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PubMed2004

Development of rapid staining protocols for laser-capture microdissection of brain vessels from human and rat coupled to gene expression analyses.

Laser-capture microdissection (LCM) is a technique that enables selective extraction of desired cells from heterogeneous tissues compatible with subsequent molecular analyses. The specific visualization of desired cell types prior to LCM is essential for achieving selective capture. We have developed rapid and selective staining protocols for LCM extraction of microvessels from human and rat brain. Vessels in human and rat brain sections were visualized by a 2 min exposure to fluorescein-labeled lectins Ulex Europeaus Agglutinin I (UEA I) and Ricinus Communis Agglutinin I (RCA I), respectively. Immunohistochemical staining for the endothelial-specific marker, Factor VIII-related antigen (FVIII-rAg), co-localized with that for either UEA I or RCA I, confirming the selective staining of vascular structures with these lectins. Both brain vessels and perivascular parenchyma were captured using LCM, followed by RNA isolation. RT-PCR analyses demonstrated the enrichment of LCM-captured vessels and parenchyma in FVIII-rAg and GFAP mRNA, respectively. LCM-captured human vessels also expressed the tight junction-specific gene, zonula occludens 1 (ZO-1). LCM extraction of vessels from brain sections can be used to perform molecular fingerprinting of neurovascular unit in various brain pathologies.

Animals↗

PubMed2005

Anatomic analysis of the Genial Bone Advancement Trephine System's effectiveness at capturing the genial tubercle and its muscular attachments.

OBJECTIVE: The Genial Bone Advancement Trephine (GBAT) System (Stryker Leibinger Inc, Kalamazoo, MI) is a 1-step system that advances the genial tubercle for treatment of retrolingual obstruction in obstructive sleep apnea. The purpose of this project was to anatomically analyze the effectiveness of the GBAT System to capture the genial tubercle and its muscular attachments in the circular trephine. METHODS: Eight cadaveric heads (ages 54-89 years; 3 female and 5 male) were examined, and Panorex radiographs (Stryker Leibinger Inc, Kalamazoo, MI) were obtained. Seven specimens were selected on the basis of mandibular height and dentition to undergo the procedure. The procedure was performed using the GBAT system with the 14-mm trephine according to the stepwise instructions. The mandibles were dissected, and analysis of the location of the osteotomy with respect to the genial tubercle, genioglossus muscles, geniohyoid muscles, and mandibular dentition was performed. RESULTS: All 7 of the specimens had complete capture of the genial tubercle. The mean percentage of the genioglossus muscle captured in the circular trephine was 85% (50%-100%). All specimens had preservation of the posterior muscle fibers. The mean percentage of the geniohyoid muscle captured was 78% (15%-100%). No tooth roots were transected or contained in the bone plug. CONCLUSIONS: The GBAT System is an effective 1-step system for capturing and advancing the genial tubercle and its muscular attachments in the treatment of OSA.

Aged↗

PubMed2004

Emergence of vertebrate retroviruses and envelope capture.

Retroviruses are members of the superfamily of retroelements, mobile genetic elements that transpose via an RNA intermediate. However, retroviruses are distinct from other retroelements in that their "transposition" is not confined to single cells but extends to neighboring cells and organisms. As such, the "transposition" of these elements is defined as infection. It appears that a key step in the conversion of a retrotransposon into a retrovirus is the modular acquisition or capture of an envelope glycoprotein (Env) which facilitates dissemination from its initial host cell. Here we present several examples of retroviruses for which envelope capture has been identified. Indeed, capture may explain the notable conservation of env sequences among otherwise phylogenetically distant retroviruses. In a recent example, sequence homologies reported between the env of the phylogenetically distant murine leukemia viruses (MLV) and human T cell leukemia viruses (HTLV) argue in favor of an env capture by the latter. Env acquisition can provide new adaptive properties to replication-competent viruses in addition to altering their host range. Also, the captured env can alter the spectrum of physiological affects of infection in new host cells and organisms. The elucidation of such envelope exchanges and properties thereof should contribute significantly to the clarification of retroviral phylogeny, insight into retroviral pathogenesis, and to the discovery of new retroviruses.

