Detection of a miRNA biomarker for cancer diagnosis using SERS tags and magnetic separation.

Detection of miR-29a, a biomarker of cancers, using SERS tags and magnetic separation is described. The assay was designed to detect the miR-29a sequence by taking the complementary sequence and splitting it into a capture and detection probe. The SERS tags comprised the highly Raman active molecule 4-mercaptobenzoic acid (4-MBA) and DNA detection probes assembled onto the surface of gold nanorods (AuNRs) through the self-assembly process. The capture DNA conjugated magnetic nanoparticles (MNPs) were applied as capture probes. The detection was based on the hybridisation and sandwich complex formation. The resultant hybridisation-dependent complexes were recovered and enriched from the samples by magnetic separation. The enriched solution containing target miRNA hybridised with capture probes were dropped on a foil-covered slide to form a droplet for SERS analysis. A characteristic spectrum of 4-MBA was observed to indicate the presence of the miR-29a in the samples. The sensitivity of the assay is examined by measuring the SERS signal of the samples containing different concentrations of the miR-29a. The SERS intensity appears to increase with the concentration of miR-29a. The limit of detection (LOD) was found to be 10 pM without any amplification process. In addition, the selectivity and feasibility of the assay in complex media are evaluated with the non-target miRNAs comprising different sequences from the target miR-29a. The system was capable of detecting the target miR-29a specifically with high selectivity. These results suggest that this solution-based SERS platform has a significant capability for simple, sensitive, and selective miR-29a analysis.

Immunogenicity of a Two-Dose Human Papillomavirus Vaccine Schedule in HIV-Infected Adolescents with Immune Reconstitution.

HIV-infected patients are at increased risk of human papillomavirus (HPV) acquisition and HPV-associated diseases. This study set out to determine whether a two-dose (2D) HPV vaccination schedule was sufficient in HIV-infected adolescents with immune reconstitution (IR) following antiretroviral treatment. Participants aged 9-15 years who had CD4 cell counts > 500 cells/mm3 and HIV-1 RNA < 40 copies/mL for at least one year were assigned to the 2D schedule, while older participants or those without IR received a three-dose (3D) schedule. Antibodies to HPV-16 and -18 were measured using a pseudovirion-based neutralization assay. A total of 96 subjects were enrolled; 31.3% and 68.7% received the 2D and 3D schedule, respectively. Of these, 66.7% and 57.6% of the 2D and 3D participants, respectively, were male. The seroconversion rates for HPV-16 and HPV-18 were 100% in all cases, except for HPV-18 in males who received the 3D schedule (97.4%). In males, the anti-HPV-16 geometric mean titers (GMTs) were 6859.3 (95% confidence interval, 4394.3-10,707.1) and 7011.1 (4648.8-10,573.9) in the 2D and 3D groups (p = 0.946), respectively, and the anti-HPV-18 GMTs were 2039.3 (1432.2-2903.8) and 2859.8 (1810.0-4518.4) in the 2D and 3D (p = 0.313) groups, respectively. In females, the anti-HPV-16 GMTs were 15,758.7 (8868.0-28,003.4) and 26,241.6 (16,972.7-40,572.3) in the 2D and 3D groups (p = 0.197), respectively, and the anti-HPV-18 GMTs were 5971.4 (3026.8-11,780.6) and 9993.1 (5950.8-16,781.1) in the 2D and 3D groups (p = 0.271), respectively. In summary, a 2D schedule is as immunogenic in young adolescents with IR as a 3D schedule in older subjects and those without IR.

Ultrasensitive detection of lung cancer-associated miRNAs by multiple primer-mediated rolling circle amplification coupled with a graphene oxide fluorescence-based (MPRCA-GO) sensor.

MicroRNAs (miRNAs) play important roles in gene regulation and have been reported as biomarkers in cancer diagnosis. Herein, we develop an isothermal miRNA detection platform based on the highly efficient, multiple primer-mediated rolling circle amplification method coupled with a graphene oxide-based fluorescence (MPRCA-GO) assay, using lung cancer-associated miRNAs (miR-21 and miR-210) and a reference miRNA (miR-16) as model targets. The combination of the designed ssDNA probe and T4 RNA ligase (T4 Rnl2) used in the MPRCA-GO assay allowed for single-base mismatch discrimination. In addition, the superfluorescence quenching ability of GO allowed for rapid fluorescence detection. The developed platform had a limit of detection as low as 0.87 fM and could detect target miRNAs in cancer cell lines and human serums. Therefore, the MPRCA-GO sensor has the potential for single nucleotide polymorphism (SNP) analysis and applications in clinical diagnostics.

