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This work reports an electrofluorochromic strategy on the basis of electric field control of fluorescent signal generation on bipolar electrodes (BPEs) for visualizing cancer cell surface glycoprotein (mucin 1). The device included two separate cells: anodic sensing cell and cathodic reporting cell, which were connected by a screen-printing electrode patterned on poly(ethylene terephthalate) (PET) membrane. In the sensing cell, anti-MUC1 antibody immobilized on a chitosan-multiwalled carbon nanotube (CS-MWCNT)-modified anodic BPE channel was used for capturing mucin-1 (MUC1) or MCF-7 cancer cells. Then ferrocene (Fc)-labeled mucin 1 aptamers were introduced through hybridization. Under an applied voltage, the ferrocene was oxidized and the electroactive molecules of 1,4-benzoquinone (BQ) in the cathodic reporting cell were reduced according to electroneutrality. This produced a strongly basic 1,4-benzoquinone anion radical (BQ•–), which turned on the fluorescence of pH-responsive fluorescent molecules of ...

This work proposed an electrochemiluminescence (ECL) immunosensor for quantitative monitoring of cardiac troponin I (cTnI) in plasma with soybean peroxidase (SBP) labeled-antibody as signal amplifier. The ECL sandwich immunosensor was constructed by covalent binding anti-cTnI capture antibody (Ab1) to polyethylenimine-functionalized graphene matrix, which was obtained by a simple hydrothermal reaction between polyethylenimine (PEI) and graphene oxide (GO). After that, the SBP-labeled detection antibody (SBP-Ab2), synthesized by NaIO4 method, was immobilized on the surface of electrode through sandwich immunoreaction. The SBP on electrode surface displayed strong and stable ECL signal of luminol in the presence of H2O2, which could be used for cTnI detection with a concentration range of 5-30,000pg/mL and a detection limit of 3.3pg/mL. This proposed SBP-modified immunosensor displayed high sensitivity, selectivity and accuracy, and was expected not only to detect cTnI in practical human plasma sample but also to be used in other biomarkers detection.

Background aims Cytokine-induced killer cells (CIKs) were shown to be a promising tool in the quest for new therapeutic approaches in the setting of metastatic solid tumors refractory to standard treatments. However, there is a practical clinical problem of different expansion rates and cell function as individual variability exists. Stimulatory molecular 4-1BB could promote division and survival of T cells and enhance effector activity including cytokine production. This study aimed to invest the contribution of co-stimulation signal to CIKs production for exploring new strategies, which increase the expansion and reliability of CIKs generation to improve access to CIKs therapy. Methods We studied the larger-scale expansion of CIKs cultured with engineered cells for costimulatory enhancement (ECCE) consisting of K562 cells that expressed 4-1BBL in heavily pretreated patients with solid tumor. The proliferation and cytotoxic capacity of CIKs were evaluated. Phenotypes of CIKs were analyzed using flow cytometry. Cytokine levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α were detected using enzyme-linked immunosorbent assay (ELISA). Results The proliferation and cytotoxic activity of CIKs were significantly up-regulated by ECCE. The percentages of CD3 + CD8 + and CD3 + CD56 − CIKs were significantly increased while the percentage of CD3 + CD56 + CIKs was decreased. In addition, the secretion of IFN-γ and TNF-α by CIKs could also be enhanced significantly when ECCE were added into the culture system. Conclusions This study suggests that ECCE may improve the efficacy of CIKs therapy and make CIKs therapy possible for the patients whose CIKs would be hard to be cultured using conventional methods.

