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PubMed2003

Protein profiling of complete mole and normal placenta using ProteinChip analysis on laser capture microdissected cells.

INTRODUCTION: Surface-enhanced laser desorption/ionization mass spectrometry (SELDI-MS) is a novel method for biomarker discovery that can provide a rapid protein expression profile from a variety of biological samples. Since SELDI-MS requires a small amount of biological material, this technique is ideal for analyzing proteins isolated from microdissected tissue samples. The current study was undertaken to investigate potential differences in protein expression between normal and molar trophoblast procured by laser capture microdissection (LCM) utilizing SELDI ProteinChip array technology. Further knowledge of protein expression in complete mole may advance our understanding of the pathogenesis of gestational trophoblastic diseases. MATERIALS AND METHODS: Laser capture microdissected trophoblast cells from nine fresh complete moles were analyzed and compared to the trophoblast cells from 10 fresh normal placentas of comparable gestational age, using SELDI ProteinChip to identify potential differences in protein expression. RESULTS: Three metal binding polypeptides were identified with the estimated molecular weights of 11.3, 13.8, and 14.0 kDa, which appeared in significantly lower levels in complete mole as compared to normal trophoblast cells (P < 0.001, P < 0.03, and P < 0.01). DISCUSSION: While further characterization of these protein peaks is important and necessary, our current work clearly demonstrates that the combined technology of SELDI and LCM is effective in distinguishing protein expression between normal placenta and complete mole. Further knowledge of protein expression in complete mole may advance our understanding of molecular mechanisms and improve management in gestational trophoblastic diseases.

Dissection↗

PubMed2002

Expression of constitutively active CREB protein facilitates the late phase of long-term potentiation by enhancing synaptic capture.

Restricted and regulated expression in mice of VP16-CREB, a constitutively active form of CREB, in hippocampal CA1 neurons lowers the threshold for eliciting a persistent late phase of long-term potentiation (L-LTP) in the Schaffer collateral pathway. This L-LTP has unusual properties in that its induction is not dependent on transcription. Pharmacological and two-pathway experiments suggest a model in which VP16-CREB activates the transcription of CRE-driven genes and leads to a cell-wide distribution of proteins that prime the synapses for subsequent synapse-specific capture of L-LTP by a weak stimulus. Our analysis indicates that synaptic capture of CRE-driven gene products may be sufficient for consolidation of LTP and provides insight into the molecular mechanisms of synaptic tagging and synapse-specific potentiation.

Animals↗

PubMed2001

An antigen capture assay for the measurement of serum clusterin concentrations.

We have developed and validated a robust antigen capture assay for the measurement of serum clusterin. Increased clusterin expression, and alterations in serum clusterin levels have been associated with a number of disease states. In particular, clusterin has been shown to be associated with tissue regression and apoptosis in the rat ventral prostate in response to androgen ablation or administration of anti-androgens. The object of this study was to determine if changes in human serum clusterin can be used as a diagnostic or prognostic marker to monitor the response to hormonal therapy in patients with prostate cancer, and to determine if clusterin concentrations increase with the progression towards androgen independence. The antigen capture assay was used for an extensive analysis of human serum clusterin concentration in fasting males, and to determine if there is any relationship between clusterin and age or cholesterol levels. The average clusterin level in serum is 101+/-42 microg/ml (n=96). There is no correlation to age or serum cholesterol levels. Analysis of serum clusterin levels in patients with newly diagnosed prostate cancer (n=5), hormone responsive tumors (n=5), and hormone refractory disease (n=5), demonstrates that no significant changes in serum clusterin levels accompany the progression of prostatic disease, or response to hormone therapy.

Antigens↗

PubMed2003

Behavioral data management using Visual Basics for Applications to automate data capture and analysis.

