (bioluminescence) detection. Performance will be evaluated and validated using a prototype integrated optical-PET imaging system, OPET. Multimodality probes for optical-PET imaging will be based on diabodies that are dually labeled with 18F for PET detection and fused to luciferases for optical imaging. 1) Two sets of fusion proteins will be built, targeting the cell surface markers CEA or HER2.more » Coelenterazine-based luciferases and variant forms will be evaluated in combination with native substrate and analogs, in order to obtain two distinct probes recognizing different targets with different spectral signatures. 2) Diabody-luciferase fusion proteins will be labeled with 18F using amine reactive [18F]-SFB produced using a novel microwave-assisted, one-pot method. 3) Sitespecific, chemoselective radiolabeling methods will be devised, to reduce the chance that radiolabeling will inactivate either the target-binding properties or the bioluminescence properties of the diabody-luciferase fusion proteins. 4) Combined optical and PET imaging of these dual modality probes will be evaluated and validated in vitro and in vivo using a prototype integrated optical-PET imaging system, OPET. Each imaging modality has its strengths and weaknesses. Development and use of dual modality probes allows optical imaging to benefit from the localization and quantitation offered by the PET mode, and enhances the PET imaging by enabling simultaneous detection of more than one probe.« less

Pre-Clinical Tests of an Integrated CMOS Biomolecular Sensor for Cardiac Diseases Diagnosis.

PubMed

Lee, Jen-Kuang; Wang, I-Shun; Huang, Chi-Hsien; Chen, Yih-Fan; Huang, Nien-Tsu; Lin, Chih-Ting

2017-11-26

Coronary artery disease and its related complications pose great threats to human health. In this work, we aim to clinically evaluate a CMOS field-effect biomolecular sensor for cardiac biomarkers, cardiac-specific troponin-I (cTnI), N -terminal prohormone brain natriuretic peptide (NT-proBNP), and interleukin-6 (IL-6). The CMOS biosensor is implemented via a standard commercialized 0.35 μm CMOS process. To validate the sensing characteristics, in buffer conditions, the developed CMOS biosensor has identified the detection limits of IL-6, cTnI, and NT-proBNP as being 45 pM, 32 pM, and 32 pM, respectively. In clinical serum conditions, furthermore, the developed CMOS biosensor performs a good correlation with an enzyme-linked immuno-sorbent assay (ELISA) obtained from a hospital central laboratory. Based on this work, the CMOS field-effect biosensor poses good potential for accomplishing the needs of a point-of-care testing (POCT) system for heart disease diagnosis.

Advancements in optical techniques and imaging in the diagnosis and management of bladder cancer.

PubMed

Rose, Tracy L; Lotan, Yair

2018-03-01

Accurate detection and staging is critical to the appropriate management of urothelial cancer (UC). The use of advanced optical techniques during cystoscopy is becoming more widespread to prevent recurrent nonmuscle invasive bladder cancer. Standard of care for muscle-invasive UC includes the use of computed tomography and/or magnetic resonance imaging, but staging accuracy of these tests remains imperfect. Novel imaging modalities are being developed to improve current test performance. Positron emission tomography/computed tomography has a role in the initial evaluation of select patients with muscle-invasive bladder cancer and in disease recurrence in some cases. Several novel immuno-positron emission tomography tracers are currently in development to address the inadequacy of current imaging modalities for monitoring of tumor response to newer immune-based treatments. This review summaries the current standards and recent advances in optical techniques and imaging modalities in localized and metastatic UC. Copyright © 2018 Elsevier Inc. All rights reserved.

Diagnosis of schistosomiasis japonica with interfacial co-assembly-based multi-channel electrochemical immunosensor arrays

NASA Astrophysics Data System (ADS)

Deng, Wangping; Xu, Bin; Hu, Haiyan; Li, Jianyong; Hu, Wei; Song, Shiping; Feng, Zheng; Fan, Chunhai

2013-05-01

Schistosomiasis control remains to be an important and challenging task in the world. However, lack of quick, simple, sensitive and specific sero-diagnostic test is still a hurdle in the control practice. The commonly employed enzyme-linked immuno-sorbent assay (ELISA) relies on the native soluble egg antigen (SEA) that is limited in supply. Here we developed an electrochemical immunosensor array (ECISA) assay with an interfacial co-assembly strategy. A recombinant Schistosoma japonicum (Sj) calcium-binding protein (SjE16) was used as a principal antigen, while the SEA as a minor, co-assembling agent, with a ratio of 8:1 (SjE16: SEA, Sj16EA), which was co-immobilized on a disposable 16-channel screen-printed carbon electrode array. A portable electrochemical detector was employed to detect antibodies in serum samples. The sensitivity of ECISA reached 100% with minimal cross-reactions. Therefore, we have demonstrated that this rapid, sensitive and specific ECISA technique has the potential to perform large-scale on-site screening of Sj infection.

Review of methods used for identification of biothreat agents in environmental protection and human health aspects.

PubMed

Mirski, Tomasz; Bartoszcze, Michał; Bielawska-Drózd, Agata; Cieślik, Piotr; Michalski, Aleksander J; Niemcewicz, Marcin; Kocik, Janusz; Chomiczewski, Krzysztof

2014-01-01

Modern threats of bioterrorism force the need to develop methods for rapid and accurate identification of dangerous biological agents. Currently, there are many types of methods used in this field of studies that are based on immunological or genetic techniques, or constitute a combination of both methods (immuno-genetic). There are also methods that have been developed on the basis of physical and chemical properties of the analytes. Each group of these analytical assays can be further divided into conventional methods (e.g. simple antigen-antibody reactions, classical PCR, real-time PCR), and modern technologies (e.g. microarray technology, aptamers, phosphors, etc.). Nanodiagnostics constitute another group of methods that utilize the objects at a nanoscale (below 100 nm). There are also integrated and automated diagnostic systems, which combine different methods and allow simultaneous sampling, extraction of genetic material and detection and identification of the analyte using genetic, as well as immunological techniques.

Development of screen-printed tryptophan-kynurenine immunosensor for in vitro assay of kynurenine-mediated immunosuppression effect of cancer cells on activated T-cells.

PubMed

Karami, Pari; Majidi, Mir Reza; Johari-Ahar, Mohammad; Barar, Jaleh; Omidi, Yadollah

2017-06-15

The development of analytical methods that respond to the emerging need to perform rapid 'in situ' analyses of human metabolic pathways (HMPs) demonstrates disposable screen-printed electrodes (SPEs) as an alternative to the traditional tools. In the kynurenine pathway, one of the important HMPs, increased production of kynurenine (Kyn) as a main catabolite of tryptophan (Trp) degradation is involved in the immuno-editing process supporting cancer cells in escaping from the human immune system. In the current study, we demonstrate the development of a screen-printed potentiometric immunosensor for in vitro evaluation of Trp consumption and Kyn production controlled by cancer cells in response to the activated T-lymphocytes. To engineer this immunosensor, uniform layer of carboxylated multiwall carbon nanotubes (MWCNT) was deposited on gold screen-printed electrode (AuSPE), and afterwards monoclonal antibody (mAb) specific to l-kynurenine was covalently conjugated with the MWCNT modified AuSPE. The engineered immunosensor was examined in monitoring Trp consumption and Kyn production in metastatic (Calu-6, NCI-H1299, and HT29) and nonmetastatic (HepG2 and 1321NI) cancer cell lines. Without applying preparation and separation steps, this Trp-Kyn immunosensor offers an improved limit of detection (0.5nM and 120nM for Kyn and Trp detection, respectively) and a broad linear range of detection (LRD: 0.001-1µM and 1-100µM for Kyn, and 0.1-300µM for Trp detection). However, this immunosensor was successfully used for in situ analysis of Kyn that are produced during immuno-editing process in cell culture media, and could reveal that Trp consumption and Kyn production by highly metastatic cancer cells (HT29) were significantly higher than nonmetastatic HepG2 cancer cells. Owing to the screen-printed nature, such kind of biosensors have capability of being integrated into lab-on-a-chip (LOC), microfluidics, and micro total analysis systems. Copyright © 2016 Elsevier B.V. All rights reserved.

An overview of adjuvants utilized in prophylactic vaccine formulation as immunomodulators.

PubMed

Chauhan, Nidhi; Tiwari, Sukirti; Iype, Tessy; Jain, Utkarsh

2017-05-01

Development of efficient and cost effective vaccines have been recognized as the primary concern to improve the overall healthcare in a country. In order to achieve this goal, more improved and powerful adjuvants need to be developed. Lacking in the self-adjuvanting immuno-modulatory constituents, vaccines exhibit lower immunogenicity. Combining potent adjuvants with vaccines is the most appropriate method to enhance the efficacy of the vaccines. Hence, this review is focussed on the most potent adjuvants for the formulation of vaccines. Areas covered: This review focuses on Oil-based emulsions, Mineral compounds, Liposomes, Bacterial products, ISCOMs and most recently used nanomaterials as adjuvants for enhancing the antigenicity of vaccines. Furthermore, this review explains the immunological response elicited by various particles. Moreover, case studies are incorporated providing an in depth analyses of various adjuvant-containing vaccines which are currently used. Expert commentary: Enhanced fundamental knowledge about the adjuvants and their immuno-stimulatory capabilities and delivery mechanisms will facilitate the rational designing of prophylactic vaccines with better efficacy.

Development and Validation of an Immuno-PET Tracer as a Companion Diagnostic Agent for Antibody-Drug Conjugate Therapy to Target the CA6 Epitope.

PubMed

Ilovich, Ohad; Natarajan, Arutselvan; Hori, Sharon; Sathirachinda, Ataya; Kimura, Richard; Srinivasan, Ananth; Gebauer, Mathias; Kruip, Jochen; Focken, Ingo; Lange, Christian; Carrez, Chantal; Sassoon, Ingrid; Blanc, Veronique; Sarkar, Susanta K; Gambhir, Sanjiv S

2015-07-01

To develop and compare three copper 64 ((64)Cu)-labeled antibody fragments derived from a CA6-targeting antibody (huDS6) as immuno-positron emission tomography (immuno-PET)-based companion diagnostic agents for an antibody-drug conjugate by using huDS6. Three antibody fragments derived from huDS6 were produced, purified, conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and evaluated in the following ways: (a) the affinity of the fragments and the DOTA conjugates was measured via flow cytometry, (b) the stability of the labeled fragments was determined ex vivo in human serum over 24 hours, and (c) comparison of the in vivo imaging potential of the fragments was evaluated in mice bearing subcutaneous CA6-positive and CA6-negative xenografts by using serial PET imaging and biodistribution. Isotype controls with antilysozyme and anti-DM4 B-Fabs and blocking experiments with an excess of either B-Fab or huDS6 were used to determine the extent of the antibody fragment (64)Cu-DOTA-B-Fab binding specificity. Immunoreactivity and tracer kinetics were evaluated by using cellular uptake and 48-hour imaging experiments, respectively. Statistical analyses were performed by using t tests, one-way analysis of variance, and Wilcoxon and Mann-Whitney tests. The antibody fragment (64)Cu-DOTA-B-Fab was more than 95% stable after 24 hours in human serum, had an immunoreactivity of more than 70%, and allowed differentiation between CA6-positive and CA6-negative tumors in vivo as early as 6 hours after injection, with a 1.7-fold uptake ratio between tumors. Isotype and blocking studies experiments showed tracer-specific uptake in antigen-positive tumors, despite some nonspecific uptake in both tumor models. Three antibody fragments were produced and examined as potential companion diagnostic agents. (64)Cu-DOTA-B-Fab is a stable and effective immuno-PET tracer for CA6 imaging in vivo.

Development and Validation of an Immuno-PET Tracer as a Companion Diagnostic Agent for Antibody-Drug Conjugate Therapy to Target the CA6 Epitope

PubMed Central

Ilovich, Ohad; Natarajan, Arutselvan; Hori, Sharon; Sathirachinda, Ataya; Kimura, Richard; Srinivasan, Ananth; Gebauer, Mathias; Kruip, Jochen; Focken, Ingo; Lange, Christian; Carrez, Chantal; Sassoon, Ingrid; Blanc, Veronique; Sarkar, Susanta K.

2015-01-01

Purpose To develop and compare three copper 64 (64Cu)–labeled antibody fragments derived from a CA6-targeting antibody (huDS6) as immuno-positron emission tomography (immuno-PET)–based companion diagnostic agents for an antibody-drug conjugate by using huDS6. Materials and Methods Three antibody fragments derived from huDS6 were produced, purified, conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and evaluated in the following ways: (a) the affinity of the fragments and the DOTA conjugates was measured via flow cytometry, (b) the stability of the labeled fragments was determined ex vivo in human serum over 24 hours, and (c) comparison of the in vivo imaging potential of the fragments was evaluated in mice bearing subcutaneous CA6-positive and CA6-negative xenografts by using serial PET imaging and biodistribution. Isotype controls with antilysozyme and anti-DM4 B-Fabs and blocking experiments with an excess of either B-Fab or huDS6 were used to determine the extent of the antibody fragment 64Cu-DOTA-B-Fab binding specificity. Immunoreactivity and tracer kinetics were evaluated by using cellular uptake and 48-hour imaging experiments, respectively. Statistical analyses were performed by using t tests, one-way analysis of variance, and Wilcoxon and Mann-Whitney tests. Results The antibody fragment 64Cu-DOTA-B-Fab was more than 95% stable after 24 hours in human serum, had an immunoreactivity of more than 70%, and allowed differentiation between CA6-positive and CA6-negative tumors in vivo as early as 6 hours after injection, with a 1.7-fold uptake ratio between tumors. Isotype and blocking studies experiments showed tracer-specific uptake in antigen-positive tumors, despite some nonspecific uptake in both tumor models. Conclusion Three antibody fragments were produced and examined as potential companion diagnostic agents. 64Cu-DOTA-B-Fab is a stable and effective immuno-PET tracer for CA6 imaging in vivo. © RSNA, 2015 Online supplemental material is available for this article. PMID:25734548

A novel method for detection of phosphorylation in single cells by surface enhanced Raman scattering (SERS) using composite organic-inorganic nanoparticles (COINs).

PubMed

Shachaf, Catherine M; Elchuri, Sailaja V; Koh, Ai Leen; Zhu, Jing; Nguyen, Lienchi N; Mitchell, Dennis J; Zhang, Jingwu; Swartz, Kenneth B; Sun, Lei; Chan, Selena; Sinclair, Robert; Nolan, Garry P

2009-01-01

Detection of single cell epitopes has been a mainstay of immunophenotyping for over three decades, primarily using fluorescence techniques for quantitation. Fluorescence has broad overlapping spectra, limiting multiplexing abilities. To expand upon current detection systems, we developed a novel method for multi-color immuno-detection in single cells using "Composite Organic-Inorganic Nanoparticles" (COINs) Raman nanoparticles. COINs are Surface-Enhanced Raman Scattering (SERS) nanoparticles, with unique Raman spectra. To measure Raman spectra in single cells, we constructed an automated, compact, low noise and sensitive Raman microscopy device (Integrated Raman BioAnalyzer). Using this technology, we detected proteins expressed on the surface in single cells that distinguish T-cells among human blood cells. Finally, we measured intracellular phosphorylation of Stat1 (Y701) and Stat6 (Y641), with results comparable to flow cytometry. Thus, we have demonstrated the practicality of applying COIN nanoparticles for measuring intracellular phosphorylation, offering new possibilities to expand on the current fluorescent technology used for immunoassays in single cells.

A Novel Method for Detection of Phosphorylation in Single Cells by Surface Enhanced Raman Scattering (SERS) using Composite Organic-Inorganic Nanoparticles (COINs)

PubMed Central

Shachaf, Catherine M.; Elchuri, Sailaja V.; Koh, Ai Leen; Zhu, Jing; Nguyen, Lienchi N.; Mitchell, Dennis J.; Zhang, Jingwu; Swartz, Kenneth B.; Sun, Lei; Chan, Selena; Sinclair, Robert; Nolan, Garry P.

2009-01-01

Background Detection of single cell epitopes has been a mainstay of immunophenotyping for over three decades, primarily using fluorescence techniques for quantitation. Fluorescence has broad overlapping spectra, limiting multiplexing abilities. Methodology/Principal Findings To expand upon current detection systems, we developed a novel method for multi-color immuno-detection in single cells using “Composite Organic-Inorganic Nanoparticles” (COINs) Raman nanoparticles. COINs are Surface-Enhanced Raman Scattering (SERS) nanoparticles, with unique Raman spectra. To measure Raman spectra in single cells, we constructed an automated, compact, low noise and sensitive Raman microscopy device (Integrated Raman BioAnalyzer). Using this technology, we detected proteins expressed on the surface in single cells that distinguish T-cells among human blood cells. Finally, we measured intracellular phosphorylation of Stat1 (Y701) and Stat6 (Y641), with results comparable to flow cytometry. Conclusions/Significance Thus, we have demonstrated the practicality of applying COIN nanoparticles for measuring intracellular phosphorylation, offering new possibilities to expand on the current fluorescent technology used for immunoassays in single cells. PMID:19367337

Large-Scale Evaluation of the Immuno-Mycologics Lateral Flow and Enzyme-Linked Immunoassays for Detection of Cryptococcal Antigen in Serum and Cerebrospinal Fluid

PubMed Central

Hansen, Jessica; Slechta, E. Susan; Gates-Hollingsworth, Marcellene A.; Neary, Brandon; Barker, Adam P.; Bauman, Sean; Kozel, Thomas R.

2013-01-01

Cryptococcosis is a systemic infection caused by the pathogenic yeasts Cryptococcus neoformans and C. gattii. Detection of cryptococcal capsular antigen (CrAg) in serum and cerebrospinal fluid (CSF) plays an important diagnostic role. We prospectively compared the new Immuno-Mycologics Inc. (IMMY) lateral flow assay (LFA) and enzyme immunoassay (EIA) to our current CrAg test (Premier EIA; Meridian Bioscience Inc.). Discordant samples were retested with the latex-Cryptococcus antigen test (IMMY) and using serotype-specific monoclonal antibodies (MAbs). A total of 589 serum and 411 CSF specimens were tested in parallel. Qualitative agreement across assays was 97.7%. In all, 56 (41 serum and 15 CSF) samples were positive and 921 (527 serum and 394 CSF) samples were negative by all three assays. The 23 discrepant specimens were all Meridian EIA negative. Of 23 discordant specimens, 20 (87.0%) were positive by both the IMMY LFA and EIA, 2 were LFA positive only, and 1 was EIA positive only. Eleven discrepant specimens had adequate volume for latex agglutination (LA) testing; 8 were LA positive, and 3 were LA negative. LA-negative samples (2 CSF samples and 1 serum) had low IMMY LFA/EIA titers (≤1:10). Serotype-specific MAb analysis of the LA-positive samples suggested that these specimens contained CrAg epitopes similar to those of serotype C strains. In conclusion, the IMMY assays showed excellent overall concordance with the Meridian EIA. Assay performance differences were related to issues of analytic sensitivity and possible serotype bias. Incomplete access to patient-level data combined with low specimen volumes limited our ability to fully resolve discrepant results. PMID:23114703

Large-scale evaluation of the immuno-mycologics lateral flow and enzyme-linked immunoassays for detection of cryptococcal antigen in serum and cerebrospinal fluid.

PubMed

Hansen, Jessica; Slechta, E Susan; Gates-Hollingsworth, Marcellene A; Neary, Brandon; Barker, Adam P; Bauman, Sean; Kozel, Thomas R; Hanson, Kimberly E

2013-01-01

Cryptococcosis is a systemic infection caused by the pathogenic yeasts Cryptococcus neoformans and C. gattii. Detection of cryptococcal capsular antigen (CrAg) in serum and cerebrospinal fluid (CSF) plays an important diagnostic role. We prospectively compared the new Immuno-Mycologics Inc. (IMMY) lateral flow assay (LFA) and enzyme immunoassay (EIA) to our current CrAg test (Premier EIA; Meridian Bioscience Inc.). Discordant samples were retested with the latex-Cryptococcus antigen test (IMMY) and using serotype-specific monoclonal antibodies (MAbs). A total of 589 serum and 411 CSF specimens were tested in parallel. Qualitative agreement across assays was 97.7%. In all, 56 (41 serum and 15 CSF) samples were positive and 921 (527 serum and 394 CSF) samples were negative by all three assays. The 23 discrepant specimens were all Meridian EIA negative. Of 23 discordant specimens, 20 (87.0%) were positive by both the IMMY LFA and EIA, 2 were LFA positive only, and 1 was EIA positive only. Eleven discrepant specimens had adequate volume for latex agglutination (LA) testing; 8 were LA positive, and 3 were LA negative. LA-negative samples (2 CSF samples and 1 serum) had low IMMY LFA/EIA titers (≤1:10). Serotype-specific MAb analysis of the LA-positive samples suggested that these specimens contained CrAg epitopes similar to those of serotype C strains. In conclusion, the IMMY assays showed excellent overall concordance with the Meridian EIA. Assay performance differences were related to issues of analytic sensitivity and possible serotype bias. Incomplete access to patient-level data combined with low specimen volumes limited our ability to fully resolve discrepant results.

Optimized protocol for quantitative multiple reaction monitoring-based proteomic analysis of formalin-fixed, paraffin embedded tissues

PubMed Central

Kennedy, Jacob J.; Whiteaker, Jeffrey R.; Schoenherr, Regine M.; Yan, Ping; Allison, Kimberly; Shipley, Melissa; Lerch, Melissa; Hoofnagle, Andrew N.; Baird, Geoffrey Stuart; Paulovich, Amanda G.

2016-01-01

Despite a clinical, economic, and regulatory imperative to develop companion diagnostics, precious few new biomarkers have been successfully translated into clinical use, due in part to inadequate protein assay technologies to support large-scale testing of hundreds of candidate biomarkers in formalin-fixed paraffin embedded (FFPE) tissues. While the feasibility of using targeted, multiple reaction monitoring-mass spectrometry (MRM-MS) for quantitative analyses of FFPE tissues has been demonstrated, protocols have not been systematically optimized for robust quantification across a large number of analytes, nor has the performance of peptide immuno-MRM been evaluated. To address this gap, we used a test battery approach coupled to MRM-MS with the addition of stable isotope labeled standard peptides (targeting 512 analytes) to quantitatively evaluate the performance of three extraction protocols in combination with three trypsin digestion protocols (i.e. 9 processes). A process based on RapiGest buffer extraction and urea-based digestion was identified to enable similar quantitation results from FFPE and frozen tissues. Using the optimized protocols for MRM-based analysis of FFPE tissues, median precision was 11.4% (across 249 analytes). There was excellent correlation between measurements made on matched FFPE and frozen tissues, both for direct MRM analysis (R2 = 0.94) and immuno-MRM (R2 = 0.89). The optimized process enables highly reproducible, multiplex, standardizable, quantitative MRM in archival tissue specimens. PMID:27462933

0-6676 : Rapid field detection of moisture content for base and subgrade : [project summary].

DOT National Transportation Integrated Search

2013-08-01

Properly applying water during compaction of : roadway base and subgrade materials is : important for achieving adequate compaction. : Construction specifications determine the : required water content, and field measurement : historically takes plac...

Component-resolved microarray analysis of IgE sensitization profiles to Felis catus major allergen molecules in Russian cat-allergic patients.

PubMed

Dolgova, Anna Sergeevna; Sudina, Anna Evgenevna; Cherkashina, Anna Sergeevna; Stukolova, Olga Alekseevna

We aimed to determine the profile of IgE reactivity to three major cat allergens, Fel d 1, Fel d 2 and Fel d 4, in cat-allergic patients in the Moscow region in Russia. sIgE levels to recombinant proteins expressed in Escherichia coli (Fel d 1 and Fel d 4) and to Fel d 2 protein purified from cat serum were measured using a microarray method developed in our laboratory. Sera from 174 anonymous subjects with a positive reaction (≥0.35 IU/mL) to cat dander extract (e1, ImmunoCAP) and 56 negative controls were used for IgE testing. Fel d 1 was recognized by 92.5%, Fel d 2 by 29.9% and Fel d 4 by 39.1% of the tested patient sera. The sensitivity to these three proteins was approximately 98% compared to cat dander extract (correlation coefficient to ImmunoCAP is 0.94 with PPV = 0.99 and NPV = 0.95). These predictive values appeared to be even more statistically significant than the divergence between the ISAC IgE test and the extract-based singleplex ImmunoCAP. The combination of the three investigated proteins (Fel d 1, Fel d 2 and Fel d 4) is suitable for in vitro molecular (serological) diagnosis of cat allergy in this region as a complement to cat dander extract. Moreover, with this method, we found distinction between Fel d 2 and other Feline sIgEs formation.

High-dose methotrexate-based immuno-chemotherapy for elderly primary CNS lymphoma patients (PRIMAIN study).

PubMed

Fritsch, K; Kasenda, B; Schorb, E; Hau, P; Bloehdorn, J; Möhle, R; Löw, S; Binder, M; Atta, J; Keller, U; Wolf, H-H; Krause, S W; Heß, G; Naumann, R; Sasse, S; Hirt, C; Lamprecht, M; Martens, U; Morgner, A; Panse, J; Frickhofen, N; Röth, A; Hader, C; Deckert, M; Fricker, H; Ihorst, G; Finke, J; Illerhaus, G

2017-04-01

To investigate immuno-chemotherapy for elderly immuno-competent patients (⩾65 years) with newly diagnosed primary central nervous system lymphoma, we conducted a multicentre single-arm trial. One cycle consisted of rituximab (375 mg/m 2 , days 1, 15, 29), high-dose methotrexate (3 g/m 2 days 2, 16, 30), procarbazine (60 mg/m 2 days 2-11) and lomustine (110 mg/m 2 , day 2)-R-MPL protocol. Owing to infectious complications, we omitted lomustine during the study and consecutive patients were treated with the R-MP protocol. Three cycles were scheduled and repeated on day 43. Subsequently, patients commenced 4 weekly maintenance treatment with procarbazine (100 mg for 5 days). Primary end point was complete remission (CR) after 3 cycles. We included 107 patients (69 treated with R-MPL and 38 with R-MP). In all, 38/107 patients achieved CR (35.5%) and 15 (14.0%) achieved partial remission. R-MP was associated with a lower CR rate (31.6%) compared with R-MPL (37.7%), but respective 2-year progression-free survival (All 37.3%; R-MP 34.9%; R-MPL 38.8%) and overall survival (All 47.0%; R-MP 47.7%; R-MPL 46.0%) rates were similar. R-MP was associated with less ⩾grade 3 toxicities compared with R-MPL (71.1% vs 87.0%). R-MP is more feasible while still associated with similar efficacy compared with R-MPL and warrants further improvement in future studies.

Measuring adverse events in helicopter emergency medical services: establishing content validity.

PubMed

Patterson, P Daniel; Lave, Judith R; Martin-Gill, Christian; Weaver, Matthew D; Wadas, Richard J; Arnold, Robert M; Roth, Ronald N; Mosesso, Vincent N; Guyette, Francis X; Rittenberger, Jon C; Yealy, Donald M

2014-01-01

We sought to create a valid framework for detecting adverse events (AEs) in the high-risk setting of helicopter emergency medical services (HEMS). We assembled a panel of 10 expert clinicians (n = 6 emergency medicine physicians and n = 4 prehospital nurses and flight paramedics) affiliated with a large multistate HEMS organization in the Northeast US. We used a modified Delphi technique to develop a framework for detecting AEs associated with the treatment of critically ill or injured patients. We used a widely applied measure, the content validity index (CVI), to quantify the validity of the framework's content. The expert panel of 10 clinicians reached consensus on a common AE definition and four-step protocol/process for AE detection in HEMS. The consensus-based framework is composed of three main components: (1) a trigger tool, (2) a method for rating proximal cause, and (3) a method for rating AE severity. The CVI findings isolate components of the framework considered content valid. We demonstrate a standardized process for the development of a content-valid framework for AE detection. The framework is a model for the development of a method for AE identification in other settings, including ground-based EMS.

Content-Based Covert Group Detection in Social Networks

DTIC Science & Technology

2017-09-06

these features to learn a model that could be applied to the holdout set. This model was then used to test on the remaining posts . Accuracy: 51.4...Content-based Covert Group Detection in Social Networks The views, opinions and/or findings contained in this report are those of the author(s) and...should not contrued as an official Department of the Army position, policy or decision, unless so designated by other documentation. 9. SPONSORING

Immuno-PET Imaging and Radioimmunotherapy of 64Cu-/177Lu-Labeled Anti-EGFR Antibody in Esophageal Squamous Cell Carcinoma Model.

PubMed

Song, In Ho; Lee, Tae Sup; Park, Yong Serk; Lee, Jin Sook; Lee, Byung Chul; Moon, Byung Seok; An, Gwang Il; Lee, Hae Won; Kim, Kwang Il; Lee, Yong Jin; Kang, Joo Hyun; Lim, Sang Moo

2016-07-01

Immuno-PET provides valuable information about tumor location, phenotype, susceptibility to therapy, and treatment response, especially to targeted radioimmunotherapy. In this study, we prepared antiepidermal growth factor receptor (EGFR) antibody via identical chelator, 3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-trience-3,6,9,-triacetic acid (PCTA), labeled with (64)Cu or (177)Lu to evaluate the EGFR expression levels using immuno-PET and the feasibility of radioimmunotherapy in an esophageal squamous cell carcinoma (ESCC) model. Cetuximab was conjugated with p-SCN-Bn-PCTA and radiolabeled with (64)Cu or (177)Lu. In vitro EGFR expression levels were determined and compared using flow cytometry and cell binding assay. In vivo EGFR expression levels were evaluated via immuno-PET imaging of (64)Cu-cetuximab and biodistribution analysis. Micro-SPECT/CT imaging, biodistribution, and radioimmunotherapy studies of (177)Lu-cetuximab were performed in the ESCC model. Therapeutic responses were monitored using (18)F-FDG PET and immunohistochemical staining. (64)Cu- or (177)Lu-labeled antibodies showed high radiolabeling yield (>98%), stability (>90%), and favorable immunoreactivity. In vitro EGFR status measured by cell binding assay was correlated with the flow cytometry data. Immuno-PET, micro-SPECT/CT, and biodistribution demonstrated specific uptake in ESCC tumors depending on the EGFR expression levels. Tumor accumulation of (64)Cu- and (177)Lu-cetuximab was peaked at 48 and 120 h, respectively. Radioimmunotherapy with (177)Lu-cetuximab showed significant inhibition of tumor growth (P < 0.01) and marked reduction of (18)F-FDG SUV compared with that of control (P < 0.05). Terminal deoxynucleotidyl transferase dUTP nick-end labeling positivity and Ki-67 staining indices increased and decreased, respectively, in the radioimmunotherapy group compared with other groups (P < 0.01). (64)Cu-cetuximab immuno-PET represented EGFR expression levels in ESCC tumors, and (177)Lu-cetuximab radioimmunotherapy effectively inhibited the tumor growth. The diagnostic and therapeutic convergence radiopharmaceutical (64)Cu-/(177)Lu-PCTA-cetuximab may be useful as a diagnostic tool in patient selection and a potent radioimmunotherapy agent in EGFR-positive ESCC tumors. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Semiselective Optoelectronic Sensors for Monitoring Microbes

NASA Technical Reports Server (NTRS)

Tabacco, Mary Beth; Chuang, Han; Taylor,Laura; Russo, Jaime

2003-01-01

Sensor systems are under development for use in real-time detection and quantitation of microbes in water without need for sampling. These systems include arrays of optical sensors; miniature, portable electronic data-acquisition circuits; and optoelectronic interfaces between the sensor arrays and data-acquisition circuits. These systems are intended for original use in long-term, inline monitoring of waterborne micro-organisms in water-reclamation systems aboard future spacecraft. They could also be adapted to similar terrestrial uses with respect to municipal water supplies, stored drinking water, and swimming water; for detecting low-level biological contamination in biotechnological, semiconductor, and pharmaceutical process streams; and in verifying the safety of foods and beverages. In addition, they could be adapted to monitoring of airborne microbes and of surfaces (e.g., to detect and/or quantitate biofilms). The designs of the sensors in these systems are based partly on those of sensors developed previously for monitoring airborne biological materials. The designs exploit molecular- recognition and fluorescence-spectroscopy techniques, such that in the presence of micro-organisms of interest, fluorescence signals change and the changes can be measured. These systems are characterized as semiselective because they respond to classes of micro-organisms and can be used to discriminate among the classes. This semiselectivity is a major aspect of the design: It is important to distinguish between (1) the principle of detection and quantitation of classes of micro-organisms by use of these sensors and (2) the principle of detection and quantitation of individual microbiological species by means of prior immuno-diagnostic and/or molecular-biology techniques. Detection of classes (in contradistinction to species) is particularly valuable when the exact nature of a contaminant is unknown.