Animals↗

PubMed2000

Synergistic effects in antigen capture ELISA using three monoclonal antibodies directed at different epitopes of the same antigen.

Using a panel of monoclonal antibodies (mAb) against human myoglobin (Mb), we have shown that the sensitivity of antigen-capture enzyme-linked immunosorbent assay (ELISA) may be significantly increased by the simultaneous immobilization on a solid phase of two co-operating capture mAbs. This method ("a three-site ELISA") uses three mAbs at different epitopes of the same antigen (two capture/one tracer), unlike the traditional two-site assay, using one capture and one tracer mAbs. We established two-site and three-site ELISA assays for Mb, by varying capture and tracer mAbs. Three-site assays showed 4-6 fold increase in sensitivity, if compared with two-site assays. The model for the effect has been suggested, according to which in three-site ELISA the high-affinity cyclic configurations may be formed by an antigen, two-capture mAbs and the surface of solid phase.

Animals↗

PubMed2000

Sinus escape-capture bigeminy and sinus extrasystolic bigeminy.

Blocking conduction between the sinus node and the atria (SA block) can be responsible for symptomatic rhythm problems. However, in atrial escape-capture bigeminy with SA block, when atrial escape P waves originate in a site within or close to the sinus node, the diagnosis of SA block is not easy. Electrocardiograms were selected from 7 people with atrial bigeminy because (1) all atrial deflections (P waves) were almost the same in shape and in length of PR intervals, (2) comparatively long PP intervals alternated with comparatively short PP intervals, and (3) occasionally the atrial bigeminy changed to normal regular sinus rhythm in which 2 or more sinus P waves were found in succession. An attempt is made to clarify the mechanism for these cases. When regular sinus rhythm changed to bigeminal rhythm, the long PP interval introduced the bigeminy in 3 cases, indicating the presence of "sinus" escape-capture bigeminy; whereas the short PP interval introduced the bigeminy in the other 4 cases, indicating the presence of "sinus" extrasystolic bigeminy. In cases of sinus escape-capture bigeminy associated with SA block, the cases may occasionally be diagnosed wrongly as ordinary sinus arrhythmia not associated with SA block. Therefore, it seems that sinus escape-capture bigeminy is not so rare as is generally believed. Patients with SA block often require implantation of the artificial pacemaker. Thus, the authors believe that differentiation of sinus escape-capture bigeminy from other forms of "sinus" bigeminy is clinically important.

Adult↗

PubMed2000

Mapping of B-cell epitopes in rabbits immunised with various gag antigens for the production of HIV-1 gag capture ELISA reagents.

An HIV-1 p24 capture enzyme linked immunosorbent assay (ELISA) was developed and used in a study of B-cell epitopes in rabbits immunised with different gag p24 antigens. Rabbits were immunised with virion HIV-1/Lai, baculovirus recombinant p24, Escherichia coli recombinant p24-15 and a mixture of synthetic peptides representing sequences of HIV-1 gag p24 protein, respectively. Five out of nine rabbits developed antibodies that could be used for an antigen capture ELISA. No significant differences in IgG titers to the whole gag protein were seen when comparing rabbits immunised with four different antigens. Three major common linear epitope regions were mapped in the rabbits immunised with virion HIV-1/Lai and baculovirus recombinant p24. The rabbit immunised with HIV-1 gag peptides had the broadest linear epitope reactive responses whereas animals immunised with E. coli recombinant antigen had the most restricted linear epitope response. The capture ELISA method thus developed using the different rabbit anti-p24 IgG preparations was shown to capture isolates from HIV-1 subtypes or clades A to G. Only rabbits immunised with virion HIV-1/Lai and baculovirus recombinant p24 developed IgG that was capable of efficiently capturing HIV-1 p24 in ELISA, indicating the importance of preparing antibodies able to recognise native or discontinuous and linear antigen configurations.