An immunosensing biochip for simultaneous detection of three biomarkers related to acute myocardial infarction (AMI) was developed based on anionic soybean peroxidase (SBP) functionalized nanoprobe and chemiluminescent imaging. The nanoprobes (Ab2-SiO2-SBP) were fabricated by co-immobilization of SBP and one of the detection polyclonal antibodies, anti-cardiac troponin I antigen (anti-cTnI), anti-creatine kinase-MB (anti-CK-MB) and anti-myoglobin (anti-Myo), on the silica nanoparticle surface. The detection sensitivity was enhanced since the large surface area of silica carriers increased the loading of SBP for per sandwiched immunoreaction. The immunosensing biochip designed as 3 × 8 wells array was constructed by simultaneously immobilizing three capture monoclonal antibodies on the same one microtiter well with 2 × 3 active spots. In the presence of target protein, the nanoprobes will be attached onto the spots with high specificity through the sandwiched immunoreactions, which triggered the chemiluminescence (CL) signals on each sensing site of the microtiter plates and allowed to CL imaging of three biomarkers in one well at the same time. Therefore, the proposed biochip was a promising convenient strategy for simultaneous detection of cTnI, CK-MB and Myo, which showed potential application for multianalyte determination in clinical diagnostics.

Bright blue luminescent graphene quantum dots (GQDs) with major graphitic structured nanocrystals and a photoluminescence (PL) quantum yield of 15.5% were synthesized and used to monitor DNA damage. The GQDs were prepared by ultraviolet irradiation without using a chemical agent. The as-prepared GQDs showed excitation-dependent PL and stable electrochemiluminescence (ECL) behaviors. Gold nanoparticles (AuNPs) were linked with a probe of single-stranded DNA (cp53 ssDNA) to form AuNPs-ssDNA. The ECL signal of the GQDs could be quenched by non-covalent binding of the AuNPs-ssDNA to the GQDs, due to the occurrence of an electrochemiluminescence resonance energy transfer between the GQDs and the AuNPs. When AuNPs-ssDNA was then hybridized with target p53 DNA to form AuNPs-dsDNA, the non-covalent interaction between the GQDs and the ds-DNA weakened and the ECL of the GQDs recovered. This engendered an ECL sensor for the detection of target p53 ssDNA, with a detection limit of 13 nM. The resultant ECL sensor could be used for DNA damage detection based on its different bonding ability to damaged target p53 ssDNA and cp53 ssDNA linked AuNPs. The presented method could be expanded to the development of other ECL biosensors, for the quantification of nucleic acids, single nucleotide polymorphisms or other aptamer-specific biomolecules.

An immunogold chromatographic assay was developed for quantitative determination of human chorionic gonadotropin (HCG) antigen. The monoclonal antibody to beta-HCG antigen (Mab II) conjugated gold nanoparticles (GNPs) were sprayed onto a conjugation pad for specific binding with the target protein to form an immunocomplex. The monoclonal antibody to alfa-HCG antigen (Mab I) was immobilized on the test line (T zone) of the nitrocellulose membrane (NC membrane) to capture the immunocomplex of gold nanoparticle labeled Mab II and HCG protein. Therefore, GNPs would aggregate on the test line of the NC membrane in the presence of HCG, which could be easily distinguished by the naked-eye. As for quantitative detection, the gray value of the red color in the T zone was proportional to the corresponding sample concentration. The gray value versus logarithm concentration curve presented a good linear relationship in the range of 10–600 ng mL−1. The duration of the assay was within 15 min and no professional large-scale analytical instrument was necessary for quantification. When applied in human serum analysis, the strips could reach the requirements of the clinic tests.

The hepatitis C virus (HCV) alternate reading frame protein (ARFP or F protein) of the HCV 1b genotype is a double-frameshift product of the HCV core protein (Core). The discovery of HCV F protein challenges various biological functions attributed to Core. However, the specific characteristics of the host cellular immune response to F protein during HCV infection have yet to be fully elucidated. Therefore, the present study investigated the cytokine response to HCV Core or F protein in peripheral blood mononuclear cells (PBMCs) and plasmacytoid dendritic cells (PDCs) from patients with chronic HCV and healthy donors in vitro. The results demonstrated that the levels of interferon (IFN)-α, analyzed by an enzyme-linked immunosorbent assay, secreted by PBMCs in patients positive for the anti-F protein antibody, were lower than those of patients negative for the anti-F protein antibody. Moreover, the frequency of PDCs in patients negative for the anti-F protein antibody, were higher than in the group positive for the anti-F protein antibody. Furthermore, HCV F protein and Core not only inhibited specific unmethylated CpG oligonucleotide sequences of type A (CpG‑A)-induced IFN-α production by PBMCs and PDCs, but also upregulated the production of interleukin (IL)-10 by PBMCs in patients with chronic HCV and healthy controls. Notably, following neutralization of IL-10 in the media and in vitro Core or F protein stimulation, levels of IFN-α were increased. Moreover, the results revealed that the roles of F protein and Core were similar with regard to the induction of apoptosis of PDCs in patients with chronic HCV. These findings suggest that F protein may inhibit PBMC IFN-α secretion by regulating the production of IL-10, and may contribute to an increase in the rates of apoptosis in PDCs. In conclusion, the results have revealed a potential involvement of F protein in the mechanisms of chronic hepatitis C.