Part-per-trillion level detection of estradiol by competitive fluorescence immunoassay using DNA/dye conjugate as antibody multiple labels.

PubMed

Zhu, Shengchao; Zhang, Qin; Guo, Liang-Hong

2008-08-22

Fluorescent organic dyes are currently the standard signal-generating labels used in microarray quantification. However, new labeling strategies are needed to meet the demand for high sensitivity in the detection of low-abundance proteins and small molecules. In this report, a long-chain DNA/dye conjugate was used to attach multiple fluorescence labels on antibodies to improve signal intensity and immunoassay sensitivity. Compared with the 30 base-pair (bp) oligonucleotide used in our previous work [Q. Zhang, L.-H. Guo, Bioconjugate Chem. 18 (2007) 1668-1672], conjugation of a 219 bp DNA in solution with a fluorescent DNA binder SYBR Green I resulted in more than sixfold increase in signal intensity, consistent with the increase in bp number. In a direct immunoassay for the detection of goat anti-mouse IgG in a mouse IgG-coated 96-well plate, the long DNA conjugate label also produced higher fluorescence than the short one, accompanied by about 15-fold improvement in the detection limit. To demonstrate its advantage in real applications, the DNA/dye conjugate was employed in the competitive immunoassay of 17beta-estradiol, a clinically and environmentally important analyte. The biotin-terminated DNA was attached to biotinylated anti-estradiol antibody through the biotin/streptavidin/biotin bridge after the immuno-reaction was completed, followed by conjugation with SYBR Green I. The limit of detection for 17beta-estradiol is 1.9 pg mL(-1), which is 200-fold lower than the assay using fluorescein-labeled antibodies. The new multiple labeling strategy uses readily available reagents, and is also compatible with current biochip platform. It has great potential in the sensitive detection of protein and antibody microarrays.

Rapid field detection of moisture content for base and subgrade : technical report.

DOT National Transportation Integrated Search

2015-03-01

Mixing and compacting soil and flexible base pavement materials at the proper moisture content is critical : for obtaining adequate compaction and meeting construction specification requirements. This project sought : to evaluate rapid non-nuclear te...

Rapid quantitative detection of glucose content in glucose injection by reaction headspace gas chromatography.

PubMed

Xie, Wei-Qi; Gong, Yi-Xian; Yu, Kong-Xian

2017-10-20

This work investigates an automated technique for rapid detecting the glucose content in glucose injection by reaction headspace gas chromatography (HS-GC). This method is based on the oxidation reaction of glucose in glucose injection with potassium dichromate. The carbon dioxide (CO 2 ) formed from the oxidation reaction can be quantitatively detected by GC. The results show that the relative standard deviation (RSD) of the present method was within 2.91%, and the measured glucose contents in glucose injection closely match those quantified by the reference method (relative differences <6.45%). The new HS-GC technique is rapid, practical and can be used to the batch detection of the glucose content in glucose injection related applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Near-Infrared Spectrum Detection of Wheat Gluten Protein Content Based on a Combined Filtering Method.

PubMed

Cai, Jian-Hua

2017-09-01

To eliminate the random error of the derivative near-IR (NIR) spectrum and to improve model stability and the prediction accuracy of the gluten protein content, a combined method is proposed for pretreatment of the NIR spectrum based on both empirical mode decomposition and the wavelet soft-threshold method. The principle and the steps of the method are introduced and the denoising effect is evaluated. The wheat gluten protein content is calculated based on the denoised spectrum, and the results are compared with those of the nine-point smoothing method and the wavelet soft-threshold method. Experimental results show that the proposed combined method is effective in completing pretreatment of the NIR spectrum, and the proposed method improves the accuracy of detection of wheat gluten protein content from the NIR spectrum.

Systemic study on the safety of immuno-deficient nude mice treated by atmospheric plasma-activated water

NASA Astrophysics Data System (ADS)

Dehui, XU; Qingjie, CUI; Yujing, XU; Bingchuan, WANG; Miao, TIAN; Qiaosong, LI; Zhijie, LIU; Dingxin, LIU; Hailan, CHEN; Michael, G. KONG

2018-04-01

Cold atmospheric-pressure plasma is a new technology, widely used in many fields of biomedicine, especially in cancer treatment. Cold plasma can selectively kill a variety of tumor cells, and its biological safety in clinical trials is also very important. In many cases, the patient’s immune level is relatively low, so we first studied the safety assessment of plasma treatment in an immuno-compromised animal model. In this study, we examined the safety of immuno-deficient nude mice by oral lavage treatment of plasma-activated water, and studied the growth status, main organs and blood biochemical indexes. Acute toxicity test results showed that the maximum dose of plasma treatment for 15 min had no lethal effect and other acute toxicity. There were no significant changes in body weight and survival status of mice after 2 min and 4 min of plasma-activated water (PAW) treatment for 2 weeks. After treatment, the major organs, including heart, liver, spleen, lung and kidney, were not significantly changed in organ coefficient and tissue structure. Blood biochemical markers showed that blood neutrophils and mononuclear cells were slightly increased, and the others remained unchanged. Liver function, renal function, electrolytes, glucose metabolism and lipid metabolism were not affected by different doses of PAW treatment. The above results indicate that PAW treatment can be used to treat immuno-deficient nude mice without significant safety problems.

Mucosal interplay among commensal and pathogenic bacteria: lessons from flagellin and Toll-like receptor 5.

PubMed

Rumbo, Martin; Nempont, Clément; Kraehenbuhl, Jean-Pierre; Sirard, Jean-Claude

2006-05-22

Toll-like receptors (TLR) detect pathogen-associated molecular patterns (PAMP) and play a crucial role in triggering immunity. Due to their large surfaces in direct contact with the environment, mucosal tissues are the major sites of PAMP-TLR signalling. How innate and adaptive immunity are triggered through flagellin-TLR5 interaction is the main focus of the review. In view of recent reports on genetic polymorphism, we will summarize the impact of TLR5 on the susceptibility to mucosal infections and on various immuno-pathologies. Finally, the contribution of TLRs in the induction and maintenance of mucosal homeostasis and commensal discrimination is discussed.

Toxicokinetics of Sulfur Mustard and its DNA-Adducts in the Hairless Guinea Pig - DNA-Adducts as a Measure for Epithelial Damage.

DTIC Science & Technology

1996-03-01

guinea pigs for the intravenous (i.v.), respiratory and percutaneous routes. A highly sensitive method for bioanalysis of the intact agent in blood and tissues was developed, involving gas chromatography with mass-spectrometric detection. Deuterated sulfur mustard (D8-SM) is used as the internal standard. 7-SM-guanine is measured with an immuno-slot-blot assay. In this midterm report the first results on the i.v. toxicokinetics of SM and 7-SM-guanine in hairless guinea pigs are presented. The 96-h i.v. LD50 appeared to be 8.2 mg/kg (95% confidence

Outdoor, indoor, and human breath content measurements of ammonia by tunable diode laser spectroscopy technique

NASA Astrophysics Data System (ADS)

Moskalenko, Konstantin L.; Nadezhdinskii, Alexander I.

1996-10-01

Trace contents of ammonia in outdoor, indoor and exhaled air were measured on the base of high resolution absorption spectra. Tunable diode laser system developed for this purpose possesses approximately one second time constant, approximately 200 cm3 sample volume, 5 ppb sensitivity. The calibration of unit was based on measurements of relative intensities of sQ(3,1)...sQ(3,3) absorption lines of v2s and following calculation on the base of a priori data on strength and broadening coefficients of detected lines. Measured indoor contents of ammonia was in 5-10 times higher than outdoor contents. Approximately two times drop in NH3 room content after 6 p.m. was detected. Obtained behavior of ammonia content in respiration right after the smoking demonstrates that the removing of ammonia from lungs has the ventilation character. Measured contents of NH3 in human respiration was ranged between 120 and 220 ppb. The absence of ammonia content differences from respiration of smoking and non smoking persons demonstrates that the accumulation of NH3 by human organism seems to be rather negligible for a short time exposure, e.g. like smoking.

Using llama derived single domain antibodies to target botulinum neurotoxins

NASA Astrophysics Data System (ADS)

Swain, Marla D.; Anderson, George P.; Bernstein, Rachael D.; Liu, Jinny L.; Goldman, Ellen R.

2010-04-01

Llama serum contains both conventional IgG as well as unique forms of antibody that contain only heavy chains where antigen binding is mediated through a single variable domain. These variable domains can be expressed recombinantly and are referred to as single domain antibodies (sdAb). SdAb are among the smallest known naturally derived antigen binding fragments, possess good solubility, thermal stability, and can refold after heat and chemical denaturation. Llamas were immunized with either BoNT A or B toxoid and phage display libraries prepared. Single domain antibodies (sdAb) that were able to detect botulinum neurotoxin (BoNT) serotypes A and B were selected from their respective libraries. Here, the binders obtained by panning the BoNT B library on either BoNT B toxoid or BoNT B complex toxoid coated plates or BoNT B toxin coupled microspheres are described. Using these panning methods, we selected for binders that showed specificity for BoNT B. Phage displayed binders were screened, moved to a protein expression vector and soluble sdAb was produced. Using a Luminex flow cytometer binders were evaluated in direct binding assays. We have exploited the unique properties of sdAb and used them as biological recognition elements in immuno-based sensors that can detect BoNT B.

Identifying the site of spin trapping in proteins by a combination of liquid chromatography, ELISA, and off-line tandem mass spectrometry.

PubMed

Lardinois, Olivier M; Detweiler, Charles D; Tomer, Kenneth B; Mason, Ronald P; Deterding, Leesa J

2008-03-01

An off-line mass spectrometry method that combines immuno-spin trapping and chromatographic procedures has been developed for selective detection of the nitrone spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) covalently attached to proteins, an attachment which occurs only subsequent to DMPO trapping of free radicals. In this technique, the protein-DMPO nitrone adducts are digested to peptides with proteolytic agents, peptides from the enzymatic digest are separated by HPLC, and enzyme-linked immunosorbent assays (ELISA) using polyclonal anti-DMPO nitrone antiserum are used to detect the eluted HPLC fractions that contain DMPO nitrone adducts. The fractions showing positive ELISA signals are then concentrated and characterized by tandem mass spectrometry (MS/MS). This method, which constitutes the first liquid chromatography-ELISA-mass spectrometry (LC-ELISA-MS)-based strategy for selective identification of DMPO-trapped protein residues in complex peptide mixtures, facilitates location and preparative fractionation of DMPO nitrone adducts for further structural characterization. The strategy is demonstrated for human hemoglobin, horse heart myoglobin, and sperm whale myoglobin, three globin proteins known to form DMPO-trappable protein radicals on treatment with H(2)O(2). The results demonstrate the power of the new experimental strategy to select DMPO-labeled peptides and identify sites of DMPO covalent attachments.

Heightened sense for sensing: recent advances in pathogen immunoassay sensing platforms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fischer, N; Tarasow, T; Tok, J B

2007-01-09

As part of its own defense mechanism, most bacteria have developed an innate ability to enable toxic secretion to ward off potential predators or invaders. However, this naturally occurring process has been abused since over production of the bacteria's toxin molecules could render them as potential bioweapons. As these processes (also known as ''black biology'') can be clandestinely performed in a laboratory, the threat of inflicting enormous potential damage to a nation's security and economy is invariably clear and present. Thus, efficient detection of these biothreat agents in a timely and accurate manner is highly desirable. A wealth of publicationsmore » describing various pathogen immuno-sensing advances has appeared over the last few years, and it is not the intent of this review article to detail each reported approach. Instead, we aim to survey a few recent highlights in hopes of providing the reader an overall sense of the breath of these sensing systems and platforms. Antigen targets are diverse and complex as they encompass proteins, whole viruses, and bacterial spores. The signaling processes for these reported immunoassays are usually based on colorimetric, optical, or electrochemical changes. Of equal interest is the type of platform in which the immunoassay can be performed. A few platforms suitable for pathogen detection are described.« less

Microfabricated Renewable Beads-Trapping/Releasing Flow Cell for Rapid Antigen-Antibody Reaction in Chemiluminescent Immunoassay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu, Zhifeng; Shao, Guocheng; Wang, Jun

2011-04-01

A filter pillar-array microstructure was coupled with a pneumatic micro-valve to fabricate a reusable miniaturized beads-trapping/releasing flow cell, in which trapping and releasing beads can be conveniently realized by switching the micro-valve. This miniaturized device was suitable to construct automatic fluidic system for “renewable surface analysis”. The renewable surface strategy based on pneumatic micro-valve enabled capture of beads in beads chamber prior to each assay, and release of the used beads after the assay. Chemiluminescent competitive immunoassay of 3,5,6-trichloropyridinol (TCP) was performed as a model to demonstrate the application potential of this reusable miniaturized flow cell. The whole fluidic assaymore » process including beads trapping, immuno-binding, beads washing, beads releasing and signal collection could be completed in 10 min. Immunoassay of TCP using this miniaturized device showed a linear range of 0.20-70 ng/mL with a limit of detection of 0.080 ng/mL. The device had been successfully used for detection of TCP spiked in rat serum with average recovery of 97%. This investigation provides a rapid, sensitive, reusable, low-cost and automatic miniaturized device for solid-phase biochemical analysis for various purposes.« less

Investigating free radical generation in HepG2 cells using immuno-spin trapping.

PubMed

Horinouchi, Yuya; Summers, Fiona A; Ehrenshaft, Marilyn; Kawazoe, Kazuyoshi; Tsuchiya, Koichiro; Tamaki, Toshiaki; Mason, Ronald P

2014-10-01

Oxidative stress can induce the generation of free radicals, which are believed to play an important role in both physiological and pathological processes and a number of diseases such as cancer. Therefore, it is important to identify chemicals which are capable of inducing oxidative stress. In this study, we evaluated the ability of four environmental chemicals, aniline, nitrosobenzene (NB), N,N-dimethylaniline (DMA) and N,N-dimethyl-4-nitrosoaniline (DMNA), to induce free radicals and cellular damage in the hepatoma cell line HepG2. Cytotoxicity was assessed using lactate dehydrogenase (LDH) assays and morphological changes were observed using phase contrast microscopy. Free radicals were detected by immuno-spin trapping (IST) in in-cell western experiments or in confocal microscopy experiments to determine the subcellular localization of free radical generation. DMNA induced free radical generation, LDH release and morphological changes in HepG2 cells whereas aniline, NB and DMA did not. Confocal microscopy showed that DMNA induced free radical generation mainly in the cytosol. Preincubation of HepG2 cells with N-acetylcysteine and 2,2'-dipyridyl significantly prevented free radical generation upon subsequent incubation with DMNA, whereas preincubation with apocynin and dimethyl sulfoxide did not. These results suggest that DMNA induces oxidative stress and that reactive oxygen species, metals and free radical generation play a critical role in DMNA-induced cytotoxicity. Copyright © 2014. Published by Elsevier Inc.

Translational Immuno- and Neuro-imaging Demonstrate Corneal Neuro-immune Crosstalk

PubMed Central

Hamrah, Pedram; Seyed-Razavi, Yashar; Yamaguchi, Takefumi

2017-01-01

Corneal immuno- and neuro-imaging approaches facilitate in vivo analyses of the cornea, including high-resolution imaging of corneal immune cells and nerves. This approach facilitates the analyses of underlying immune and nerve alterations not detected by clinical slit-lamp examination alone. In this review, we describe recent work performed in our translational ocular immunology center with a focus on ‘bench-to-bedside’ and ‘bedside-to-bench’ research. The ability to visualize dendritiform immune cells (DCs) in patients with laser in vivo confocal microscopy (IVCM), recently discovered in the central murine cornea, has allowed us to demonstrated their utility as a potential surrogate biomarker for inflammatory ocular surface diseases. This biomarker for inflammation allows the measurement of therapeutic efficacy of anti-inflammatory drugs and its utility as an endpoint in clinical trials with high inter-observer agreement. IVCM image analyses from our studies demonstrated a significant increase in DC density and size in ocular disease, a positive correlation between DC density and clinical signs and symptoms of disease and pro-inflammatory tear cytokines, and a strong negative correlation between DC density and subbasal nerve density. In conjunction with pre-clinical research investigating the inflammatory state in a partial or fully denervated cornea, our results indicated that corneal nerves are directly involved in the regulation of homeostasis and immune privilege in the cornea. PMID:27631352

One-chip biosensor for simultaneous disease marker/calibration substance measurement in human urine by electrochemical surface plasmon resonance method.

PubMed

Nakamoto, Kohei; Kurita, Ryoji; Niwa, Osamu

2010-12-15

We have developed a miniaturized electrochemical surface plasmon resonance biosensor for measuring two biomolecules that have very different molecular sizes, one is transferrin (MW=75 kDa) as a disease marker protein, the other is creatinine (MW=113) as a calibration marker for the accurate measurement of human urinary samples. The sensor has a PDMS based microchannel that is 2 mm wide and 20 μm deep. Two gold films were integrated in the microchannel; one was modified with anti-transferrin antibody for immuno-reaction, and the other was modified with osmium-poly-vinylpyridine wired horseradish peroxidase (Os-gel-HRP). We further immobilized a tri-enzyme layer of creatininase, creatinase and sarcosine oxidase in order to measure creatinine by converting it to hydrogen peroxide in the upstream channel. We measured the transferrin concentration from the refractive index change involved in an immuno-complex formation, and we were simultaneously able to measure creatinine by employing the refractive index change in the Os-gel-HRP caused by oxidation with the hydrogen peroxide produced from creatinine by the tri-enzyme. The effects of ascorbic acid and uric acid in urine samples were sufficiently eliminated by adding ascorbate oxidase and uricase to the urine samples during sampling. We were able to measure two analyte concentrations within 15 min by one simple injection of 50 μL of diluted human urine into our sensor. The detectable transferrin and creatinine ranges were 20 ng/mL to 10 μg/mL, and 10 μM to 10 mM, respectively, which are sufficient levels for clinical tests. Finally, we compared the results obtained using our sensor with those obtained with a conventional immunoassay and the Jaffe method. We obtained a similar trend that can reduce the fluctuation in the urinary transferrin concentration from three different samples by calibrating the creatinine concentration. Copyright © 2010 Elsevier B.V. All rights reserved.

Depletion of highly abundant proteins in blood plasma by ammonium sulfate precipitation for 2D-PAGE analysis.

PubMed

Mahn, Andrea; Ismail, Maritza

2011-11-15

Ammonium sulfate precipitation (ASP) was explored as a method for depleting some highly abundant proteins from blood plasma, in order to reduce the dynamic range of protein concentration and to improve the detection of low abundance proteins by 2D-PAGE. 40% ammonium sulfate saturation was chosen since it allowed depleting 39% albumin and 82% α-1-antitrypsin. ASP-depletion showed high reproducibility in 2D-PAGE analysis (4.2% variation in relative abundance of albumin), similar to that offered by commercial affinity-depletion columns. Besides, it allowed detecting 59 spots per gel, very close to the number of spots detected in immuno-affinity-depleted plasma. Thus, ASP at 40% saturation is a reliable depletion method that may help in proteomic analysis of blood plasma. Finally, ASP-depletion seems to be complementary to hydrophobic interaction chromatography (HIC)-depletion, and therefore an ASP-step followed by a HIC-step could probably deplete the most highly abundant plasma proteins, thus improving the detection of low abundance proteins by 2D-PAGE. Copyright © 2011 Elsevier B.V. All rights reserved.

Development of an RNA-based kit for easy generation of TCR-engineered lymphocytes to control T-cell assay performance.

PubMed

Bidmon, Nicole; Kind, Sonja; Welters, Marij J P; Joseph-Pietras, Deborah; Laske, Karoline; Maurer, Dominik; Hadrup, Sine Reker; Schreibelt, Gerty; Rae, Richard; Sahin, Ugur; Gouttefangeas, Cécile; Britten, Cedrik M; van der Burg, Sjoerd H

2018-07-01

Cell-based assays to monitor antigen-specific T-cell responses are characterized by their high complexity and should be conducted under controlled conditions to lower multiple possible sources of assay variation. However, the lack of standard reagents makes it difficult to directly compare results generated in one lab over time and across institutions. Therefore TCR-engineered reference samples (TERS) that contain a defined number of antigen-specific T cells and continuously deliver stable results are urgently needed. We successfully established a simple and robust TERS technology that constitutes a useful tool to overcome this issue for commonly used T-cell immuno-assays. To enable users to generate large-scale TERS, on-site using the most commonly used electroporation (EP) devices, an RNA-based kit approach, providing stable TCR mRNA and an optimized manufacturing protocol were established. In preparation for the release of this immuno-control kit, we established optimal EP conditions on six devices and initiated an extended RNA stability study. Furthermore, we coordinated on-site production of TERS with 4 participants. Finally, a proficiency panel was organized to test the unsupervised production of TERS at different laboratories using the kit approach. The results obtained show the feasibility and robustness of the kit approach for versatile in-house production of cellular control samples. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Effects of an immuno-enhanced diet containing antioxidants in esophageal cancer surgery following neoadjuvant therapy.

PubMed

Aiko, S; Kumano, I; Yamanaka, N; Tsujimoto, H; Takahata, R; Maehara, T

2012-02-01

Neoadjuvant therapy-induced immunological deterioration may be a key factor in postoperative morbidity in patients with esophageal cancer. This study aimed to determine the effects of perioperative feeding with an immuno-enhanced diet on immune competence in patients treated with neoadjuvant therapy followed by surgery. Because an immuno-enhanced diet that contained several antioxidants was used, perioperative oxidative stress and the effects of the immuno-enhanced diet on this stress were also investigated. Of 39 patients with esophageal cancer who underwent similar surgical procedures, 26 patients who received chemotherapy or chemoradiation therapy before surgery were randomly divided into two groups: group 1 (n= 14) was given an immuno-enhanced diet for 5 days before surgery, and group 2 (n= 12) received no enteral feeding products before surgery. Group 3 (n= 13) consisted of patients that did not receive neoadjuvant therapy and received no enteral feeding products before surgery. Several markers for coagulation and fibrinolysis were determined and immunological assessments were performed for each patient. To measure reactive oxygen metabolites and the total antioxidant capacity, diacron-reactive oxygen metabolites (d-ROMs) and OXY-adsorbent tests were performed using a free radical elective evaluator. Significant depression in lymphocyte numbers was observed in groups 1 and 2 before and early after surgery as compared to group 3. Numbers of B cells, CD4/CD8 ratio, and phytohemagglutinin-induced lymphocyte transformation tests were also significantly decreased in groups 1 and 2 on postoperative day 1. Fibrin and fibrinogen degradation products were significantly elevated in group 2 compared to group 1. d-ROMs and OXY-adsorbent test values were elevated before surgery and were decreased transiently early after surgery. Compared to groups 2 and 3, d-ROMs values were significantly lower in group 1 patients throughout the postoperative period, while OXY-adsorbent test values were significantly higher in group 2 patients. Oxidative index was significantly suppressed in group 1 compared to group 3. No significant intergroup differences were observed with regard to morbidity after surgery. Although the baseline levels of immunological function might have been different because of less-advanced cancer stages in group 3, neoadjuvant therapy significantly affected several immunological parameters. Preoperative administration of an immuno-enhanced diet did not significantly prevent neoadjuvant therapy-induced immunological deterioration prior to esophageal cancer surgery. Patients with esophageal cancer had elevated levels of oxidant and antioxidant activities before surgery, which were transiently decreased early after surgery. Although the underlying mechanisms for these perioperative changes are unclear, this study showed that an immuno-enhanced diet containing several antioxidants may reduce oxidative stress following esophageal cancer surgery. After these mechanisms are studied further, oxidative stress control may become another tool for perioperative management to reduce morbidity after esophageal cancer surgery. © 2011 Copyright the Authors. Journal compilation © 2011, Wiley Periodicals, Inc. and the International Society for Diseases of the Esophagus.

Micromotor-based lab-on-chip immunoassays

NASA Astrophysics Data System (ADS)

García, Miguel; Orozco, Jahir; Guix, Maria; Gao, Wei; Sattayasamitsathit, Sirilak; Escarpa, Alberto; Merkoçi, Arben; Wang, Joseph

2013-01-01

Here we describe the first example of using self-propelled antibody-functionalized synthetic catalytic microengines for capturing and transporting target proteins between the different reservoirs of a lab-on-a-chip (LOC) device. A new catalytic polymer/Ni/Pt microtube engine, containing carboxy moieties on its mixed poly(3,4-ethylenedioxythiophene) (PEDOT)/COOH-PEDOT polymeric outermost layer, is further functionalized with the antibody receptor to selectively recognize and capture the target protein. The new motor-based microchip immunoassay operations are carried out without any bulk fluid flow, replacing the common washing steps in antibody-based protein bioassays with the active transport of the captured protein throughout the different reservoirs, where each step of the immunoassay takes place. A first microchip format involving an `on-the-fly' double-antibody sandwich assay (DASA) is used for demonstrating the selective capture of the target protein, in the presence of excess of non-target proteins. A secondary antibody tagged with a polymeric-sphere tracer allows the direct visualization of the binding events. In a second approach the immuno-nanomotor captures and transports the microsphere-tagged antigen through a microchannel network. An anti-protein-A modified microengine is finally used to demonstrate the selective capture, transport and convenient label-free optical detection of a Staphylococcus aureus target bacteria (containing proteinA in its cell wall) in the presence of a large excess of non-target (Saccharomyces cerevisiae) cells. The resulting nanomotor-based microchip immunoassay offers considerable potential for diverse applications in clinical diagnostics, environmental and security monitoring fields.Here we describe the first example of using self-propelled antibody-functionalized synthetic catalytic microengines for capturing and transporting target proteins between the different reservoirs of a lab-on-a-chip (LOC) device. A new catalytic polymer/Ni/Pt microtube engine, containing carboxy moieties on its mixed poly(3,4-ethylenedioxythiophene) (PEDOT)/COOH-PEDOT polymeric outermost layer, is further functionalized with the antibody receptor to selectively recognize and capture the target protein. The new motor-based microchip immunoassay operations are carried out without any bulk fluid flow, replacing the common washing steps in antibody-based protein bioassays with the active transport of the captured protein throughout the different reservoirs, where each step of the immunoassay takes place. A first microchip format involving an `on-the-fly' double-antibody sandwich assay (DASA) is used for demonstrating the selective capture of the target protein, in the presence of excess of non-target proteins. A secondary antibody tagged with a polymeric-sphere tracer allows the direct visualization of the binding events. In a second approach the immuno-nanomotor captures and transports the microsphere-tagged antigen through a microchannel network. An anti-protein-A modified microengine is finally used to demonstrate the selective capture, transport and convenient label-free optical detection of a Staphylococcus aureus target bacteria (containing proteinA in its cell wall) in the presence of a large excess of non-target (Saccharomyces cerevisiae) cells. The resulting nanomotor-based microchip immunoassay offers considerable potential for diverse applications in clinical diagnostics, environmental and security monitoring fields. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr32400h

Measuring Adverse Events in Helicopter Emergency Medical Services: Establishing Content Validity

PubMed Central

Patterson, P. Daniel; Lave, Judith R.; Martin-Gill, Christian; Weaver, Matthew D.; Wadas, Richard J.; Arnold, Robert M.; Roth, Ronald N.; Mosesso, Vincent N.; Guyette, Francis X.; Rittenberger, Jon C.; Yealy, Donald M.

2015-01-01

Introduction We sought to create a valid framework for detecting Adverse Events (AEs) in the high-risk setting of Helicopter Emergency Medical Services (HEMS). Methods We assembled a panel of 10 expert clinicians (n=6 emergency medicine physicians and n=4 prehospital nurses and flight paramedics) affiliated with a large multi-state HEMS organization in the Northeast U.S. We used a modified Delphi technique to develop a framework for detecting AEs associated with the treatment of critically ill or injured patients. We used a widely applied measure, the Content Validity Index (CVI), to quantify the validity of the framework’s content. Results The expert panel of 10 clinicians reached consensus on a common AE definition and four-step protocol/process for AE detection in HEMS. The consensus-based framework is composed of three main components: 1) a trigger tool, 2) a method for rating proximal cause, and 3) a method for rating AE severity. The CVI findings isolate components of the framework considered content valid. Conclusions We demonstrate a standardized process for the development of a content valid framework for AE detection. The framework is a model for the development of a method for AE identification in other settings, including ground-based EMS. PMID:24003951

Rapid diagnosis of tuberculosis in dromedary camel (Camelus dromedarius) using lateral flow assay-based kit.

PubMed

Ranjan, Rakesh; Narnaware, Shirish D; Nath, Kashi; Sawal, R K; Patil, N V

2018-04-01

Accurate early antemortem diagnosis of tuberculosis in dromedary camels is difficult due to the lack of reliable diagnostic test. The present study aimed to evaluate a lateral flow assay-based kit (rapid assay kit) in tuberculosis diagnosis that employs immuno-chromatographic detection of antibodies in serum, plasma, or whole blood. In a dromedary camel herd comprising 337 animals located at Bikaner, Rajasthan, India, 50 adult weak camels (11 males and 39 females) were tested by applying a single intradermal tuberculin test (SIDT) and rapid assay kit. A total of 14 animals (2 males, 12 females) were found positive in rapid assay. In SIDT, four animals revealed a positive reaction in the neck region and seven animals in the tail base. Another male animal was found SIDT positive but negative in rapid assay; it died after 12 months. Nine rapid assay positive animals died asymptomatically in 1- to 11-month period revealing postmortem tuberculosis lesions that were confirmed by Ziehl-Neelsen staining and histopathology. No tuberculous lesion was evident in the animal found positive in SIDT alone. Results of the present study indicated that serological tests like rapid assay kit can serve as a reliable test for antemortem diagnosis of tuberculosis in dromedary camel.

Automated Selection of Hotspots (ASH): enhanced automated segmentation and adaptive step finding for Ki67 hotspot detection in adrenal cortical cancer.

PubMed

Lu, Hao; Papathomas, Thomas G; van Zessen, David; Palli, Ivo; de Krijger, Ronald R; van der Spek, Peter J; Dinjens, Winand N M; Stubbs, Andrew P

2014-11-25

In prognosis and therapeutics of adrenal cortical carcinoma (ACC), the selection of the most active areas in proliferative rate (hotspots) within a slide and objective quantification of immunohistochemical Ki67 Labelling Index (LI) are of critical importance. In addition to intratumoral heterogeneity in proliferative rate i.e. levels of Ki67 expression within a given ACC, lack of uniformity and reproducibility in the method of quantification of Ki67 LI may confound an accurate assessment of Ki67 LI. We have implemented an open source toolset, Automated Selection of Hotspots (ASH), for automated hotspot detection and quantification of Ki67 LI. ASH utilizes NanoZoomer Digital Pathology Image (NDPI) splitter to convert the specific NDPI format digital slide scanned from the Hamamatsu instrument into a conventional tiff or jpeg format image for automated segmentation and adaptive step finding hotspots detection algorithm. Quantitative hotspot ranking is provided by the functionality from the open source application ImmunoRatio as part of the ASH protocol. The output is a ranked set of hotspots with concomitant quantitative values based on whole slide ranking. We have implemented an open source automated detection quantitative ranking of hotspots to support histopathologists in selecting the 'hottest' hotspot areas in adrenocortical carcinoma. To provide wider community easy access to ASH we implemented a Galaxy virtual machine (VM) of ASH which is available from http://bioinformatics.erasmusmc.nl/wiki/Automated_Selection_of_Hotspots . The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_216.

Evaluation of Immunomagnetic Separation Method for the Recovery of Hepatitis A Virus and GI.1 and GII.4 Norovirus Strains Seeded on Oyster and Mussel.