Rolling circle amplification and graphene-based sensor-on-a-chip for sensitive detection of serum circulating miRNAs.

In this study, we developed a simple multiplex miRNA detection platform based on rolling circle amplification and the fluorescence quenching property of reduced graphene oxide. The detection platform could be applied on a microfluidics chip with a mobile system controller to eliminate contamination and to facilitate potential use in remote areas. As a proof of concept, two fluorescence-labeled ssDNA tags were used for detection of miR-29a and miR-144*, two miRNAs that are highly expressed in the blood circulation of some patients with cancer or tuberculosis. The circular ssDNA probes in this study were designed to have an advantage over padlock probes as they can be prepared in advance. Our multiplex miRNA detection platform exhibited high sensitivity and selectivity, with a limit of detection of 0.05 pmol. In addition, our platform could detect target miRNAs from the total miRNA population extracted from human serum or a cancer cell line. These results indicated that our miRNA sensor has the potential to provide simple and high throughput miRNA analysis for disease diagnosis and prognosis.

Identification of reference genes for circulating long noncoding RNA analysis in serum of cervical cancer patients.

Circulating lncRNAs have attracted considerable attention as potential noninvasive biomarkers for diagnosing cancers. RT-qPCR is the canonical technique for detecting circulating RNA and depends largely on stable reference genes for data normalization. However, no systematic evaluation of reference genes for serum lncRNA has been reported for cervical cancer. Here, we profiled and validated lncRNA expression from serum of cervical cancer patients and controls using microarrays and RT-qPCR. We identified lncRNA RP11-204K16.1, XLOC_012542, and U6 small nuclear RNA as the most stable reference genes based on geNorm, NormFinder, BestKeeper, delta Ct, and RefFinder. These genes were suitable also for samples from different age groups or with hemolysis. Additionally, we discovered lncRNA AC017078.1 and XLOC_011152 as candidate biomarkers, whose expression was down-regulated in cervical cancer. Our findings could aid research on circulating lncRNA and the discovery of blood-based biomarkers for cervical cancer diagnosis.

Anti-EpCAM scFv gadolinium chelate: a novel targeted MRI contrast agent for imaging of colorectal cancer.

OBJECTIVES: The development of targeted contrast agents for magnetic resonance imaging (MRI) facilitates enhanced cancer imaging and more accurate diagnosis. In the present study, a novel contrast agent was developed by conjugating anti-EpCAM humanized scFv with gadolinium chelate to achieve target specificity. MATERIALS AND METHODS: The material design strategy involved site-specific conjugation of the chelating agent to scFv. The scFv monomer was linked to maleimide-DTPA via unpaired cysteine at the scFv C-terminus, followed by chelation with gadolinium (Gd). Successful scFv-DTPA conjugation was achieved at 1:10 molar ratio of scFv to maleimide-DTPA at pH 6.5. The developed anti-EpCAM-Gd-DTPA MRI contrast agent was evaluated for cell targeting ability, in vitro serum stability, cell cytotoxicity, relaxivity, and MR contrast enhancement. RESULTS: A high level of targeting efficacy of anti-EpCAM-Gd-DTPA to an EpCAM-overexpressing HT29 colorectal cell was demonstrated by confocal microscopy. Good stability of the contrast agent was obtained and no cytotoxicity was observed in HT29 cells after 48 h incubation with 25-100 µM of Gd. Favorable imaging was obtained using anti-EpCAM-Gd-DTPA, including 1.8-fold enhanced relaxivity compared with Gd-DTPA, and MR contrast enhancement observed after binding to HT29. CONCLUSION: The potential benefit of this contrast agent for in vivo MR imaging of colorectal cancer, as well as other EpCAM positive cancers, is suggested and warrants further investigation.

Paper-based immunosensor with signal amplification by enzyme-labeled anti-p16INK4a multifunctionalized gold nanoparticles for cervical cancer screening.

The aim of this study was to develop a paper-based immunosensor for cervical cancer screening, with signal amplification by multifunctionalized gold nanoparticles (AuNPs). The AuNPs were functionalized with a highly specific antibody to the p16INK4a cancer biomarker. The signal was amplified using a combination of the peroxidase activity of horseradish peroxidase (HRP) enzyme-antibody conjugate and the peroxidase-like activity of the AuNPs. The immune complex of p16INK4a protein and multifunctionalized AuNPs was deposited on the nitrocellulose membrane, and a positive result was generated by catalytic oxidation of peroxidase enzyme substrate 3,3',5,5'-Tetramethylbenzidine (TMB). The entire reaction occurred on the membrane within 30 min. Evaluation in clinical samples revealed 85.2% accuracy with a kappa coefficient of 0.69. This proof of concept study demonstrates the successful development of a highly accurate, paper-based immunosensor that is easy to interpret using the naked eye and that is suitable for cervical cancer screening in low-resource settings.