Many researchers are familiar with the spreadsheet capabilities of Microsoft Excel, but have never explored using customized VISUAL BASIC FOR APPLICATIONS (VBA) macros embedded in the program. At the United States Army Medical Research Institute of Chemical Defense (USAMRICD), the implementation of VBA program code to carry out repetitive operations has resulted in a tremendous savings in both the time and manpower required to reliably capture, analyze, and plot data from research protocols. A set of "template" workbooks was developed and is used to organize data from different types of studies. At the heart of the concept is a "setup information" worksheet onto which the user enters information about the study (i.e. the number of subjects, groups, graphs). Clicking a control button on this worksheet launches the VBA code that creates worksheets for each subject, group and chart specified. A "pairmatch" function allows the user to create groups either randomly or by pair matching based on user-specified variables. Controls are provided that run VBA program code to parse subject data files into the proper worksheets and perform group averages. Charts are updated automatically whenever group averages change so that the researcher always has an up-to-date plot available. The ability to reformat the captured data onto a "statistical output" worksheet allows data to be imported easily to statistical software packages. The concept is being used for several different types of studies at USAMRICD and has significantly reduced not only amount of time spent on data management, but also the number of data entry errors. Readers interested in acquiring an electronic copy of the startle workbook example, which contains the complete VBA code, should send the request to the authors at reseco@erols.com or maurice.sipos@us.army.mil.

Acoustic Stimulation↗

PubMed2001

Development of dengue IgM-capture enzyme-linked immunosorbent assay with higher sensitivity using monoclonal detection antibody.

An IgM-capture enzyme-linked immunosorbent assay (IgM-ELISA) is used widely for serodiagnosis of dengue. A dengue IgM-ELISA with higher sensitivity has been developed. In the new ELISA, anti-dengue IgM antibody, which had been captured on the solid phase, was reacted with tetravalent dengue viral antigens, and detected by a flavivirus group specific monoclonal antibody, D1-4G2-4-15 (4G2). Reaction of 4G2 to viral antigens was similar to that of dengue patients' IgG. Non-specific reaction of 4G2 to the control antigen, which was prepared from uninfected cell culture fluid of mosquito C6/36 cells, was much lower than that of patients' IgG. Thus, specificity of the ELISA with 4G2 was much higher than that with patients' IgG, and lower levels of specific IgM was detected in the serum samples. These results suggest that the modified dengue IgM-ELISA with monoclonal antibody 4G2 has many advantages over the original "in-house" ELISA.

Antibodies, Monoclonal↗

PubMed2001

HHV-8 ELISA based on a one-step affinity capture of biotinylated K8.1 antigen.

The immunogenic envelope antigen gp35-37 of human herpesvirus-8 (HHV-8) is encoded by orfK8.1. An ELISA is described using streptavidin capture of bacterially expressed and biotinylated recombinant K8.1 antigen. The antigen capture strategy provides a simple and reliable method, which does not require high yield production and purification of the recombinant antigen before the serological assay. The specificity and sensitivity of the K8.1 ELISA were validated by gp35-37 envelope antigen Western blot and anti-lytic membrane immunofluorescence assay using lytically induced HHV-8 infected BCBL-1 cells. Under the established ELISA conditions, eight of the 10 Kaposi's sarcoma patients and five of the 180 healthy blood donors had IgG antibodies to K8.1 envelope antigen.

Biotin↗

PubMed2001

Second-generation Hybrid capture test and Amplicor monitor test generate highly correlated hepatitis B virus DNA levels.

The performance of the Digene Hybrid Capture II HBV DNA Test HC II and the Roche Cobas Amplicor Monitor Test (Cobas-HBV) was evaluated on 252 serum samples. One hundred and seventy-three samples were HBV DNA positive and 75 HBV DNA negative by both assays. Four samples were HBV DNA positive by Cobas-HBV only. Linear regression analysis showed that the HBV DNA concentrations obtained from both assays were significantly related (n=173, r=0.976, P<0.0001). The results of the study show that Hybrid capture II and Cobas-HBV could be used equally in the management for patients with chronic HBV infection.

Automation↗

PubMed2003

IgG-sandwich and IgM-capture enzyme-linked immunosorbent assay for the detection of antibody to Rift Valley fever virus in domestic ruminants.