Animals↗

PubMed1999

Motion-transparent inducers have different effects on induced motion and motion capture.

To assess the relationship among the underlying mechanisms of induced motion, motion capture, and motion transparency, directions of the former two illusions in the presence of motion-transparent inducers were examined. Two random-dot patterns (inducers) were superimposed upon a stationary disk (target), and moved in orthogonal directions. Either a high-contrast target (for induced motion) or a low-contrast target (for motion capture) was used. The task was to report the perceived direction of the target. The depth order of inducers was controlled either by adding binocular disparity or by asking the subject to report subjective depth order. For induced motion, the target appeared to move in the direction opposite to the inducer that had a disparity closer to the target; when there was no difference in disparity, induced motion occurred oppositely to the 'vector sum' of the inducers' directions. For motion capture, the target was captured by the inducer that subjectively appeared behind. These results suggest that the underlying mechanism of motion capture utilizes the output from the process for motion transparency, whereas induced motion has no clear relationship to the output of the process for motion transparency.

Contrast Sensitivity↗

PubMed2000

Influence of a poly-ethylene glycol spacer on antigen capture by immobilized antibodies.

The use of spacers to distance an immobilized antibody from the surface of a support matrix introduces flexibility, which can reduce steric interferences between antibodies leading to a higher antigen capture efficiency. In this paper we investigated the use of a spacer molecule, poly-ethylene glycol (PEG), between the matrix surface and antibodies for the capture of Bacillus globigii, E. coli O157:H7, and ovalbumin. The antigen capture efficiency was determined using a surface ELISA method. Antibodies against the antigens were covalently immobilized either directly or via PEG to glass surfaces using a one-step EDC reaction. The amount of antibody immobilized was determined before blocking the nonspecific binding sites with bovine serum albumin. Antibodies immobilized via a PEG spacer showed a higher capture efficiency compared to direct immobilization, which was more pronounced with large antigens. Antibodies immobilized on glass supports were stable at 65 degrees C for at least 80 min, and the capture efficiency increased with heating at 65 degrees C for 20 min.

Animals↗

PubMed2001

Production and characterization of monoclonal antibodies detecting chicken interleukin-2 and the development of an antigen capture enzyme-linked immunosorbent assay.

Eleven monoclonal antibodies (mAbs) which are specific for chicken interleukin-2 (chIL-2) were produced and characterized by enzyme-linked immunosorbent assay (ELISA), Western blotting and neutralizing assays. These mAbs were used to develop a mAb-based antigen capture ELISA specific for chicken IL-2 detection. Anti-IL-2 mAbs bound specifically to E. coli-derived rchIL-2 in ELISA and identified a 16kDa IL-2 polypeptide band in Western blot. Several mAbs were shown to neutralize the biological activities of both rchIL-2 and native chicken IL-2 as measured by concanavalin A (ConA)-induced lymphocyte proliferation assay, IL-2 bioassay, and natural killer cell assay. Among the neutralizing mAbs, the mAb chIL-2/11 was most potent in neutralizing IL-2 activity. To develop a sensitive ELISA for the detection of chicken IL-2, an antigen capture ELISA was developed using the mAb chIL-2/16 as the antigen capture antibody and rabbit anti-IL-2 peptide antibody as the detection antibody. Using the mAb-based antigen capture ELISA, significant correlation between the level of IL-2 detected in bioassays and in ELISA was observed. These results showed that the mAb-based antigen capture ELISA is less time-consuming and more reliable compared to a conventional IL-2 bioassay for chicken IL-2. These neutralizing mAbs will facilitate basic immunobiological studies of the role of IL-2 in normal and disease states in chickens.

