ABSTRACT
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.
Subject(s)
Antigens, Viral/analysis , Gold , Hepacivirus/isolation & purification , Hepatitis C/diagnosis , Immunoassay/methods , Nanoparticles , Viral Core Proteins/analysis , Humans , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
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.
Subject(s)
Biosensing Techniques , Bluetongue virus/isolation & purification , Animals , Antibodies, Monoclonal/analysis , Bluetongue/diagnosis , Fiber Optic Technology/methods , Livestock/virology , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/metabolism , Chemical Warfare Agents/analysis , DNA, Single-Stranded/metabolism , Magnetite Nanoparticles/statistics & numerical data , Ricin/analysis , Gold/chemistry , Humans , Immunosorbent Techniques , Magnetite Nanoparticles/chemistry , Observer Variation , Ricin/immunology , Sensitivity and SpecificityABSTRACT
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.
Subject(s)
Bluetongue virus/isolation & purification , Bluetongue/diagnosis , Clinical Laboratory Techniques/methods , Nanoparticles , Veterinary Medicine/methods , Viral Core Proteins/analysis , Virology/methods , Animals , Antibodies, Viral , Antigens, Viral/analysis , Bluetongue/virology , Immunoassay/methods , Polymerase Chain Reaction/methods , Sensitivity and Specificity , SheepABSTRACT
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.
Subject(s)
Immunoassay/methods , Ricin/analysis , Antigens/analysis , Antigens/chemistry , DNA Barcoding, Taxonomic , Gold , Limit of Detection , Magnetics , Metal Nanoparticles , Plant Proteins/analysis , Plant Proteins/chemistry , Reproducibility of Results , Ricin/chemistryABSTRACT
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.
Subject(s)
Bluetongue virus/isolation & purification , Bluetongue/diagnosis , Clinical Laboratory Techniques/methods , DNA Barcoding, Taxonomic/methods , Nanoparticles , Veterinary Medicine/methods , Virology/methods , Animals , Antibodies, Monoclonal , Antibodies, Viral , Bluetongue virus/genetics , Bluetongue virus/immunology , Fluorescence , Immunoassay/methods , Immunomagnetic Separation/methods , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Sensitivity and Specificity , Serum/virology , Sheep , Viral Core Proteins/analysis , Viral Core Proteins/immunologyABSTRACT
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.