The recent occurrence of the first confirmed outbreaks of Rift Valley fever in humans and livestock outside the African region, namely in the Kingdom of Saudi Arabia and Yemen, is of global medical and veterinary concern. Disadvantages of classical techniques for serological diagnosis of Rift Valley fever include health risk to laboratory personnel, restrictions for their use outside endemic areas and inability to distinguish between different classes of immunoglobulins. We report on the development and validation of sandwich and capture ELISAs (both based on inactivated antigen) for detection of IgG and IgM antibody to Rift Valley fever virus in bovine, caprine and ovine sera. Compared to virus neutralisation and haemagglutination-inhibition tests, the IgG sandwich ELISA was more sensitive in detection of the earliest immunological responses to infection or vaccination with Rift Valley fever virus. Its sensitivity and specificity derived from field data sets ranged in different ruminant species from 99.05 to 100% and from 99.1 to 99.9%, respectively. The specificity of IgM-capture ELISA varied between different species from 97.4 to 99.4%; its sensitivity was 100% in sheep tested 5-42 days post-infection. Our results in field-collected, experimental and post-vaccination sera demonstrate that these assays will be useful for epidemiological surveillance and control programmes, import/export veterinary certification, early diagnosis of infection, and for monitoring of immune response in vaccinated animals. As highly accurate and safe tests, they have the potential to replace traditional diagnostic methods, which pose biohazard risks limiting their use outside of endemic areas to high containment facilities.

Animals↗

PubMed1997

Detection of parvovirus B19-specific IgM by antibody capture radioimmunoassay.

A solid phase IgM-capture radioimmunoassay (MACRIA) for the detection of parvovirus B19-IgM is described (Cohen et al., 1983, J. Hyg. Camb. 91, 113-130). IgM from a dilution of patients serum is 'captured' onto a solid phase coated by anti-human IgM. To determine whether any of the IgM is specific for parvovirus B19, B19 antigen is added followed by a detector system. In the MACRIA described here the detector system comprises a mouse monoclonal antibody to parvovirus B19 and a 125I-labelled anti-mouse antibody. A calibration curve derived from a standard B19 IgM serum is used to quantify B19 IgM using a single dilution of test sera. The purpose of the protocol is the diagnosis of recent acute infection with parvovirus B19.

Animals↗

PubMed1997

The development of an antigen capture polymerase chain reaction assay to detect and type human enteroviruses.

Antigen capture polymerase chain reaction (AC-PCR) is a technique that combines the advantages of PCR with those of antibody mediated methods, to detect and type human enteroviruses. Virus particles are captured by specific antisera and RNA is released by heat denaturation to generate the substrate for reverse transcription and PCR. Use of this technique results in purification of human enteroviruses from tissue culture and 10% faecal samples in a serotype-specific manner allowing both rapid detection and a direct correlation between serological and genetic typing methods. The sensitivity of AC-PCR was comparable with that of PCR protocols employing a conventional organic solvent based extraction procedure.

Animals↗

PubMed1998

Detection and identification of human papilloma viral DNA, types 16, 18, and 33, by a combination of polymerase chain reaction and a colorimetric solid phase capture hybridisation assay.

A colorimetric microplate hybridization assay was developed previously to simplify detection procedures of DNA fragments resulting from polymerase chain reactions (PCR). This format has now been adapted for the simultaneous detection and identification of three human papillomavirus (HPV), types 16, 18 and 33, associated frequently with cervical cancer. This post-PCR detection system uses three type-specific capture oligonucleotides linked covalently to a single microplate well and three type-specific multibiotinylated oligonucleotidic probes for detection. It therefore offers a double specificity; the first is conferred by pairs of primers, specific of each type of virus tested, and the second, by the sets of capture and detection probes which are complementary to internal regions of the amplified DNA fragments. The detection format outperformed agarose gel electrophoresis of amplified DNA products in sensitivity and specificity. The rapidity and simplicity of this hybridisation system would justify its use in routine diagnostic examination of cervical specimens (smears and biopsies).

Colorimetry↗

PubMed1999

Purification of full-length enterovirus cDNA by solid phase hybridization capture facilitates amplification of complete genomes.