PubMed

Ha, Ji-Hyoung; Choi, Changsun; Ha, Sang-Do

2014-12-01

Outbreaks of viral diseases are frequently associated with the consumption of minimally processed shellfish. Among the viruses in these outbreaks, hepatitis A virus (HAV) and human norovirus (NoV) have been increasingly reported as the most common food-borne pathogens. These viruses must be concentrated in tested samples in order to be detected. In this study, a method for the detection of NoV and HAV in shellfish using an immuno-magnetic separation (IMS) procedure combined with reverse transcriptase (RT)-PCR was developed. The IMS/RT-PCR method was applied to investigate the recovery rates of HAV, NoV GI.1, and GII.4 from oyster and mussel. Based on IMS/RT-PCR results, recovery rates for HAV from oyster and mussel test samples were 2.4 and 1.1%, respectively. The NoV GI.1 recovery rates from oyster and mussel samples were 4.9-9.2% (mean 6.9%) and 4.3-8.6% (mean 6.2%), respectively, and the NoV GII.4 recovery rates were 8.8 and 8.5%, respectively. These results verified that HAV, NoV GI.1, and GII.4 can be detected in all the test samples using the IMS/RT-PCR method, although the three inoculated viruses were recovered with low efficiency. In conclusion, the IMS/RT-PCR method can be used to efficiently and rapidly detect viruses such as HAV and NoV in shellfish such as oyster and mussel.

The Relationship Between Human Nucleolar Organizer Regions and Nucleoli, Probed by 3D-ImmunoFISH.

PubMed

van Sluis, Marjolein; van Vuuren, Chelly; McStay, Brian

2016-01-01

3D-immunoFISH is a valuable technique to compare the localization of DNA sequences and proteins in cells where three-dimensional structure has been preserved. As nucleoli contain a multitude of protein factors dedicated to ribosome biogenesis and form around specific chromosomal loci, 3D-immunoFISH is a particularly relevant technique for their study. In human cells, nucleoli form around transcriptionally active ribosomal gene (rDNA) arrays termed nucleolar organizer regions (NORs) positioned on the p-arms of each of the acrocentric chromosomes. Here, we provide a protocol for fixing and permeabilizing human cells grown on microscope slides such that nucleolar proteins can be visualized using antibodies and NORs visualized by DNA FISH. Antibodies against UBF recognize transcriptionally active rDNA/NORs and NOP52 antibodies provide a convenient way of visualizing the nucleolar volume. We describe a probe designed to visualize rDNA and introduce a probe comprised of NOR distal sequences, which can be used to identify or count individual NORs.

Plant microRNAs as novel immunomodulatory agents

PubMed Central

Cavalieri, Duccio; Rizzetto, Lisa; Tocci, Noemi; Rivero, Damariz; Asquini, Elisa; Si-Ammour, Azeddine; Bonechi, Elena; Ballerini, Clara; Viola, Roberto

2016-01-01

An increasing body of literature is addressing the immuno-modulating functions of miRNAs which include paracrine signaling via exosome-mediated intercellular miRNA. In view of the recent evidence of intake and bioavailability of dietary miRNAs in humans and animals we explored the immuno-modulating capacity of plant derived miRNAs. Here we show that transfection of synthetic miRNAs or native miRNA-enriched fractions obtained from a wide range of plant species and organs modifies dendritic cells ability to respond to inflammatory agents by limiting T cell proliferation and consequently dampening inflammation. This immuno-modulatory effect appears associated with binding of plant miRNA on TLR3 with ensuing impairment of TRIF signaling. Similarly, in vivo, plant small RNAs reduce the onset of severity of Experimental Autoimmune Encephalomyelities by limiting dendritic cell migration and dampening Th1 and Th17 responses in a Treg-independent manner. Our results indicate a potential for therapeutic use of plant miRNAs in the prevention of chronic-inflammation related diseases. PMID:27167363

Demonstration of correlative atomic force and transmission electron microscopy using actin cytoskeleton

PubMed Central

Yamada, Yutaro; Konno, Hiroki; Shimabukuro, Katsuya

2017-01-01

In this study, we present a new technique called correlative atomic force and transmission electron microscopy (correlative AFM/TEM) in which a targeted region of a sample can be observed under AFM and TEM. The ultimate goal of developing this new technique is to provide a technical platform to expand the fields of AFM application to complex biological systems such as cell extracts. Recent advances in the time resolution of AFM have enabled detailed observation of the dynamic nature of biomolecules. However, specifying molecular species, by AFM alone, remains a challenge. Here, we demonstrate correlative AFM/TEM, using actin filaments as a test sample, and further show that immuno-electron microscopy (immuno-EM), to specify molecules, can be integrated into this technique. Therefore, it is now possible to specify molecules, captured under AFM, by subsequent observation using immuno-EM. In conclusion, correlative AFM/TEM can be a versatile method to investigate complex biological systems at the molecular level. PMID:28828286

Proteomics Insights into Autophagy.

PubMed

Cudjoe, Emmanuel K; Saleh, Tareq; Hawkridge, Adam M; Gewirtz, David A

2017-10-01

Autophagy, a conserved cellular process by which cells recycle their contents either to maintain basal homeostasis or in response to external stimuli, has for the past two decades become one of the most studied physiological processes in cell biology. The 2016 Nobel Prize in Medicine and Biology awarded to Dr. Ohsumi Yoshinori, one of the first scientists to characterize this cellular mechanism, attests to its importance. The induction and consequent completion of the process of autophagy results in wide ranging changes to the cellular proteome as well as the secretome. MS-based proteomics affords the ability to measure, in an unbiased manner, the ubiquitous changes that occur when autophagy is initiated and progresses in the cell. The continuous improvements and advances in mass spectrometers, especially relating to ionization sources and detectors, coupled with advances in proteomics experimental design, has made it possible to study autophagy, among other process, in great detail. Innovative labeling strategies and protein separation techniques as well as complementary methods including immuno-capture/blotting/staining have been used in proteomics studies to provide more specific protein identification. In this review, we will discuss recent advances in proteomics studies focused on autophagy. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modeling the economic outcomes of immuno-oncology drugs: alternative model frameworks to capture clinical outcomes.

PubMed

Gibson, E J; Begum, N; Koblbauer, I; Dranitsaris, G; Liew, D; McEwan, P; Tahami Monfared, A A; Yuan, Y; Juarez-Garcia, A; Tyas, D; Lees, M

2018-01-01

Economic models in oncology are commonly based on the three-state partitioned survival model (PSM) distinguishing between progression-free and progressive states. However, the heterogeneity of responses observed in immuno-oncology (I-O) suggests that new approaches may be appropriate to reflect disease dynamics meaningfully. This study explored the impact of incorporating immune-specific health states into economic models of I-O therapy. Two variants of the PSM and a Markov model were populated with data from one clinical trial in metastatic melanoma patients. Short-term modeled outcomes were benchmarked to the clinical trial data and a lifetime model horizon provided estimates of life years and quality adjusted life years (QALYs). The PSM-based models produced short-term outcomes closely matching the trial outcomes. Adding health states generated increased QALYs while providing a more granular representation of outcomes for decision making. The Markov model gave the greatest level of detail on outcomes but gave short-term results which diverged from those of the trial (overstating year 1 progression-free survival by around 60%). Increased sophistication in the representation of disease dynamics in economic models is desirable when attempting to model treatment response in I-O. However, the assumptions underlying different model structures and the availability of data for health state mapping may be important limiting factors.

Utilization of a DNA enzyme immunoassay for the detection of proviral DNA of human immunodeficiency virus type 1 by polymerase chain reaction.

PubMed

Zella, D; Cavicchini, A; Cattaneo, E; Cimarelli, A; Bertazzoni, U

1995-02-01

The detection of proviral DNA by Polymerase Chain Reaction (PCR) is regarded as an important tool in the diagnosis of HIV-1 infection, specially among adults at risk of AIDS and children born to seropositive mothers. However, application of PCR in routine testing is hampered by the need to use radioactive probes. In this study, a non-radioactive test based on a microtiter plate (DNA Enzyme ImmunoAssay, DEIA) was used for the detection of proviral sequences of HIV-1 in peripheral blood cells of different patients. The results of the PCR-DEIA assay were compared to those obtained by liquid hybridization (PCR-LH), virus isolation (VI) and Western blot (WB). The study population included 92 patients belonging to three different groups: seropositive subjects with a well-defined clinical status and WB profile; adults at risk of infection with negative or indeterminate WB; children born to seropositive mothers with still unestablished HIV-1 infection. In the seropositive subjects, both PCR-LH and PCR-DEIA confirmed infection and gave the same results as WB. In adults at risk of infection, PCR with both methods anticipated the seroconversion in one patient with indeterminate WB and confirmed the absence of infection among seronegative and other indeterminate patients. In children born to seropositive mothers, both PCR systems as well as VI permitted an early diagnosis of infection, as confirmed by the clinical follow-up. This study has shown that in subjects at risk of AIDS and in children born to seropositive mothers, the non-isotopic DEIA method presents the same sensitivity and specificity for the detection of HIV-1 infection as the radioactive procedure. The DEIA method appears to be particularly useful for the detection of PCR products in routine diagnostic analyses.

Tumeur de Frantz: deux nouveaux cas

PubMed Central

Bellarbi, Salma; Sina, Mohamed; Jahid, Ahmed; Zouaidia, Fouad; Bernoussi, Zakia; Mahassini, Najat

2013-01-01

A travers cet article, nous détaillons les caractéristiques clinico-pathologiques et discutons l'histogenèse de la tumeur de Frantz. Deux patients opérés pour tumeur de Frantz. Ils ont eu un traitement chirurgical seul. L'étude morphologique était couplée à un examen immuno-histochimique (IHC) utilisant les anticorps anti CD10, anti- vimentine, anti-énolase neuronale spécifique (NSE), anti-synaptophysine, anti-chromogranine A et anti-cytokératine. Un immuno-marquage à l'anti-oestrogène et l'anti-progestérone a été réalisé dans un cas. Il s'agissait d'une femme âgée de 45ans et d'un garçon de 12 ans. Les aspects échographiques et scannographiques étaient non spécifiques. Une exérèse chirurgicale complète a été réalisée dans les deux cas. L'analyse histologique évoquait une tumeur de Frantz. Le diagnostic a été retenu après étude immuno-histohimique. L'évolution était favorable sans récidive avec respectivement un recul de 18 et 16 mois. La tumeur de Frantz est une entité rare. Son diagnostic repose sur l'examen anatomopathologique complété par l'étude immuno-histochimique. Son pronostic est excellent après résection chirurgicale. PMID:23503717

Comparison of six Aspergillus-specific IgG assays for the diagnosis of chronic pulmonary aspergillosis (CPA).

PubMed

Page, Iain D; Richardson, Malcolm D; Denning, David W

2016-02-01

Chronic pulmonary aspergillosis (CPA) is estimated to affect 3 million persons worldwide. Aspergillus-specific IgG is a key component in CPA diagnosis. We aimed to establish the optimal diagnostic cut offs for CPA and the comparative performance of six assays in this context. Sera from 241 patients with CPA and 100 healthy blood donors were tested using five Aspergillus-specific IgG assays plus precipitin testing using Microgen Aspergillus antigens. Receiver operating characteristic (ROC) curve area under the curve (AUC) results were as follows: ThermoFisher Scientific ImmunoCAP 0.996 (95% confidence interval 0.992-1), Siemens Immulite 0.991 (0.982-1), Serion 0.973 (0.960-0.987), Dynamiker 0.918 (0.89-0.946) and Genesis 0.902 (0.871-0.933). Optimal CPA diagnostic cut-offs were; ImmunoCAP 20 mg/L (96% sensitivity, 98% specificity), Immulite 10 mg/L (96% sensitivity, 98% specificity), Serion 35 U/ml (90% sensitivity, 98% specificity), Dynamiker 65 AU/ml (77% sensitivity, 97% specificity) and Genesis 20 U/ml (75% sensitivity, 99% specificity). The precipitin test was 59% sensitive and 100% specific. ImmunoCAP and Immulite were statistically significantly superior to the other assays. Precipitins testing performed poorly. The currently accepted ImmunoCAP cut-off of 40 mg/L appears sub-optimal for CPA diagnosis and may require revision in this context. Copyright © 2015 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Influence of correlation between HLA-G polymorphism and Interleukin-6 (IL6) gene expression on the risk of schizophrenia.

PubMed

Shivakumar, Venkataram; Debnath, Monojit; Venugopal, Deepthi; Rajasekaran, Ashwini; Kalmady, Sunil V; Subbanna, Manjula; Narayanaswamy, Janardhanan C; Amaresha, Anekal C; Venkatasubramanian, Ganesan

2018-07-01

Converging evidence suggests important implications of immuno-inflammatory pathway in the risk and progression of schizophrenia. Prenatal infection resulting in maternal immune activation and developmental neuroinflammation reportedly increases the risk of schizophrenia in the offspring by generating pro-inflammatory cytokines including IL-6. However, it is not known how prenatal infection can induce immuno-inflammatory responses despite the presence of immuno-inhibitory Human Leukocyte Antigen-G (HLA-G) molecules. To address this, the present study was aimed at examining the correlation between 14 bp Insertion/Deletion (INDEL) polymorphism of HLA-G and IL-6 gene expression in schizophrenia patients. The 14 bp INDEL polymorphism was studied by PCR amplification/direct sequencing and IL-6 gene expression was quantified by using real-time RT-PCR in 56 schizophrenia patients and 99 healthy controls. We observed significantly low IL6 gene expression in the peripheral mononuclear cells (PBMCs) of schizophrenia patients (t = 3.8, p = .004) compared to the controls. In addition, schizophrenia patients carrying Del/Del genotype of HLA-G 14 bp INDEL exhibited significantly lower IL6 gene expression (t = 3.1; p = .004) than the Del/Ins as well as Ins/Ins carriers. Our findings suggest that presence of "high-expressor" HLA-G 14 bp Del/Del genotype in schizophrenia patients could attenuate IL-6 mediated inflammation in schizophrenia. Based on these findings it can be assumed that HLA-G and cytokine interactions might play an important role in the immunological underpinnings of schizophrenia. Copyright © 2017 Elsevier Ltd. All rights reserved.

Review on shilajit used in traditional Indian medicine.

PubMed

Wilson, Eugene; Rajamanickam, G Victor; Dubey, G Prasad; Klose, Petra; Musial, Frauke; Saha, F Joyonto; Rampp, Thomas; Michalsen, Andreas; Dobos, Gustav J

2011-06-14

Shilajit is a multi-component natural occurring mineral substance used in Ayurveda and Siddha systems of medicine which originated in India. Its source can be traced to the mountainous regions, where the hilly tribes first identified its beneficial use. Shilajit is aptly referred to as 'rasayana'/'rasayanam' in Ayurveda and Siddha literature which means rejuvenator because it prevents ailment and enhances the quality of life. An attempt has been put forth to review shilajit pertaining to its origin, synonyms, varieties, physical properties, chemical constituents, therapeutic properties and important biological properties to affirm its rasayana property. All relevant information on shilajit was collected from classical texts including pharmacopoeias, formularies, etc. Moreover, select doctoral thesis from Banaras Hindu University, Varanasi and Gujarat Ayurved University, Jamnagar were also scanned. Published papers on shilajit were collected from important databases for biomedical sciences. Amongst, the various biological properties of shilajit, antioxidant activity and immuno-modulatory activity were focused as it is closely related to its rasayana potential. This review finds that shilajit is used in twenty Sastric formulations and twenty-four proprietary drugs for extraneous indications. Even-though, there is a long history of use of shilajit in traditional Indian materia medica, shilajit unfortunately lacks scientific evaluation and systematic documentation. In vivo antioxidant activity of shilajit has been studied at an irrelevant dose and without using a positive control. The immuno-modulatory activity does not stand the test of critical assessment and currently may be considered as unproven. Based on the earlier studies, the bioactivity of shilajit lacks substantial evidence. Nevertheless, further studies are imperative to overcome the lacuna in establishing the antioxidant property of shilajit and more specific assays are needed to vouch shilajit as an immuno-modulator which may be of use to establish its rasayana potential. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Sesame allergy: role of specific IgE and skin-prick testing in predicting food challenge results.

PubMed

Permaul, Perdita; Stutius, Lisa M; Sheehan, William J; Rangsithienchai, Pitud; Walter, Jolan E; Twarog, Frank J; Young, Michael C; Scott, Jordan E; Schneider, Lynda C; Phipatanakul, Wanda

2009-01-01

There are conflicting data regarding the diagnostic value of sesame-specific IgE and sesame skin test. Currently, there are no established thresholds that predict clinical reactivity. We examined the correlation of sesame ImmunoCAP and skin-prick test (SPT) results with oral challenge outcomes in children suspected of having a sesame food allergy. We conducted a retrospective chart review of children, aged 2-12 years, receiving a sesame ImmunoCAP level, SPT, and food challenge from January 2004 to August 2008 at Children's Hospital Boston and affiliated allergy clinics. Food challenges were conducted in cases of questionable clinical history or a negative ImmunoCAP and/or negative SPT despite a convincing history. Thirty-three oral sesame challenges were conducted. Of the 33 challenges performed, 21% (n = 7) failed and 79% (n = 26) passed. A sesame-specific IgE level of > or = 7 kU(A)/L showed specificity of >90%. An SPT wheal size of > or = 6 mm showed specificity of >90%. Receiver operator characteristic (ROC) curve analysis for sesame-specific IgE revealed an area under the curve (AUC) of 0.56. ROC curve analysis for SPT wheal size revealed an AUC of 0.67. To our knowledge, this study represents the largest number of sesame challenges performed to evaluate the diagnostic value of both sesame-specific IgE and SPT. Based on our sample, both tests are not good predictors of true sesame allergy as determined by an oral challenge. We were unable to establish a threshold with a 95% positive predictive value for both sesame-specific IgE and SPT.

Insights into the immuno-molecular biology of Angiostrongylus vasorum through transcriptomics--prospects for new interventions.

PubMed

Ansell, Brendan R E; Schnyder, Manuela; Deplazes, Peter; Korhonen, Pasi K; Young, Neil D; Hall, Ross S; Mangiola, Stefano; Boag, Peter R; Hofmann, Andreas; Sternberg, Paul W; Jex, Aaron R; Gasser, Robin B

2013-12-01

Angiostrongylus vasorum is a metastrongyloid nematode of dogs and other canids of major clinical importance in many countries. In order to gain first insights into the molecular biology of this worm, we conducted the first large-scale exploration of its transcriptome, and predicted essential molecules linked to metabolic and biological processes as well as host immune responses. We also predicted and prioritized drug targets and drug candidates. Following Illumina sequencing (RNA-seq), 52.3 million sequence reads representing adult A. vasorum were assembled and annotated. The assembly yielded 20,033 contigs, which encoded proteins with 11,505 homologues in Caenorhabditis elegans, and additional 2252 homologues in various other parasitic helminths for which curated data sets were publicly available. Functional annotation was achieved for 11,752 (58.6%) proteins predicted for A. vasorum, including peptidases (4.5%) and peptidase inhibitors (1.6%), protein kinases (1.7%), G protein-coupled receptors (GPCRs) (1.5%) and phosphatases (1.2%). Contigs encoding excretory/secretory and immuno-modulatory proteins represented some of the most highly transcribed molecules, and encoded enzymes that digest haemoglobin were conserved between A. vasorum and other blood-feeding nematodes. Using an essentiality-based approach, drug targets, including neurotransmitter receptors, an important chemosensory ion channel and cysteine proteinase-3 were predicted in A. vasorum, as were associated small molecular inhibitors/activators. Future transcriptomic analyses of all developmental stages of A. vasorum should facilitate deep explorations of the molecular biology of this important parasitic nematode and support the sequencing of its genome. These advances will provide a foundation for exploring immuno-molecular aspects of angiostrongylosis and have the potential to underpin the discovery of new methods of intervention. © 2013.

Switch-on fluorescence scheme for antibiotics based on a magnetic composite probe with aptamer and hemin/G-quadruplex coimmobilized nano-Pt-luminol as signal tracer.

PubMed

Miao, Yang-Bao; Gan, Ning; Ren, Hong-Xia; Li, Tianhua; Cao, Yuting; Hu, Futao; Chen, Yinji

2016-01-15

A selective and facile fluorescence "switch-on" scheme is developed to detect antibiotics residues in food, using chloramphenicol (CAP) as model, based on a novel magnetic aptamer probe (aptamer-Pt-luminol nanocomposite labeled with hemin/G-quadruplex). Firstly, the composite probe is prepared through the immuno-reactions between the capture beads (anti-dsDNA antibody labeled on magnetic Dynabeads) and the nanotracer (nano-Pt-luminol labeled with double-strand aptamer, as ds-Apt, and hemin/G-quadruplex). When the composite probe is mixed with CAP, the aptamer preferentially reacted with CAP to decompose the double-strand aptamer to ssDNA, which cannot be recognized by the anti-dsDNA antibody on the capture probes. Thus, after magnetic separation, the nanotracer can be released into the supernatant. Because the hemin/G-quadruplex and PtNPs in nanotracer can catalyze luminol-H2O2 system to emit fluorescence. Thus a dual-amplified "switch-on" signal appeared, of which intensity is proportional to the concentration of CAP between 0.001 and 100ng mL(-1) with detection limit of 0.0005ng mL(-1) (S/N=3). Besides, our method has good selectivity and was employed for CAP detection in real milk samples. The results agree well with those from conventional gas chromatograph-mass spectrometer (GC-MS). The switch-on signal is produced by one-step substitution reaction between aptamer in nanotracer and target. When the analyte is changed, the probe can be refabricated only by changing the corresponding aptamer. Thus, all features above prove our strategy to be a facile, feasible and selective method in antibiotics screening for food safety. Copyright © 2015 Elsevier B.V. All rights reserved.

A sensitive electrochemical impedance immunosensor for determination of malachite green and leucomalachite green in the aqueous environment.

PubMed

Zhu, Dan; Li, Qiangqiang; Pang, Xiumei; Liu, Yue; Wang, Xue; Chen, Gang

2016-08-01

Application of malachite green (MG) and leucomalachite green (LMG) in fish farm water causes an environmental problem. This study proposes for the first time a sensitive and convenient electrochemical impedance spectroscopy (EIS) method for determining MG and LMG by a bovine serum albumin-decorated gold nanocluster (BSA-AuNC)/antibody composite film-based immunosensor. In order to improve the analytical performance, the glassy carbon electrode (GCE) was modified with 1, 4-phenylenediamine to form a stable layer, and then, BSA-AuNCs were covalently bound to the GCE. An adequate quantity of the polyclonal antibody of LMG was immobilized onto the surface of the BSA-AuNCs by the chemical reaction of EDC/NHS. The sensors can respond to the specific target based on specific covalent bonding. The experimental parameters, such as the pH, incubating concentration, and time, have been investigated and optimized. The calibration curve for LMG was linear in the range of 0.1~10.0 ng/mL with the limit of detection (LOD) 0.03 ng/mL. Furthermore, the sum of MG and LMG was detected in fish farm water by MG reduction. The recovery was between 89.7 % and 99.2 % in spiked samples. The EC sensor method was also compared with the ELISA method and validated by the LC-MS/MS method, which proves its great promise as a field instrument for the rapid monitoring of MG and LMG pollution. Graphical abstract 1, 4-Phenylenediamine and BSA-AuNC/antibody-decorated glassy carbon electrodes have been used for the impedimetric detection of the sum of malachite green and leucomalachite green via specific immuno-binding.

Recent mycotoxin survey data and advanced mycotoxin detection techniques reported from China: a review.

PubMed

Selvaraj, Jonathan Nimal; Wang, Yan; Zhou, Lu; Zhao, Yueju; Xing, Fuguo; Dai, Xiaofeng; Liu, Yang

2015-01-01

Mycotoxin contamination in agro-food systems has been a serious concern over the last few decades in China, where the Ministry of Health has set maximum limits for mycotoxins in different agro-products. Overall survey data show that aflatoxin contamination in infant cereals, edible oils, raw milk, ginger and its related products are far below Chinese regulatory limits. The absence of aflatoxin M1 contamination in infant milk powders indicates a high standard of control. Aflatoxins in liquorice roots and lotus seeds have been reported for the first time. For deoxynivalenol, high levels were found in wheat grown in the Yangtze Delta region, which is more prone to rainfall, supporting Fusarium infection. The emerging mycotoxins beauvericins and enniatins have been reported in the medicinal herbs in China. Ochratoxin A in wine was below the European Union regulatory limits, but fumonisins in maize need to be monitored and future regulatory control considered. Overall from all the survey data analysed in this review, it can be concluded that 92% of the samples analysed had mycotoxin levels below the Chinese regulatory limits. In terms of detection techniques in recent years, immuno-based assays have been developed largely due to their excellent sensitivity and ease of use. Assays targeting multiple mycotoxins like aflatoxins, ochratoxin A, zearalenone and deoxynivalenol have been reported using microarrays and suspension arrays targeting in particular maize, rice and peanuts. Aptamer-based assays against ochratoxin A and aflatoxins B1 and B2 have been developed involving fluorescence detection; and surface plasmon resonance immunosensors have been developed targeting wine, maize, wheat, wild rye, hay and peanut oil with high sensitivity (> 0.025 ng l(-1)). Commercialisation of these technologies is much needed for wider usage in the coming years.

Major quality trait analysis and QTL detection in hexaploid wheat in humid rain-fed agriculture.

PubMed

Li, H M; Tang, Z X; Zhang, H Q; Yan, B J; Ren, Z L

2013-05-21

Humid rain-fed agriculture is a special environment for wheat (Triticum aestivum) culture that tends to negatively affect wheat yield and quality. To identify quality characters of wheat in a humid environment, we conducted quality analysis and quantitative trait loci (QTL) detection in a recombinant inbred line whose parent had a high level of quality for several years. We found that high-quality wheat had less gluten content and lower protein content. Apparently, wheat quality and associated quantity traits were in a dynamic state of equilibrium. We detected 83 QTL for 10 wheat quality traits in this recombinant inbred line population. Nine QTL were detected in both evaluation years; Q.DT.scau-2A, linked to Xwmc522-2A, was detected at the same genetic location in both years. Other QTL for different traits were detected simultaneously in more than one location. Consequently, there appeared to be pleiotropic genes that control wheat quality. Based on previous studies and our research on QTL analysis of grain protein content, we conclude that there must be one or more genes for grain protein content on chromosome 6B, whose expression was little affected by environment. We constructed a consensus map and projected the QTL on it. It was useful for choosing optimal markers for marker-assisted breeding and map-based cloning.

Development of Rapid Diagnostic Kit for Identification of Hanwoo (Korean Native Cattle) Brand Meat by Detecting BIO-TAG

PubMed Central

Park, Sung Kwon; Lee, Myung Hoon; Cho, Soo Hyun

2014-01-01

This study was performed to develop a rapid immuno-assay kit, by using a specific antigen to detect Hanwoo brand meat. We selected a synthetic antigen specific to our target antibody, named BIO-TAG (Tyr-D-Ala-Phe), by utilizing a computer-based analysis and literature review. BIO-TAG tagged with adjuvant was subcutaneously injected in sheep and Hanwoo. The serum and meat juice of the immunized or non-immunized animal were then analyzed, to measure the titer of antibody by ELISA and Western blot. The amount of antibodies against the BIO-TAG increased (p<0.05) in serum by vaccination. Furthermore, meat juice from the immunized Hanwoo showed greater (p<0.05) antibody titer, compared with those from non-immunized groups. To optimze the dilution factor, we performed dot-ELISA, with various combination levels of BIO-TAG. Results from dot-ELISA showed that 2 mg/mL BIO-TAG was sufficient to distinguish the immunized meat from non-immunized groups. These results support our hypothesis that simple immunization of Hanwoo generates a sufficient amount of antibodies to be detectable in the meat juice by means of the immune-assay. Therefore, specific Hanwoo brand meat can be more precisely identified by our rapid diagnostic kit. This technology can deter possible fraud of counterfeit meat brands in the Korean domestic market with ease and rapidity; and offers a new tool that guarantees consumers high quality Hanwoo brand beef. PMID:26761175

Detection of Goss's Wilt Pathogen Clavibacter michiganensis subsp. nebraskensis in Maize by Loop-Mediated Amplification.

PubMed

Yasuhara-Bell, Jarred; de Silva, Asoka; Heuchelin, Scott A; Chaky, Jennifer L; Alvarez, Anne M

2016-03-01

The Goss's wilt pathogen, Clavibacter michiganensis subsp. nebraskensis, can cause considerable losses in maize (Zea mays) production. Diagnosis of Goss's wilt currently is based on symptomology and identification of C. michiganensis subsp. nebraskensis, following isolation on a semiselective medium and/or serological testing. In an effort to provide a more efficient identification method, a loop-mediated amplification (LAMP) assay was developed to detect the tripartite ATP-independent periplasmic (TRAP)-type C4-dicarboxylate transport system large permease component and tested using strains of C. michiganensis subsp. nebraskensis, all other C. michiganensis subspecies and several genera of nontarget bacteria. Only strains of C. michiganensis subsp. nebraskensis reacted positively with the LAMP assay. The LAMP assay was then used to identify bacterial isolates from diseased maize. 16S rDNA and dnaA sequence analyses were used to confirm the identity of the maize isolates and validate assay specificity. The Cmm ImmunoStrip assay was included as a presumptive identification test of C. michiganensis subsp. nebraskensis at the species level. The Cmn-LAMP assay was further tested using symptomatic leaf tissue. The Cmn-LAMP assay was run in a hand-held real-time monitoring device (SMART-DART) and performed equally to in-lab quantitative polymerase chain reaction equipment. The Cmn-LAMP assay accurately identified C. michiganensis subsp. nebraskensis and has potential as a field test. The targeted sequence also has potential application in other molecular detection platforms.

An electrochemical immunosensor for cardiac Troponin I using electrospun carboxylated multi-walled carbon nanotube-whiskered nanofibres.

PubMed

Rezaei, Babak; Shoushtari, Ahmad Mousavi; Rabiee, Mohammad; Uzun, Lokman; Mak, Wing Cheung; Turner, Anthony P F

2018-05-15

A sandwich-type nanostructured immunosensor based on carboxylated multi-walled carbon nanotube (CMWCNT)-embedded whiskered nanofibres (WNFs) was developed for detection of cardiac Troponin I (cTnI). WNFs were directly fabricated on glassy carbon electrodes (GCE) by removing the sacrificial component (polyethylene glycol, PEG) after electrospinning of polystyrene/CMWCNT/PEG nanocomposite nanofibres, and utilised as a transducer layer for enzyme-labeled amperometric immunoassay of cTnI. The whiskered segments of CMWCNTs were activated and utilised to immobilise anti-cTnT antibodies. It was observed that the anchored CMWCNTs within the nanofibres were suitably stabilised with excellent electrochemical repeatability. A sandwich-type immuno-complex was formed between cTnI and horseradish peroxidase-conjugated anti-cTnI (HRP-anti-cTnI). The amperometric responses of the immunosensor were studied using cyclic voltammetry (CV) through an enzymatic reaction between hydrogen peroxide and HRP conjugated to the secondary antibody. The nanostructured immunosensor delivered a wide detection range for cTnI from the clinical borderline for a normal person (0.5-2ngmL -1 ) to the concentration present in myocardial infarction patients (> 20ngmL -1 ), with a detection limit of ~ 0.04ngmL -1 . It also showed good reproducibility and repeatability for three different cTnI concentration (1, 10 and 25ngmL -1 ) with satisfactory relative standard deviations (RSD). Hence, the proposed nanostructured immunosensor shows potential for point-of-care testing. Copyright © 2018 Elsevier B.V. All rights reserved.

Ultrasensitive Detection of Ricin Toxin in Multiple Sample Matrixes Using Single-Domain Antibodies.

PubMed

Gaylord, Shonda T; Dinh, Trinh L; Goldman, Ellen R; Anderson, George P; Ngan, Kevin C; Walt, David R

2015-07-07

Ricin is an extremely potent ribosomal inactivating protein listed as a Category B select agent. Although ricin intoxication is not transmittable from person to person, even a single ricin molecule can lead to cell necrosis because it inactivates 1500 ribosomes/min. Since there is currently no vaccine or therapeutic treatment for ricin intoxication, ultrasensitive analytical assays capable of detecting ricin in a variety of matrixes are urgently needed to limit exposure to individuals as well as communities. In this paper, we present the development and application of a single-molecule array (Simoa) for the detection of ricin toxin in human urine and serum. Single-domain antibodies (sdAbs), among the smallest engineered binding fragments, were chemically coupled to the surface of paramagnetic beads for the sensitive detection of ricin toxin. The Simoa was able to detect ricin at levels of 10 fg/mL, 100 fg/mL, and 1 pg/mL in buffer, urine and serum, respectively, in a fraction of the assay time need using immuno-polymerase chain reaction (IPCR). Using a fully automated state-of-the-art platform, the Simoa HD-1 analyzer, the assay time was reduced to 64 min.

An improved RT-IPCR for detection of pyrene and related polycyclic aromatic hydrocarbons.