A novel strategy for selective collection and detection of breast cancer cells (MCF-7) based on aptamer-cell interaction was developed. Mucin 1 protein (MUC1) aptamer (Apt1) was covalently conjugated to magnetic beads to capture MCF-7 cell through affinity interaction between Apt1 and MUC1 protein that overexpressed on the surface of MCF-7 cells. Meanwhile, a nano-bio-probe was constructed by coupling of nucleolin aptamer AS1411 (Apt2) to CdTe quantum dots (QDs) which were homogeneously coated on the surfaces of monodispersed silica nanoparticles (SiO2 NPs). The nano-bio-probe displayed similar optical and electrochemical performances to free CdTe QDs, and remained high affinity to nucleolin overexpressed cells through the interaction between AS1411 and nucleolin protein. Photoluminescence (PL) and square-wave voltammetric (SWV) assays were used to quantitatively detect MCF-7 cells. Improved selectivity was obtained by using these two aptamers together as recognition elements simultaneously, compared to using any single aptamer. Based on the signal amplification of QDs coated silica nanoparticles (QDs/SiO2), the detection sensitivity was enhanced and a detection limit of 201 and 85 cells mL(-1) by PL and SWV method were achieved, respectively. The proposed strategy could be extended to detect other cells, and showed potential applications in cell imaging and drug delivery.

The majority of hepatocellular carcinoma (HCC) patients have a poor prognosis with current therapies, and new approaches are urgently needed. We have developed a novel therapeutic cancer vaccine platform based on tumor cell derived autophagosomes (DRibbles) for cancer immunotherapy. We here evaluated the effectiveness of DRibbles-pulsed dendritic cell (DC) immunization to induce anti-tumor immunity in BALB/c mouse HCC and humanized HCC mouse models generated by transplantation of human HCC cells (HepG2) into BALB/c-nu mice. DRibbles were enriched from H22 or BNL cells, BALB/c-derived HCC cell lines, by inducing autophagy and blocking protein degradation. DRibbles-pulsed DC immunization induced a specific T cell response against HCC and resulted in significant inhibition of tumor growth compared to mice treated with DCs alone. Anti- tumor efficacy of the DCs-DRibbles vaccine was also demonstrated in a humanized HCC mouse model. The results indicated that HCC/DRibbles-pulsed DCs immunotherapy might be useful for suppressing the growth of residual tumors after primary therapy of human HCC.

Th1 and Th2 cytokine response has been confirmed to be correlated with the pathogenesis of HCV infection. The aim of the study is to investigate the Th1 and Th2 cytokine profiles induced by HCV alternate reading frame protein (F protein) in chronic hepatitis C patients. We assessed the immune responses specific to HCV F protein in 55 chronic HCV patients. IFN-γ, IL-2, IL-4 and IL-5 secretion by peripheral blood mononuclear cells (PBMC) post F protein stimulation were compared among HCV patients and healthy donors. Finally, the associations between HCV F protein and HLA class II alleles were explored. We found that the seroprevalence of anti-F antibodies in HCV-related hepatocellular carcinoma (HCC) patients was significantly higher than that of patients without HCC, but such a significant difference in humoral immune responses to F protein was not observed in HCV 1b-infected- and non-HCV 1b-infected-patients. Additionally, the PBMC proliferation of HCC patients was significantly lower than that of patients without HCC. Furthermore, F protein stimulation of PBMCs from F-seropositive patients resulted in Th2 biased cytokine responses (significantly decreased IFN-γ and/or IL-2 and significantly increased IL-4 and/or IL-5 levels) that reportedly may contribute to HCC progression and pathogenesis. However, no significant difference in the association between HCV F protein and HLA-DRB1*0201, 0301, 0405, 1001 and HLA-DQB1*0201, 0401, 0502, 0602 was observed in this study. These findings suggest that F protein may contribute to the HCV-associated bias in Th1/Th2 responses of chronic hepatitis C patients including the progress of HCC pathogenesis.