Amino Acid Sequence↗

PubMed2002

A novel multiplex RT-PCR probe capture hybridization (RT-PCR-ELISA) for simultaneous detection of six viroids in four genera: Apscaviroid, Hostuviroid, Pelamoviroid, and Pospiviroid.

A rapid and sensitive assay was developed for the detection and identification of viroids by standard or multiplex reverse transcription-polymerase chain reaction (RT-PCR)-probe capture hybridization (RT-PCR-ELISA). The assay was applied successfully for the detection and identification of the following six viroid species from infected tissues: Potato spindle tuber viroid (Pospiviroid), Peach latent mosaic viroid (Pelamoviroid), Apple scar skin viroid (Apscaviroid), Apple dimple fruit viroid (Apscaviroid), Pear blister canker viroid (Apscaviroid), and Hop stunt viroid (Hostuviroid). Total RNA was obtained from infected tissue by the Qiagen RNeasy kit and, then viroid cDNA was synthesized using viroid specific complementary DNA primer. To identify and differentiate the amplicons of the six viroids, each amplicon was digoxigenin (DIG)-labelled during the amplification process, and then detected by a colorimetric system using a biotinylated cDNA capture probe specific for each viroid. The results revealed that each capture probe hybridized only to its complementary DIG-labelled amplicon. Thus the six viroids can be detected and differentiated in a multiplex RT-PCR-ELISA assay. In the multiplex assay, cDNAs of six viroids were synthesized simultaneously in one tube, DIG-labelled during amplification, then a portion of the DIG-labelled amplified products was hybridized with selected capture probe. All the six viroid capture probes hybridized to their respective complementary DIG-labelled RT-PCR-amplified product. These findings are important for viroid detection and identification for studying host-viroid interactions and for management and control viroid diseases.

DNA Probes↗

PubMed1998

Rapid screening of embryonated chicken eggs for bluetongue virus infection with an antigen capture enzyme linked immunosorbent assay.

The sensitivity and specificity of an antigen capture ELISA have been compared with virus isolation in cell culture. Bluetongue virus (BLU) (serotype 23) from the blood of a sheep was titrated by inoculating embryonated chicken eggs (ECEs) and detecting viral antigen in chicken embryo livers using an antigen capture enzyme linked immunosorbent assay (ELISA) (Stanislawek et al., 1996. Detection by ELISA of bluetongue antigen directly in the blood of experimentally infected sheep. Vet. Microbiol. 52, 1-12). Five days after inoculation of ECEs with lysed red blood cells from the infected sheep the embryo livers were harvested and homogenised. The supernatant from the homogenate was used in the antigen capture ELISA to determine which livers were infected and the virus titre calculated as CEID50/ml packed red blood cells. These results were compared with a standard cell culture isolation protocol which passaged the liver homogenate supernatant through Aedes albopictus cells and up to three passages in BHK21 cells. The antigen capture ELISA showed 100% sensitivity and specificity with no false negatives or false positives when compared to cell culture isolation of the virus. The major advantage of the combination of ECE inoculation and antigen capture ELISA is the reduction in the time to less than 7 days from a maximum of 35 days for the ECE/cell culture system. The procedure is easy to undertake, cost effective and does not require expensive specialist cell culture facilities.

Aedes↗

PubMed1998

Development and evaluation of a novel antigen capture assay for the detection of classical swine fever virus antigens.