PubMed

Meng, X Y; Li, Y S; Zhou, Y; Sun, Y; Qiao, B; Si, C C; Hu, P; Lu, S Y; Ren, H L; Liu, Z S; Qiu, H J; Liu, J Q

2016-04-15

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous homogeneous chemicals which are well known by carcinogens, mutagens and endocrine disorder. Here, an improved real-time immuno-PCR (RT-IPCR) was developed for detection of pyrene and its homologs in water samples. The PAHs in sample compete with pyrene-modified DNA to bind with monoclonal antibody (McAb) coated on PCR plate. The reporter DNA was exponentially amplified by real-time PCR instrument using Fast Start universal SYBR Green Master (ROX) kit. Only two reaction steps were needed to accomplish the detection. The assay had a good linear range from 5 pmol L(-1) to 5 nmol L(-1) with a detection limit of 3.5 pmol L(-1). For application assay, the average recoveries from tap water, lake water and mineral water were 98.4%, 98.2% and 99.7%, respectively which showed a good correlation (R(2)=0.9906) with those from GC-MS. The results indicated that the improved RT-IPCR seems to be a potential method for simple and ultrasensitive detection of pyrene and some homologues in environment water samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Video content analysis of surgical procedures.

PubMed

Loukas, Constantinos

2018-02-01

In addition to its therapeutic benefits, minimally invasive surgery offers the potential for video recording of the operation. The videos may be archived and used later for reasons such as cognitive training, skills assessment, and workflow analysis. Methods from the major field of video content analysis and representation are increasingly applied in the surgical domain. In this paper, we review recent developments and analyze future directions in the field of content-based video analysis of surgical operations. The review was obtained from PubMed and Google Scholar search on combinations of the following keywords: 'surgery', 'video', 'phase', 'task', 'skills', 'event', 'shot', 'analysis', 'retrieval', 'detection', 'classification', and 'recognition'. The collected articles were categorized and reviewed based on the technical goal sought, type of surgery performed, and structure of the operation. A total of 81 articles were included. The publication activity is constantly increasing; more than 50% of these articles were published in the last 3 years. Significant research has been performed for video task detection and retrieval in eye surgery. In endoscopic surgery, the research activity is more diverse: gesture/task classification, skills assessment, tool type recognition, shot/event detection and retrieval. Recent works employ deep neural networks for phase and tool recognition as well as shot detection. Content-based video analysis of surgical operations is a rapidly expanding field. Several future prospects for research exist including, inter alia, shot boundary detection, keyframe extraction, video summarization, pattern discovery, and video annotation. The development of publicly available benchmark datasets to evaluate and compare task-specific algorithms is essential.

Influence of drying methods over in vitro antitumoral effects of exopolysaccharides produced by Agaricus blazei LPB 03 on submerged fermentation.

PubMed

Fernandes, M B A; Habu, S; de Lima, M A; Thomaz-Soccol, V; Soccol, C R

2011-03-01

Agaricus blazei is a mushroom that belongs to the Brazilian biodiversity and is considered as an important producer of bioactive compounds beneficial to human health. Studies have demonstrated that these compounds present immuno-modulatory, antioxidant and antitumor properties. In order to compare the most used method for fungal polysaccharide drying, lyophilization with other industrial-scale methods, the aim of this work was to submit A. blazei LPB 03 polysaccharide extracts to vaucum, spray and freeze drying, and evaluate the maintenance of its antitumoral effects in vitro. Exopolysaccharides produced by A. blazei LPB 03 on submerged fermentation were extracted with ethanol and submitted to drying processes. The efficiency represents the water content that was removed during the drying process. The resultant dried products showed water content around 3% and water activity less than 0.380, preventing therefore the growth of microorganisms and reactions of chemical degradation. Exopolysaccharide extracts dried by vacuum and spray dryer did not showed any significant cytotoxic effect on cell viability of Wistar mice macrophages. Content of total sugars and protein decrease after drying, nevertheless, 20 mg/ml of exopolysaccharides dried by spray dryer reached 33% of inhibition rate over Ehrlich tumor cells in vitro.

A novel framework for intelligent surveillance system based on abnormal human activity detection in academic environments.

PubMed

Al-Nawashi, Malek; Al-Hazaimeh, Obaida M; Saraee, Mohamad

2017-01-01

Abnormal activity detection plays a crucial role in surveillance applications, and a surveillance system that can perform robustly in an academic environment has become an urgent need. In this paper, we propose a novel framework for an automatic real-time video-based surveillance system which can simultaneously perform the tracking, semantic scene learning, and abnormality detection in an academic environment. To develop our system, we have divided the work into three phases: preprocessing phase, abnormal human activity detection phase, and content-based image retrieval phase. For motion object detection, we used the temporal-differencing algorithm and then located the motions region using the Gaussian function. Furthermore, the shape model based on OMEGA equation was used as a filter for the detected objects (i.e., human and non-human). For object activities analysis, we evaluated and analyzed the human activities of the detected objects. We classified the human activities into two groups: normal activities and abnormal activities based on the support vector machine. The machine then provides an automatic warning in case of abnormal human activities. It also embeds a method to retrieve the detected object from the database for object recognition and identification using content-based image retrieval. Finally, a software-based simulation using MATLAB was performed and the results of the conducted experiments showed an excellent surveillance system that can simultaneously perform the tracking, semantic scene learning, and abnormality detection in an academic environment with no human intervention.

The Innovative Medicines Initiative moves translational immunology forward.

PubMed

Goldman, Michel; Wittelsberger, Angela; De Magistris, Maria-Teresa

2013-02-01

The Innovative Medicines Initiative (IMI) was established in 2008 as a public-private partnership between the European Union and the European Federation of Pharmaceutical Industries and Associations with the mission to promote the development of novel therapies through collaborative efforts based on the concept of pre-competitive research. Several consortia supported by IMI are dedicated to immuno-inflammatory disorders, immune-based biopharmaceuticals and vaccines. Herein, we present the key principles underlying IMI, briefly review the status of projects related to translational immunology, and present future topics of interest to immunologists.

Blurry-frame detection and shot segmentation in colonoscopy videos

NASA Astrophysics Data System (ADS)

Oh, JungHwan; Hwang, Sae; Tavanapong, Wallapak; de Groen, Piet C.; Wong, Johnny

2003-12-01

Colonoscopy is an important screening procedure for colorectal cancer. During this procedure, the endoscopist visually inspects the colon. Human inspection, however, is not without error. We hypothesize that colonoscopy videos may contain additional valuable information missed by the endoscopist. Video segmentation is the first necessary step for the content-based video analysis and retrieval to provide efficient access to the important images and video segments from a large colonoscopy video database. Based on the unique characteristics of colonoscopy videos, we introduce a new scheme to detect and remove blurry frames, and segment the videos into shots based on the contents. Our experimental results show that the average precision and recall of the proposed scheme are over 90% for the detection of non-blurry images. The proposed method of blurry frame detection and shot segmentation is extensible to the videos captured from other endoscopic procedures such as upper gastrointestinal endoscopy, enteroscopy, cystoscopy, and laparoscopy.

Fast methods for analysis of neurotransmitters from single cell and monitoring their releases in central nervous system by capillary electrophoresis, fluorescence microscopy and luminescence imaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Ziqiang

1999-12-10

Fast methods for separation and detection of important neurotransmitters and the releases in central nervous system (CNS) were developed. Enzyme based immunoassay combined with capillary electrophoresis was used to analyze the contents of amino acid neurotransmitters from single neuron cells. The release of amino acid neurotransmitters from neuron cultures was monitored by laser induced fluorescence imaging method. The release and signal transduction of adenosine triphosphate (ATP) in CNS was studied with sensitive luminescence imaging method. A new dual-enzyme on-column reaction method combined with capillary electrophoresis has been developed for determining the glutamate content in single cells. Detection was based onmore » monitoring the laser-induced fluorescence of the reaction product NADH, and the measured fluorescence intensity was related to the concentration of glutamate in each cell. The detection limit of glutamate is down to 10 -8 M level, which is 1 order of magnitude lower than the previously reported detection limit based on similar detection methods. The mass detection limit of a few attomoles is far superior to that of any other reports. Selectivity for glutamate is excellent over most of amino acids. The glutamate content in single human erythrocyte and baby rat brain neurons were determined with this method and results agreed well with literature values.« less

Gold Nanocages for Biomedical Applications**

PubMed Central

Skrabalak, Sara E.; Chen, Jingyi; Au, Leslie; Lu, Xianmao; Li, Xingde; Xia, Younan

2008-01-01

Nanostructured materials provide a promising platform for early cancer detection and treatment. Here we highlight recent advances in the synthesis and use of Au nanocages for such biomedical applications. Gold nanocages represent a novel class of nanostructures, which can be prepared via a remarkably simple route based on the galvanic replacement reaction between Ag nanocubes and HAuCl4. The Au nanocages have a tunable surface plasmon resonance peak that extends into the near-infrared, where the optical attenuation caused by blood and soft tissue is essentially negligible. They are also biocompatible and present a well-established surface for easy functionalization. We have tailored the scattering and absorption cross-sections of Au nanocages for use in optical coherence tomography and photothermal treatment, respectively. Our preliminary studies show greatly improved spectroscopic image contrast for tissue phantoms containing Au nanocages. Our most recent results also demonstrate the photothermal destruction of breast cancer cells in vitro by using immuno-targeted Au nanocages as an effective photo-thermal transducer. These experiments suggest that Au nanocages may be a new class of nanometer-sized agents for cancer diagnosis and therapy. PMID:18648528

Predicting IGF-1R therapy response in bone sarcomas: immuno-SPECT imaging with radiolabeled R1507

PubMed Central

Fleuren, Emmy D.G.; Versleijen-Jonkers, Yvonne M.H.; van de Luijtgaarden, Addy C.M.; Molkenboer-Kuenen, Janneke D.M.; Heskamp, Sandra; Roeffen, Melissa H.S.; van Laarhoven, Hanneke W.M.; Houghton, Peter J.; Oyen, Wim J.G.; Boerman, Otto C.; van der Graaf, Winette T.A.

2011-01-01

Purpose To investigate whether 111In-R1507 immuno-SPECT, a novel non-invasive, in vivo screening method to visualize membranous Insulin-like Growth Factor 1 Receptor (IGF-1R) expression and accessibility, can be used to predict IGF-1R treatment (R1507) responsein bone sarcomas. Experimental design BALB/c nude mice were subcutaneously implanted with IGF-1R-expressing human bone sarcoma xenografts (OS-1, EW-5 and EW-8) which demonstrated high, modest or no response, respectively, to R1507, a monoclonal antibody targeting the extracellular domain of IGF-1R. An IGF-1R-negative tumor (OS-33), unresponsive to IGF-1R inhibitors, was examined as well. Mice were injected with indium-111 labeled R1507 (111In-R1507). Biodistribution and immuno-SPECT/CT imaging studies were performed 1, 3 and 7 days p.i. in mice with OS-1 and EW-5 xenografts and 3 days p.i. in mice with EW-8 and OS-33 xenografts. Results Biodistribution studies showed specific accumulation of 111In-R1507 in OS-1 and EW-5 xenografts (27.5±6.5%ID/g and 14.0±2.8%ID/g, 3 days p.i., respectively). Most importantly, 111In-R1507 uptake in IGF-1R-positive, but unresponsive, EW-8 xenografts (6.5±1.5%ID/g, 3 days p.i.) was similar to that of the IGF-1R-negative OS-33 tumor (5.5±0.6%ID/g, 3 days p.i.). Uptake in normal tissues was low and non-specific. Corresponding immuno-SPECT images clearly discriminated between high, modest and non-responding tumors by demonstrating a homogeneous (OS-1), heterogeneous (EW-5) or non-specific (EW-8 and OS-33)tumor uptake of 111In-R1507. Conclusions 111In-R1507 immuno-SPECT is an excellent method to visualize membranous IGF-1R expression and target accessibility in vivo in human bone sarcoma xenografts and may serve as an independent marker to predict IGF-1R therapy (R1507) responsein bone sarcoma patients. PMID:22038993

16S rRNA and Omp31 Gene Based Molecular Characterization of Field Strains of B. melitensis from Aborted Foetus of Goats in India

PubMed Central

Singh, Ajay; Gupta, Vivek Kumar; Kumar, Amit; Singh, Vikas Kumar; Nayakwadi, Shivasharanappa

2013-01-01

Brucellosis is a reemerging infectious zoonotic disease of worldwide importance. In human, it is mainly caused by Brucella melitensis, a natural pathogen for goats. In India, a large number of goats are reared in semi-intensive to intensive system within the close vicinity of human being. At present, there is no vaccination and control strategy for caprine brucellosis in the country. Thus, to formulate an effective control strategy, the status of etiological agent is essential. To cope up with these, the present study was conducted to isolate and identify the prevalent Brucella species in caprine brucellosis in India. The 30 samples (fetal membrane, fetal stomach content and vaginal swabs) collected throughout India from the aborted fetus of goats revealed the isolation of 05 isolates all belonging to Brucella melitensis biovars 3. All the isolates produced amplification products of 1412 and 720 bp in polymerase chain reaction with genus and species specific 16S rRNA and omp31 gene based primers, respectively. Moreover, the amplification of omp31 gene in all the isolates confirmed the presence of immuno dominant outer membrane protein (31 kDa omp) in all the field isolates of B. melitensis in aborted foetus of goats in India. These findings can support the development of omp31 based specific serodiagnostic test as well as vaccine for the control of caprine brucellosis in India. PMID:24453799

Controlled-rate freezer cryopreservation of highly concentrated peripheral blood mononuclear cells results in higher cell yields and superior autologous T-cell stimulation for dendritic cell-based immunotherapy.

PubMed

Buhl, Timo; Legler, Tobias J; Rosenberger, Albert; Schardt, Anke; Schön, Michael P; Haenssle, Holger A

2012-11-01

Availability of large quantities of functionally effective dendritic cells (DC) represents one of the major challenges for immunotherapeutic trials against infectious or malignant diseases. Low numbers or insufficient T-cell activation of DC may result in premature termination of treatment and unsatisfying immune responses in clinical trials. Based on the notion that cryopreservation of monocytes is superior to cryopreservation of immature or mature DC in terms of resulting DC quantity and immuno-stimulatory capacity, we aimed to establish an optimized protocol for the cryopreservation of highly concentrated peripheral blood mononuclear cells (PBMC) for DC-based immunotherapy. Cryopreserved cell preparations were analyzed regarding quantitative recovery, viability, phenotype, and functional properties. In contrast to standard isopropyl alcohol (IPA) freezing, PBMC cryopreservation in an automated controlled-rate freezer (CRF) with subsequent thawing and differentiation resulted in significantly higher cell yields of immature and mature DC. Immature DC yields and total protein content after using CRF were comparable with results obtained with freshly prepared PBMC and exceeded results of standard IPA freezing by approximately 50 %. While differentiation markers, allogeneic T-cell stimulation, viability, and cytokine profiles were similar to DC from standard freezing procedures, DC generated from CRF-cryopreserved PBMC induced a significantly higher antigen-specific IFN-γ release from autologous effector T cells. In summary, automated controlled-rate freezing of highly concentrated PBMC represents an improved method for increasing DC yields and autologous T-cell stimulation.

Qualitative and Semiquantitative Analysis of Doping Products Seized at the Swiss Border.

PubMed

Weber, Christina; Krug, Oliver; Kamber, Matthias; Thevis, Mario

2017-05-12

Substances developed for therapeutic use are also known to be misused by athletes as doping agents and, outside of regulated sport, for image-enhancement. This has generated a market for counterfeit doping substances. Counterfeit doping agents may be of poor pharmaceutical quality and therefore constitute health risks to consumers. This study aims to investigate the pharmaceutical quality of 1,190 doping products seized at the Swiss border. Swiss customs authorities seize incoming shipments potentially containing doping agents. Qualitative and semiquantitative analyses were performed in order to test for prohibited doping substances. The main analytical methods utilized for characterizing confiscated compounds were liquid chromatography-high resolution mass spectrometry, polyacrylamide gel electrophoresis with subsequent in-gel tryptic digestion and identification of peptidic compounds using nanoliquid chromatography-tandem mass spectrometry, and electrochemiluminescence immuno assay. For 889 (75%) of the analyzed products, the label suggested the content of anabolic agents, for 146 samples (12%) peptide hormones or growth factors, and for 113 items (9%) antiestrogens, aromatase inhibitors or other metabolic modulators. For the majority of the investigated products, the pharmaceutical quality was an unsatisfactory standard: nonapproved substances were detected and less than 20% of the products contained the claimed substance in the respective amount. A comprehensive sample of confiscated doping products was analyzed, allowing for monitoring of developments regarding the use of doping substances in Switzerland and for anticipating future trends and challenges in sports drug testing. An alarming number of tested products was of substandard pharmaceutical quality.

Development for equipment of the milk macromolecules content detection

NASA Astrophysics Data System (ADS)

Ding, Guochao; Li, Weimin; Shang, Tingyi; Xi, Yang; Gao, Yunli; Zhou, Zhen

Developed an experimental device for rapid and accurate detection of milk macromolecular content. This device developed based on laser scattered through principle, the principle use of the ingredients of the scattered light and transmitted light ratio characterization of macromolecules. Peristaltic pump to achieve automatic input and output of the milk samples, designing weak signal detection amplifier circuit for detecting the ratio with ICL7650. Real-time operating system μC / OS-II is the core design of the software part of the whole system. The experimental data prove that the device can achieve a fast real-time measurement of milk macromolecules.

Fiber-optic array using molecularly imprinted microspheres for antibiotic analysis.

PubMed

Carrasco, Sergio; Benito-Peña, Elena; Walt, David R; Moreno-Bondi, María C

2015-05-01

In this article we describe a new class of high-density optical microarrays based on molecularly imprinted microsphere sensors that directly incorporate specific recognition capabilities to detect enrofloxacin (ENRO), an antibiotic widely used for both human and veterinary applications. This approach involves the preparation of highly cross-linked polymer microspheres by thermal precipitation-polymerization in the presence and absence of the target analyte ENRO to generate either molecularly imprinted (MIP) or non-imprinted polymer (NIP) microspheres, respectively. Each polymer type of tailor-made microsphere is fluorescently encoded with either coumarin-30 or tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(ii) dichloride [Ru(dip) 3 ]Cl 2 to enable the microspheres to be distinguished. The new MIP-based sensing platform utilizes an optical fiber bundle containing approximately 50 000 individual 3.1 μm diameter fibers that are chemically etched to create microwells in which MIP and NIP microspheres can be deposited and imaged using an epi-fluorescence microscope. The method enables multiplexed detection by independently addressing both types of beads through their separate light channels. The unique response to the presence of ENRO is manifested on the basis of a competitive immunoassay. A red-fluorescent dye-tagged ENRO, labeled with BODIPY® TR Cadaverine, competes with ENRO for specific binding sites. The developed immuno-like assay displayed a limit of detection (LOD) of 0.04 μM (10% binding inhibition) and a dynamic range of 0.29-21.54 μM (20-80% binding inhibition). The selectivity of the assay was evaluated by measuring the cross-reactivity of other fluoroquinolones (ciprofloxacin, norfloxacin, danofloxacin, and flumequine) and non-related antibiotics (penicillin G and doxycycline). This work demonstrates, for the first time, the applicability of MIPs, as an alternative to biomolecule receptors, for the development of multiplexed detection fiber-optic microarrays paving the way for a new generation of biomimetic sensors.

Morphological effects of MMPs inhibitors on the dentin bonding

PubMed Central

Li, He; Li, Tianbo; Li, Xiuying; Zhang, Zhimin; Li, Penglian; Li, Zhenling

2015-01-01

Matrix metalloproteinases (MMPs) have been studied extensively, and MMP inhibitors have been used as dental pretreatment agents prior to dentin bonding because they reduce collagen fiber degradation and improve bonding strength. However, morphologic characteristics of the collagen network after etching and of the post-adhesive dentin hybrid layers (DHL) after MMP inhibitors pretreatment have not been evaluated. Thus, we investigated demineralized dentin pretreated with chlorhexidine (CHX) and minocycline (MI) in an etch- and -rinse adhesive system with field emission scanning electron microscopy (FESEM) and immuno-gold labeling markers to observe the collagen network and DHL. FESEM revealed after CHX and MI, a demineralized dentin surface and improved collagen network formation, reduced collagen degradation, and distinct gold-labeling signals. Applying adhesive after either MMP inhibitor created a better dentin interface as evidenced by immuno-gold staining, better adhesive penetration, and higher DHL quality. With microtensile bond strength tests (µTBS) we estimated bonding strength using µTBS data. Immediate µTBS was enhanced with MMP inhibitor application to the bonding surface, and the CHX group was significantly different than non-treated etched surfaces, but no significant change was detected in the MI group. Surface micromorphology of the fractured dentin resin restoration showed that the CHX group had a better resin and dentin tube combination. Both MMP inhibitors created uniform resin coverage. Thus, morphologic results and µTBS data suggest that CHX and MI can inhibit MMP activity, improve immediate bonding strength, and enhance dentin bonding stability with an etch- and -rinse adhesive system. PMID:26379873

The Susceptibilities of Respiratory Syncytial Virus to Nucleolin Receptor Blocking and Antibody Neutralization Are Dependent upon the Method of Virus Purification

PubMed Central

Bilawchuk, Leanne M.; Jensen, Lionel D.; Marchant, David J.

2017-01-01

Respiratory Syncytial Virus (RSV) that is propagated in cell culture is purified from cellular contaminants that can confound experimental results. A number of different purification methods have been described, including methods that utilize fast protein liquid chromatography (FPLC) and gradient ultracentrifugation. Thus, the constituents and experimental responses of RSV stocks purified by ultracentrifugation in sucrose and by FPLC were analyzed and compared by infectivity assay, Coomassie stain, Western blot, mass spectrometry, immuno-transmission electron microscopy (TEM), and ImageStream flow cytometry. The FPLC-purified RSV had more albumin contamination, but there was less evidence of host-derived exosomes when compared to ultracentrifugation-purified RSV as detected by Western blot and mass spectrometry for the exosome markers superoxide dismutase [Cu-Zn] (SOD1) and the tetraspanin CD63. Although the purified virus stocks were equally susceptible to nucleolin-receptor blocking by the DNA aptamer AS1411, the FPLC-purified RSV was significantly less susceptible to anti-RSV polyclonal antibody neutralization; there was 69% inhibition (p = 0.02) of the sucrose ultracentrifugation-purified RSV, 38% inhibition (p = 0.03) of the unpurified RSV, but statistically ineffective neutralization in the FPLC-purified RSV (22% inhibition; p = 0.30). The amount of RSV neutralization of the purified RSV stocks was correlated with anti-RSV antibody occupancy on RSV particles observed by immuno-TEM. RSV purified by different methods alters the stock composition and morphological characteristics of virions that can lead to different experimental responses. PMID:28771197

Analytical techniques for steroid estrogens in water samples - A review.

PubMed

Fang, Ting Yien; Praveena, Sarva Mangala; deBurbure, Claire; Aris, Ahmad Zaharin; Ismail, Sharifah Norkhadijah Syed; Rasdi, Irniza

2016-12-01

In recent years, environmental concerns over ultra-trace levels of steroid estrogens concentrations in water samples have increased because of their adverse effects on human and animal life. Special attention to the analytical techniques used to quantify steroid estrogens in water samples is therefore increasingly important. The objective of this review was to present an overview of both instrumental and non-instrumental analytical techniques available for the determination of steroid estrogens in water samples, evidencing their respective potential advantages and limitations using the Need, Approach, Benefit, and Competition (NABC) approach. The analytical techniques highlighted in this review were instrumental and non-instrumental analytical techniques namely gas chromatography mass spectrometry (GC-MS), liquid chromatography mass spectrometry (LC-MS), enzyme-linked immuno sorbent assay (ELISA), radio immuno assay (RIA), yeast estrogen screen (YES) assay, and human breast cancer cell line proliferation (E-screen) assay. The complexity of water samples and their low estrogenic concentrations necessitates the use of highly sensitive instrumental analytical techniques (GC-MS and LC-MS) and non-instrumental analytical techniques (ELISA, RIA, YES assay and E-screen assay) to quantify steroid estrogens. Both instrumental and non-instrumental analytical techniques have their own advantages and limitations. However, the non-instrumental ELISA analytical techniques, thanks to its lower detection limit and simplicity, its rapidity and cost-effectiveness, currently appears to be the most reliable for determining steroid estrogens in water samples. Copyright © 2016 Elsevier Ltd. All rights reserved.

An unusual case of occupational asthma in a part time magician. He has got an allergy surprise from his top hat!

PubMed

Liccardi, Gennaro; Billeri, L; Foglia, M; Sapio, C; De Giglio, M A R; D'Amato, G

2014-09-01

In this report we describe a case of respiratory allergy induced by an unusual occupational exposure to rabbit. The patient worked as a part-time magician in theatres and private parties and the most popular performance of his show was to pull out a white rabbit from a top hat. Unfortunately, a few minutes after the extraction of rabbit from top hat, the patient experienced the onset of upper and lower airway symptoms, and in some occasions he was forced to stop the show and to use short acting β₂agonists and intramuscular steroids. The results of SPT and evaluation of serological specific IgE (ImmunoCAP and ImmunoCAP ISAC IgE) revealed allergic sensitization to rabbit (Oryctolagus cuniculus) dander as well as to Parietaria and dust mites. ImmunoCAP ISAC IgE excluded allergic sensitization to other cross-reacting animal allergens. Rabbit constitutes a reliable risk factor for allergic sensitization in individuals working as professional / part-time magicians or as animators in some recreational settings (resorts, parties, charity shows, etc).

Analysis of hepatocyte growth factor immunostaining in the placenta of HIV-infected normotensive versus preeclamptic pregnant women.

PubMed

Cele, S B; Odun-Ayo, F; Onyangunga, O A; Moodley, J; Naicker, T

2018-08-01

Hepatocyte Growth Factor (HGF) plays a role in the migration and morphogenesis of different cell types and tissues. Preeclampsia (PE) is associated with deficient trophoblast invasion and placental insufficiency; hence HGF production is expected to be compromised. This study therefore aimed to immunolocalize and morphometrically analyse placental HGF in normotensive versus PE pregnancies stratified by HIV status and gestational age. Normotensive (N; n = 40) and preeclamptic (PE; n = 80) women were stratified by HIV status (HIV- and HIV+), and gestational age i.e. early onset of PE (EOPE; <34 weeks) and late onset of PE (LOPE; ≥34 weeks). Placental tissues were stained using conventional immunohistochemistry, performed using mouse anti-human HGF antibody. Morphometric image analysis was performed using Zeiss Axio-Vision software. HGF was immuno-localized within the syncytiotrophoblast, cytotrophoblast, endothelial and fibroblast-like cell populations of both conducting and exchange villi. Based on pregnancy type, HGF immunoexpression within the conducting villi was significantly different between Nvs EOPE (p = 0.0372) and EOPE vs LOPE (p = 0.0006). Within the exchange villi, no significant difference of HGF immunostaining was noted between N vs EOPE and N vs LOPE. A down-regulation of HGF immuno-expression was observed in LOPE compared to other groups within both villi types, albeit non-significant. Based on HIV status, no significant difference in HGF immuno-expression was demonstrated between HIV- vs HIV + within the exchange and conducting villi. However, the expression of HGF in HIV- group was elevated in both villi types. Across the groups, a significant difference was found between N+ vs EOPE- (p  = 0.0207), EOPE+ vs LOPE- (p = 0.0036) and EOPE- vs LOPE- (p = 0.0016) of the conducting villi while no significant difference was found within the exchange villi. This novel study demonstrates that HGF was two-fold higher in conducting compared to exchange villi irrespective of the pregnancy type. HIV infection does not influence HGF expression within the conducting and exchange villi. The HGF/c-MET receptor complex may modulate the ligand expression within the placenta. Copyright © 2018 Elsevier B.V. All rights reserved.

Molecular and immunological approaches in quantifying the air-borne food allergen tropomyosin in crab processing facilities.

PubMed

Kamath, Sandip D; Thomassen, Marte R; Saptarshi, Shruti R; Nguyen, Hong M X; Aasmoe, Lisbeth; Bang, Berit E; Lopata, Andreas L

2014-09-01

Tropomyosin is a cross-reactive allergenic protein present in ingested shellfish species. Exposure and sensitization to this protein via inhalation is particularly important in the crustacean processing industry where workers are continuously exposed to the aerosolized form of this allergen. The aim of this study was to develop an antibody-based immunoassay to enable the specific and sensitive quantification of aerosolized tropomyosin present in the environment of two crab processing facilities. Anti-tropomyosin antibody was generated in rabbits against tropomyosins from four different crustacean species. These antibodies were purified using recombinant tropomyosin using an immuno-affinity column. The recombinant tropomyosin was also used as an allergen standard for the sandwich ELISA. In order to quantify aerosolized tropomyosin, air collection was performed in the personal breathing zone of 80 workers during two crab processing activities, edible crab (Cancer pagurus) and king crab (Paralithodes camtschaticus) using polytetrafluoroethylene filters. The purified antibody was able to detect tropomyosin selectively from different crustaceans but not from vertebrate sources. The limit of detection (LOD) for the developed sandwich ELISA was 60 picogram/m(3) and limit of quantitation (LOQ) 100 picogram/m(3). Immunoassay validation was based on linearity (R(2) 0.999), matrix interference test (78.8±6.5%), intra-assay CV (9.8%) and inter-assay CV (11%). The novel immunoassay was able to successfully identify working activities, which generated low, medium or high concentrations of the aerosolized food allergen. We describe an IgG antibody-based immunoassay for quantification of the major food allergen tropomyosin, with high sensitivity and specificity. This modified immunological approach can be adapted for the detection of other aerosolized food allergens, assisting in the identification of high-risk allergen exposure areas in the food industry. Copyright © 2014 Elsevier GmbH. All rights reserved.

The relationship between red meat allergy and sensitization to gelatin and galactose-alpha-1,3-galactose

PubMed Central

Mullins, Raymond James; James, Hayley; Platts-Mills, Thomas A.E.; Commins, Scott

2012-01-01

Background We have observed patients clinically allergic to red meat and meat-derived gelatin. Objective We describe a prospective evaluation of the clinical significance of gelatin sensitization, the predictive value of a positive test and an examination of the relationship between allergic reactions to red meat and sensitization to gelatin and alpha-Gal. Methods Adult patients evaluated 1997-2011 for suspected allergy/anaphylaxis to medication, insect venom or food were skin tested with gelatin colloid. In vitro (ImmunoCap) testing was undertaken where possible. Results Positive gelatin tests were observed in 40/1335 individuals; 30/40 patients with red meat allergy (12 also clinically allergic to gelatin); 2/2 with gelatin colloid anaphylaxis; 4/172 with idiopathic anaphylaxis (all responded to intravenous gelatin challenge of 0.02 to 0.4g); 4/368 with drug allergy. Testing was negative in all patients with venom allergy (n=241), non-meat food allergy (n=222), and miscellaneous disorders (n=290). ImmunoCap was positive to alpha-Gal in 20/24 meat allergics and in 20/22 with positive gelatin skin tests. The results of gelatin skin testing and anti-alpha-Gal IgE were strongly correlated (r=0.46; P<0.01). Alpha-Gal was detected in bovine gelatin colloids at concentrations of ~ 0.44 to 0.52ug/gm gelatin by inhibition radioimmunoassay. Conclusion Most patients allergic to red meat were sensitized to gelatin and a subset was clinically allergic to both. The detection of alpha-Gal in gelatin and correlation between the results of alpha-Gal and gelatin testing raises the possibility that alpha-Gal IgE may be the target of reactivity to gelatin. The pathogenic relationship between tick bites and sensitization to red meat, alpha-Gal and gelatin (with or without clinical reactivity) remains uncertain. PMID:22480538

CNV Workshop: an integrated platform for high-throughput copy number variation discovery and clinical diagnostics.