Objective The aim of the present study was to further evaluate the clinical value of single or joint of golgi protein 73 (GP73) and alphafetoprotein (AFP) in diagnosis of hepatocellular (HCC).

The efficacy of interferon α (IFNα) therapy for chronic hepatitis B (CHB) patients is about 40% and often associates with adverse side-effects, thus identification of an easy accessible biomarker that can predict the outcome of IFNα treatment for individual CHB patients would be greatly helpful. Recent reports by us and others show that microRNAs encoded by human cytomegalovirus (HCMV) were readily detected in human serum and can interfere with lymphocyte responses required by IFNα therapeutic effect. We thus postulate that differential expression profile of serum HCMV miRNAs in CHB patients may serve as indicator to predict the efficacy of IFNα treatment for CHB patients. Blood was drawn from 56 individual CHB patients prior to IFNα treatment. By quantifying 13 HCMV miRNAs in serum samples, we found that the levels of HCMV-miR-US4-1 and HCMV-miR-UL-148D were significantly higher in IFNα-responsive group than in IFNα-non-responsive group. In a prospective study of 96 new CHB patients, serum level of HCMV-miR-US4-1 alone classified those who were and were not responsive to IFN-α treatment with correct rate of 84.00% and 71.74%, respectively. In conclusion, our results demonstrate that serum HCMV-miR-US4-1 can serve as a novel biomarker for predicting the outcome of IFNα treatment in CHB patients.

BACKGROUD: HIV-infected homosexual men are more frequently diagnosed with Kaposi's sarcoma. With the increase of HIV-infected homosexual men in China, we urgently need to know the KS-related human herpesvirus (KSHV/HHV-8) seroprevalence in this population. To investigate the HHV-8 prevalence among HIV-positive homosexual men, we recruited 183 patients naive of antiretroviral therapy (ART) whose blood samples present HIV-antibody positive confirmed by western blot. METHODS: HIV viral load was tested using Cobas TaqMan HIV-1 test Version 2.0, CD4 T cell counts were tested using a Flow cytometry instrument. All HIV-positive blood samples were collected and screened for KSHV. Use of an immunofluorescence (IFA) test for HHV-8-Specific antibodies (anti-LANA) in the plasma. HHV-8 DNA in whole blood cells of IFA-positive subjects was quantified with Real-time polymerase chain reaction (RT-PCR). RESULTS: ALL samples showed HIV RNA positive. CD4+ T cell count was 23% cases (42/183) showed ≤200 cells/μL, 51.3% cases (94/183) showed 201-500 cells/μL and 25.7% cases (47/183) showed ≥501 cells/μL. Immunofluorescence (IFA) test demonstrated a HHV-8 prevalence of 50.8% (93/183), among which 20.4% of the cases (19/93) was HHV-8 DNA positive. HHV-8 infection have no difference among different age groups (p=0.96). Similarly, HHV-8 infection have no significant difference among different HIV viral load groups (p=0.08).However, HHV-8 infection among different CD4+ T cell count showed significant difference (P=0.0004). CONCLUSION: This study showed a high seroprevalence of human herpesvirus 8 infection in HIV- positive homosexual men.