The aim of the study was to develop a method for the selective purification of full-length enterovirus single strand (ss) cDNA for subsequent amplification of complete enterovirus genomes by long distance PCR. As a model system we have used the prototype strain of echovirus 5 (EV5). Due to inefficient first strand cDNA synthesis using EV5 RNA as template, only a few molecules of EV5 sscDNA were completely reverse transcribed and no amplification products were observed when long distance polymerase chain reaction (LD-PCR) was used for amplification of complete EV5 genomes. To purify the complete EV5 cDNA present, an oligonucleotide, derived from the conserved 5' end of an enterovirus genome, was immobilized on paramagnetic beads and complete EV5 sscDNA was captured and purified from the less than full-length cDNAs. LD-PCR using the purified EV5 cDNA resulted in amplification of complete EV5 genomes. Transfection of the EV5 RNA transcribed from these uncloned amplicons resulted in production of replicating viruses. This demonstrates that solid phase hybridization capture of sscDNA is an efficient method that can be used for enrichment and purification of full-length enterovirus sscDNAs.

Cell Line↗

PubMed1999

Serotyping of foot-and-mouth disease virus by antigen capture reverse transcriptase/polymerase chain reaction.

The technique of capturing of foot-and-mouth disease virus (FMDV) from clinical material in microcentrifuge tubes coated with type-specific antibodies and amplifying the viral sequences by RT/PCR in the same tube, promoted the detection and serotyping of FMDV with high sensitivity and specificity. The efficiency of antigen capturing and shelf life of the coated tubes was improved by glutaraldehyde fixation of antibodies to the tubes. Virus in infected tissues, even after storage for 25-30 years at 70 degrees C, could be successfully typed by this method. Conserved sequences flanking the variable region of immunoreactive VP1 gene of FMDV were used as primers in the assay and hence the nucleotide sequence analysis of the product could reveal the strain variation. The test has been found to be at least 125-fold more sensitive than type specific ELISA and of comparable sensitivity as other protocols for detection of FMDV by RT/PCR.

Animals↗

PubMed2000

Serological diagnosis of varicella-zoster virus in sera with antibody-capture enzyme-linked immunosorbent assay of IgM.

Evaluation was made of three enzyme-linked immunosorbent assay (ELISA) formats; varicella-zoster virus (VZV) indirect ELISA; VZV IgM capture using biotin and VZV IgM capture using peroxidase, for the detection of VZV-specific IgM antibodies in human sera. It was observed that there was no significant difference in sensitivity of detection using the three formats but there were important practical differences in the number of steps and hence time for assay completion between the three assay formats. All assays showed some cross-reactivity with sera containing anti-HSV1 antibodies.

Antibodies, Viral↗

PubMed2000

Laboratory and field studies of an antigen capture ELISA for bluetongue virus.

An improved bluetongue antigen capture ELISA (BTACE) technique was evaluated for its ability to detect the full range of 24 bluetongue (BLU) serotypes. The BTACE detected all 24 serotypes in cell culture fluids, including eight serotypes where the representative strains originated from both Australia and also from the South African reference collection. The amount of infectious virus required to obtain a positive BTACE result varied between 100-1000 TCID50. This was approximately 10-fold more sensitive than the antigen capture test described previously (Hosseini, M., Hawkes, R.A., Kirkland, P.D., Dixon, R., 1998. J. Virol. Methods 75, 39-46.). The BTACE method was compared with conventional passage in cell culture to detect the presence of virus in the tissues of embryonated chicken eggs (ECEs) which had been inoculated intravenously with the blood of sheep and cattle infected experimentally with the eight Australian serotypes of BLU (1, 3, 9, 15, 16, 20, 21, and 23). The BTACE method was at least as sensitive as the conventional cell culture detecting virus in ECEs, obviating the need for prolonged cell culture passage to detect the virus. A comparison of the amount of antigen detected in different embryo tissues indicated that liver homogenates gave the highest positive to negative ratios in the BTACE and were selected as the specimen of choice. In studies of sheep infected with all 24 South African reference BLU serotypes this new BTACE was able to detect viraemia with all serotypes. Finally, the BTACE was validated in surveillance programs for BLU in both New South Wales, Australia and in Yunnan Province, People's Republic of China. Blood samples from sentinel cattle were inoculated into ECEs. Homogenised ECE livers were tested by BTACE and those positive were passaged subsequently in cell culture for virus isolation and identification. This protocol led to the efficient isolation of field isolates of many serotypes. The high sensitivity and broad reactivity of the method indicates that it should be valuable for BLU diagnosis and surveillance programs.

