An improved gold nanoparticle probe-based assay for HCV core antigen ultrasensitive detection.

A gold nanoparticle probe-based assay (GNPA) was developed for ultrasensitive detection of Hepatitis C virus (HCV) core antigen. In the GNPA, after anti-HCV core antigen polyclonal antibodies and single-stranded barcode signal DNA were labeled on gold nanoparticle probe (NP), DNA enzyme was used to degrade the unbound barcode DNAs. The anti-HCV core antigen monoclonal antibodies were coated on magnetic microparticles probe (MMP). Then the NP-HCV core antigen-MMP sandwich immuno-complex was formed when the target antigen protein was added and captured. Magnetically separated, the immuno-complex containing the single-stranded barcode signal DNA was characterized by TaqMan probe based real-time fluorescence PCR. A detection limit of 1 fg/ml was determined for the HCV core antigen which is magnitude greater than that of ELISA (2ng/ml). The coefficients of variation (CV) of intra-assay and inter-assay respectively ranged from 0.22-2.62% and 1.92-3.01%. The improved GNPA decreased the interference of unbound barcode DNAs and may be an new way for HCV core antigen detection.

Establishment of evanescent wave fiber-optic immunosensor method for detection bluetongue virus.

The evanescent wave fiber immunosensors (EWFI) technique was developed for the real-time rapidly sensitive and specific detection of the monoclonal antibody 3E2 of BTV. The outer-core protein VP7 of BTV was labled on the surface of the exposed fiber-optic core. The monoclonal antibody 3E2 of BTV VP7 were added and then the goat ant-rat IgG conjugated with Cy3 was captured. After the 532nm pulse (excitation source) reached the fiber probe, evanescent wave was generated, which excited the Cy3 bound to the immuno-complex and produced the fluorescent signal, which was changed into electrical signals read through computer. The preliminary results suggested that a detection limit of 10ng/ml was measured for the monoclonal antibody 3E2, which is equal to the sensitivity of ELISA. The 3E2 sample was specifically detected through the EWFI assay in 15min, and the fiber can be recycled at least ten times through TEA solution condition. This developed EWFI was a real-time rapidly sensitive and specific way for the detection of BTV antibodies.

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

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.

Development of a highly sensitive gold nanoparticle probe-based assay for bluetongue virus detection.

A simple gold nanoparticle (GNP) probe based assay (GNPA) that was modified from a bio-barcode assay (BCA) technique, was developed for ultra-sensitive, rapid detection of the bluetongue virus (BTV) VP7 outer-core protein. This assay captures the VP7 target antigen using the GNP probe coated with anti-VP7 polyclonal antibodies and single-stranded signal DNA. Magnetic microparticle (MMP) probes coated with anti-VP7 monoclonal antibodies were then added to form a sandwich immuno-complex. The single-stranded signal DNA coated onto the GNP probe present in the immuno-complex could then be detected by PCR and real-time fluorescence PCR using a TaqMan probe. The assay has a purified VP7 detection limit of 10(-2)fg/ml which is 8 orders of magnitude greater than that of conventional antigen capture ELISAs and 1 order of magnitude more sensitive than that of a conventional BCA. These results indicate that the GNPA is a highly sensitive method for easy detection of BTV proteins and that it can be modified as needed to measure the presence of other proteins.

Noninvasive molecular imaging of interferon beta activation in mouse liver.