Diagnosis of hepatitis B virus (HBV)-positive hepatocellular carcinoma (HCC), particularly HCC independent of cirrhosis etiology, presents a great challenge because of a lack of biomarkers. Here we test the hypothesis that expression profiles of microRNAs (miRNAs) in serum can serve as biomarkers for diagnosis of HBV infection and HBV-positive HCC. We recruited 513 subjects (210 controls and 135 HBV-, 48 hepatitis C virus (HCV)-, and 120 HCC-affected individuals) and employed a strategy of initial screening by Solexa sequencing followed by validation with TaqMan probe-based quantitative reverse transcription-PCR assay. First, because of a close link between chronic hepatitis B and HCC, we compared miRNA expression profiles in HBV serum with that in control serum and successfully obtained 13 miRNAs that were differentially expressed in HBV serum. This 13-miRNA–based biomarker accurately discriminated not only HBV cases from controls and HCV cases, but also HBV-positive HCC cases from control and HBV cases. Second, we directly compared miRNA expressions in HCC serum with those in controls and identified 6 miRNAs that were significantly upregulated in HCC samples. Interestingly, 2 of these miRNAs, miR-375 and miR-92a, were also identified by our first approach as HBV specific. When we employed 3 of these miRNAs (miR-25, miR-375, and let-7f) as biomarkers, we could clearly separate HCC cases from controls, and miR-375 alone had an ROC of 0.96 (specificity: 96%; sensitivity: 100%) in HCC prediction. In conclusion, our study demonstrates for the first time that serum miRNA profiles can serve as novel and noninvasive biomarkers for HBV infection and HBV-positive HCC diagnosis. Cancer Res; 70(23); 9798–807. ©2010 AACR.

Here, we describe a new approach for electrochemiluminescence (ECL) assay with Ru(bpy)(3)(2+)-encapsulated silica nanoparticle (SiO(2)@Ru) as labels. A water-in-oil (W/O) microemulsion method was employed for one-pot synthesis of SiO(2)@Ru nanoparticles. The as-synthesized SiO(2)@Ru nanoparticles have a narrow size distribution, which allows reproducible loading of Ru(bpy)(3)(2+) inside the silica shell and of alpha-fetoprotein antibody (anti-AFP), a model antibody, on the silica surface with glutaraldehyde as linkage. The silica shell effectively prevents leakage of Ru(bpy)(3)(2+) into the aqueous solution due to strong electrostatic interaction between the positively charged Ru(bpy)(3)(2+) and the negatively charged surface of silica. The porous structure of silica shell allowed the ion to move easily through the pore to exchange energy/electrons with the entrapped Ru(bpy)(3)(2+). The as-synthesized SiO(2)@Ru can be used as a label for ultrasensitive detection of biomarkers through a sandwiched immunoassay process. The calibration range of AFP concentration was 0.05-30 ng mL(-1) with linear relation from 0.05 to 20 ng mL(-1) and a detection limit of 0.035 ng mL(-1) at 3sigma. The resulting immunosensors possess high sensitivity and good analytical performance.

Transplacentally acquired maternal antibodies in infants may inhibit active immune responses to vaccines. In this study, we compared the immunogenicity of the recombinant hepatitis B vaccine, which was intramuscularly injected at 0, 1, and 6 months of age, in 71 infants born to mothers with positive or negative antibody against hepatitis B surface antigen (anti-HBs). Forty-one infants born to anti-HBs positive mothers were all positive at birth. At 2 months after the second injection, anti-HBs in 30 infants with negative maternal antibody was significantly higher than that in 41 infants with positive maternal anti-HBs (191.1 mIU/ml vs. 96.2 mIU/ml, P = 0.018). At one month after the full immunization, the anti-HBs levels had no statistical difference between maternal anti-HBs negative and positive groups, but the antibody response in infants with high maternal anti-HBs (>1000 mIU/ml) was significantly inhibited. Nevertheless, all infants had anti-HBs higher than the protective level. In conclusion, passively acquired maternal anti-HBs in infants may to some extent impair the antibody response to hepatitis B vaccine. The long-term efficacy of hepatitis B vaccine in infants with high titers of maternal anti-HBs remains to be further evaluated.