EpCAM expression in squamous cell carcinoma of the uterine cervix detected by monoclonal antibody to the membrane-proximal part of EpCAM.

BACKGROUND: Epithelial cell adhesion molecule (EpCAM) is a promising biomarker for squamous cell carcinoma (SCC) of the uterine cervix, because it is over-expressed in various cancers of epithelial origin. However, EpCAM expression reported in previous immunohistochemistry (IHC) studies was inconsistent. We hypothesize that the membrane-distal part of EpCAM may be lost during tissue preparation, leaving only the membrane-proximal part of EpCAM available for antibody binding and IHC staining. METHODS: Two new anti-EpCAM MAbs to the membrane-proximal part (WC-2) and the membrane-distal part (WC-1) of EpCAM were generated and characterized. WC-2 was selected for its ability to detect EpCAM in cervical tissues by IHC. One hundred thirty-five archival paraffin-embedded tissues previously diagnosed as cervical SCC (n=44), high-grade (HSIL) (n=43), or low-grade (LSIL) (n=48) squamous intraepithelial lesions were examined. IHC score was collected, recorded, and analyzed for distribution, intensity, and percentage of cancer cells stained for EpCAM. RESULTS: EpCAM expression was consistently detected on cervical tissues by WC-2, but not by WC-1. EpCAM was expressed with high IHC score in the majority of cervical SCC (37/44), but not in normal epithelial area adjacent to SCC. EpCAM was also highly expressed on precancerous lesion of the cervix, particularly in HSIL. More importantly, EpCAM expression could be used to distinguish between HSIL and LSIL, according to staining distribution. HSIL tissues displayed EpCAM expression in two-thirds to full thickness of the epithelium, while in LSIL the staining was limited to the lower one-third of the thickness. The IHC score of EpCAM expression was strongly correlated with cervical cancer and grades of precancerous lesions (r=0.875, p<0.001). CONCLUSION: Only the anti-EpCAM MAb to the membrane-proximal part is able to detect EpCAM on paraffin-embedded cervical cancer tissues. A strong positive correlation between EpCAM expression level and the grades of SILs provides the possibility that EpCAM can be used to predict prognosis and severity in these patients.

Production, characterization, and in vitro effects of a novel monoclonal antibody against Mig-7.

Development of new cancer therapies based on specific recognition of molecules in cancer cells is a significant challenge, as this requires identification of such molecules (molecular targets) and subsequent development of high-affinity, selective binders (targeting molecules). While several molecular targets for cancer therapies are currently under evaluation in clinical trials, greater selectivity for cancer cells over normal cells is required to enhance efficacy. Migration-inducing gene 7 (Mig-7), a membrane protein found in various types of carcinoma cells, is a cancer-specific biomarker and a promising molecular target for targeted cancer therapies. The purpose of this study was to produce and characterize a novel monoclonal antibody (mAb) raised against an N-terminal peptide of human Mig-7 (Mig-7(1-30)). The Mig-7(1-30) peptide was conjugated with a KLH carrier protein for immunization, and the mAb specific to Mig-7 (STmAb-1) was produced using hybridoma technology. Western blot analysis showed that STmAb-1 specifically reacted with a 23-kDa Mig-7 protein expressed in cancer cell lines, and, crucially, not with primary human fibroblasts. The affinity constant (Kaff) of STmAb-1, as measured by non-competitive enzyme immunoassay, was 1.31 × 10(9) M(-1), indicating high mAb affinity against Mig-7. Immunofluorescence assays demonstrated that STmAb-1 could specifically recognize Mig-7 expressed in cancer cell lines, but not in primary human fibroblasts and keratinocytes. Moreover, STmAb-1 inhibited the growth of MCF7 and HeLa cell lines in contrast to primary human fibroblasts, highlighting its potential usefulness in the development of new cancer therapeutics.

Design and Generation of Humanized Single-chain Fv Derived from Mouse Hybridoma for Potential Targeting Application.