BACKGROUND/AIMS: Interferon beta (IFN-ß) is the priming cytokine in the interferons (IFNs) response that plays essential roles in innate immune system. Only very few studies on IFN activation in animals have been reported before, therefore, we embarked to develop a novel method to dynamically examine IFN-ß activation in mouse liver by noninvasive molecular imaging. METHODS: Interferon beta promoter-directed firefly luciferase gene was integrated into chromosomes of hepatocytes by hydrodynamic injection. Mouse hepatitis virus type 3 (MHV-3) and polyinosinic-polycytidylic acid [poly(I:C)] were used to stimulate the activation of IFN-ß. Luciferase activity was used as an indicator of the IFN-ß promoter activity in vitro and in vivo. The expression level of IFN-ß in the sera and firefly luciferase in the liver was assessed by ELISA and bioluminescence imaging respectively. Western blot was used for detecting proteins expression. RESULTS: A rapid and elevated luciferase expression in the mouse liver induced by poly (I:C) and MHV-3 was detected by bioluminescence imaging. The detectable level of IFN-ß in the sera was not induced by MHV-3. Moreover, IFN-ß activation was significantly inhibited by the hepatitis C virus (HCV) NS3/4A protease in mouse liver. These results were consistent with IFN-ß production in the sera. Therefore, a novel visual method to monitor IFN-ß promoter activity was established in the current study. CONCLUSION: This novel sensitive method can be used for not only assessing IFN-ß activation or inhibition in the liver under different conditions, but also screening drug candidates of stimulating or inhibiting of IFN-ß production.

A nanoparticle-based bio-barcode assay for ultrasensitive detection of ricin toxin.

The ultrasensitive bio-barcode amplification assay (BCA) technique was developed for the specific detection of the A chain of ricin toxin. The target antigen A chain was first captured by gold nanoparticles (GNPs) coated with polyclonal antibodies. Magnetic microparticles (MMPs) coated with A chain monoclonal antibody were then added to form a sandwich immuno-complex. After the immuno-complex was formed, signal DNA annealed to DNA strands covalently bound to the GNPs were released by heating and characterized by PCR and real-time fluorescence PCR. A detection limit of 1fg/ml was measured for A chain, six orders of magnitude more sensitive than that of conventional antigen-capture ELISA. The coefficient of variation (CV) of intra-assay and inter-assay ranged from 3.39% to 6.84%. The BCA can detect the A chain in milk and water mimic samples. In the following work it is demonstrated that this assay is a highly sensitive method for the detection of ricin proteins that could be adapted to measure other proteins.

Nanoparticle-based bio-barcode assay for the detection of bluetongue virus.

The ultrasensitive bio-barcode amplification assay (BCA) technique was developed for the specific detection of the outer-core protein VP7 of bluetongue virus (BTV). The target antigen VP7 was first captured by gold nanoparticles (GNPs) coated with polyclonal antibodies. Magnetic microparticles (MMP) coated with VP7 monoclonal antibody were then added to form a sandwich immuno-complex. After the immuno-complex was formed, signal DNA annealed to DNA strands covalently bound to the GNPs were released by heating and characterized by PCR and real-time fluorescence PCR. A detection limit of 0.1fg/ml was measured for purified VP7, seven orders of magnitude more sensitive than that of conventional antigen capture ELISA. The BCA demonstrated the same enhanced sensitivity for detecting BTV in serum samples from sheep. In the following work it is demonstrated that this assay is a highly sensitive method for the detection of BTV proteins that could be adapted to measure other proteins.

Detection and quantitation of bluetongue virus serotypes by a TaqMan probe-based real-time RT-PCR and differentiation from epizootic hemorrhagic disease virus.

Twenty-four serotypes of bluetongue virus (BTV) have been recognized world wide. Reliable and quantitative assays of virus universal detection are essential for fighting against BT. A real-time reverse transcription-polymerase chain reaction (RT-PCR) with a TaqMan fluorescence probe has been developed for detection of the NS1 gene of different BTV serotypes. In BHK-21 cells, in the assay detected BTV1-22 specifically, and had no cross-reactivity with the closely related epizootic hemorrhagic disease virus (EHDV) serotypes 1-5. The limit of sensitivity of the assay was 0.1 TCID(50)/ml for BTV-1 and 10(2) copies for the control R121/pGEM. Accurate quantitation can be achieved with samples containing between 10(2) and 10(6) copies. The coefficient of variation (CV) of intra-assay and inter-assay ranged from 2.17% to 5.60%. The developed real-time RT-PCR assay showed good coincident rate (99.2%) with duplex RT-PCR in 122 whole blood clinical samples from sheep. Therefore, the real-time RT-PCR can be a reliable method for detection of various serotypes of BTV.