Early and accurate diagnosis is considered the key issue to prevent the further spread of viruses and facilitate influenza therapy. Herein, we report a colorimetric immunosensor for influenza A virus (IAV) based on gold nanoparticles (AuNPs) modified with monoclonal anti-hemagglutinin antibody (mAb). The immunosensor allows for a fast, simple, and selective detection of IAV. In this assay, influenza-specific antibodies are conjugated to AuNPs to create mAb–AuNP probes. Since IAV has multiple recognition sites for probes on the surface, the mAb–AuNP probes can be specifically arranged on the virus surface due to their very specific antigen recognition. In this case, this aggregation of the mAb–AuNP probes produces a red shift in the absorption spectrum due to plasmon coupling between adjacent AuNPs, and it can be detected with the naked eye as a color change from red to purple and quantified with the absorption spectral measurements. The aggregate formation is also confirmed with transmission electron microscopy (TEM) imaging and dynamic light scattering (DLS). Under the optimal conditions, the present immunoassay can sensitively measure H3N2 IAV (A/Brisbane/10/2007) with a detection limit of 7.8 hemagglutination units (HAU). This proposed immunosensor revealed high specificity, accuracy, and good stability. Notably, it is a single-step detection using AuNP probes and UV-vis spectrophotometer for readout, and no additional amplification, e.g., enzymatic, is needed to read the result. This assay depends on an ordered AuNP structure covering the virus surface and can be applied to any virus pathogen by incorporating the appropriate pathogen-specific antibody.

It is generally believed that most tumor antigens are passively released from either health or dying tumor cells as intact soluble antigens, peptide fragments complexed with heat shock proteins (HSPs), or packaged in secretary vesicles in the form of microparticles or exosomes. The passive release of tumor antigens is generally non-inflammatory and non-immunogenic; however, results from others and our laboratories suggest that autophagy is critically involved in immunogenic cell death.

This work reports a fluorescent dye insertion approach for detection of DNA damage. The capture DNA with overhanging 3'-terminus was immobilized on silicon surface to hybridize with target DNA. The intercalation of cyanine dye of thiazole orange (TO) to the double helix structure of DNA (dsDNA) allowed intense enhancement of fluorescence signal. The DNA damage with chemicals led to poor intercalation of TO into double helix structure, resulting in the decrease of the fluorescence signal. This signal decrease could be further enhanced by exonuclease III (Exo III). With this approach, the target DNA could be detected down to 47 fM. Seven chemicals were chosen as models to monitor DNA damage. The results suggested that the present strategy could be developed to detect DNA damage, to classify the damaging mechanism with chemicals and to estimate the toxic effect of chemicals.

Although T cells are the primary and most-studied targets of the Human Immunodeficiency Virus (HIV), B cells, especially memory B lymphocytes, are also chronically depleted in the course of HIV disease. Although the lack of CD4+ T cell help may explain these deficiencies, intrinsic defects in B lymphocytes appear to contribute to B cell depletion and reduced antibody (Ab) production in the setting of HIV, especially of some antigens eliciting T cell-independent responses. The gut mucosal barrier is disrupted in HIV disease, resulting in increased systemic exposure to microbial products such as Toll-Like Receptor (TLR) agonists. The association of enhanced systemic levels of TLR agonists and B cell dysfunction in HIV disease is not understood. This review discusses the potential role of microbial TLR agonists in the B cell depletion, enhanced autoantibody production and impaired responses to vaccination observed in HIV-infected hosts. Increased microbial translocation in HIV infection may drive B cells to produce autoantibodies and increase susceptibilities of B cells to apoptosis through activation-induced cell death. Determining the mechanisms of B cell perturbations in HIV disease will inform the design of novel strategies of improve immune responses to vaccines, reduce opportunistic infections and slow disease progression.

Apoptosis is involved in the pathology of a variety of diseases. The measurement of apoptosis will help us to evaluate the onset of disease and the effect of therapeutic interventions. In addition, the increased demand for understanding the early stages of apoptosis is pushing the envelope for solutions in early instance real-time monitoring of death kinetics. Here we present a novel electrochemiluminescent cytosensing strategy to quantitate apoptotic cell numbers, screen some anticancer drugs, and evaluate their effects on hepatocarcinoma cell line (HepG2) cells by utilizing the human antiphosphatidyl serine antibody (APSA) conjugated Ru(bpy)(3)(2+)-encapsulated silica nanoparticle (APSA-SiO(2)@Ru) as the detection probe. HepG2 cells were easily immobilized on the arginine-glycine-aspartic acid-serine (RGDS)-multiwalled carbon nanotubes (RGDS-MWCNTs) nanocomposite by the specific combination of RGD domains with integrin receptors on the cell surface. Then APSA-SiO(2)@Ru was introduced to the surface of apoptosis cells through the specific interaction between APSA and phosphatidylserine (PS) that distributed on the outer membrane of apoptotic cells. On the basis of the signal amplification of the APSA-SiO(2)@Ru nanoprobe, the cytosensor could respond as low as 800 cells mL(-1), showing very high sensitivity. In addition, the dynamic alterations of surface PS expression on HepG2 cells in response to drugs and the cell heterogeneity were also demonstrated. The strategy presented a promising platform for highly sensitive cytosensing and convenient screening of some clinically available anticancer drugs.