PubMed

Gai, Xiaowu; Perin, Juan C; Murphy, Kevin; O'Hara, Ryan; D'arcy, Monica; Wenocur, Adam; Xie, Hongbo M; Rappaport, Eric F; Shaikh, Tamim H; White, Peter S

2010-02-04

Recent studies have shown that copy number variations (CNVs) are frequent in higher eukaryotes and associated with a substantial portion of inherited and acquired risk for various human diseases. The increasing availability of high-resolution genome surveillance platforms provides opportunity for rapidly assessing research and clinical samples for CNV content, as well as for determining the potential pathogenicity of identified variants. However, few informatics tools for accurate and efficient CNV detection and assessment currently exist. We developed a suite of software tools and resources (CNV Workshop) for automated, genome-wide CNV detection from a variety of SNP array platforms. CNV Workshop includes three major components: detection, annotation, and presentation of structural variants from genome array data. CNV detection utilizes a robust and genotype-specific extension of the Circular Binary Segmentation algorithm, and the use of additional detection algorithms is supported. Predicted CNVs are captured in a MySQL database that supports cohort-based projects and incorporates a secure user authentication layer and user/admin roles. To assist with determination of pathogenicity, detected CNVs are also annotated automatically for gene content, known disease loci, and gene-based literature references. Results are easily queried, sorted, filtered, and visualized via a web-based presentation layer that includes a GBrowse-based graphical representation of CNV content and relevant public data, integration with the UCSC Genome Browser, and tabular displays of genomic attributes for each CNV. To our knowledge, CNV Workshop represents the first cohesive and convenient platform for detection, annotation, and assessment of the biological and clinical significance of structural variants. CNV Workshop has been successfully utilized for assessment of genomic variation in healthy individuals and disease cohorts and is an ideal platform for coordinating multiple associated projects. Available on the web at: http://sourceforge.net/projects/cnv.

Detection of classical enterotoxins and identification of enterotoxin genes in Staphylococcus aureus from milk and dairy products.

PubMed

Morandi, S; Brasca, M; Lodi, R; Cremonesi, P; Castiglioni, B

2007-09-20

Milk and dairy products are frequently contaminated with enterotoxigenic Staphylococcus aureus, which is often involved in staphylococcal food poisoning. The distribution of genes encoding staphylococcal enterotoxins (SE) in S. aureus isolated from bovine, goat, sheep and buffalo milk and dairy products was verified by the presence of the corresponding SE production. A total of 112 strains of S. aureus were tested for SE production by immuno-enzymatic (SEA-SEE) and reversed passive latex agglutination (SEA-SED) methods, while multiplex-PCR was applied for SE genes (sea, sec, sed, seg, seh, sei, sej and sel). Of the total strains studied, 67% were detected to have some SE genes (se), but only 52% produced a detectable amount of the classic antigenic SE types. The bovine isolates frequently had enterotoxin SEA, SED and sej, while SEC and sel predominated in the goat and sheep strains. The results demonstrated (i) marked enterotoxigenic S. aureus strain variations, in accordance with strain origin and (ii) the two methods resulted in different information but concurred on the risk of foodstuff infection by S. aureus.

Identification of Trypanosome Proteins in Plasma from African Sleeping Sickness Patients Infected with T. b. rhodesiense

PubMed Central

Enyaru, John C.; Carr, Steven A.; Pearson, Terry W.

2013-01-01

Control of human African sleeping sickness, caused by subspecies of the protozoan parasite Trypanosoma brucei, is based on preventing transmission by elimination of the tsetse vector and by active diagnostic screening and treatment of infected patients. To identify trypanosome proteins that have potential as biomarkers for detection and monitoring of African sleeping sickness, we have used a ‘deep-mining” proteomics approach to identify trypanosome proteins in human plasma. Abundant human plasma proteins were removed by immunodepletion. Depleted plasma samples were then digested to peptides with trypsin, fractionated by basic reversed phase and each fraction analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This sample processing and analysis method enabled identification of low levels of trypanosome proteins in pooled plasma from late stage sleeping sickness patients infected with Trypanosoma brucei rhodesiense. A total of 254 trypanosome proteins were confidently identified. Many of the parasite proteins identified were of unknown function, although metabolic enzymes, chaperones, proteases and ubiquitin-related/acting proteins were found. This approach to the identification of conserved, soluble trypanosome proteins in human plasma offers a possible route to improved disease diagnosis and monitoring, since these molecules are potential biomarkers for the development of a new generation of antigen-detection assays. The combined immuno-depletion/mass spectrometric approach can be applied to a variety of infectious diseases for unbiased biomarker identification. PMID:23951171

Identification of Trypanosome proteins in plasma from African sleeping sickness patients infected with T. b. rhodesiense.

PubMed

Eyford, Brett A; Ahmad, Rushdy; Enyaru, John C; Carr, Steven A; Pearson, Terry W

2013-01-01

Control of human African sleeping sickness, caused by subspecies of the protozoan parasite Trypanosoma brucei, is based on preventing transmission by elimination of the tsetse vector and by active diagnostic screening and treatment of infected patients. To identify trypanosome proteins that have potential as biomarkers for detection and monitoring of African sleeping sickness, we have used a 'deep-mining" proteomics approach to identify trypanosome proteins in human plasma. Abundant human plasma proteins were removed by immunodepletion. Depleted plasma samples were then digested to peptides with trypsin, fractionated by basic reversed phase and each fraction analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This sample processing and analysis method enabled identification of low levels of trypanosome proteins in pooled plasma from late stage sleeping sickness patients infected with Trypanosoma brucei rhodesiense. A total of 254 trypanosome proteins were confidently identified. Many of the parasite proteins identified were of unknown function, although metabolic enzymes, chaperones, proteases and ubiquitin-related/acting proteins were found. This approach to the identification of conserved, soluble trypanosome proteins in human plasma offers a possible route to improved disease diagnosis and monitoring, since these molecules are potential biomarkers for the development of a new generation of antigen-detection assays. The combined immuno-depletion/mass spectrometric approach can be applied to a variety of infectious diseases for unbiased biomarker identification.

In Vivo Imaging of Immuno-Spin Trapped Radicals With Molecular Magnetic Resonance Imaging in a Diabetic Mouse Model

PubMed Central

Towner, Rheal A.; Smith, Nataliya; Saunders, Debra; Henderson, Michael; Downum, Kristen; Lupu, Florea; Silasi-Mansat, Robert; Ramirez, Dario C.; Gomez-Mejiba, Sandra E.; Bonini, Marcelo G.; Ehrenshaft, Marilyn; Mason, Ronald P.

2012-01-01

Oxidative stress plays a major role in diabetes. In vivo levels of membrane-bound radicals (MBRs) in a streptozotocin-induced diabetic mouse model were uniquely detected by combining molecular magnetic resonance imaging (mMRI) and immunotrapping techniques. An anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antibody (Ab) covalently bound to an albumin (BSA)-Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)-biotin MRI contrast agent (anti-DMPO probe), and mMRI, were used to detect in vivo levels of DMPO-MBR adducts in kidneys, livers, and lungs of diabetic mice, after DMPO administration. Magnetic resonance signal intensities, which increase in the presence of a Gd-based molecular probe, were significantly higher within the livers, kidneys, and lungs of diabetic animals administered the anti-DMPO probe compared with controls. Fluorescence images validated the location of the anti-DMPO probe in excised tissues via conjugation of streptavidin-Cy3, which targeted the probe biotin moiety, and immunohistochemistry was used to validate the presence of DMPO adducts in diabetic mouse livers. This is the first report of noninvasively imaging in vivo levels of MBRs within any disease model. This method can be specifically applied toward diabetes models for in vivo assessment of free radical levels, providing an avenue to more fully understand the role of free radicals in diabetes. PMID:22698922

Applications of immunomagnetic capture and time-resolved fluorescence detection for Salmonella enteriditis in liquid eggs

NASA Astrophysics Data System (ADS)

Tu, Shu-I.; Gehring, Andrew; Paoli, George

2008-04-01

An immuno sandwich method was evaluated for the detection of Salmonella in liquid eggs. Liquid eggs spiked with different out-break strains of Salmonella were mixed with proper enrichment media and incubated at 37 C for 4 to 20 h. After enrichment, immunomagnetic beads (IMB) coated with anti Salmonella antibodies were used to capture the bacteria. Samarium (Sm) labeled anti Salmonella antibodies were then used to form sandwiched complexes with IMB captured bacteria. Sandwiched Salmonella were then treated with Sm-chelator to allow the measurement of the released Sm by time-resolved fluorescence (TRF). The processes ranging from IMB capture to Sm chelation were performed using an automated KingFisher apparatus. With this approach, the presence of ~ 1 CFU of outbreak strains of Salmonella Enteritidis per egg (~50 g of liquid eggs) could be detected after enrichment for 20 h at 37 C. For higher levels of Salmonella Enteritidis contamination, e.g., 10 CFU per 50 g of liquid eggs, the enrichment time could be reduced to 5 h at 37 C. The results demonstrated that a combination of IMB capture and TRF measurement could be a rapid and sensitive method for Salmonella Enteritidis detection in liquid eggs.

Spatial detection of tv channel logos as outliers from the content

NASA Astrophysics Data System (ADS)

Ekin, Ahmet; Braspenning, Ralph

2006-01-01

This paper proposes a purely image-based TV channel logo detection algorithm that can detect logos independently from their motion and transparency features. The proposed algorithm can robustly detect any type of logos, such as transparent and animated, without requiring any temporal constraints whereas known methods have to wait for the occurrence of large motion in the scene and assume stationary logos. The algorithm models logo pixels as outliers from the actual scene content that is represented by multiple 3-D histograms in the YC BC R space. We use four scene histograms corresponding to each of the four corners because the content characteristics change from one image corner to another. A further novelty of the proposed algorithm is that we define image corners and the areas where we compute the scene histograms by a cinematic technique called Golden Section Rule that is used by professionals. The robustness of the proposed algorithm is demonstrated over a dataset of representative TV content.

Design, fabrication, and characterization of polymeric bioMEMS for the detection of feline immunodeficiency virus (FIV)

NASA Astrophysics Data System (ADS)

Cohen, Brian; Gadre, Anand; Kaloyeros, Alain E.

2007-02-01

This project comprises the development of a novel polymeric BioMEMS device capable of rapidly detecting FIV in a minimally invasive manner. FIV severely inhibits the infected feline from mounting an immune response, and causes susceptibility to other types of diseases. Vaccines against FIV do exist, but have some strong limitations to their effectiveness; so early detection is the best method to combat the spread of the disease. Current testing methods look for antibodies to the FIV protein p24 in feline blood using established Enzyme Linked ImmunoSorbent Assay (ELISA) protocols. The focus of this research is to design and construct a device that can detect antibodies to p24 in a salivary sample by non-intrusive electrochemical means. The device is constructed upon a silicon substrate with gold microelectrodes coated with polypyrrole (PPy), an electrically conducting and biocompatible polymer. In the current phase of the research, the PPy deposition process has been optimized with regards to film thickness, uniformity and conductivity. Microfluidic channels have been fabricated using SU-8, an epoxy based polymer that enables the test sample and other solutions to pass freely through the device. The PPy will be coated with anti-FIV p24 antibodies that can capture FIV p24 antigens present in a salivary sample. Future research will involve the analysis of PPy/antibody interaction and its effect on functionality. The capture of such antigens will interfere with a reduction-oxidation (redox) reaction in a subsequently added ionic solution. This interference will change the characteristic resistance of the solution yielding a qualitative test for the presence of the viral antigens in the sample and hence determining the occurrence of infection.

In vitro characterization of high purity factor IX concentrates for the treatment of hemophilia B.

PubMed

Limentani, S A; Gowell, K P; Deitcher, S R

1995-04-01

This study employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoblotting to assess the purity of seven high purity factor IX concentrates: Aimafix (Aima), AlphaNine-SD (Alpha Therapeutic), Factor IX VHP (Biotransfusion), Immunine (Immuno), Mononine (Armour Pharmaceutical), Nanotiv (Kabi Pharmacia), and 9MC (Blood Products Laboratory). The mean specific activity of these products ranged from 68 U factor IX/mg (Aimafix) to 246 U factor IX/mg (Mononine). SDS-PAGE analysis showed that the highest purity product, Mononine, had a single contaminating band under non-reducing conditions. Two additional bands were detected when this product was analyzed under reducing conditions. All other products had multiple contaminating bands that were more apparent under reducing than non-reducing conditions. The immunoblot for factor IX showed a dominant factor IX band for all products. In addition, visible light chain of factor IX was detected for AlphaNine-SD, Factor IX VHP, Immunine, Mononine, Nanotiv, and 9MC, suggesting that the factor IX in these products had undergone partial activation to factor IXa. Another contaminating band was visible at 49,500 for all of the products except 9MC. In addition to this band, high molecular weight contaminants were apparent for some products, most notably AlphaNine-SD. The identity of these bands is unknown. Immunoblotting failed to demonstrate factor VII as a contaminant of any of the high purity products, although factor VIIa could be detected in some lots of Immunine, Nanotiv, and 9MC by a clot-based assay. Factor X contaminated Aimafix, AlphaNine-SD, Factor IX VHP, Immunine, Nanotiv, and 9MC, but activation products of factor X were not detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Scanning EM of non-heavy metal stained biosamples: Large-field of view, high contrast and highly efficient immunolabeling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuipers, Jeroen; Boer, Pascal de; Giepmans, Ben N.G., E-mail: b.n.g.giepmans@umcg.nl

Scanning electron microscopy (SEM) is increasing its application in life sciences for electron density measurements of ultrathin sections. These are traditionally analyzed with transmission electron microscopy (TEM); by most labs, SEM analysis still is associated with surface imaging only. Here we report several advantages of SEM for thin sections over TEM, both for structural inspection, as well as analyzing immuno-targeted labels such as quantum dots (QDs) and gold, where we find that QD-labeling is ten times more efficient than gold-labeling. Furthermore, we find that omitting post-staining with uranyl and lead leads to QDs readily detectable over the ultrastructure, but undermore » these conditions ultrastructural contrast was even almost invisible in TEM examination. Importantly, imaging in SEM with STEM detection leads to both outstanding QDs and ultrastructural contrast. STEM imaging is superior over back-scattered electron imaging of these non-contrasted samples, whereas secondary electron detection cannot be used at all. We conclude that examination of ultrathin sections by SEM, which may be immunolabeled with QDs, will allow rapid and straightforward analysis of large fields with more efficient labeling than can be achieved with immunogold. The large fields of view routinely achieved with SEM, but not with TEM, allows straightforward raw data sharing using virtual microscopy, also known as nanotomy when this concerns EM data in the life sciences. - Highlights: • High resolution and large fields of view via nanotomy or virtual microscopy. • Highly relevant for EM‐datasets where information density is high. • Sample preparation with low contrast good for STEM, not TEM. • Quantum dots now stand out in STEM‐based detection. • 10 Times more efficient labeling with quantum dots compared to gold.« less

Virus load in pigs affected with different clinical forms of classical swine fever.

PubMed

Rout, M; Saikumar, G

2012-04-01

Classical swine fever (CSF) is an endemic disease in India, but the real magnitude of the problem is not known as only outbreaks of acute CSF are reported and many cases of chronic and clinically inapparent forms of the disease, which manifest a confusing clinical picture, remain undiagnosed. The real status of classical swine fever virus (CSFV) infection can only be known by testing pigs with highly specific and sensitive diagnostic assays. To obtain the baseline prevalence of CSFV infection among pigs in an endemic region where no vaccination was being performed, a real-time PCR assay was used to detect viral genetic material in tissue samples collected from a slaughterhouse in the northern state of Uttar Pradesh in India. In total, 1120 slaughtered pigs were examined for the presence of CSF suggestive pathological lesions and tissues from suspected cases were tested for the presence of CSFV antigen and nucleic acids by indirect immuno-peroxidase test and real-time PCR, respectively. Based on the detection of viral genetic material in the tonsils, the prevalence of CSFV infection among slaughtered pigs was found to be 7.67%. Pigs detected positive for viral genome by quantitative real-time PCR assay when categorized into different forms of CSF, depending upon the pathological lesions observed, the viral load in the tonsils of some of the pigs with chronic or clinically inapparent form of the disease was similar to that detected in pigs with acute CSF. The results of the study suggested that the risk posed by pigs with chronic disease or those infected but showing no clinical disease may be relatively higher as they can transmit the virus to new susceptible hosts over a longer period of time. © 2011 Blackwell Verlag GmbH.

Learning to Detect Vandalism in Social Content Systems: A Study on Wikipedia

NASA Astrophysics Data System (ADS)

Javanmardi, Sara; McDonald, David W.; Caruana, Rich; Forouzan, Sholeh; Lopes, Cristina V.

A challenge facing user generated content systems is vandalism, i.e. edits that damage content quality. The high visibility and easy access to social networks makes them popular targets for vandals. Detecting and removing vandalism is critical for these user generated content systems. Because vandalism can take many forms, there are many different kinds of features that are potentially useful for detecting it. The complex nature of vandalism, and the large number of potential features, make vandalism detection difficult and time consuming for human editors. Machine learning techniques hold promise for developing accurate, tunable, and maintainable models that can be incorporated into vandalism detection tools. We describe a method for training classifiers for vandalism detection that yields classifiers that are more accurate on the PAN 2010 corpus than others previously developed. Because of the high turnaround in social network systems, it is important for vandalism detection tools to run in real-time. To this aim, we use feature selection to find the minimal set of features consistent with high accuracy. In addition, because some features are more costly to compute than others, we use cost-sensitive feature selection to reduce the total computational cost of executing our models. In addition to the features previously used for spam detection, we introduce new features based on user action histories. The user history features contribute significantly to classifier performance. The approach we use is general and can easily be applied to other user generated content systems.

Anaphylaxis to pork kidney is related to IgE antibodies specific for galactose-alpha-1,3-galactose.

PubMed

Morisset, M; Richard, C; Astier, C; Jacquenet, S; Croizier, A; Beaudouin, E; Cordebar, V; Morel-Codreanu, F; Petit, N; Moneret-Vautrin, D A; Kanny, G

2012-05-01

Carbohydrate-specific IgE antibodies present on nonprimate mammalian proteins were incriminated recently in delayed meat anaphylaxis. The aim of this study was to explore whether anaphylaxis to mammalian kidney is also associated with galactose-α-1,3-galactose (αGal)-specific IgE. Fourteen patients with anaphylaxis to pork or beef kidney underwent prick tests to meat and kidney. Some patients also underwent skin tests to Erbitux(®) (cetuximab). IgE antibodies to αGal, swine urine proteins, beef and pork meat, serum albumin proteins, cat, and rFel d 1 were measured by ImmunoCAP(®). The αGal levels were estimated in meats and kidney by ELISA inhibition assay. Cross-reactivity between αGal and pork kidney was studied with the ImmunoCAP(®) inhibition assay. Among the 14 patients, 12 presented with anaphylactic shock. Reactions occurred within 2 h from exposure in 67% of patients. Associated risk factors were observed in 10 cases, and alcohol was the main cofactor. Three patients underwent an oral challenge to pork kidney, and anaphylaxis occurred after ingestion of small quantities (1-2 g). Prick tests to kidney were positive in 54% of patients. All tested patients showed positive skin tests to Erbitux(®). All patients tested positive for IgE to αGal, with levels ranging from 0.4 to 294 kU/l. IgE binding to αGal was inhibited by raw pork kidney extract (mean, 77%; range, 55-87%), which showed a high amount of αGal determinants. Pork or beef kidney anaphylaxis is related to αGal IgE. Its peculiar severity could be due to an elevated content of αGal epitopes in kidney. © 2012 John Wiley & Sons A/S.

Structural and immunological characterization of hydroxyl radical modified human IgG: Clinical correlation in rheumatoid arthritis

NASA Astrophysics Data System (ADS)

Islam, Sidra; Mir, Abdul Rouf; Arfat, Mir Yasir; Khan, Farzana; Zaman, Masihuz; Ali, Asif; Moinuddin

2018-04-01

Structural alterations in proteins under oxidative stress have been widely implicated in the immuno-pathology of various disorders. This study has evaluated the extent of damage in the conformational characteristics of IgG by hydroxyl radical (OHrad) and studied its implications in the immuno-pathology of rheumatoid arthritis (RA). Using various biophysical and biochemical techniques, changes in aromatic microenvironment of the IgG and the protein aggregation became evident after treatment with OHrad . The SDS-PAGE study confirmed the protein aggregation while far ultraviolet circular dichroism spectroscopy (Far-UV CD) and fourier transform infrared spectroscopy (FTIR) inferred towards the alterations in secondary structure of IgG under OHrad stress. Dynamic light scattering showed that the modification increased the hydrodynamic radius and polydispersity of IgG. The free arginine and lysine content reduced upon modification. OHrad induced aggregation was confirmed by enhanced thioflavin-T (ThT) fluorescence and red shift in the congo red (CR) absorbance. The study on experimental animals reiterates the earlier findings of enhanced immunogenicity of OHrad treated IgG (OHrad -IgG) compared to that of native IgG. OHrad -IgG strongly interacted with the antibodies derived from the serum of 80 rheumatoid arthritis (RA) patients. The overwhelming and strong tendency of OHrad -IgG to bind the antibodies derived from the serum of RA patients points towards the modification of IgG under patho-physiological conditions in RA that generate neo-epitopes and eventually cause the generation of auto antibodies that circulate in the patient sera. Further studies on this aspect may possibly lead to the development of a biomarker for RA.

High-purity capture and release of circulating exosomes using an exosome-specific dual-patterned immunofiltration (ExoDIF) device.

PubMed

Kang, Yoon-Tae; Kim, Young Jun; Bu, Jiyoon; Cho, Young-Ho; Han, Sae-Won; Moon, Byung-In

2017-09-21

We present a microfluidic device for the capture and release of circulating exosomes from human blood. The exosome-specific dual-patterned immunofiltration (ExoDIF) device is composed of two distinct immuno-patterned layers, and is capable of enhancing the chance of binding between the antibody and exosomes by generating mechanical whirling, thus achieving high-throughput exosome isolation with high specificity. Moreover, follow-up recovery after the immuno-affinity based isolation, via cleavage of a linker, enables further downstream analysis. We verified the performance of the present device using MCF-7 secreted exosomes and found that both the concentration and proportion of exosome-sized vesicles were higher than in the samples obtained from the conventional exosome isolation kit. We then isolated exosomes from the human blood samples with our device to compare the exosome level between cancer patients and healthy donors. Cancer patients show a significantly higher exosome level with higher selectivity when validating the exosome-sized vesicles using both electron microscopy and nanoparticle tracking analysis. The captured exosomes from cancer patients also express abundant cancer-associated antigens, the epithelial cell adhesion molecule (EpCAM) on their surface. Our simple and rapid exosome recovery technique has huge potential to elucidate the function of exosomes in cancer patients and can thus be applied for various exosome-based cancer research studies.

Comparing efficacy of reduced-toxicity allogeneic hematopoietic cell transplantation with conventional chemo-(immuno) therapy in patients with relapsed or refractory CLL: a Markov decision analysis.

PubMed

Kharfan-Dabaja, M A; Pidala, J; Kumar, A; Terasawa, T; Djulbegovic, B

2012-09-01

Despite therapeutic advances, relapsed/refractory CLL, particularly after fludarabine-based regimens, remains a major challenge for which optimal therapy is undefined. No randomized comparative data exist to suggest the superiority of reduced-toxicity allogeneic hematopoietic cell transplantation (RT-allo-HCT) over conventional chemo-(immuno) therapy (CCIT). By using estimates from a systematic review and by meta-analysis of available published evidence, we constructed a Markov decision model to examine these competing modalities. Cohort analysis demonstrated superior outcome for RT-allo-HCT, with a 10-month overall life expectancy (and 6-month quality-adjusted life expectancy (QALE)) advantage over CCIT. Although the model was sensitive to changes in base-case assumptions and transition probabilities, RT-allo-HCT provided superior overall life expectancy through a range of values supported by the meta-analysis. QALE was superior for RT-allo-HCT compared with CCIT. This conclusion was sensitive to change in the anticipated state utility associated with the post-allogeneic HCT state; however, RT-allo-HCT remained the optimal strategy for values supported by existing literature. This analysis provides a quantitative comparison of outcomes between RT-allo-HCT and CCIT for relapsed/refractory CLL in the absence of randomized comparative trials. Confirmation of these findings requires a prospective randomized trial, which compares the most effective RT-allo-HCT and CCIT regimens for relapsed/refractory CLL.

Modeling the economic outcomes of immuno-oncology drugs: alternative model frameworks to capture clinical outcomes

PubMed Central

Gibson, EJ; Begum, N; Koblbauer, I; Dranitsaris, G; Liew, D; McEwan, P; Tahami Monfared, AA; Yuan, Y; Juarez-Garcia, A; Tyas, D; Lees, M

2018-01-01

Background Economic models in oncology are commonly based on the three-state partitioned survival model (PSM) distinguishing between progression-free and progressive states. However, the heterogeneity of responses observed in immuno-oncology (I-O) suggests that new approaches may be appropriate to reflect disease dynamics meaningfully. Materials and methods This study explored the impact of incorporating immune-specific health states into economic models of I-O therapy. Two variants of the PSM and a Markov model were populated with data from one clinical trial in metastatic melanoma patients. Short-term modeled outcomes were benchmarked to the clinical trial data and a lifetime model horizon provided estimates of life years and quality adjusted life years (QALYs). Results The PSM-based models produced short-term outcomes closely matching the trial outcomes. Adding health states generated increased QALYs while providing a more granular representation of outcomes for decision making. The Markov model gave the greatest level of detail on outcomes but gave short-term results which diverged from those of the trial (overstating year 1 progression-free survival by around 60%). Conclusion Increased sophistication in the representation of disease dynamics in economic models is desirable when attempting to model treatment response in I-O. However, the assumptions underlying different model structures and the availability of data for health state mapping may be important limiting factors. PMID:29563820

Quantification of (1→4)-β-d-Galactans in Compression Wood Using an Immuno-Dot Assay

PubMed Central

Chavan, Ramesh R.; Fahey, Leona M.; Harris, Philip J.

2015-01-01

Compression wood is a type of reaction wood formed on the underside of softwood stems when they are tilted from the vertical and on the underside of branches. Its quantification is still a matter of some scientific debate. We developed a new technique that has the potential to do this based on the higher proportions of (1→4)-β-d-galactans that occur in tracheid cell walls of compression wood. Wood was milled, partially delignified, and the non-cellulosic polysaccharides, including the (1→4)-β-d-galactans, extracted with 6 M sodium hydroxide. After neutralizing, the solution was serially diluted, and the (1→4)-β-d-galactans determined by an immuno-dot assay using the monoclonal antibody LM5, which specifically recognizes this polysaccharide. Spots were quantified using a dilution series of a commercially available (1→4)-β-d-galactan from lupin seeds. Using this method, compression and opposite woods from radiata pine (Pinus radiata) were easily distinguished based on the amounts of (1→4)-β-d-galactans extracted. The non-cellulosic polysaccharides in the milled wood samples were also hydrolysed using 2 M trifluoroacetic acid followed by the separation and quantification of the released neutral monosaccharides by high performance anion exchange chromatography. This confirmed that the compression woods contained higher proportions of galactose-containing polysaccharides than the opposite woods. PMID:27135316

Development of solid - based paper strips for rapid diagnosis of Pseudorabies infection.

PubMed

Joon Tam, Yew; Mohd Lila, Mohd Azmi; Bahaman, Abdul Rani

2004-12-01

Pseudorabies (Aujeszky's disease) is an economically significant disease of swine known to cause central nervous disorders, respiratory disease, reproductive failure and mortality in infected pigs. In attempts to eradicate the disease from becoming endemic, early detection is important to prevent further economic losses and to allow for detection and removal of infected pigs in domestic herds. Thus, a rapid and sensitive technique is necessary for the detection of the virus. For rapid and simple examination, an immuno - chromatographic lateral - flow assay system based on immunologic recognition of specific pseudorabies virus antigen was developed by utilising, as signal generator, colloidal gold conjugated to secondary antibody to detect primary or sample antibody in the sera of pseudorabies infected animals. The pseudorabies virus used as a capture antigen in the test strip was first cultivated in VERO cell culture and then purified by sucrose gradient separation to produce the viral protein concentration of 3.8 mg/ml. The standard pseudorabies antigens reacted well with the hyperimmune serum (HIS). The antibody detection system is basically composed of colloidal gold - labelled antibodies fixed on a conjugate pad, and the complementary pseudorabies antigen immobilised onto a nitrocellulose membrane forming capture zone. If the target antibody is present in a specimen, the colloidal gold-labelled antibody will form a complex with the antibody sample. Subsequently, the formed complex will migrate to the capture zone and is then bound to the solid phase via antigen - antibody interaction. As a result, a signal marker is generated by the accumulation of colloidal gold for detection confirmation. The results obtained demonstrated that the optimum combination of pseudorabies antigen needed as the capture reagent and gold conjugate as secondary antibody recognition marker was at a concentration of 0.38mg/ml and at 1:10 dilution factor respectively. The sensitivity of the solid - based test strip towards pseudorabies antibodies was high with a detection limit of 1 to 10,000 - dilution factor. The specificity of the assay was 100% with no cross - reaction being observed with other sera or antibodies. Accurate reading time needed for confirmation of the assay can be completed in 5 min with a whole blood sample of 25 microl. The colloidal gold - labelled antibody is stable at room temperature for 6 months or more (data not shown). Findings from this study indicated that the solid - based test strip assay system provided high sensitivity and specificity for the detection of pseudorabies at low levels of antibody concentration. The assay was rapid, simple, cheap, and does not require any sophisticated equipment. Thus, the solid based test strip will be a useful serological screening technique or for rapid diagnosis of an infectious disease in target populations of animals characterised by heterogeneous antibody responses.

Development of an antigen-based rapid diagnostic test for the identification of blowfly (Calliphoridae) species of forensic significance.

PubMed

McDonagh, Laura; Thornton, Chris; Wallman, James F; Stevens, Jamie R

2009-06-01

In this study we examine the limitations of currently used sequence-based approaches to blowfly (Calliphoridae) identification and evaluate the utility of an immunological approach to discriminate between blowfly species of forensic importance. By investigating antigenic similarity and dissimilarity between the first instar larval stages of four forensically important blowfly species, we have been able to identify immunoreactive proteins of potential use in the development of species-specific immuno-diagnostic tests. Here we outline our protein-based approach to species determination, and describe how it may be adapted to develop rapid diagnostic assays for the 'on-site' identification of blowfly species.

Evaluation of information-theoretic similarity measures for content-based retrieval and detection of masses in mammograms.

PubMed

Tourassi, Georgia D; Harrawood, Brian; Singh, Swatee; Lo, Joseph Y; Floyd, Carey E

2007-01-01

The purpose of this study was to evaluate image similarity measures employed in an information-theoretic computer-assisted detection (IT-CAD) scheme. The scheme was developed for content-based retrieval and detection of masses in screening mammograms. The study is aimed toward an interactive clinical paradigm where physicians query the proposed IT-CAD scheme on mammographic locations that are either visually suspicious or indicated as suspicious by other cuing CAD systems. The IT-CAD scheme provides an evidence-based, second opinion for query mammographic locations using a knowledge database of mass and normal cases. In this study, eight entropy-based similarity measures were compared with respect to retrieval precision and detection accuracy using a database of 1820 mammographic regions of interest. The IT-CAD scheme was then validated on a separate database for false positive reduction of progressively more challenging visual cues generated by an existing, in-house mass detection system. The study showed that the image similarity measures fall into one of two categories; one category is better suited to the retrieval of semantically similar cases while the second is more effective with knowledge-based decisions regarding the presence of a true mass in the query location. In addition, the IT-CAD scheme yielded a substantial reduction in false-positive detections while maintaining high detection rate for malignant masses.

Evaluation of information-theoretic similarity measures for content-based retrieval and detection of masses in mammograms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tourassi, Georgia D.; Harrawood, Brian; Singh, Swatee

The purpose of this study was to evaluate image similarity measures employed in an information-theoretic computer-assisted detection (IT-CAD) scheme. The scheme was developed for content-based retrieval and detection of masses in screening mammograms. The study is aimed toward an interactive clinical paradigm where physicians query the proposed IT-CAD scheme on mammographic locations that are either visually suspicious or indicated as suspicious by other cuing CAD systems. The IT-CAD scheme provides an evidence-based, second opinion for query mammographic locations using a knowledge database of mass and normal cases. In this study, eight entropy-based similarity measures were compared with respect to retrievalmore » precision and detection accuracy using a database of 1820 mammographic regions of interest. The IT-CAD scheme was then validated on a separate database for false positive reduction of progressively more challenging visual cues generated by an existing, in-house mass detection system. The study showed that the image similarity measures fall into one of two categories; one category is better suited to the retrieval of semantically similar cases while the second is more effective with knowledge-based decisions regarding the presence of a true mass in the query location. In addition, the IT-CAD scheme yielded a substantial reduction in false-positive detections while maintaining high detection rate for malignant masses.« less

Development and Evaluation of Recombinant Nucleocapsid Protein Based Diagnostic ELISA for Detection of Nipah Virus Infection in Pigs.