[The construction of reporter plasmid of ULBP1 and preliminary studying on the influence of NS3/4A on transcription of ULBP1].

AIM: To construct a report vector of ULBP1 promoter gene and preliminary study on the influence of NS3/4A on transcription of ULBP1. METHODS: ULBP1 core promoter sequence was amplified by PCR, and inserted into pGL3-enhance vector, constructing ULBP1 reporter plasmid pGL3-ULBP1; The HCV protease NS3/4A gene was subcloned into pcDNA3-Flag vector (pcDNA3-Flag-NS3/4A), and the expression of this plasmid was demonstrated by Western blot. The influence of inhibition by NS3/4A on the level of ULBP1 transcription was tested by assaying the Luciferase activity in cells transfected with pGL3-ULBP1 with Luminometer. RESULTS: The reporter plasmid of ULBP1 promoter gene and the eukaryotic expression plasmid Flag-NS3/4A have been constructed and expressed successfully; and the protease NS3/4A inhibit the level of ULPB transcription. CONCLUSION: The protease NS3/4A of HCV down-regulates ULBP1 expression by inhibiting the transcription of ULBP1.

[Epidemiological survey on the infection of hepatitis E virus among pigs in Henan province].

OBJECTIVE: To investigate hepatitis E virus (HEV) infection among pigs in Henan province. METHODS: A total of 623 swine sera, collected from 5 districts, were divided into two groups, under 3-month of age and over 3-month of age. They were tested for HEV antigen and antibody by using ELISAs, respectively. The sera positive for HEV antigen were tested for HEV RNA with RT-PCR. The positive products of RT-PCR were cloned and sequenced. RESULTS: The positive rates of anti-HEV antibody of the groups under 3-month and over 3-month of age were 90.27% and 92.55%, respectively, without statistical difference, while those of HEV antigen were 15.93% and 5.69%, respectively, with significant difference. The positive rates of anti-HEV antibody and HEV antigen were significantly different among different districts. HEV RNA was detectable in 5 of 47 HEV antigen positive samples. The sequence analysis showed that in 4 of 5 specimens the sequence belonged to genotype 4 while in the remaining one the sequence was genotype 1. CONCLUSION: The prevalence rate of HEV infection in pigs was high in Henan province and the rate differed in different districts.

[Secretable eukaryotic expression of recombinant chicken Iglambda and preparation of monoclonal antibodies against chicken Iglambda].

AIM: To construct the eukaryotic expression vector for chicken Iglambda light chain, to express it on COS7 cells and to prepare the monoclonal antibodies against chicken Iglambda. METHODS: The cDNA of chicken Iglambda light chain with signal peptide sequence was amplified and then inserted into eukaryotic expression plasmid pcDNA3 after double enzyme cutting. The constructed recombinant vector was transfected into COS7 cells by lipofectamin and the secretable eukaryotic expression of chicken Iglambda light chain was verified by Western blot. The monoclonal antibodies (mAbs) against chicken Iglambda light chain were prepared by immunizing BALB/c mice with 2 x 10(6) chicken B cells and by cell fusion technology. RESULTS: The eukaryotic expression vector was successfully constructed. Western blot demonstrated that chicken Iglambda light chain existed in the cultural supernatant. The hybridoma lines secreting anti-Iglambda mAbs were screened by indirect ELISA. The specific reactivity between anti-Iglambda mAbs and recombinant chicken Iglambda light chain was detected by Western blot. CONCLUSION: The secreted recombinant chicken Iglambda light chain and anti-Iglambda mAbs provide a basis for further study of the functions of chicken Iglambda.