The implementation of hepatitis B surface antigen (HBsAg) screening tests has significantly enhanced blood transfusion safety. However, the transmission of HBsAg-negative blood components can still occur in the acute phase of infection during the seronegative window period or during chronic stages of infection such as occult hepatitis virus B infection (OBI). OBI, characterized by the presence of HBV infection without detectable HBsAg, is capable to elude the routine detection with HBV serologic markers and harbor a potential risk of HBV transmission through blood transfusion or organ transplantation. Here, we test the hypothesis that OBI patients have a differentially expressed profile of microRNA (miRNA) in serum, and this unique serum miRNA signature can serve as a biomarker to detect OBI. Employing TaqMan probe-based quantitative reverse transcription polymerase chain reaction (qRT-PCR), we assessed the expression level of miRNAs in serum samples. To control for miRNA quantitation, we added an exogenous plant miRNA, MIR156a, into the samples before RNA extraction and used it as an internal control. After screening 13 previously identified HBV-specific serum miRNAs, we obtained four miRNAs, let-7c, miR-23b, miR-122, and miR-150, which are differentially expressed in OBI sera compared to healthy control sera. This 4-serum miRNA signature shows a high level of accuracy in discriminating both OBI (AUC = 0.999) and HBV (AUC = 0.989) cases from the non-infected controls. Cluster analysis also demonstrates that this 4-miRNA signature can clearly separate OBI patients from the control group. Our results demonstrate for the first time that a profile of serum miRNAs can serve as a sensitive and accurate biomarker for OBI detection.

To study the preparation of hepatitis C virus (HCV) diagnosis microarray and its accuracy in diagnosis of gene type of hepatitis C virus.Probe and primer and primers were designed in 5'-untranslated region and C region of hepatitis C virus gene. The probes were synthesized by DNA synthesizer. Solutions of probe of the final concentration of 50 micromol/L were made by dissolving the probes into sodium carbonate buffer. Hepatitis C virus genotype array spotting was performed by pin-based spotting robot PixSys5500 with CMP3 pin. The gene chips were prepared by spotting the probes onto the specially treated glass sliders. Sixty HCV RNA positive serum samples were obtained from the in-patients of the Nanjing Second Hospital (experimental group), and 60 HCV RNA negative serum samples were obtained from the healthy people undergoing physical examination (control group). Quantitative examination of serum HCV RNA was made by fluorescent quantitation PCR. The HCV RNA in the serum specimens of the experimental group (with the HCV RNA concentration of more than 500 copies/ml) and of the control group (with the HCV RNA concentration of less than 500 copies/ml) was isolated and purified, underwent reversed transcription and nested PCR to be amplified, and then genotyped by gene microarray and HCV RNA sequencing. During the experiment, double blind method was used.Tested by the gene microarray, the serum specimens in the experimental group were all HCV RNA positive, out of which 46 cases were 1b type, 3 cases were 3a type, 3 cases were 3b type, 2 cases were 2a type, 2 cases were 2b type, 2 cases were 1b + 2a type, and 2 cases were 3a type. Tested by nucleotide sequencing assay, 50 cases were 1b type, 3 cases were 3a type, 3 cases were 3b type, 2 cases were 2a type, and 2 cases were 2b type. The double-blind test results showed a coincidence rate of 93.3% in genotyping HCV by these two methods.Hepatitis gene microarray can be used in detection of serum HCV RNA and in diagnostic genotyping with great accuracy.