PubMed

Kulkarni, Diwakar D; Venkatesh, Govindarajalu; Tosh, Chakradhar; Patel, Priyanka; Mashoria, Anita; Gupta, Vandana; Gupta, Sourabh; D, Senthilkumar

2016-01-01

The recombinant viral protein-based indirect enzyme-linked immunosorbent assay (ELISA) is a cost-effective, safe, specific, and rapid tool to diagnose the viral infection. Nipah virus nucleocapsid (NiV-N) protein was expressed in Escherichia coli and purified by histidine tag-based affinity chromatography. The N protein was selected based on its immuno dominance and conservation among different NiV strains. An indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for swine sera was optimized using the recombinant NiV-N protein as an antigen along with negative and positive controls. The background reading was blocked using skim milk powder and chicken serum. A total number of 1709 swine serum samples from various states of India were tested with indirect ELISA and Western blot. The test was considered positive only when its total reactivity reading was higher than 0.2 cut-off value and the ratio of the total reactivity to the background reading was more than 2.0. Since specificity is high for Western blotting it was used as standard test for comparison of results of indirect ELISA. Sensitivity and specificity of indirect ELISA was 100% and 98.7%, respectively, in comparison with Western blotting. Recombinant N protein-based ELISA can be used in screening large number of serum samples for epidemiological investigations in developing countries where high containment laboratories are not available to handle this zoonotic virus.

Asymptomatic dystrophinopathy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morrone, A.; Hoffman, E.P.; Hoop, R.C.

1997-03-31

A 4-year-old girl was referred for evaluation for a mild but persistent serum aspartate aminotransferase (AST) elevation detected incidentally during routine blood screening for a skin infection. Serum creatine kinase activity was found to be increased. Immuno-histochemical study for dystrophin in her muscle biopsy showed results consistent with a carrier state for muscular dystrophy. Molecular work-up showed the proposita to be a carrier of a deletion mutation of exon 48 of the dystrophin gene. Four male relatives also had the deletion mutation, yet showed no clinical symptoms of muscular dystrophy (age range 8-58 yrs). Linkage analysis of the dystrophin genemore » in the family showed a spontaneous change of an STR45 allele, which could be due to either an intragenic double recombination event, or CA repeat length mutation leading to identical size alleles. To our knowledge, this is the first documentation of an asymptomatic dystrophinopathy in multiple males of advanced age. Based on molecular findings, this family would be given a diagnosis of Becker muscular dystrophy. This diagnosis implies the development of clinical symptoms, even though this family is clearly asymptomatic. This report underscores the caution which must be exercised when giving presymptomatic diagnoses based on molecular studies. 28 refs., 4 figs., 1 tab.« less

Live cell and immuno-labeling techniques to study gravitational effects on single plant cells.

PubMed

Chebli, Youssef; Geitmann, Anja

2015-01-01

The constant force of gravity plays a primordial role in the ontogeny of all living organisms. Plants, for example, develop their roots and shoots in accordance with the direction of the gravitational vector. Any change in the magnitude and/or the direction of gravity has an important impact on the development of tissues and cells. In order to understand how the gravitational force affects plant cell growth and differentiation, we established two complementary experimental procedures with which the effect of hyper-gravity on single plant cell development can be assessed. The single model cell system we used is the pollen tube or male gametophyte which, because of its rapid growth behavior, is known for its instant response to external stresses. The physiological response of the pollen tube can be assessed in a quantitative manner based on changes in the composition and spatial distribution of its cell wall components and in the precisely defined pattern of its very dynamic cytoplasmic streaming. Here, we provide a detailed description of the steps required for the immuno-localization of various cell wall components using microwave-assisted techniques and we explain how live imaging of the intracellular traffic can be achieved under hyper-gravity conditions.

Immuno-therapy of Acute Radiation Syndromes : Extracorporeal Immuno-Lympho-Plasmo-Sorption.

NASA Astrophysics Data System (ADS)

Popov, Dmitri; Maliev, Slava

Methods Results Summary and conclusions Introduction: Existing Medical Management of the Acute Radiation Syndromes (ARS) does not include methods of specific immunotherapy and active detoxication. Though the Acute Radiation Syndromes were defined as an acute toxic poisonous with development of pathological processes: Systemic Inflammatory Response Syndrome (SIRS), Toxic Multiple Organ Injury (TMOI), Toxic Multiple Organ Dysfunction Syndrome(TMODS), Toxic Multiple Organ Failure (TMOF). Radiation Toxins of SRD Group play an important role as the trigger mechanisms in development of the ARS clinical symptoms. Methods: Immuno-Lympho-Plasmo-Sorption is a type of Immuno-therapy which includes prin-ciples of immunochromato-graphy, plasmopheresis, and hemodialysis. Specific Antiradiation Antitoxic Antibodies are the active pharmacological agents of immunotherapy . Antiradia-tion Antitoxic Antibodies bind selectively to Radiation Neurotoxins, Cytotoxins, Hematotox-ins and neutralize their toxic activity. We have developed the highly sensitive method and system for extracorporeal-immune-lypmh-plasmo-sorption with antigen-specific IgG which is clinically important for treatment of the toxic and immunologic phases of the ARS. The method of extracorporeal-immune-lypmh-plasmo-sorption includes Antiradiation Antitoxic Antibodies (AAA) immobilized on microporous polymeric membranes with a pore size that is capable to provide diffusion of blood-lymph plasma. Plasma of blood or lymph of irradiated mammals contains Radiation Toxins (RT) that have toxic and antigenic properties. Radiation Toxins are Antigen-specific to Antitoxic blocking antibodies (Immunoglobulin G). Plasma diffuses through membranes with immobilized AAA and AA-antibodies bind to the polysaccharide chain of tox-ins molecules and complexes of AAA-RT that are captured on membrane surfaces. RT were removed from plasma. Re-transfusion of plasma of blood and lymph had been provided. We show a statistical significant reduction in postradiation lethality.

Relationship Between Preoperative Sarcopenia Status and Immuno-nutritional Parameters in Patients with Early-stage Non-small Cell Lung Cancer.

PubMed

Shoji, Fumihiro; Matsubara, Taichi; Kozuma, Yuka; Haratake, Naoki; Akamine, Takaki; Takamori, Shinkichi; Katsura, Masakazu; Toyokawa, Gouji; Okamoto, Tatsuro; Maehara, Yoshihiko

2017-12-01

Although the skeletal muscle in the region of the third lumbar vertebra (L3) is generally assessed in order to judge sarcopenia, not every patient with non-small cell lung cancer (NSCLC) undergoes computed tomography including the L3 region. We hypothesized that immuno-nutritional parameters could predict the existence of sarcopenia in patients with NSCLC. The aim of this study was to retrospectively investigate the correlation between preoperative sarcopenia and immuno-nutritional parameters in patients with early-stage NSCLC. We selected 147 of patients with pathological stage I NSCLC who underwent preoperative measurement of immuno-nutritional parameters and CT including the L3 region. Preoperative sarcopenia was significantly associated with female gender (p=0.0003) and poor prognosis (p=0.0322). In Kaplan-Meier analysis of overall survival (OS) by preoperative sarcopenia status, the sarcopenic group had significantly shorter OS than the non-sarcopenic group (5-year OS: 87.27% vs. 77.37%, p=0.0131, log-rank test). In multivariate analysis, the preoperative sarcopenia status (hazard ratio=5.138; 95% confidence interval=2.305-11.676; p<0.0001) was an independent prognostic factor. Preoperative sarcopenia status was significantly related to controlling nutritional status score (p=0.0071) and Geriatric Nutritional Risk Index (GNRI) (p<0.0001). Spearman's correlation test showed good significant correlation between preoperative sarcopenia status and GNRI (r=0.348, p<0.0001). The preoperative GNRI is a simple and useful predictor for existence of preoperative sarcopenia which was associated with poor outcome in patients with early-stage NSCLC. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

MPEG-7 based video annotation and browsing

NASA Astrophysics Data System (ADS)

Hoeynck, Michael; Auweiler, Thorsten; Wellhausen, Jens

2003-11-01

The huge amount of multimedia data produced worldwide requires annotation in order to enable universal content access and to provide content-based search-and-retrieval functionalities. Since manual video annotation can be time consuming, automatic annotation systems are required. We review recent approaches to content-based indexing and annotation of videos for different kind of sports and describe our approach to automatic annotation of equestrian sports videos. We especially concentrate on MPEG-7 based feature extraction and content description, where we apply different visual descriptors for cut detection. Further, we extract the temporal positions of single obstacles on the course by analyzing MPEG-7 edge information. Having determined single shot positions as well as the visual highlights, the information is jointly stored with meta-textual information in an MPEG-7 description scheme. Based on this information, we generate content summaries which can be utilized in a user-interface in order to provide content-based access to the video stream, but further for media browsing on a streaming server.

From benchmarking HITS-CLIP peak detection programs to a new method for identification of miRNA-binding sites from Ago2-CLIP data.

PubMed

Bottini, Silvia; Hamouda-Tekaya, Nedra; Tanasa, Bogdan; Zaragosi, Laure-Emmanuelle; Grandjean, Valerie; Repetto, Emanuela; Trabucchi, Michele

2017-05-19

Experimental evidence indicates that about 60% of miRNA-binding activity does not follow the canonical rule about the seed matching between miRNA and target mRNAs, but rather a non-canonical miRNA targeting activity outside the seed or with a seed-like motifs. Here, we propose a new unbiased method to identify canonical and non-canonical miRNA-binding sites from peaks identified by Ago2 Cross-Linked ImmunoPrecipitation associated to high-throughput sequencing (CLIP-seq). Since the quality of peaks is of pivotal importance for the final output of the proposed method, we provide a comprehensive benchmarking of four peak detection programs, namely CIMS, PIPE-CLIP, Piranha and Pyicoclip, on four publicly available Ago2-HITS-CLIP datasets and one unpublished in-house Ago2-dataset in stem cells. We measured the sensitivity, the specificity and the position accuracy toward miRNA binding sites identification, and the agreement with TargetScan. Secondly, we developed a new pipeline, called miRBShunter, to identify canonical and non-canonical miRNA-binding sites based on de novo motif identification from Ago2 peaks and prediction of miRNA::RNA heteroduplexes. miRBShunter was tested and experimentally validated on the in-house Ago2-dataset and on an Ago2-PAR-CLIP dataset in human stem cells. Overall, we provide guidelines to choose a suitable peak detection program and a new method for miRNA-target identification. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

From benchmarking HITS-CLIP peak detection programs to a new method for identification of miRNA-binding sites from Ago2-CLIP data

PubMed Central

Bottini, Silvia; Hamouda-Tekaya, Nedra; Tanasa, Bogdan; Zaragosi, Laure-Emmanuelle; Grandjean, Valerie; Repetto, Emanuela

2017-01-01

Abstract Experimental evidence indicates that about 60% of miRNA-binding activity does not follow the canonical rule about the seed matching between miRNA and target mRNAs, but rather a non-canonical miRNA targeting activity outside the seed or with a seed-like motifs. Here, we propose a new unbiased method to identify canonical and non-canonical miRNA-binding sites from peaks identified by Ago2 Cross-Linked ImmunoPrecipitation associated to high-throughput sequencing (CLIP-seq). Since the quality of peaks is of pivotal importance for the final output of the proposed method, we provide a comprehensive benchmarking of four peak detection programs, namely CIMS, PIPE-CLIP, Piranha and Pyicoclip, on four publicly available Ago2-HITS-CLIP datasets and one unpublished in-house Ago2-dataset in stem cells. We measured the sensitivity, the specificity and the position accuracy toward miRNA binding sites identification, and the agreement with TargetScan. Secondly, we developed a new pipeline, called miRBShunter, to identify canonical and non-canonical miRNA-binding sites based on de novo motif identification from Ago2 peaks and prediction of miRNA::RNA heteroduplexes. miRBShunter was tested and experimentally validated on the in-house Ago2-dataset and on an Ago2-PAR-CLIP dataset in human stem cells. Overall, we provide guidelines to choose a suitable peak detection program and a new method for miRNA-target identification. PMID:28108660

Immuno-detection of cleaved SNAP-25 from differentiated mouse embryonic stem cells provides a sensitive assay for determination of botulinum A toxin and antitoxin potency.

PubMed

Yadirgi, G; Stickings, P; Rajagopal, S; Liu, Y; Sesardic, D

2017-12-01

Botulinum toxin type A is a causative agent of human botulism. Due to high toxicity and ease of production it is classified by the Centres for Disease Control and Prevention as a category A bioterrorism agent. The same serotype, BoNT/A, is also the most widely used in pharmaceutical preparations for treatment of a diverse range of neuromuscular disorders. Traditionally, animals are used to confirm the presence and activity of toxin and to establish neutralizing capabilities of countermeasures in toxin neutralization tests. Cell based assays for BoNT/A have been reported as the most viable alternative to animal models, since they are capable of reflecting all key steps (binding, translocation, internalization and cleavage of intracellular substrate) involved in toxin activity. In this paper we report preliminary development of a simple immunochemical method for specifically detecting BoNT/A cleaved intracellular substrate, SNAP-25, in cell lysates of neurons derived from mouse embryonic stem cells. The assay offers sensitivity of better than 0.1LD50/ml (3fM) which is not matched by other functional assays, including the mouse bioassay, and provides serotype specificity for quantitative detection of BoNT/A and anti-BoNT/A antitoxin. Subject to formal validation, the method described here could potentially be used as a substitute for the mouse bioassay to measure potency and consistency of therapeutic products. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Inter-laboratory evaluation of the performance parameters of a Lateral Flow Test device for the detection of Bluetongue virus-specific antibodies.

PubMed

Hanon, Jean-Baptiste; Vandenberge, Valerie; Deruelle, Matthias; De Leeuw, Ilse; De Clercq, Kris; Van Borm, Steven; Koenen, Frank; Liu, Lihong; Hoffmann, Bernd; Batten, Carrie Anne; Zientara, Stéphan; Breard, Emmanuel; Van der Stede, Yves

2016-02-01

Bluetongue (BT) is a viral vector-borne disease affecting domestic and wild ruminants worldwide. In this study, a commercial rapid immuno-chromatographic method or Lateral Flow Test (LFT) device, for the detection of BT virus-specific antibodies in animal serum, was evaluated in an international inter-laboratory proficiency test. The evaluation was done with sera samples of variable background (ruminant species, serotype, field samples, experimental infections, vaccinated animals). The diagnostic sensitivity was 100% (95% C.I. [90.5-100]) and the diagnostic specificity was 95.2% (95% C.I. [76.2-99.9]). The repeatability (accordance) and reproducibility (concordance) were 100% for seropositive samples but were lower for two of the seronegative samples (45% and 89% respectively). The analytical sensitivity, evaluated by testing positive sera at increasing dilutions was better for the BT LFT compared to some commercial ELISAs. Seroconversion of an infected sheep was detected at 4 days post infection. Analytical specificity was impaired by cross-reactions observed with some of the samples seropositive for Epizootic Haemorrhagic Disease Virus (EHDV). The agreement (Cohen's kappa) between the LFT and a commercial BT competitive ELISA was 0.79 (95% CI [0.62-0.95]). Based on these results, it can be concluded that the BT LFT device is a rapid and sensitive first-line serological test that can be used in the field, especially in areas endemic for the disease where there is a lack of diagnostic facilities. Copyright © 2015 Elsevier B.V. All rights reserved.

A Method for Isolation of Extracellular Vesicles and Characterization of Exosomes from Brain Extracellular Space.

PubMed

Pérez-González, Rocío; Gauthier, Sebastien A; Kumar, Asok; Saito, Mitsuo; Saito, Mariko; Levy, Efrat

2017-01-01

Extracellular vesicles (EV), including exosomes, secreted vesicles of endocytic origin, and microvesicles derived from the plasma membrane, have been widely isolated and characterized from conditioned culture media and bodily fluids. The difficulty in isolating EV from tissues, however, has hindered their study in vivo. Here, we describe a novel method designed to isolate EV and characterize exosomes from the extracellular space of brain tissues. The purification of EV is achieved by gentle dissociation of the tissue to free the brain extracellular space, followed by sequential low-speed centrifugations, filtration, and ultracentrifugations. To further purify EV from other extracellular components, they are separated on a sucrose step gradient. Characterization of the sucrose step gradient fractions by electron microscopy demonstrates that this method yields pure EV preparations free of large vesicles, subcellular organelles, or debris. The level of EV secretion and content are determined by assays for acetylcholinesterase activity and total protein estimation, and exosomal identification and protein content are analyzed by Western blot and immuno-electron microscopy. Additionally, we present here a method to delipidate EV in order to improve the resolution of downstream electrophoretic analysis of EV proteins.

Electrical resistivity characteristics of diesel oil-contaminated kaolin clay and a resistivity-based detection method.

PubMed

Liu, Zhibin; Liu, Songyu; Cai, Yi; Fang, Wei

2015-06-01

As the dielectric constant and conductivity of petroleum products are different from those of the pore water in soil, the electrical resistivity characteristics of oil-contaminated soil will be changed by the corresponding oil type and content. The contaminated soil specimens were manually prepared by static compaction method in the laboratory with commercial kaolin clay and diesel oil. The water content and dry density of the first group of soil specimens were controlled at 10 % and 1.58 g/cm(3). Corresponding electrical resistivities of the contaminated specimens were measured at the curing periods of 7, 14, and 28 and 90, 120, and 210 days on a modified oedometer cell with an LCR meter. Then, the electrical resistivity characteristics of diesel oil-contaminated kaolin clay were discussed. In order to realize a resistivity-based oil detection method, the other group of oil-contaminated kaolin clay specimens was also made and tested, but the initial water content, oil content, and dry density were controlled at 0~18 %, 0~18 %, 1.30~1.95 g/cm(3), respectively. Based on the test data, a resistivity-based artificial neural network (ANN) was developed. It was found that the electrical resistivity of kaolin clay decreased with the increase of oil content. Moreover, there was a good nonlinear relationship between electrical resistivity and corresponding oil content when the water content and dry density were kept constant. The decreasing velocity of the electrical resistivity of oil-contaminated kaolin clay was higher before the oil content of 12 % than after 12 %, which indicated a transition of the soil from pore water-controlled into oil-controlled electrical resistivity characteristics. Through microstructural analysis, the decrease of electrical resistivity could be explained by the increase of saturation degree together with the collapse of the electrical double layer. Environmental scanning electron microscopy (ESEM) photos indicated that the diesel oil in kaolin clay normally had three kinds of effects including oil filling, coating, and bridging. Finally, a resistivity-based ANN model was established based on the database collected from the experiment data. The performance of the model was proved to be reasonably accepted, which puts forward a possible simple, economic, and effective tool to detect the oil content in contaminated clayey soils just with four basic parameters: wet density, dry density, measured moisture content, and electrical resistivity.

Application of laser to nondestructive detection of fruit quality

NASA Astrophysics Data System (ADS)

Li, Jing; Xue, Long; Liu, Muhua; Li, Zhanlong; Yang, Yong

2008-12-01

In this study, a hyperspectral imaging system using a laser source was developed and two experiments were carried out. The first experiment was detection of pesticide residue on navel orange surface. We calculated the mean intensity of regions of interest to plot the curves between 629nm to 638nm. The analysis of the mean intensity curves showed that the mean intensity can be described by a characteristic Gaussian curve equation. The coefficients a in characteristic equations of 0%, 0.1% and 0.5% fenvalerate residue images were more than 2400, 1570-2400 and less than 1570, respectively. So we suggest using equation coefficient a to detect pesticide residue on navel orange surface. The second experiment was predicting firmness, sugar content and vitamin C content of kiwi fruit. The optimal wavelength range of the kiwi fruit firmness, sugar content, vitamin C content line regressing prediction model were 680-711nm, 674-708nm, 669-701nm. The correlation coefficients (R) of prediction models for firmness, sugar content and vitamin C content were 0.898, 0.932 and 0.918. The mean errors of validation results were 0.35×105Pa, 0.32%Brix and 7mg/100g. The experimental results indicate that a hyperspectral imaging system based on a laser source can detect fruit quality effectively.

Detection of Adulterated Vegetable Oils Containing Waste Cooking Oils Based on the Contents and Ratios of Cholesterol, β-Sitosterol, and Campesterol by Gas Chromatography/Mass Spectrometry.

PubMed

Zhao, Haixiang; Wang, Yongli; Xu, Xiuli; Ren, Heling; Li, Li; Xiang, Li; Zhong, Weike

2015-01-01

A simple and accurate authentication method for the detection of adulterated vegetable oils that contain waste cooking oil (WCO) was developed. This method is based on the determination of cholesterol, β-sitosterol, and campesterol in vegetable oils and WCO by GC/MS without any derivatization. A total of 148 samples involving 12 types of vegetable oil and WCO were analyzed. According to the results, the contents and ratios of cholesterol, β-sitosterol, and campesterol were found to be criteria for detecting vegetable oils adulterated with WCO. This method could accurately detect adulterated vegetable oils containing 5% refined WCO. The developed method has been successfully applied to multilaboratory analysis of 81 oil samples. Seventy-five samples were analyzed correctly, and only six adulterated samples could not be detected. This method could not yet be used for detection of vegetable oils adulterated with WCO that are used for frying non-animal foods. It provides a quick method for detecting adulterated edible vegetable oils containing WCO.

A Randomized Phase 2 Trial of 177Lu Radiolabeled Anti-PSMA Monoclonal Antibody J591 in Patients with High-Risk Castrate, Biochemically Relapsed Prostate Cancer

DTIC Science & Technology

2014-09-01

antibody (J591) against the external portion of PSMA that binds to viable PSMA-expressing cells and is internalized. Studies utilizing J591...metastatic CRPC to bone (Rad223, Xofigo ®) leading to excitement within the field. A more tumor-targeted approach utilizing J591 is of increased...has generated renewed scientific and clinical interest. In addition, recent studies utilizing J591-based immuno- PET imaging providing additional

Development of immunodetection system for botulinum neurotoxin type B using synthetic gene based recombinant protein

PubMed Central

Jain, Swati; Ponmariappan, S.; Kumar, Om

2011-01-01

Background & objectives: Botulinum neurotoxins (A-G) are among most poisonous substances in the world, produced by obligate anaerobic bacteria Clostridum botulinum. Among the seven serotypes A, B, E and F are of human importance. In India, the prevalence of C. botulinum as well as botulism outbreaks have been reported. Due to its extreme toxicity it has been classified in the Category A of biological warfare agent. So far, there is no commercial detection system available in India to detect botulism. The present study aims to develop an immuno detection system for botulinum neurotoxin serotype B using synthetic gene approach. Methods: The truncated fragment of the botulinum neurotoxin type B from amino acid 1-450 was synthesized using PCR overlap primers; the constructed gene was cloned in the pQE30UA vector and transformed to Escherichia coli SG 13009. The recombinant protein expression was optimized using various concentration of isopropylthiogalactoside (IPTG) induction, further the expression was confirmed by Western blot analysis using anti-His antibody. Recombinant protein was purified under denatured condition using Ni-NTA affinity chromatography. Antibody was generated against the recombinant protein using alum adjuvant in BALB/c mice and tested for cross reactivity with other serotypes of C. botulinum as well as closely related clostridia. An ELISA test was developed for the detection of botulinum neurotoxin and the minimum detection limit was also estimated. Results: The recombinant protein was expressed at maximum yield at 4.3 h of post-induction with 0.5 mM IPTG concentration. The recombinant protein was purified using Ni-NTA affinity chromatography up to the homogeneity level. The polyclonal antibodies were raised in mice with a titre of 1:2048000. The developed antibody was highly specific with a sensitivity of detecting approximately 15 ng/ml of recombinant protein and not showing any cross-reactivity with other serotypes. Interpretation & conclusions: There is no commercial immunodetection system available in India to detect botulism. The developed detection system is highly specific. It will be useful for growing food industry to detect botulinum neurotoxin in food samples as well as in clinical samples. PMID:21808132

Experimental study on water content detection of traditional masonry based on infrared thermal image

NASA Astrophysics Data System (ADS)

Zhang, Baoqing; Lei, Zukang

2017-10-01

Based on infrared thermal imaging technology for seepage test of two kinds of brick masonry, find out the relationship between the distribution of one-dimensional two brick surface temperature distribution and one-dimensional surface moisture content were determined after seepage brick masonry minimum temperature zone and water content determination method of the highest point of the regression equation, the relationship between temperature and moisture content of the brick masonry reflected the quantitative and establish the initial wet masonry building disease analysis method, then the infrared technology is applied to the protection of historic buildings in.

Combination of heterogeneous criteria for the automatic detection of ethical principles on health web sites.

PubMed

Gaudinat, Arnaud; Grabar, Natalia; Boyer, Célia

2007-10-11

The detection of ethical issues of web sites aims at selection of information helpful to the reader and is an important concern in medical informatics. Indeed, with the ever-increasing volume of online health information, coupled with its uneven reliability and quality, the public should be aware about the quality of information available online. In order to address this issue, we propose methods for the automatic detection of statements related to ethical principles such as those of the HONcode. For the detection of these statements, we combine two kinds of heterogeneous information: content-based categorizations and URL-based categorizations through application of the machine learning algorithms. Our objective is to observe the quality of categorization through URL's for web pages where categorization through content has been proven to be not precise enough. The results obtained indicate that only some of the principles were better processed.

Liquid biopsy in cancer patients: advances in capturing viable CTCs for functional studies using the EPISPOT assay.

PubMed

Alix-Panabières, Catherine; Pantel, Klaus

2015-01-01

Circulating tumor cells (CTCs) in the blood of cancer patients have received increasing attention as new diagnostic tool enabling 'liquid biopsies'. In contrast to the wealth of descriptive studies demonstrating the clinical relevance of CTCs as biomarkers, the extremely low concentration of CTCs in the peripheral blood of most cancer patients challenges further functional studies. This article discusses the current possibilities to enrich and, in particular, detect viable CTCs with emphasis on the EPithelial ImmunoSPOT technology. This functional assay detects viable CTCs at the single-cell level and has been used on hundreds of patients with different tumor types including epithelial tumors (breast, prostate and colon cancer) and melanomas. Moreover, the article summarizes recent advances in the in vitro and in vivo expansion of CTCs from cancer patients. These functional analyses will contribute to identifying the biological properties of metastatic cells and reveal new therapeutic targets against disseminating cancer cells.

Defect in IgV gene somatic hypermutation in common variable immuno-deficiency syndrome.

PubMed

Levy, Y; Gupta, N; Le Deist, F; Garcia, C; Fischer, A; Weill, J C; Reynaud, C A

1998-10-27

Common Variable Immuno-Deficiency (CVID) is the most common symptomatic primary antibody-deficiency syndrome, but the basic immunologic defects underlying this syndrome are not well defined. We report here that among eight patients studied (six CVID and two hypogammaglobulinemic patients with recurrent infections), there is in two CVID patients a dramatic reduction in Ig V gene somatic hypermutation with 40-75% of IgG transcripts totally devoid of mutations in the circulating memory B cell compartment. Functional assays of the T cell compartment point to an intrinsic B cell defect in the process of antibody affinity maturation in these two cases.

Light microscopic study of periapical lesions associated with asymptomatic apical periodontitis.

PubMed

Kabak, S L; Kabak, Y S; Anischenko, S L

2005-04-01

The purpose of the study has been to evaluate the degree of chronic inflammation in tissues surrounding the apex of the tooth root in patients with apical periodontitis in the remission phase. The material included 37 apical granulomas and radicular cysts obtained as a result of apiectomy, and 20 teeth which were removed together with the focus of the periapical inflammation. Routine histological techniques, as well as the immunofluorescent and immuno-chemical methods were used to examine the material. Despite the absence of clinical symptoms in 23 of 57 cases, the morphological signs of chronic inflammation were observed in the apical area of the tooth root. Morphological signs of viral invasion of epithelial and stromal cells in the radicular cyst wall were revealed in six cases. The presence of the virus of Herpes simplex I in epithelial cells (five cases) and adenoviral invasion (one case) was confirmed by immuno-fluorescent and immuno-chemical methods. Histological examination often reveals morphological signs of an active inflammatory process in the periapical tissues of patients treated during clinical remission. In our opinion, the presence of viruses in the epithelial cells of the radicular cyst may contribute to the persistence of the active stage of the inflammatory process.

Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry Enables Multiplex, Quantitative Pharmacodynamic Studies of Phospho-Signaling*

PubMed Central

Whiteaker, Jeffrey R.; Zhao, Lei; Yan, Ping; Ivey, Richard G.; Voytovich, Uliana J.; Moore, Heather D.; Lin, Chenwei; Paulovich, Amanda G.

2015-01-01

In most cell signaling experiments, analytes are measured one Western blot lane at a time in a semiquantitative and often poorly specific manner, limiting our understanding of network biology and hindering the translation of novel therapeutics and diagnostics. We show the feasibility of using multiplex immuno-MRM for phospho-pharmacodynamic measurements, establishing the potential for rapid and precise quantification of cell signaling networks. A 69-plex immuno-MRM assay targeting the DNA damage response network was developed and characterized by response curves and determinations of intra- and inter-assay repeatability. The linear range was ≥3 orders of magnitude, the median limit of quantification was 2.0 fmol/mg, the median intra-assay variability was 10% CV, and the median interassay variability was 16% CV. The assay was applied in proof-of-concept studies to immortalized and primary human cells and surgically excised cancer tissues to quantify exposure–response relationships and the effects of a genomic variant (ATM kinase mutation) or pharmacologic (kinase) inhibitor. The study shows the utility of multiplex immuno-MRM for simultaneous quantification of phosphorylated and nonmodified peptides, showing feasibility for development of targeted assay panels to cell signaling networks. PMID:25987412

Unheated Cannabis sativa extracts and its major compound THC-acid have potential immuno-modulating properties not mediated by CB1 and CB2 receptor coupled pathways.

PubMed

Verhoeckx, Kitty C M; Korthout, Henrie A A J; van Meeteren-Kreikamp, A P; Ehlert, Karl A; Wang, Mei; van der Greef, Jan; Rodenburg, Richard J T; Witkamp, Renger F

2006-04-01

There is a great interest in the pharmacological properties of cannabinoid like compounds that are not linked to the adverse effects of Delta(9)-tetrahydrocannabinol (THC), e.g. psychoactive properties. The present paper describes the potential immuno-modulating activity of unheated Cannabis sativa extracts and its main non-psychoactive constituent Delta(9)-tetrahydrocanabinoid acid (THCa). By heating Cannabis extracts, THCa was shown to be converted into THC. Unheated Cannabis extract and THCa were able to inhibit the tumor necrosis factor alpha (TNF-alpha) levels in culture supernatants from U937 macrophages and peripheral blood macrophages after stimulation with LPS in a dose-dependent manner. This inhibition persisted over a longer period of time, whereas after prolonged exposure time THC and heated Cannabis extract tend to induce the TNF-alpha level. Furthermore we demonstrated that THCa and THC show distinct effects on phosphatidylcholine specific phospholipase C (PC-PLC) activity. Unheated Cannabis extract and THCa inhibit the PC-PLC activity in a dose-dependent manner, while THC induced PC-PLC activity at high concentrations. These results suggest that THCa and THC exert their immuno-modulating effects via different metabolic pathways.

(124)I-L19-SIP for immuno-PET imaging of tumour vasculature and guidance of (131)I-L19-SIP radioimmunotherapy.