Changes of phosphatidylserine distribution in human red blood cells during the process of loading sugars.

The plasma membrane of red blood cells permits sugars to be loaded into the cytoplasm simply by incubation in a suitable buffer solution containing the sugar. This may provide some hope for the freeze-drying of human red blood cells. However, the effect of the loading process on red blood cells has not been fully investigated. The exposure of phosphatidylserine (PS) on the surface of the cell can be recognized by macrophages and result in shortened circulation in vivo. This study evaluates the effects of the concentration, the incubation time, and the temperature of exposure of human red blood cells to extracellular trehalose or glucose. Exposure of PS was demonstrated by annexin V labeling. It was shown that the efficiency of loading of glucose was significantly greater than that of trehalose. The loading efficiency of both sugars increased with increase in extracellular sugar concentration, prolongation of incubation time, and increase of incubation temperature. The percentages of cells with exposed PS and of damaged cells were dependent on the extracellular sugar concentration, the incubation time, and the temperature. With an extracellular glucose concentration of 0.8M, the percentage of cells with exposed PS was more than 80% and significantly higher than that of red blood cells loaded with trehalose (approximate 20%, P<0.01). As the incubation time was prolonged, the percentage of PS exposure and of damaged cells also increased. After incubation for 5h, the percentage of red cells with exposed PS following loading with glucose was more than 80% and significantly higher than that of cells loaded with trehalose (40%, P<0.01). In addition, the incubation temperature had a major effect on PS exposure. The percentage of cells with PS exposure and the proportion of damaged cells increased with increase of incubation temperature. At 37 degrees C, the percentage of cells with exposed PS and of damaged cells after loading with glucose was more than 80% and significantly higher than that of cells loaded with trehalose (P<0.01). However, when the temperature was below 25 degrees C, the percentage of cells with exposed PS and of damaged cells after loading with glucose or trehalose were both less than 10%. In conclusion, the loading efficiency for glucose was higher than that for trehalose, but the lesser effect of trehalose on exposure of PS suggests that it can maintain the asymmetrical distribution of membrane phospholipids and the intracellular trehalose can increase the osmotic tolerance of cells.

[Progress in the study of lyophilization of human red blood cells--review].

Now the clinical preservation methods of human red blood cells mainly include hypothermic storage (4 degrees C) and cryopreservation (-80 degrees C or -196 degrees C). The preservation time of hypothermic storage of red blood cells is relatively short and it is easy to be contaminated by microbes. Cryopreservation greatly prolongs the storage time, but it needs heavy storage equipments. Because the protective solutions in cryopreservation contain glycerol, red blood cells need complicated washing in order to remove glycerol. These shortage methods limit their application to some special conditions, such as war or natural disasters. Compared with conventional preservation methods of red blood cells, lyophilization has many advantages such as less weight, convenient transportation, room temperature preservation, prone to be rehydrated. In this review, the progress and challenge in the development of lyophilization of red blood cells, especially application of trehalose and its mechanism in the lyophilization of red blood cells were systematically discussed. This review can provide some theoretic guidance for developing a safe, simple and efficient preservation approach of red blood cells by lyophilization.

[Preparation and characterization of non-agglutinating mAbs against membrane antigen on human erythrocytes].