An antigen-capture enzyme immunoassay (EIA) was developed to detect classical swine fever virus (CSFV) antigen directly from 10% w/v tissue suspension. The assay, based on the sandwich principle, uses a biotinylated monoclonal antibody bound to streptavidin-coated microplates as the capture system and a swine anti-CSFV antibody and rabbit anti-swine HRPO-conjugate as the detector system. The antigen-capture EIA was compared with conventional virus isolation and polymerase chain reaction (PCR) for detection of CSFV in tissues. The ability of the antigen-capture EIA to discriminate classical swine fever (CSF) from bovine viral diarrhea and African swine fever viruses was also tested. The assay was shown to detect 21 different strains of CSFV and was unreactive with tissues from uninfected animals. Signal to noise (S/N) ratios were calculated from the EIA absorbance values. Readings from samples positive by virus isolation (n = 47) averaged a S/N ratio of 5.34. In contrast, samples negative by virus isolation (n = 96) demonstrated a mean S/N ratio of 0.16. At S/N cut-off value of 1.0, all samples that yield virus isolation and PCR negative result were negative in the antigen-capture EIA. Compared with virus propagation in tissue culture using PK15 cells (followed by indirect peroxidase assay detection) and PCR, the EIA had a specificity of 98.7% and a sensitivity of 91.4%. The EIA is simple, can be performed in 4 h and lends itself to automation for screening of tissues sample from pigs suspected of CSFV infection.

African Swine Fever↗

PubMed1999

Recommendations for presentation and evaluation of capture-recapture estimates in epidemiology.

We propose 15 recommendations for approaches to capture-recapture analysis in epidemiology. We apply them to a report of such an analysis of a measles epidemic [McGilchrist et al., J Clinical Epidemiol 1996, 49: 293-296] and to comments thereon by R. C. Cormack [J Clinical Epidemiol 1999; 52: 909-914]. The latter challenged the utility of the data on the measles outbreak for any reliable capture-recapture estimates. We suggest that, adopting the perspective of W. Edwards Deming, one can only make judgments as to the reliability of capture-recapture data, methods, and derived estimates in the light of (i.e., conditional upon) their eventual intended use. Capture-recapture approaches "unreliable" from one perspective may be "reliable," and/or more appropriately, "useful" from another. We consider the utility of ancillary and ad hoc information that may be available or worth seeking to supplement a capture-recapture analysis. We use information within the study of McGilchrist et al. to illustrate how, with such ancillary information, one may overcome the main thrust of the objections of Cormack in situations in which one observes apparently anomalous or hard to understand data structures. Making certain simple assumptions we regard as plausible, we estimate the number of affected in the measles epidemic as between about 700-1300. We derive this from data on 502 cases in a Register, an ad hoc sample of 91 cases in one age group in the general population, and the report of 41 cases in both of these. Our result is only 15-30% the total implied by the estimates McGilchrist et al. derived with more complex methods and many assumptions in addition to our own. We discuss various approaches to evaluating "reliability" of our estimate conditional upon intended uses by policy makers.

Australia↗

PubMed2001

Attentional set modulates visual areas: an event-related potential study of attentional capture.

The present experiment offers event-related potential evidence suggesting that modulation of neural activity in the visual cortex underlies top-down attentional capture by irrelevant cues. Participants performed a covert visual search task where they identified the unique stimulus in a brief, four-location display. Targets defined uniquely by color or onset were run in separate blocks, encouraging observers to adopt different attentional sets in each block. In Experiment 1, a brief, white, abrupt-onset cue highlighted one of the locations 100 or 200 ms prior to the target display. In Experiment 2, the cue display consisted of three white and one red cues simultaneously presented at the four locations. In both experiments, participants were informed that there was no predictive relation between the location of the cue and that of the target. Reaction times were dependent on the location of the preceding cue (i.e. attention was captured), but only in those blocks where the cue shared the uniquely relevant target feature. Evoked potentials over the right hemisphere were modulated during the attention-capturing blocks just prior to the cue's appearance. Additionally, the N1 wave elicited by the cue was enhanced over occipital regions during the attention-capturing blocks. These findings support the notion that attentional capture with peripheral cues is not simply reflexive but is modulated by top-down processes.

Adolescent↗

PubMed1997

Evaluation of the MUREX*ICE HIV-1.O.2 capture enzyme immunoassay for early identification of HIV-1 seroreverting infants in a developing country.