PubMed

Tijink, Bernard M; Perk, Lars R; Budde, Marianne; Stigter-van Walsum, Marijke; Visser, Gerard W M; Kloet, Reina W; Dinkelborg, Ludger M; Leemans, C René; Neri, Dario; van Dongen, Guus A M S

2009-08-01

The human monoclonal antibody (MAb) fragment L19-SIP is directed against extra domain B (ED-B) of fibronectin, a marker of tumour angiogenesis. A clinical radioimmunotherapy (RIT) trial with (131)I-L19-SIP was recently started. In the present study, after GMP production of (124)I and efficient production of (124)I-L19-SIP, we aimed to demonstrate the suitability of (124)I-L19-SIP immuno-PET for imaging of angiogenesis at early-stage tumour development and as a scouting procedure prior to clinical (131)I-L19-SIP RIT. (124)I was produced in a GMP compliant way via (124)Te(p,n)(124)I reaction and using a TERIMO module for radioiodine separation. L19-SIP was radioiodinated by using a modified version of the IODO-GEN method. The biodistribution of coinjected (124)I- and (131)I-L19-SIP was compared in FaDu xenograft-bearing nude mice, while (124)I PET images were obtained from mice with tumours of <50 to approximately 700 mm(3). (124)I was produced highly pure with an average yield of 15.4 +/- 0.5 MBq/microAh, while separation yield was approximately 90% efficient with <0.5% loss of TeO(2). Overall labelling efficiency, radiochemical purity and immunoreactive fraction were for (124)I-L19-SIP: approximately 80 , 99.9 and >90%, respectively. Tumour uptake was 7.3 +/- 2.1, 10.8 +/- 1.5, 7.8 +/- 1.4, 5.3 +/- 0.6 and 3.1 +/- 0.4%ID/g at 3, 6, 24, 48 and 72 h p.i., resulting in increased tumour to blood ratios ranging from 6.0 at 24 h to 45.9 at 72 h p.i.. Fully concordant labelling and biodistribution results were obtained with (124)I- and (131)I-L19-SIP. Immuno-PET with (124)I-L19-SIP using a high-resolution research tomograph PET scanner revealed clear delineation of the tumours as small as 50 mm(3) and no adverse uptake in other organs. (124)I-MAb conjugates for clinical immuno-PET can be efficiently produced. Immuno-PET with (124)I-L19-SIP appeared qualified for sensitive imaging of tumour neovasculature and for predicting (131)I-L19-SIP biodistribution.

Stimulatory effect of Eucalyptus essential oil on innate cell-mediated immune response

PubMed Central

Serafino, Annalucia; Vallebona, Paola Sinibaldi; Andreola, Federica; Zonfrillo, Manuela; Mercuri, Luana; Federici, Memmo; Rasi, Guido; Garaci, Enrico; Pierimarchi, Pasquale

2008-01-01

Background Besides few data concerning the antiseptic properties against a range of microbial agents and the anti-inflammatory potential both in vitro and in vivo, little is known about the influence of Eucalyptus oil (EO) extract on the monocytic/macrophagic system, one of the primary cellular effectors of the immune response against pathogen attacks. The activities of this natural extract have mainly been recognized through clinical experience, but there have been relatively little scientific studies on its biological actions. Here we investigated whether EO extract is able to affect the phagocytic ability of human monocyte derived macrophages (MDMs) in vitro and of rat peripheral blood monocytes/granulocytes in vivo in absence or in presence of immuno-suppression induced by the chemotherapeutic agent 5-fluorouracil (5-FU). Methods Morphological activation of human MDMs was analysed by scanning electron microscopy. Phagocytic activity was tested: i) in vitro in EO treated and untreated MDMs, by confocal microscopy after fluorescent beads administration; ii) in vivo in monocytes/granulocytes from peripheral blood of immuno-competent or 5-FU immuno-suppressed rats, after EO oral administration, by flow cytometry using fluorescein-labelled E. coli. Cytokine release by MDMs was determined using the BD Cytometric Bead Array human Th1/Th2 cytokine kit. Results EO is able to induce activation of MDMs, dramatically stimulating their phagocytic response. EO-stimulated internalization is coupled to low release of pro-inflammatory cytokines and requires integrity of the microtubule network, suggesting that EO may act by means of complement receptor-mediated phagocytosis. Implementation of innate cell-mediated immune response was also observed in vivo after EO administration, mainly involving the peripheral blood monocytes/granulocytes. The 5-FU/EO combined treatment inhibited the 5-FU induced myelotoxicity and raised the phagocytic activity of the granulocytic/monocytic system, significantly decreased by the chemotherapic. Conclusion Our data, demonstrating that Eucalyptus oil extract is able to implement the innate cell-mediated immune response, provide scientific support for an additional use of this plant extract, besides those concerning its antiseptic and anti-inflammatory properties and stimulate further investigations also using single components of this essential oil. This might drive development of a possible new family of immuno-regulatory agents, useful as adjuvant in immuno-suppressive pathologies, in infectious disease and after tumour chemotherapy. PMID:18423004

Cerenkov luminescence imaging of medical isotopes

PubMed Central

Ruggiero, Alessandro; Holland, Jason P.; Lewis, Jason S.; Grimm, Jan

2011-01-01

The development of novel multimodality imaging agents and techniques represents the current frontier of research in the field of medical imaging science. However, the combination of nuclear tomography with optical techniques has yet to be established. Here, we report the use of the inherent optical emissions from the decay of radiopharmaceuticals for Cerenkov luminescence imaging (CLI) of tumors in vivo and correlate the results with those obtained from concordant immuno-PET studies. Methods In vitro phantom studies were used to validate the visible light emission observed from a range of radionuclides including the positron emitters 18F, 64Cu, 89Zr, and 124I; β-emitter 131I; and α-particle emitter 225Ac for potential use in CLI. The novel radiolabeled monoclonal antibody 89Zr-desferrioxamine B-[DFO-J591 for immuno-PET of prostate-specific membrane antigen (PSMA) expression was used to coregister and correlate the CLI signal observed with the immuno-PET images and biodistribution studies. Results Phantom studies confirmed that Cerenkov radiation can be observed from a range of positron-,β-, and α-emitting radionuclides using standard optical imaging devices. The change in light emission intensity versus time was concordant with radionuclide decay and was also found to correlate linearly with both the activity concentration and the measured PET signal (percentage injected dose per gram). In vivo studies conducted in male severe combined immune deficient mice bearing PSMA-positive, subcutaneous LNCaP tumors demonstrated that tumor-specific uptake of 89Zr-DFO-J591 could be visualized by both immuno-PET and CLI. Optical and immuno-PET signal intensities were found to increase over time from 24 to 96 h, and biodistribution studies were found to correlate well with both imaging modalities. Conclusion These studies represent the first, to our knowledge, quantitative assessment of CLI for measuring radiotracer uptake in vivo. Many radionuclides common to both nuclear tomographic imaging and radiotherapy have the potential to be used in CLI. The value of CLI lies in its ability to image radionuclides that do not emit either positrons or γ-rays and are, thus, unsuitable for use with current nuclear imaging modalities. Optical imaging of Cerenkov radiation emission shows excellent promise as a potential new imaging modality for the rapid, high-throughput screening of radiopharmaceuticals PMID:20554722

Effective dose to immuno-PET patients due to metastable impurities in cyclotron produced zirconium-89

NASA Astrophysics Data System (ADS)

Alfuraih, Abdulrahman; Alzimami, Khalid; Ma, Andy K.; Alghamdi, Ali; Al Jammaz, Ibrahim

2014-11-01

Immuno-PET is a nuclear medicine technique that combines positron emission tommography (PET) with radio-labeled monoclonal antibodies (mAbs) for tumor characterization and therapy. Zirconium-89 (89Zr) is an emerging radionuclide for immuno-PET imaging. Its long half-life (78.4 h) gives ample time for the production, the administering and the patient uptake of the tagged radiopharmaceutical. Furthermore, the nuclides will remain in the tumor cells after the mAbs are catabolized so that time series studies are possible without incurring further administration of radiopharmarceuticals. 89Zr can be produced in medical cyclotrons by bombarding an yttrium-89 (89Y) target with a proton beam through the 89Y(p,n)89Zr reaction. In this study, we estimated the effective dose to the head and neck cancer patients undergoing 89Zr-based immune-PET procedures. The production of 89Zr and the impurities from proton irradiation of the 89Y target in a cyclotron was calculated with the Monte Carlo code MCNPX and the nuclear reaction code TALYS. The cumulated activities of the Zr isotopes were derived from real patient data in literature and the effective doses were estimated using the MIRD specific absorbed fraction formalism. The estimated effective dose from 89Zr is 0.5±0.2 mSv/MBq. The highest organ dose is 1.8±0.2 mSv/MBq in the liver. These values are in agreement with those reported in literature. The effective dose from 89mZr is about 0.2-0.3% of the 89Zr dose in the worst case. Since the ratio of 89mZr to 89Zr depends on the cooling time as well as the irradiation details, contaminant dose estimation is an important aspect in optimizing the cyclotron irradiation geometry, energy and time.

The role of Vitamin D in immuno-inflammatory responses in Ankylosing Spondylitis patients with and without Acute Anterior Uveitis.

PubMed

Mitulescu, T C; Stavaru, C; Voinea, L M; Banica, L M; Matache, C; Predeteanu, D

2016-01-01

Hypothesis: Abnormal Vitamin D (Vit D) level could have consequences on the immuno-inflammatory processes in Ankylosing Spondylitis (AS). Aim: The purpose of this study was to analyze the role of Vitamin D in the interplay between immune and inflammation effectors in AS associated-Acute Anterior Uveitis (AAU). Methods and Results: 25-hydroxyvitamin D (Vit D), LL-37 peptide, IL-8 and Serum Amyloid A (SAA) were identified and quantified in the serum/ plasma of thirty-four AS patients [eleven AS patients presenting AAU (AAU AS patients) and twenty-three AS patients without AAU (wAAU AS patients)] and eighteen healthy individuals (Control) using enzyme-linked immunosorbent assay. Acute-phase SAA level was significantly higher in AS patients compared to Controls. Contrary with wAAU AS patients, significantly elevated levels of IL-8, and diminished levels of Vit D characterized AAU AS patients. Regarding LL-37, its level decreased concomitantly with the level of Vit D. When AS patients were subgrouped based on AAU presence or on Vit D level, important associations between immuno-inflammatory assessed markers and AS features were noticed. Generally, Vit D levels were associated indirectly with leukocytes/ neutrophils number or with ESR, CRP, and Fibrinogen levels. The levels of SAA and IL-8 associated directly with AAU or with AAU relapses, especially in AS patients with Vit D insufficiency, while SAA associated directly with infection/ inflammatory markers and with disease activity indexes or with the degree of functional limitation. Discussion: Altered levels of Vit D affect the balance between LL-37, IL-8 and SAA, suggesting an association with AAU, an extra-articular manifestation of AS. Abbreviations: Vit D = Vitamin D, AS = Ankylosing Spondylitis, AAU = Acute Anterior Uveitis, AAU AS = AS patients with AAU, wAAU AS = AS patients without AAU, SSZ = Sulphasalazine, Leu = Leukocytes, Neu = Neutrophils.

The role of Vitamin D in immuno-inflammatory responses in Ankylosing Spondylitis patients with and without Acute Anterior Uveitis

PubMed Central

Mitulescu, TC; Stavaru, C; Voinea, LM; Banica, LM; Matache, C; Predeteanu, D

2016-01-01

Hypothesis:Abnormal Vitamin D (Vit D) level could have consequences on the immuno-inflammatory processes in Ankylosing Spondylitis (AS). Aim:The purpose of this study was to analyze the role of Vitamin D in the interplay between immune and inflammation effectors in AS associated-Acute Anterior Uveitis (AAU). Methods and Results:25-hydroxyvitamin D (Vit D), LL-37 peptide, IL-8 and Serum Amyloid A (SAA) were identified and quantified in the serum/ plasma of thirty-four AS patients [eleven AS patients presenting AAU (AAU AS patients) and twenty-three AS patients without AAU (wAAU AS patients)] and eighteen healthy individuals (Control) using enzyme-linked immunosorbent assay. Acute-phase SAA level was significantly higher in AS patients compared to Controls. Contrary with wAAU AS patients, significantly elevated levels of IL-8, and diminished levels of Vit D characterized AAU AS patients. Regarding LL-37, its level decreased concomitantly with the level of Vit D. When AS patients were subgrouped based on AAU presence or on Vit D level, important associations between immuno-inflammatory assessed markers and AS features were noticed. Generally, Vit D levels were associated indirectly with leukocytes/ neutrophils number or with ESR, CRP, and Fibrinogen levels. The levels of SAA and IL-8 associated directly with AAU or with AAU relapses, especially in AS patients with Vit D insufficiency, while SAA associated directly with infection/ inflammatory markers and with disease activity indexes or with the degree of functional limitation. Discussion:Altered levels of Vit D affect the balance between LL-37, IL-8 and SAA, suggesting an association with AAU, an extra-articular manifestation of AS. Abbreviations:Vit D = Vitamin D, AS = Ankylosing Spondylitis, AAU = Acute Anterior Uveitis, AAU AS = AS patients with AAU, wAAU AS = AS patients without AAU, SSZ = Sulphasalazine, Leu = Leukocytes, Neu = Neutrophils. PMID:27713770

Micromotor-based lab-on-chip immunoassays.

PubMed

García, Miguel; Orozco, Jahir; Guix, Maria; Gao, Wei; Sattayasamitsathit, Sirilak; Escarpa, Alberto; Merkoçi, Arben; Wang, Joseph

2013-02-21

Here we describe the first example of using self-propelled antibody-functionalized synthetic catalytic microengines for capturing and transporting target proteins between the different reservoirs of a lab-on-a-chip (LOC) device. A new catalytic polymer/Ni/Pt microtube engine, containing carboxy moieties on its mixed poly(3,4-ethylenedioxythiophene) (PEDOT)/COOH-PEDOT polymeric outermost layer, is further functionalized with the antibody receptor to selectively recognize and capture the target protein. The new motor-based microchip immunoassay operations are carried out without any bulk fluid flow, replacing the common washing steps in antibody-based protein bioassays with the active transport of the captured protein throughout the different reservoirs, where each step of the immunoassay takes place. A first microchip format involving an 'on-the-fly' double-antibody sandwich assay (DASA) is used for demonstrating the selective capture of the target protein, in the presence of excess of non-target proteins. A secondary antibody tagged with a polymeric-sphere tracer allows the direct visualization of the binding events. In a second approach the immuno-nanomotor captures and transports the microsphere-tagged antigen through a microchannel network. An anti-protein-A modified microengine is finally used to demonstrate the selective capture, transport and convenient label-free optical detection of a Staphylococcus aureus target bacteria (containing proteinA in its cell wall) in the presence of a large excess of non-target (Saccharomyces cerevisiae) cells. The resulting nanomotor-based microchip immunoassay offers considerable potential for diverse applications in clinical diagnostics, environmental and security monitoring fields.

Application of MPEG-7 descriptors for content-based indexing of sports videos

NASA Astrophysics Data System (ADS)

Hoeynck, Michael; Auweiler, Thorsten; Ohm, Jens-Rainer

2003-06-01

The amount of multimedia data available worldwide is increasing every day. There is a vital need to annotate multimedia data in order to allow universal content access and to provide content-based search-and-retrieval functionalities. Since supervised video annotation can be time consuming, an automatic solution is appreciated. We review recent approaches to content-based indexing and annotation of videos for different kind of sports, and present our application for the automatic annotation of equestrian sports videos. Thereby, we especially concentrate on MPEG-7 based feature extraction and content description. We apply different visual descriptors for cut detection. Further, we extract the temporal positions of single obstacles on the course by analyzing MPEG-7 edge information and taking specific domain knowledge into account. Having determined single shot positions as well as the visual highlights, the information is jointly stored together with additional textual information in an MPEG-7 description scheme. Using this information, we generate content summaries which can be utilized in a user front-end in order to provide content-based access to the video stream, but further content-based queries and navigation on a video-on-demand streaming server.

Analytically Sensitive Protein Detection in Microtiter Plates by Proximity Ligation with Rolling Circle Amplification.

PubMed

Ebai, Tonge; Souza de Oliveira, Felipe Marques; Löf, Liza; Wik, Lotta; Schweiger, Caroline; Larsson, Anders; Keilholtz, Ulrich; Haybaeck, Johannes; Landegren, Ulf; Kamali-Moghaddam, Masood

2017-09-01

Detecting proteins at low concentrations in plasma is crucial for early diagnosis. Current techniques in clinical routine, such as sandwich ELISA, provide sensitive protein detection because of a dependence on target recognition by pairs of antibodies, but detection of still lower protein concentrations is often called for. Proximity ligation assay with rolling circle amplification (PLARCA) is a modified proximity ligation assay (PLA) for analytically specific and sensitive protein detection via binding of target proteins by 3 antibodies, and signal amplification via rolling circle amplification (RCA) in microtiter wells, easily adapted to instrumentation in use in hospitals. Proteins captured by immobilized antibodies were detected using a pair of oligonucleotide-conjugated antibodies. Upon target recognition these PLA probes guided oligonucleotide ligation, followed by amplification via RCA of circular DNA strands that formed in the reaction. The RCA products were detected by horseradish peroxidase-labeled oligonucleotides to generate colorimetric reaction products with readout in an absorbance microplate reader. We compared detection of interleukin (IL)-4, IL-6, IL-8, p53, and growth differentiation factor 15 (GDF-15) by PLARCA and conventional sandwich ELISA or immuno-RCA. PLARCA detected lower concentrations of proteins and exhibited a broader dynamic range compared to ELISA and iRCA using the same antibodies. IL-4 and IL-6 were detected in clinical samples at femtomolar concentrations, considerably lower than for ELISA. PLARCA offers detection of lower protein levels and increased dynamic ranges compared to ELISA. The PLARCA procedure may be adapted to routine instrumentation available in hospitals and research laboratories. © 2017 American Association for Clinical Chemistry.

Demonstration of IgG Subclass (IgG1 and IgG3) in Immuno-Related Hemocytopenia.

PubMed

Shao, Yuanyuan; Qi, Xiao; Fu, Rong; Liu, Hui; Wang, Yihao; Ding, Shaoxue; Wang, Huaquan; Li, Lijuan; Shao, Zonghong

2018-06-01

Immuno-related hemocytopenia (IRH) is defined as idiopathic cytopenia of undetermined significance (ICUS) patients with autoantibodies. In our previous studies, we found that IgG1 levels were increased in IRH patients and might cause the destruction of hematopoietic cells. In this study, we analyzed IgG subclasses in 30 IRH patients (male:female = 13:17, median age 32 years, range 18 - 56), 15 IRH remission patients (IRH-R) (male:female = 6:9, median age 34, range 20 - 52) and 20 normal controls (male:female = 8:12, median age 27, range 24 - 36) by Cytometric Bead Array, Flow Cytometry and Immunohistochemical staining. Levels of IgG1/IgG3 in the bone marrow supernatant of IRH patents, as well as the proportion of CD5+ B lymphocytes and Th2 cells (CD3+CD8-IL-4+) were higher than those of IRH-R patients and normal controls, and IgG1 levels had a positive correlation with the proportion of Th2 cells. In IRH patients, IgG1 and IgG3 were positive on nucleated erythrocytes and granulocytes, which were negative in IRH-R patients and healthy controls and had inverse correlations with hematopoietic function. Using immunohistochemical staining, IgG1 were also detected on bone marrow biopsies of IRH patients. The results indicated that IgG1 and IgG3 autoantibodies in IRH patients might play a key role in the IRH pathogenesis and in the abnormal immune function of IRH patients.

Characterization of Differential Toll-Like Receptor Responses below the Optical Diffraction Limit**

PubMed Central

Aaron, Jesse S.; Carson, Bryan D.; Timlin, Jerilyn A.

2013-01-01

Many membrane receptors are recruited to specific cell surface domains to form nanoscale clusters upon ligand activation. This step appears to be necessary to initiate signaling, including pathways in innate immune system activation. However, virulent pathogens such as Yersinia pestis (the causative agent of plague) are known to evade innate immune detection, in contrast to similar microbes (such as E. coli) that elicit a robust response. This disparity has been partly attributed to the structure of lipopolysaccharides (LPS) on the bacterial cell wall, which are recognized by the innate immune receptor TLR4. As such, we hypothesized that nanoscale differences would exist between the spatial clustering of TLR4 upon binding of LPS derived from Y. pestis and E. coli. Although optical imaging can provide exquisite details of the spatial organization of biomolecules, there is a mismatch between the scale at which receptor clustering occurs (<300 nm) and the optical diffraction limit (>400 nm). The last decade has seen the emergence of super-resolution imaging methods that effectively break the optical diffraction barrier to yield truly nanoscale information in intact biological samples. This study reports the first visualizations of TLR4 distributions on intact cells at image resolutions of <30 nm using a novel, dual-color stochastic optical reconstruction microscopy (STORM) technique. This methodology permits distinction between receptors containing bound LPS from those without at the nanoscale. Importantly, we also show that LPS derived from immuno-stimulatory bacteria resulted in significantly higher LPS-TLR4 cluster sizes and a nearly two-fold greater ligand/receptor colocalization as compared to immuno-evading LPS. PMID:22807232

Lipid based delivery and immuno-stimulatory systems: Master tools to combat leishmaniasis.

PubMed

Sabur, Abdus; Asad, Mohammad; Ali, Nahid

2016-11-01

Disease management of leishmaniasis is appalling due to lack of a human vaccine and the toxicity and resistance concerns with limited therapeutic drugs. The challenges in development of a safe vaccine for generation and maintenance of robust antileishmanial protective immunity through a human administrable route of immunization can be addressed through immunomodulation and targeted delivery. The versatility of lipid based particulate system for deliberate delivery of diverse range of molecules including immunomodulators, antigens and drugs have essentially found pivotal role in design of proficient vaccination and therapeutic strategies against leishmaniasis. The prospects of lipid based preventive and curative formulations for leishmaniasis have been highlighted in this review. Copyright © 2016. Published by Elsevier Inc.

Content and distribution of noncollagenous matrix proteins in bone and cementum: relationship to speed of formation and collagen packing density.

PubMed

Nanci, A

1999-06-30

The organic matrix of collagen-based calcified tissues consists of a supporting collagen meshwork and various noncollagenous matrix proteins (NCPs). Together, they contribute to determining the structure and biomechanical properties of the tissue. Their respective organization and interrelation can advantageously be examined by immunocytochemistry, an approach which allows correlation of composition with structure. The aim of this article is to review postembedding immuno- and lectin-gold-labeling data on the characterization of the noncollagenous compartment in rat and human bone and cementum, and on its relationship to collagen. The two major NCPs, bone sialoprotein and osteopontin, generally codistribute and accumulate in cement lines and in the spaces among the mineralized collagen fibrils. However, there are variations in their distribution and density of labeling throughout the tissue. Indeed, bone and cementum can form in environments that are either poor or enriched in NCPs. The amount of NCPs generally correlates with bone and cementum types and with speed of formation of the tissue and packing density of collagen fibrils. Taken together, the data suggest that production of both collagenous and noncollagenous constituents can be "modulated" during formation of collagen-based calcified tissues. It is concluded that, in addition to structural and compositional parameters, tissue dynamics must be taken into consideration in order to understand the significance of the apparent accumulation of NCPs at some sites and to determine the mechanisms of normal and pathological calcified tissue formation. Copyright 1999 Academic Press.

Multiplex real-time quantitative PCR, microscopy and rapid diagnostic immuno-chromatographic tests for the detection of Plasmodium spp: performance, limit of detection analysis and quality assurance.

PubMed

Khairnar, Krishna; Martin, Donald; Lau, Rachel; Ralevski, Filip; Pillai, Dylan R

2009-12-09

Accurate laboratory diagnosis of malaria species in returning travelers is paramount in the treatment of this potentially fatal infectious disease. A total of 466 blood specimens from returning travelers to Africa, Asia, and South/Central America with suspected malaria infection were collected between 2007 and 2009 at the reference public health laboratory. These specimens were assessed by reference microscopy, multipex real-time quantitative polymerase chain reaction (QPCR), and two rapid diagnostic immuno-chromatographic tests (ICT) in a blinded manner. Key clinical laboratory parameters such as limit of detection (LOD) analysis on clinical specimens by parasite stage, inter-reader variability of ICTs, staffing implications, quality assurance and cost analysis were evaluated. QPCR is the most analytically sensitive method (sensitivity 99.41%), followed by CARESTART (sensitivity 88.24%), and BINAXNOW (sensitivity 86.47%) for the diagnosis of malaria in returning travelers when compared to reference microscopy. However, microscopy was unable to specifically identify Plasmodia spp. in 18 out of 170 positive samples by QPCR. Moreover, the 17 samples that were negative by microscopy and positive by QPCR were also positive by ICTs. Quality assurance was achieved for QPCR by exchanging a blinded proficiency panel with another reference laboratory. The Kappa value of inter-reader variability among three readers for BINAXNOW and CARESTART was calculated to be 0.872 and 0.898 respectively. Serial dilution studies demonstrated that the QPCR cycle threshold correlates linearly with parasitemia (R(2) = 0.9746) in a clinically relevant dynamic range and retains a LOD of 11 rDNA copies/microl for P. falciparum, which was several log lower than reference microscopy and ICTs. LOD for QPCR is affected not only by parasitemia but the parasite stage distribution of each clinical specimen. QPCR was approximately 6-fold more costly than reference microscopy. These data suggest that multiplex QPCR although more costly confers a significant diagnostic advantage in terms of LOD compared to reference microscopy and ICTs for all four species. Quality assurance of QPCR is essential to the maintenance of proficiency in the clinical laboratory. ICTs showed good concordance between readers however lacked sensitivity for non-falciparum species due to antigenic differences and low parasitemia. Multiplex QPCR but not ICTs is an essential adjunct to microscopy in the reference laboratory detection of malaria species specifically due to the superior LOD. ICTs are better suited to the non-reference laboratory where lower specimen volumes challenge microscopy proficiency in the non-endemic setting.

75 FR 18907 - Notice of Opportunity for Public Comment on the Proposed Model Safety Evaluation for Plant...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-04-13

... Specifications (STS) to define a new time limit for restoring inoperable RCS leakage detection instrumentation to... contents of the facility design bases related to the operability of the RCS leakage detection... leakage detection instrumentation to operable status and establish alternate methods of monitoring RCS...

Moisture Influence Reducing Method for Heavy Metals Detection in Plant Materials Using Laser-Induced Breakdown Spectroscopy: A Case Study for Chromium Content Detection in Rice Leaves.

PubMed

Peng, Jiyu; He, Yong; Ye, Lanhan; Shen, Tingting; Liu, Fei; Kong, Wenwen; Liu, Xiaodan; Zhao, Yun

2017-07-18

Fast detection of heavy metals in plant materials is crucial for environmental remediation and ensuring food safety. However, most plant materials contain high moisture content, the influence of which cannot be simply ignored. Hence, we proposed moisture influence reducing method for fast detection of heavy metals using laser-induced breakdown spectroscopy (LIBS). First, we investigated the effect of moisture content on signal intensity, stability, and plasma parameters (temperature and electron density) and determined the main influential factors (experimental parameters F and the change of analyte concentration) on the variations of signal. For chromium content detection, the rice leaves were performed with a quick drying procedure, and two strategies were further used to reduce the effect of moisture content and shot-to-shot fluctuation. An exponential model based on the intensity of background was used to correct the actual element concentration in analyte. Also, the ratio of signal-to-background for univariable calibration and partial least squared regression (PLSR) for multivariable calibration were used to compensate the prediction deviations. The PLSR calibration model obtained the best result, with the correlation coefficient of 0.9669 and root-mean-square error of 4.75 mg/kg in the prediction set. The preliminary results indicated that the proposed method allowed for the detection of heavy metals in plant materials using LIBS, and it could be possibly used for element mapping in future work.

Critical moisture content for microbial growth in dried food-processing residues.

PubMed

Rezaei, Farzaneh; Vandergheynst, Jean S

2010-09-01

Food-processing residues are good feedstocks for biofuel and biochemical production because they have high energy content and are abundant. Year-round biofuel and biochemical production requires proper storage to prevent microbial decomposition and thermal runaway. In this study, microbial activity of tomato pomace (TP), grape pomace (GP), fermented grape pomace (FGP) and sugar beet pulp (SBP) was monitored at nine different moisture contents. Maximum and cumulative respirations for each feedstock with respect to moisture content followed a sigmoidal relationship. The critical moisture content below which no microbial activity was detected for SBP, TP, FGP and GP was 24-31, 16-21, 23-33 and 43-46% (dry basis) respectively. A logarithmic relationship was observed (R(2) = 0.94) between critical moisture content and initial water-soluble carbohydrate (WSC) content of the processing residues. The critical moisture content below which no microbial activity was detected and the relationship between critical moisture content and initial WSC content were determined in this study for four food-processing residues. Both parameters permit evaluation of the potential for deterioration of food-processing residues during storage based on moisture content and WSC content. Copyright 2010 Society of Chemical Industry.

Protective Effects of Myricetin on Acute Hypoxia-Induced Exercise Intolerance and Mitochondrial Impairments in Rats

PubMed Central

Zou, Dan; Liu, Peng; Chen, Ka; Xie, Qi; Liang, Xinyu; Bai, Qian; Zhou, Qicheng; Liu, Kai; Zhang, Ting; Zhu, Jundong; Mi, Mantian

2015-01-01

Purpose Exercise tolerance is impaired in hypoxia. The aim of this study was to evaluate the effects of myricetin, a dietary flavonoid compound widely found in fruits and vegetables, on acute hypoxia-induced exercise intolerance in vivo and in vitro. Methods Male rats were administered myricetin or vehicle for 7 days and subsequently spent 24 hours at a barometric pressure equivalent to 5000 m. Exercise capacity was then assessed through the run-to-fatigue procedure, and mitochondrial morphology in skeletal muscle cells was observed by transmission electron microscopy (TEM). The enzymatic activities of electron transfer complexes were analyzed using an enzyme-linked immuno-sorbent assay (ELISA). mtDNA was quantified by real-time-PCR. Mitochondrial membrane potential was measured by JC-1 staining. Protein expression was detected through western blotting, immunohistochemistry, and immunofluorescence. Results Myricetin supplementation significantly prevented the decline of run-to-fatigue time of rats in hypoxia, and attenuated acute hypoxia-induced mitochondrial impairment in skeletal muscle cells in vivo and in vitro by maintaining mitochondrial structure, mtDNA content, mitochondrial membrane potential, and activities of the respiratory chain complexes. Further studies showed that myricetin maintained mitochondrial biogenesis in skeletal muscle cells under hypoxic conditions by up-regulating the expressions of mitochondrial biogenesis-related regluators, in addition, AMP-activated protein kinase(AMPK) plays a crucial role in this process. Conclusions Myricetin may have important applications for improving physical performance under hypoxic environment, which may be attributed to the protective effect against mitochondrial impairment by maintaining mitochondrial biogenesis. PMID:25919288

Different properties of skin of different body sites: The root of keloid formation?

PubMed

Butzelaar, Liselotte; Niessen, Frank B; Talhout, Wendy; Schooneman, Dennis P M; Ulrich, Magda M; Beelen, Robert H J; Mink van der Molen, Aebele B

2017-09-01

The purpose of this study was to examine extracellular matrix composition, vascularization, and immune cell population of skin sites prone to keloid formation. Keloids remain a complex problem, posing esthetical as well as functional difficulties for those affected. These scars tend to develop at anatomic sites of preference. Mechanical properties of skin vary with anatomic location and depend largely on extracellular matrix composition. These differences in extracellular matrix composition, but also vascularization and resident immune cell populations might play a role in the mechanism of keloid formation. To examine this hypothesis, skin samples of several anatomic locations were taken from 24 human donors within zero to 36 hours after they had deceased. Collagen content and cross-links were determined through high-performance liquid chromatography. The expression of several genes, involved in extracellular matrix production and degradation, was measured by means of real-time PCR. (Immuno)histochemistry was performed to detect fibroblasts, collagen, elastin, blood vessels, Langerhans cells, and macrophages. Properties of skin of keloid predilections sites were compared to properties of skin from other locations (nonpredilection sites [NPS]). The results indicated that there are site specific variations in extracellular matrix properties (collagen and cross-links) as well as macrophage numbers. Moreover, predilection sites (PS) for keloid formation contain larger amounts of collagen compared to NPS, but decreased numbers of macrophages, in particular classically activated CD40 positive macrophages. In conclusion, the altered (histological, protein, and genetic) properties of skin of keloid PS may cause a predisposition for and contribute to keloid formation. © 2017 by the Wound Healing Society.

Studies on Antiviral and Immuno-Regulation Activity of Low Molecular Weight Fucoidan from Laminaria japonica

NASA Astrophysics Data System (ADS)

Sun, Taohua; Zhang, Xinhui; Miao, Ying; Zhou, Yang; Shi, Jie; Yan, Meixing; Chen, Anjin

2018-06-01

The antiviral activity in vitro and in vivo and the effect of the immune system of two fucoidan fractions with low molecular weight and different sulfate content from Laminaria japonica (LMW fucoidans) were investigated in order to examine the possible mechanism. In vitro, I-type influenza virus, adenovirus and Parainfluenza virus I were used to infect Hep-2, Hela and MDCK cells, respectively. And 50% tissue culture infective dose was calculated to detect the antiviral activity of two LMW fucoidans. The results indicated that compared with the control group, 2 kinds of LMW fucoidans had remarkable antiviral activity in vitro in middle and high doses, while at low doses, the antiviral activity of 2 kinds of LMW fucoidans was not statistically different from that in the blank control group. And there was no statistically difference between two LMW fucoidans in antiviral activity. In vivo, LMW fucoidans could prolong the survival time of virus-infected mice, and could improve the lung index of virus-infected mice significantly, which have statistical differences with the control group significantly ( p < 0.01). However, the survival time of the two LMW fucoidans was not statistically significant ( p > 0.05). In this study, it was shown that both of two LMW fucoidans (LF1, LF2) could increase the thymus index, spleen index, phagocytic index, phagocytosis coefficient and half hemolysin value in middle and high doses, which suggested that LMW fucoidans could play an antiviral role by improving the quality of immune organs, improving immune cell phagocytosis and humoral immunity.