Single-chain variable antibody fragments (scFvs) are attractive candidates for targeted immunotherapy in several human diseases. In this study, a concise humanization strategy combined with an optimized production method for humanizing scFvs was successfully employed. Two antibody clones, one directed against the hemagglutinin of H5N1 influenza virus, the other against EpCAM, a cancer biomarker, were used to demonstrate the validity of the method. Heavy chain (VH) and light chain (VL) variable regions of immunoglobulin genes from mouse hybridoma cells were sequenced and subjected to the construction of mouse scFv 3-D structure. Based on in silico modeling, the humanized version of the scFv was designed via complementarity-determining region (CDR) grafting with the retention of mouse framework region (FR) residues identified by primary sequence analysis. Root-mean-square deviation (RMSD) value between mouse and humanized scFv structures was calculated to evaluate the preservation of CDR conformation. Mouse and humanized scFv genes were then constructed and expressed in Escherichia coli. Using this method, we successfully generated humanized scFvs that retained the targeting activity of their respective mouse scFv counterparts. In addition, the humanized scFvs were engineered with a C-terminal cysteine residue (hscFv-C) for site-directed conjugation for use in future targeting applications. The hscFv-C expression was extensively optimized to improve protein production yield. The protocol yielded a 20-fold increase in production of hscFv-Cs in E. coli periplasm. The strategy described in this study may be applicable in the humanization of other antibodies derived from mouse hybridoma.

Ultrasensitive electrochemical immunosensor based on dual signal amplification process for p16(INK4a) cervical cancer detection in clinical samples.

The p16(INK4a) (p16) is a cyclin-dependent kinase inhibitor, which has been evaluated in several studies as a diagnostic marker of cervical cancer. Immunostaining using p16 specific antibody has confirmed an over-expression of p16 protein in cervical cancer cells and its association with disease progression. This article reports an ultrasensitive electrochemical immunosensor for specific detection of p16 and demonstrates its performance for detection of solubilized p16 protein in cell lysates obtained from patients. Sandwich-based immunoreaction couple with double signal amplification strategy based on catalytic enlargement of particle tag was used for high sensitivity and specificity. The conditions were optimized to create an immunoassay protocol. Disposable screen-printed electrode modified with capture antibodies (Ab1) was selected for further implementation towards point-of-care diagnostics. Small gold nanoparticles (15 nm diameter) conjugated with detection antibodies (Ab2) were found to better serve as a detection label due to limited interference with antigen-antibody interaction. Double signal enhancement was performed by sequential depositions of gold and silver layers. This gave the sensitivity of 1.78 µA mL(ng GST-p16)(-1) cm(-2) and detection limit of 1.3 ng mL(-1) for GST-p16 protein which is equivalent to 0.49 ng mL(-1) for p16 protein and 28 cells for HeLa cervical cancer cells. In addition to purified protein, the proposed immunosensor effectively detected elevated p16 level in cervical swab samples obtained from 10 patients with positive result from standard Pap smear test, indicating that an electrochemical immunosensors hold an excellent promise for detection of cervical cancer in clinical setting.

Hybrid Nanomaterial Complexes for Advanced Phage-guided Gene Delivery.

Developing nanomaterials that are effective, safe, and selective for gene transfer applications is challenging. Bacteriophages (phage), viruses that infect bacteria only, have shown promise for targeted gene transfer applications. Unfortunately, limited progress has been achieved in improving their potential to overcome mammalian cellular barriers. We hypothesized that chemical modification of the bacteriophage capsid could be applied to improve targeted gene delivery by phage vectors into mammalian cells. Here, we introduce a novel hybrid system consisting of two classes of nanomaterial systems, cationic polymers and M13 bacteriophage virus particles genetically engineered to display a tumor-targeting ligand and carry a transgene cassette. We demonstrate that the phage complex with cationic polymers generates positively charged phage and large aggregates that show enhanced cell surface attachment, buffering capacity, and improved transgene expression while retaining cell type specificity. Moreover, phage/polymer complexes carrying a therapeutic gene achieve greater cancer cell killing than phage alone. This new class of hybrid nanomaterial platform can advance targeted gene delivery applications by bacteriophage.

Simultaneous discrimination and detection of influenza A(H1N1)pdm09 and seasonal influenza A viruses using a rapid immunogold biosensor.