Animals↗

PubMed2000

Anomalies on capture nets of Hydropsyche slossonae larvae (Trichoptera; Hydropsychidae), a potential indicator of chronic toxicity of malathion (organophosphate insecticide).

A laboratory study on the sublethal effects of malathion on the net-spinning behavior of the caddisfly larvae Hydropsyche slossonae was conducted in order to assess the potential of net anomalies as an indicator of chronic exposure to organophosphorus insecticides. Two anomalies were identified after chronic exposure to 0.01, 0.05, 0.1, 0.5 and 1.0 µg l(-1) malathion. The first was a distortion of the midline meshes where the normal diamond shape structure was disrupted and the meshes were separated by extra strands (called 'midline' anomaly). The second aberration observed was a significant decrease in net symmetry. Both anomalies were highly correlated to the toxic action of malathion, i.e. inhibition of the acetylcholinesterase enzyme (AChE). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of capture nets did not show any modification of silk polypeptides after exposure to malathion, indicating that net distortions were not related to silk composition. Both anomalies seem to represent the symptoms of the specific toxic action of malathion; nevertheless, they can serve as an index of the physiological condition of the larvae, especially the midline anomaly. The symmetry of the nets decreased significantly after exposure to 0.5 and 1.0 µg l(-1). However, the toxicity curves (EC(50)) showed that the sensitivity threshold for the midline anomaly ranged from 0.11 to 0.28 µg l(-1), which reflect more realistic exposure to concentrations expected to occur in the field. Hence, the use of capture net anomalies of hydropsychid larvae could represent a valuable indicator of sublethal toxicity induced by malathion and other organophosphorus insecticides in running waters.

Journal Article↗

PubMed2002

Immuno-capture PCR for detection of Aeromonas hydrophila.

In this report, we describe the use of universal primer PCR (UPPCR) for the detection of 16S ribosomal RNA (rRNA) genes from Aeromonas hydrophila captured by anti-A. hydrophila antibody coupled to a microplate. The approach combining immuno-capture with UPPCR provides a quick, sensitive, and reproducible way for the detection of bacterial cells.

Aeromonas hydrophila↗

PubMed2003

Tracking of injected and resident (previously injected) bacterial cells in groundwater using ferrographic capture.

A high-resolution bacterial tracking technique, ferrographic capture, was used to enumerate fluorescent-stained bacterial cells that were injected into groundwater during a field experiment. The goal of the experiment was to investigate whether detachment of previously injected stained resident cells attached to aquifer sediment was enhanced in the presence of the newly injected mobile cells. This injection was an improvement on past experiments in that the attached (resident) cells were stained, allowing their concentrations to be enumerated directly by ferrographic capture (upon detachment). Contrary to expectations based on previous experiments, enhanced detachment of stained resident cells did not occur upon the arrival of injected cells. Consistent with previous experiments, however, was the observation of ephemeral increases in unstained cell concentrations coincident with the arrival of the stained injected cells. The ephemeral pulses of unstained cells were previously speculated to represent enhanced detachment of unstained indigenous cells in response to hydrodynamic collision with injected cells. The lack of enhanced detachment of stained resident cells in the present experiments indicates that increased concentrations of unstained cells may have occurred by mechanisms other than hydrodynamic collision. Visually observed variations in stain intensity indicated that increased unstained cell concentrations may have resulted from cell division at the low-concentration fringe of the injected plume.

Aerobiosis↗