Evolution of Gluten Content in Cereal-Based Gluten-Free Products: An Overview from 1998 to 2016

PubMed Central

Bustamante, María Ángeles; Fernández-Gil, María Pilar; Churruca, Itziar; Miranda, Jonatan; Lasa, Arrate; Navarro, Virginia; Simón, Edurne

2017-01-01

The treatment of Celiac disease consists in a strict lifelong gluten-free (GF) diet. As the ingestion of small amounts can have damaging complications, there has been an ongoing discussion regarding the safe threshold for dietary residual gluten. The aim was to analyze the evolution of gluten content in cereal-based GF foodstuffs (n = 3141) from 1998 to 2016 measured by the enzyme-linked immunosorbent assay (ELISA) technique. Eight categories were defined: flours, breakfast cereals/bars, bakery, pasta, breads, dough, snacks, and yeasts, and these were divided into GF labeled-foods (GF-L) or reportedly GF foodstuffs, but not certified (GF-NC). Gluten-detection was decreased over time in line with the evolving European regulations about food information and gluten content claims. This decline started sooner in GF-L products than in GF-NC. As a whole, gluten was detected in 371 samples, with breakfast cereals/bars being the most contaminated group. Snacks and yeasts changed from being high gluten-detected samples to being totally GF over the years. The downside is that, of contaminated samples, those in the low levels of gluten detection range have decreased while flour samples containing over 100 mg/kg gluten have risen in the 2013–2016 period. Obtained data confirm that GF cereal-based foods are becoming safer but gluten control must be maintained. PMID:28054938

Evolution of Gluten Content in Cereal-Based Gluten-Free Products: An Overview from 1998 to 2016.

PubMed

Bustamante, María Ángeles; Fernández-Gil, María Pilar; Churruca, Itziar; Miranda, Jonatan; Lasa, Arrate; Navarro, Virginia; Simón, Edurne

2017-01-03

The treatment of Celiac disease consists in a strict lifelong gluten-free (GF) diet. As the ingestion of small amounts can have damaging complications, there has been an ongoing discussion regarding the safe threshold for dietary residual gluten. The aim was to analyze the evolution of gluten content in cereal-based GF foodstuffs ( n = 3141) from 1998 to 2016 measured by the enzyme-linked immunosorbent assay (ELISA) technique. Eight categories were defined: flours, breakfast cereals/bars, bakery, pasta, breads, dough, snacks, and yeasts, and these were divided into GF labeled-foods (GF-L) or reportedly GF foodstuffs, but not certified (GF-NC). Gluten-detection was decreased over time in line with the evolving European regulations about food information and gluten content claims. This decline started sooner in GF-L products than in GF-NC. As a whole, gluten was detected in 371 samples, with breakfast cereals/bars being the most contaminated group. Snacks and yeasts changed from being high gluten-detected samples to being totally GF over the years. The downside is that, of contaminated samples, those in the low levels of gluten detection range have decreased while flour samples containing over 100 mg/kg gluten have risen in the 2013-2016 period. Obtained data confirm that GF cereal-based foods are becoming safer but gluten control must be maintained.

Updates in the Development of ImmunoRNases for the Selective Killing of Tumor Cells

PubMed Central

Jordaan, Sandra; Akinrinmade, Olusiji A.; Nachreiner, Thomas; Cremer, Christian; Naran, Krupa; Chetty, Shivan; Barth, Stefan

2018-01-01

Targeted cancer therapy includes, amongst others, antibody-based delivery of toxic payloads to selectively eliminate tumor cells. This payload can be either a synthetic small molecule drug composing an antibody-drug conjugate (ADC) or a cytotoxic protein composing an immunotoxin (IT). Non-human cytotoxic proteins, while potent, have limited clinical efficacy due to their immunogenicity and potential off-target toxicity. Humanization of the cytotoxic payload is essential and requires harnessing of potent apoptosis-inducing human proteins with conditional activity, which rely on targeted delivery to contact their substrate. Ribonucleases are attractive candidates, due to their ability to induce apoptosis by abrogating protein biosynthesis via tRNA degradation. In fact, several RNases of the pancreatic RNase A superfamily have shown potential as anti-cancer agents. Coupling of a human RNase to a humanized antibody or antibody derivative putatively eliminates the immunogenicity of an IT (now known as a human cytolytic fusion protein, hCFP). However, RNases are tightly regulated in vivo by endogenous inhibitors, controlling the ribonucleolytic balance subject to the cell’s metabolic requirements. Endogenous inhibition limits the efficacy with which RNase-based hCFPs induce apoptosis. However, abrogating the natural interaction with the natural inhibitors by mutation has been shown to significantly enhance RNase activity, paving the way toward achieving cytolytic potency comparable to that of bacterial immunotoxins. Here, we review the immunoRNases that have undergone preclinical studies as anti-cancer therapeutic agents. PMID:29510557

Updates in the Development of ImmunoRNases for the Selective Killing of Tumor Cells.

PubMed

Jordaan, Sandra; Akinrinmade, Olusiji A; Nachreiner, Thomas; Cremer, Christian; Naran, Krupa; Chetty, Shivan; Barth, Stefan

2018-03-05

Targeted cancer therapy includes, amongst others, antibody-based delivery of toxic payloads to selectively eliminate tumor cells. This payload can be either a synthetic small molecule drug composing an antibody-drug conjugate (ADC) or a cytotoxic protein composing an immunotoxin (IT). Non-human cytotoxic proteins, while potent, have limited clinical efficacy due to their immunogenicity and potential off-target toxicity. Humanization of the cytotoxic payload is essential and requires harnessing of potent apoptosis-inducing human proteins with conditional activity, which rely on targeted delivery to contact their substrate. Ribonucleases are attractive candidates, due to their ability to induce apoptosis by abrogating protein biosynthesis via tRNA degradation. In fact, several RNases of the pancreatic RNase A superfamily have shown potential as anti-cancer agents. Coupling of a human RNase to a humanized antibody or antibody derivative putatively eliminates the immunogenicity of an IT (now known as a human cytolytic fusion protein, hCFP). However, RNases are tightly regulated in vivo by endogenous inhibitors, controlling the ribonucleolytic balance subject to the cell's metabolic requirements. Endogenous inhibition limits the efficacy with which RNase-based hCFPs induce apoptosis. However, abrogating the natural interaction with the natural inhibitors by mutation has been shown to significantly enhance RNase activity, paving the way toward achieving cytolytic potency comparable to that of bacterial immunotoxins. Here, we review the immunoRNases that have undergone preclinical studies as anti-cancer therapeutic agents.

Nebulised amphotericin B-polymethacrylic acid nanoparticle prophylaxis prevents invasive aspergillosis

PubMed Central

Shirkhani, Khojasteh; Teo, Ian; Armstrong-James, Darius; Shaunak, Sunil

2015-01-01

Aspergillus species are the major life threatening fungal pathogens in transplant patients. Germination of inhaled fungal spores initiates infection, causes severe pneumonia, and has a mortality of > 50%. This is leading to the consideration of pre-exposure prophylaxis to prevent infection. We made a very low MWt amphotericin B-polymethacrylic acid nanoparticle. It was not toxic to lung epithelial cells or monocyte-derived-macrophages in-vitro, or in an in-vivo transplant immuno-suppression mouse model of life threatening invasive aspergillosis. Three days of nebuliser based prophylaxis delivered the nanoparticle effectively to lung and prevented both fungal growth and lung inflammation. Protection from disease was associated with > 99% killing of the Aspergillus and a 90% reduction in lung TNF-α; the primary driver of tissue destructive immuno-pathology. This study provides in-vivo proof-of-principle that very small and cost-effective nanoparticles can be made simply, and delivered safely and effectively to lung by the aerosol route to prevent fungal infections. From the Clinical Editor Aspergillus is an opportunistic pathogen, which affects immunocompromised patients. One novel way to help fight against this infection is pre-exposure prophylaxis. The authors here made PMA based anionic hydrogels carrying amphotericin B, with mucoadhesive behavior. They showed that aerosol route of the drug was very effective in protecting against the disease in an in-vivo model and should provide a stepping-stone towards clinical trials in the future. PMID:25791815

Nebulised amphotericin B-polymethacrylic acid nanoparticle prophylaxis prevents invasive aspergillosis.

PubMed

Shirkhani, Khojasteh; Teo, Ian; Armstrong-James, Darius; Shaunak, Sunil

2015-07-01

Aspergillus species are the major life threatening fungal pathogens in transplant patients. Germination of inhaled fungal spores initiates infection, causes severe pneumonia, and has a mortality of >50%. This is leading to the consideration of pre-exposure prophylaxis to prevent infection. We made a very low MWt amphotericin B-polymethacrylic acid nanoparticle. It was not toxic to lung epithelial cells or monocyte-derived-macrophages in-vitro, or in an in-vivo transplant immuno-suppression mouse model of life threatening invasive aspergillosis. Three days of nebuliser based prophylaxis delivered the nanoparticle effectively to lung and prevented both fungal growth and lung inflammation. Protection from disease was associated with >99% killing of the Aspergillus and a 90% reduction in lung TNF-α; the primary driver of tissue destructive immuno-pathology. This study provides in-vivo proof-of-principle that very small and cost-effective nanoparticles can be made simply, and delivered safely and effectively to lung by the aerosol route to prevent fungal infections. Aspergillus is an opportunistic pathogen, which affects immunocompromised patients. One novel way to help fight against this infection is pre-exposure prophylaxis. The authors here made PMA based anionic hydrogels carrying amphotericin B, with mucoadhesive behavior. They showed that aerosol route of the drug was very effective in protecting against the disease in an in-vivo model and should provide a stepping-stone towards clinical trials in the future. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Synergistic effect of EMF-BEMER-type pulsed weak electromagnetic field and HPMA-bound doxorubicin on mouse EL4 T-cell lymphoma.

PubMed

Říhová, Blanka; Etrych, Tomáš; Šírová, Milada; Tomala, Jakub; Ulbrich, Karel; Kovář, Marek

2011-12-01

We have investigated the effects of low-frequency pulsed electromagnetic field (LF-EMF) produced by BEMER device on experimental mouse T-cell lymphoma EL4 growing on conventional and/or athymic (nude) mice. Exposure to EMF-BEMER slowed down the growth of tumor mass and prolonged the survival of experimental animals. The effect was more pronounced in immuno-compromised nude mice compared to conventional ones. Acceleration of tumor growth was never observed. No measurable levels of Hsp 70 or increased levels of specific anti-EL4 antibodies were detected in the serum taken from experimental mice before and at different intervals during the experiment, i.e. before solid tumor appeared, at the time of its aggressive growth, and at the terminal stage of the disease. A significant synergizing antitumor effect was seen when EL4 tumor-bearing mice were simultaneously exposed to EMF-BEMER and treated with suboptimal dose of synthetic HPMA copolymer-based doxorubicin, DOX(HYD)-HPMA. Such a combination may be especially useful for heavily treated patients suffering from advanced tumor and requiring additional aggressive chemotherapy which, however, at that time could represent almost life-threatening way of medication.

A qPCR-Based Tool to Diagnose the Presence of Harmful Cyanobacteria and Cyanotoxins in Drinking Water Sources.

PubMed

Chiu, Yi-Ting; Chen, Yi-Hsuan; Wang, Ting-Shaun; Yen, Hung-Kai; Lin, Tsair-Fuh

2017-05-20

Harmful cyanobacteria have been an important concern for drinking water quality for quite some time, as they may produce cyanotoxins and odorants. Microcystis and Cylindrospermopsis are two common harmful cyanobacterial genera detected in freshwater lakes and reservoirs, with microcystins (MCs) and cylindrospermopsin (CYN) as their important metabolites, respectively. In this study, two sets of duplex qPCR systems were developed, one for quantifying potentially-toxigenic Microcystis and Microcystis , and the other one for cylindrospermopsin-producing cyanobacteria and Cylindrospermopsis . The duplex qPCR systems were developed and validated in the laboratory by using 338 samples collected from 29 reservoirs in Taiwan and her offshore islands. Results show that cell numbers of Microcystis and Cylindorspermopsis enumerated with microscopy, and MCs and CYN concentrations measured with the enzyme-linked immuno-sorbent assay method, correlated well with their corresponding gene copies determined with the qPCR systems (range of coefficients of determination R² = 0.392-0.740). The developed qPCR approach may serve as a useful tool for the water industry to diagnose the presence of harmful cyanobacteria and the potential presence of cyanotoxins in source waters.

Functionalized vertically aligned ZnO nanorods for application in electrolyte-insulator-semiconductor based pH sensors and label-free immuno-sensors

NASA Astrophysics Data System (ADS)

Kumar, Narendra; Senapati, Sujata; Kumar, Satyendra; Kumar, Jitendra; Panda, Siddhartha

2016-04-01

Vertically aligned ZnO nanorods were grown on a SiO2/Si surface by optimization of the temperature and atmosphere for annealing of the seed. The seed layer annealed at 500 °C in vacuum provided well separated and uniform seeds which also provided the best condition to get densely packed, uniformly distributed, and vertically aligned nanorods. These nanorods grown on the substrates were used to fabricate electrolyte-insulator-semiconductor (EIS) devices for pH sensing. Etching of ZnO at acidic pH prevents the direct use of nanorods for pH sensing. Therefore, the nanorods functionalised with 3-aminopropyltriethoxysilane (APTES) were utilized for pH sensing and showed the pH sensitivity of 50.1 mV/pH. APTES is also known to be used as a linker to immobilize biomolecules (such as antibodies). The EIS device with APTES functionalized nanorods was used for the label free detection of prostate-specific antigen (PSA). Finally, voltage shifts of 23 mV and 35 mV were observed with PSA concentrations of 1 ng/ml and 100 ng/ml, respectively.

A false positive newborn screening result due to a complex allele carrying two frequent CF-causing variants.

PubMed

Bergougnoux, Anne; Boureau-Wirth, Amandine; Rouzier, Cécile; Altieri, Jean-Pierre; Verneau, Fanny; Larrieu, Lise; Koenig, Michel; Claustres, Mireille; Raynal, Caroline

2016-05-01

The detection of two frequent CFTR disease-causing variations in the context of a newborn screening program (NBS) usually leads to the diagnosis of cystic fibrosis (CF) and a relevant genetic counseling in the family. In the present study, CF-causing variants p.Phe508del (F508del) and c.3140-26A>G (3272-26A>G) were identified on a neonate with positive ImmunoReactive Trypsinogen test by the Elucigene™ CF30 kit. The CF diagnosis initially suggested, despite three inconclusive Sweat Chloride Tests (SCT), was finally ruled out after the familial segregation study combined with a negative SCT. Haplotype studies, based on the comparison of 80 p.Phe508del haplotypes, suggested a probable de novo occurrence of c.3140-26A>G on the p.Phe508del ancestral allele in this family. This false positive case emphasizes the importance of SCT in the NBS strategy. Moreover, it raises the need for familial segregation studies in CF and in overall molecular diagnosis strategy of autosomal recessive diseases. Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

A qPCR-Based Tool to Diagnose the Presence of Harmful Cyanobacteria and Cyanotoxins in Drinking Water Sources

PubMed Central

Chiu, Yi-Ting; Chen, Yi-Hsuan; Wang, Ting-Shaun; Yen, Hung-Kai; Lin, Tsair-Fuh

2017-01-01

Harmful cyanobacteria have been an important concern for drinking water quality for quite some time, as they may produce cyanotoxins and odorants. Microcystis and Cylindrospermopsis are two common harmful cyanobacterial genera detected in freshwater lakes and reservoirs, with microcystins (MCs) and cylindrospermopsin (CYN) as their important metabolites, respectively. In this study, two sets of duplex qPCR systems were developed, one for quantifying potentially-toxigenic Microcystis and Microcystis, and the other one for cylindrospermopsin-producing cyanobacteria and Cylindrospermopsis. The duplex qPCR systems were developed and validated in the laboratory by using 338 samples collected from 29 reservoirs in Taiwan and her offshore islands. Results show that cell numbers of Microcystis and Cylindorspermopsis enumerated with microscopy, and MCs and CYN concentrations measured with the enzyme-linked immuno-sorbent assay method, correlated well with their corresponding gene copies determined with the qPCR systems (range of coefficients of determination R2 = 0.392−0.740). The developed qPCR approach may serve as a useful tool for the water industry to diagnose the presence of harmful cyanobacteria and the potential presence of cyanotoxins in source waters. PMID:28531121

An Asymmetry in the Automatic Detection of the Presence or Absence of a Frequency Modulation within a Tone: A Mismatch Negativity Study

PubMed Central

Timm, Jana; Weise, Annekathrin; Grimm, Sabine; Schröger, Erich

2011-01-01

The infrequent occurrence of a transient feature (deviance; e.g., frequency modulation, FM) in one of the regular occurring sinusoidal tones (standards) elicits the deviance related mismatch negativity (MMN) component of the event-related brain potential. Based on a memory-based comparison, MMN reflects the mismatch between the representations of incoming and standard sounds. The present study investigated to what extent the infrequent exclusion of an FM is detected by the MMN system. For that purpose we measured MMN to deviances that either consisted of the exclusion or inclusion of an FM at an early or late position within the sound that was present or absent, respectively, in the standard. According to the information-content hypothesis, deviance detection relies on the difference in informational content of the deviant relative to that of the standard. As this difference between deviants with FM and standards without FM is the same as in the reversed case, comparable MMNs should be elicited to FM inclusions and exclusions. According to the feature-detector hypothesis, however, the deviance detection depends on the increased activation of feature detectors to additional sound features. Thus, rare exclusions of the FM should elicit no or smaller MMN than FM inclusions. In passive listening condition, MMN was obtained only for the early inclusion, but not for the exclusions nor for the late inclusion of an FM. This asymmetry in automatic deviance detection seems to partly reflect the contribution of feature detectors even though it cannot fully account for the missing MMN to late FM inclusions. Importantly, the behavioral deviance detection performance in the active listening condition did not reveal such an asymmetry, suggesting that the intentional detection of the deviants is based on the difference in informational content. On a more general level, the results partly support the “fresh-afferent” account or an extended memory-comparison based account of MMN. PMID:21852979

Design of Moisture Content Detection System

NASA Astrophysics Data System (ADS)

Wang, W. C.; Wang, L.

In this paper, a method for measuring the moisture content of grain was presented based on single chip microcomputer and capacitive sensor. The working principle of measuring moisture content is introduced and a concentric cylinder type of capacitive sensor is designed, the signal processing circuits of system are described in details. System is tested in practice and discussions are made on the various factors affecting the capacitive measuring of grain moisture based on the practical experiments, experiment results showed that the system has high measuring accuracy and good controlling capacity.

Microchip ELISA coupled with cell phone to detect ovarian cancer HE4 biomarker in urine.

PubMed

Wang, ShuQi; Akbas, Ragip; Demirci, Utkan

2015-01-01

Ovarian cancer is a leading cause of death from gynecologic cancers in the USA, and early diagnosis can potentially increase 5-year survival rate. Detection of biomarkers derived from hyperplasia of epithelial tissue by enzyme-linked immunosorbent assay (ELISA) proves to be a practical way of early diagnosis of ovarian cancer. However, ELISA is commonly performed in a laboratory setting, and it cannot be used in a clinical setting for on-site consultation. We have shown a microchip ELISA that detects HE4, an ovarian cancer biomarker, from urine using a cell phone integrated with a mobile application for imaging and data analysis. In microchip ELISA, HE4 from urine was first absorbed on the surface; the primary and secondary antibodies were subsequently anchored on the surface via immuno-reaction; and addition of substrate led to color development because of enzymatic labeling. The microchip after color development was imaged using a cell phone, and the color intensity was analyzed by an integrated mobile application. By comparing with an ELISA standard curve, the concentration of HE4 was reported on the cell phone screen. The presented microchip ELISA coupled with a cell phone is portable as opposed to traditional ELISA, and this method can facilitate the detection of ovarian cancer at the point-of-care (POC).

Magnetic Particles Coupled to Disposable Screen Printed Transducers for Electrochemical Biosensing

PubMed Central

Yáñez-Sedeño, Paloma; Campuzano, Susana; Pingarrón, José M.

2016-01-01

Ultrasensitive biosensing is currently a growing demand that has led to the development of numerous strategies for signal amplification. In this context, the unique properties of magnetic particles; both of nano- and micro-size dimensions; have proved to be promising materials to be coupled with disposable electrodes for the design of cost-effective electrochemical affinity biosensing platforms. This review addresses, through discussion of selected examples, the way that nano- and micro-magnetic particles (MNPs and MMPs; respectively) have contributed significantly to the development of electrochemical affinity biosensors, including immuno-, DNA, aptamer and other affinity modes. Different aspects such as type of magnetic particles, assay formats, detection techniques, sensitivity, applicability and other relevant characteristics are discussed. Research opportunities and future development trends in this field are also considered. PMID:27681733

PubMed Central

Montplaisir, S.; Côté, P. P.; Martineau, B.; Roche, A. J.; Mongeau, J. G.; Robitaille, P.

1976-01-01

The demonstration by immuno-fluorescence of antibodies on the surface of urinary bacteria, a new method of determining the site of a urinary tract infection, was found to be as valuable in children as it is in adults. A clear correlation exists between a positive test result and renal parenchymal infection on one hand, and a negative result and lower urinary tract infection on the other. Moreover, immunoglobulins were still detectable in original positive urine samples that had been standing at 4degrees C for 7 weeks. The constant finding of IgA on bacteria suggests a particular synthesis for this class of immunoglobulin. A pathophysiologic role for complement would appear to be excluded by the facts that the serum concentrations of C3 were normal and that C3 was invariably absent from the bacterial surface. Images FIG. 1 PMID:793701

Antibodies and antimatter: the resurgence of immuno-PET.

PubMed

Wu, Anna M

2009-01-01

The completion of the human genome, coupled with parallel major research efforts in proteomics and systems biology, has led to a flood of information on the roles of individual genes and proteins in normal physiologic processes and their disruptions in disease. In practical terms, this information has opened the door to increasingly targeted therapies as specific molecular markers are identified and validated. The ongoing transition from empiric to molecular medicine has engendered a need for corresponding molecular diagnostics, including noninvasive molecular imaging. Convergence of knowledge regarding key biomarkers that define normal biologic processes and disease with protein and imaging technology makes this an opportune time to revisit the combination of antibodies and PET, or immuno-PET.

[Schimke immuno-osseous dysplasia. A pediatric disease reaches adulthood].

PubMed

Lücke, Thomas; Kanzelmeyer, Nele; Franke, Doris; Hartmann, Hans; Ehrich, Jochen H H; Das, Anibh M

2006-03-15

Schimke immuno-osseous dysplasia (SIOD) is a rare autosomal recessive multisystemic disorder caused by mutations of the SMARCAL 1 gene (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1). Main clinical features are: disproportional growth deficiency due to spondyloepiphyseal dysplasia, nephrotic syndrome with focal and segmental glomerulosclerosis, and defective cellular immunity. Patients with severe SIOD have life-limiting complications like cerebral ischemia due to vaso-occlusive processes. Only a few patients reached adulthood. The clinical course of four adult SIOD patients is presented. Even patients with severe SIOD can reach adulthood. Therefore, doctors working in the field of internal medicine and family doctors should be familiar with the clinical picture of SIOD.

16 kDa heat shock protein from heat-inactivated Mycobacterium tuberculosis is a homodimer - suitability for diagnostic applications with specific llama VHH monoclonals.

PubMed

Srivastava, Saurabh K; Ruigrok, Vincent J B; Thompson, Natalie J; Trilling, Anke K; Heck, Albert J R; van Rijn, Cees; Beekwilder, Jules; Jongsma, Maarten A

2013-01-01

The 16 kDa heat shock protein (HSP) is an immuno-dominant antigen, used in diagnosis of infectious Mycobacterium tuberculosis (M.tb.) causing tuberculosis (TB). Its use in serum-based diagnostics is limited, but for the direct identification of M.tb. bacteria in sputum or cultures it may represent a useful tool. Recently, a broad set of twelve 16 kDa specific heavy chain llama antibodies (VHH) has been isolated, and their utility for diagnostic applications was explored. To identify the epitopes recognized by the nine (randomly selected from a set of twelve 16 kDa specific VHH antibodies) distinct VHH antibodies, 14 overlapping linear epitopes (each 20 amino acid long) were characterized using direct and sandwich ELISA techniques. Seven out of 14 epitopes were recognized by 8 out of 9 VHH antibodies. The two highest affinity binders B-F10 and A-23 were found to bind distinct epitopes. Sandwich ELISA and SPR experiments showed that only B-F10 was suitable as secondary antibody with both B-F10 and A-23 as anchoring antibodies. To explain this behavior, the epitopes were matched to the putative 3D structure model. Electrospray ionization time-of-flight mass spectrometry and size exclusion chromatography were used to determine the higher order conformation. A homodimer model best explained the differential immunological reactivity of A-23 and B-F10 against heat-treated M.tb. lysates. The concentrations of secreted antigens of M.tb. in sputum are too low for immunological detection and existing kits are only used for identifying M.tb. in cultures. Here we describe how specific combinations of VHH domains could be used to detect the intracellular HSP antigen. Linked to methods of pre-concentrating M.tb. cells prior to lysis, HSP detection may enable the development of protein-based diagnostics of sputum samples and earlier diagnosis of diseases.

Prolactin variants in ram adenohypophyses vary with season

NASA Technical Reports Server (NTRS)

Stroud, C. M.; Deaver, D. R.; Peters, J. L.; Loeper, D. C.; Toth, B. E.; Derr, J. A.; Hymer, W. C.

1992-01-01

Secretion of PRL in sheep is affected by photoperiod being highest during the spring and summer, lowest in fall and winter. The objectives of this study were to determine if 1) the production of variant forms of PRL, and 2) immuno- and bioactivities of PRL (iPRL and bPRL) differ during times of the year selected to represent periods of low, transitional and high PRL secretion. Twelve mature rams were maintained on pasture and killed in October, December, and April (n = 4/month). Individual pituitary glands were dispersed, cells obtained, and fixed for immunocytochemical flow cytometry, extracted with 0.01 N NaHCO3 or cultured in serum-free, defined media. The Mr of PRL extracted from cells immediately following dispersion ranged from 14-140K, with significantly more bands greater than 40K being detected from rams sacrificed in December than from those killed in October and April (P less than 0.01). No bands of PRL greater than 25K were observed when samples were reduced with beta-mercaptoethanol prior to electrophoresis, indicating that the high Mr forms were disulfide-linked aggregates. Culture media from October and April contained variants of PRL that ranged from 22-40K but those greater than 25K were generally not observed from cells harvested during December. Extracts of cells after 24 h in culture contained fewer high Mr species during December than had been present in initial extracts from that month. In contrast, during April more high Mr intracellular forms were present after culture than had been detected prior to culture during that month. The percentage of lactotrophs averaged 50.0 +/- 2.5, 47.4 +/- 5.7, and 59.4 +/- 5.5 for October, December, and April, respectively. Initial lactotroph content (pg/lactotroph) of iPRL was higher (P = 0.06) in April (46.0 +/- 17.0) when compared to October and December (8.0 +/- 2.0 and 20.0 +/- 10.0, respectively). In contrast, the bPRL content of initial extracts was higher (P = 0.05) in December (267.0 +/- 68.0) than in October (101.0 +/- 35.0), but not than in April (190.0 +/- 70.0). Although iPRL and bPRL concentrations in culture media were similar for the 3 months, the intracellular iPRL (P less than 0.001) and bPRL (P less than 0.0001) content after culture was greatest during April. In summary, in addition to the well-documented seasonal changes in blood concentrations of PRL, different molecular forms of PRL were found within the pituitary at different times of the year and seasonal variations in iPRL and bPRL did not occur in parallel.

Early pregnancy factor (EPF) as a marker for detecting subclinical embryonic loss in clomiphene citrate-treated women.

PubMed

Shahani, S K; Moniz, C L; Gokral, J S; Meherji, P K

1995-05-01

A discrepancy exists between the apparently normal ovulation and the pregnancy rates in women treated with clomiphene citrate (CC). Our previous studies have indicated that immuno-suppressive "early pregnancy factor" (EPF) is a novel marker to detect subclinical embryonic loss in infertile women. In the present study EPF was used as a marker to detect subclinical embryonic loss in women treated with CC with/without gonadotropins. In some of the women treated with CC, conception was assisted by artificial insemination with husband's semen (AIH). Our results have indicated that fertilization occurred (EPF + ve) in 47.7% (52/109) of women treated with CC with/without gonadotropins; 13.46% (7/52) retained the fetus and continued pregnancy till full term, whereas 78.9% (41/52) did not retain the fetuses. In the group where after stimulation, conception was assisted by AIH, fertilization was observed in 38.24% (26/68), retention in 11.54% (3/26) but subclinical embryonic loss was observed in 80.8% (21/26) cases. Thus, our results have indicated that subclinical embryonic loss may account for some of the discrepancy observed between the apparently normal ovulation and the pregnancy rates in women treated with clomiphene citrate.

Screening of colorectal cancer: present and future.

PubMed

Maida, Marcello; Macaluso, Fabio Salvatore; Ianiro, Gianluca; Mangiola, Francesca; Sinagra, Emanuele; Hold, Georgina; Maida, Carlo; Cammarota, Giovanni; Gasbarrini, Antonio; Scarpulla, Giuseppe

2017-12-01

Colorectal cancer (CRC) is the third most common cancer in males and second in females, and the fourth most common cause of cancer death worldwide. Currently, about 60-70% of diagnosed cases in symptomatic patients are detected at an advanced stage of disease. Earlier stage detection through the use of screening strategies would allow for better outcomes in terms of reducing the disease burden. Areas covered: The aim of this paper is to review the current published evidence from literature which assesses the performance and effectiveness of different screening tests for the early detection of CRC. Expert commentary: Adequate screening strategies can reduce CRC incidence and mortality. In the last few decades, several tests have been proposed for CRC screening. To date, there is still insufficient evidence to identify which approach is definitively superior, and no screening strategy for CRC can therefore be defined as universally ideal. The best strategy would be the one that can be economically viable and to which the patient can adhere best to over time. The latest guidelines suggest colonoscopy every 10 years or annual fecal immuno-chemical test (FIT) for people with normal risk, while for individuals with high risk or hereditary syndromes specific recommendations are provided.

Immunolocalisation of two tropinone reductases in potato (Solanum tuberosum L.) root, stolon, and tuber sprouts.

PubMed

Kaiser, Heike; Richter, Ute; Keiner, Ronald; Brabant, Anja; Hause, Bettina; Dräger, Birgit

2006-12-01

Tropinone reductases (TRs) are essential enzymes in the tropane alkaloid biosynthesis, providing either tropine for hyoscyamine and scopolamine formation or providing pseudotropine for calystegines. Two cDNAs coding for TRs were isolated from potato (Solanum tuberosum L.) tuber sprouts and expressed in E. coli. One reductase formed pseudotropine, the other formed tropine and showed kinetic properties typical for tropine-forming tropinone reductases (TRI) involved in hyoscyamine formation. Hyoscyamine and tropine are not found in S. tuberosum plants. Potatoes contain calystegines as the only products of the tropane alkaloid pathway. Polyclonal antibodies raised against both enzymes were purified to exclude cross reactions and were used for Western-blot analysis and immunolocalisation. The TRI (EC 1.1.1.206) was detected in protein extracts of tuber tissues, but mostly in levels too low to be localised in individual cells. The function of this enzyme in potato that does not form hyoscyamine is not clear. The pseudotropine-forming tropinone reductase (EC 1.1.1.236) was detected in potato roots, stolons, and tuber sprouts. Cortex cells of root and stolon contained the protein; additional strong immuno-labelling was located in phloem parenchyma. In tuber spouts, however, the protein was detected in companion cells.