A rapid immunogold biosensor for the simultaneous discrimination of influenza A(H1N1)pdm09 and seasonal influenza A viruses was developed successfully. Monoclonal antibodies (mAbs) that were specific for the hemagglutinin protein of the A(H1N1)pdm09 virus were produced, and the best mAb pairs were selected. Using an mAb that was specific for the influenza A nucleoprotein, a rapid immunogold biosensor for the discrimination and detection of A(H1N1)pdm09/seasonal influenza viruses was developed. When tested with 72 virus isolates, the system achieved 100 % detection of the A(H1N1)pdm09 virus without cross-reactivity against seasonal influenza A (H1, H3 subtypes) and B viruses, parainfluenza viruses, respiratory syncytial viruses, and adenoviruses. The detection limits for A(H1N1)pdm09 and seasonal strains were 5 × 10(2)-7.5 × 10(3) and 1 × 10(3)-7.5 × 10(5) TCID50/mL, respectively. When tested with 49 clinical specimens, the specificity was high (100 %). The sensitivity for the detection of A(H1N1)pdm09 and seasonal strains was 90 % and 100 %, respectively, which correlated with the results of real-time reverse transcription polymerase chain reaction as a reference method. The ability of the system to detect and discriminate the A(H1N1)pdm09 strain from the seasonal strains suggests that this method may be beneficial for investigation of outbreaks and diagnostic applications. Furthermore, this method might be a useful platform for developing a rapid diagnostic system for the simultaneous discrimination of other influenza virus subtypes during future outbreaks.

Multispecies detection of antibodies to influenza A viruses by a double-antigen sandwich ELISA.

A double-antigen sandwich ELISA was developed for the detection of antibodies to influenza A viruses. A recombinant nucleoprotein (rNP) of influenza A virus was used as a capture antigen and an HRP-conjugate for detecting the antibodies. A total of 125 serum samples from birds of different species including chickens, geese, open-billed storks, Khaki Campbell ducks, lesser whistling ducks, and pigeons with known antibodies were tested by ELISA. The sensitivity and the specificity of ELISA were found to be 98% and 97.3%, respectively. The assay was able to detect the presence of influenza A antibodies as early as the fourth day post-inoculation in ducks infected experimentally with influenza A (H5N1) virus. Excellent agreement (97.6%) was obtained between this sandwich ELISA and the hemagglutination inhibition (HI) tests (kappa=0.95). The double-antigen sandwich ELISA correlated well with a commercial avian influenza (AI) multispecies ELISA and was slightly more sensitive than the AI multispecies ELISA. These findings indicate that the double-antigen sandwich ELISA based on rNP may offer an effective screening method for serodiagnosis of influenza A virus. The double-antigen sandwich ELISA also enables the detection of antibodies to influenza A viruses in different species without the need for species-specific secondary antibodies.

Cell-mediated immune responses to GAD and beta-casein in type 1 diabetes mellitus in Thailand.

We measured the cell-mediated immune response to GAD and bovine beta-casein in 38 type 1 and 37 type 2 diabetic patients who visited diabetic clinics or who were hospitalized in Bangkok, Thailand, and in 43 normal controls, by using a lymphoproliferation assay. Positive results against GAD were found in 29/38 (76.3%) type 1, 6/37 (16.2%) type 2 diabetic patients and 1/43 (2.3%) normal controls. Positive results against bovine beta-casein were found in 18/38 (47.4%), 5/37 (13.5%) and 1/43 (2.3%) of these subjects, respectively. The frequencies of the positive results and the magnitude of the responses to both antigens in type 1 diabetic patients were significantly higher than those in the other two groups (P<0.001). In addition, the prevalence of a positive lymphoproliferative response to these antigens in type 1 diabetic patients was higher than that of anti-GAD antibody positivity in the same group of subjects (26.3%). Thus, the prevalence of positive lymphoproliferative response to GAD in type 1 diabetic patients studied was higher than the prevalence of other autoimmune markers previously reported in type 1 diabetic patients in Thailand.

Hepatitis C virus nonstructural 3 protein: recombinant NS3 protein of the Thai isolates as an antigen in a diagnostic assay.

Nonstructural 3 (NS3) protein of hepatitis C virus (HCV) is one of the antigens commonly used in diagnostic assays for antibody to hepatitis C virus. However, immune response to the NS3 protein from one genotype may not cross-react with that from other genotypes. In the development of an anti-HCV assay, the NS3 genes from genotypes 1 and 3 commonly found in Thailand were amplified and cloned into a bacterial expression system. These recombinant NS3 proteins were immunogenic and reacted with plasma samples of Thai patients infected with various HCV genotypes. Interestingly, the NS3 proteins from the Thai genotypes could react with 3 plasma samples from HCV infected Thai blood donors, which could not bind to the NS3.1 protein in the commercial HCV immunoblot kit using antigen from HCV genotype 1. This finding supports our prior observation that the appropriate HCV antigens used in a diagnostic assay should be derived from the virus genotypes commonly found in that geographical region.