AIM: To prepare monoclonal antibodies (mAbs) against membrane antigens on human erythrocytes and characterize their properties. METHODS: BALB/c mice were immunized with the membrane antigens of human type O erythrocytes. The splenocytes of the immunized mice were fused with Sp2/0 myeloma cells by hybridoma technique. The antibodies to common antigen on human erythrocytes were screened by hexadimethrine bromide (polybrene) test tube method and then the agglutinating antibodies (complete antibodies) were weeded out by slide hemagglutination assay. The hybridoma cells secreting non-agglutinating antibodies (incomplete antibodies) were cloned by limiting dilution method. The stability of the obtained hybridoma cells and the properties of the mAbs were identified. RESULTS: One hybridoma cell 2E8 was obtained, which secreted non-agglutinating antibody. The mAb 2E8 belonged to IgG1, could agglutinate H antigen, and had no species cross-agglutination reaction. The titers of culture supernatant and ascites of 2E8 were 1:1,024 and 1:64x10(6), respectively. When the affinity of mAb 2E8 was evaluated by agglutination reaction, erythrocytes began to agglutinate after 7 seconds and the clots exceeded 1 mm(2) in 3 minutes. CONCLUSION: The non-agglutinating mAb against H antigen was prepared successfully. The mAb 2E8 has good titer, affinity and specificity, which lays the foundation for preparation of bispecific antibodies (BsAb).

Fluorescence microplate reader measurement of tissue susceptibility to lipid peroxidation.

This unit describes a method for the measurement of cellular membrane antioxidant capacity or susceptibility of tissue samples to lipid peroxidation using a fluorescence microplate reader. The assay is simple and has the advantage of monitoring susceptibility to lipid peroxidation in a large number of samples in real time.

Vitamin E therapy in Parkinson's disease.

Though the etiology is not well understood, late-onset Parkinson's disease (PD) appears to result from several key factors including exposure to unknown environmental toxicants, toxic endogenous compounds and genetic alterations. A plethora of scientific evidence suggest that these environmental and endogenous factors cause PD by producing mitochondrial (mito) oxidative stress and damage in the substantia nigra, leading to cell death. Thus assuming a critical role for mito oxidative stress in PD, therapies to treat or prevent PD must target these mito and protect them against oxidative damage. The focus of this article is to briefly review the experimental and clinical evidence for the role of environmental toxicants and mito oxidative stress/damage in PD as well as discuss the potential protective role of mito d-alpha-tocopherol (T) enrichment and vitamin E therapy in PD. New experimental data are presented that supports the enrichment of mito with T as a critical event in cytoprotection against toxic mito-derived oxidative stress. We propose that chronic, high dose vitamin E dietary supplementation or parenteral vitamin E administration (e.g. vitamin E succinate) may serve as a successful therapeutic strategy for the prevention or treatment of PD (by enriching substantia nigra mito with protective levels of T).

[Study on the origin of Rana temporaria for quality Oviduetus Ranae].

OBJECTIVE: To determine the origin of Rana temporaria for quality Oviduetus Ranae in the light of historical documents and modern researches on the classification of Rana temporaria chensinensis. METHOD: Works of Chinese meteria medica of all ages, related historical documents and reports from home and abroad on researches of R. temporaria chensinensis were consulted, sorted out, analyzed and summarized. RESULT: The original Shange recorded in the works of Chinese meteria medica is R. temporaria chensinensis, which is the independent species, not one of species of European forest frogs. R. temporaria chensinensis is divided into 4 subspecies: R. temporaria chensinensis, Lanzhou, Kangding, and Changbaishan. The origin of R. temporaria is Changbaishan subspecies of R. temporaria chensinensis. CONCLUSION: Changbaishan subspecies of R. temporaria chensinensis is determined as the origin for quality Oviduetus Ranae.

Thenoyltrifluoroacetone, a potent inhibitor of carboxylesterase activity.

Thenoyltrifluoroacetone (TTFA), a conventional mitochondrial complex II inhibitor, was found to inhibit purified porcine liver carboxylesterase non-competitively with a K(i) of 0.61x10(-6)M and an IC(50) of 0.54x10(-6)M. Both rat plasma and liver mitochondrial esterases were inhibited in a concentration-dependent fashion. Results indicate that TTFA is a potent inhibitor of carboxylesterase activity, in addition to its ability to inhibit mitochondrial complex II activity. Therefore, caution is warranted in using TTFA as a mitochondrial complex inhibitor in combination with esterase substrates, such as fluorescence probes or vitamin E esters.