BACKGROUND: A simple, inexpensive serological assay is required for the early determination of HIV infection status among infants born to HIV-1-seropositive women in developing countries. OBJECTIVES: To evaluate the use of a commercially available capture enzyme immunoassay (EIA), the MUREX*ICE HIV-1.O.2, for the early identification of seroreverting, uninfected infants. STUDY DESIGN: Infants with a clearly defined HIV-1 infection status, as determined by polymerase chain reaction results and/or seroreactivity at 18 months, were tested for antibodies to HIV. The time to seroreversion using the capture EIA was compared with the results obtained using an indirect assay, the GENELAVIA MIXT EIA. RESULTS: Seroreverting infants were identified earlier with the capture than the indirect EIA; all of the uninfected infants were seronegative at 12 months with the capture EIA while 100% seroreversion was only seen at 18 months with the indirect EIA. CONCLUSIONS: In general, the capture EIA identified seroreverting infants 3-6 months earlier than the indirect EIA. However, caution must be exercised in interpreting seroreactivity in a breast-fed population where HIV infection may occur in a child who has previously seroreverted.

Africa↗

PubMed1998

Evaluation of CAPTURE CMV solid phase testing over the allowable shelf-life of red blood cells (adenine-saline added).

BACKGROUND: CAPTURE CMV (IMMUCOR Inc., Norcross, GA) is a solid-phase screening test used to detect IgM and IgG antibodies to cytomegalovirus (CMV). CAPTURE CMV is licensed for testing whole blood (WB) in citrate phosphate dextrose (CPD) preserved segments of units of red blood cells (RBC) only up to 7 days of storage. We determined if CAPTURE CMV could produce consistent results in CPD preserved WB segments from RBC adenine-saline added (ASA) for their 42 day shelf-life. METHODS: Ten CMV-seropositive and 10 CMV-seronegative RBC (ASA) tested by CAPTURE CMV during the first week of storage were studied. Segments were tested weekly for 6 weeks. RESULTS: All 10 units that initially tested as CMV-seropositive remained strongly seropositive. All 10 units initially CMV-seronegative, remained seronegative. CONCLUSION: CAPTURE CMV testing provides consistent results over the entire shelf-life of RBC (ASA).

Adenine↗

PubMed1998

Highly sensitive and specific ELISA with monoclonal antibody capture to measure Dermatophagoides farinae 1-specific IgE.

BACKGROUND: Dermatophagoides farinae (Der f) is a major allergen in Der f extract. Measurement of Der f I-specific IgE has not been commonly used, because of the difficulty in obtaining large amounts of purified Der f 1. OBJECTIVES: To improve the diagnosis of dust mite allergy, we wanted to develop an ELISA to measure Der f 1-specific IgE in which purified Der f 1 is not required. METHODS: Using a monoclonal antibody (mAb) against Der f 1, a mAb-capture ELISA was developed. Microplates coated with the mAb were sequentially incubated with crude Der f extract, serum samples, goat anti-human IgE, and conjugated rabbit anti-goat IgG. A conventional ELISA using purified Der f 1 as capture (Der f 1-ELISA) was developed to compare it with the mAb-capture ELISA. RESULTS: There was a significant correlation between the two ELISAs (r = 0.89, P < .001) with a sensitivity of 0.8 U/mL. Both the assays were inhibited by the crude Der f extract in a dose-dependent manner. Using the two ELISAs, serum Der f 1-specific IgE was evaluated in 12 allergic patients with positive skin tests to Der f extract, 14 allergic patients with negative skin tests to Der f extract, and 15 healthy subjects. The mean Der f 1-IgE and the positivity rate of Der f I-IgE were higher in the mite-allergic group and lower in the healthy group when the mAb-ELISA was used (P < .01) than when the Der f 1-ELISA was used (P < .05). CONCLUSIONS: Der f 1-specific IgE can be measured by mAb-capture ELISA using crude Der f extract. The mAb-ELISA is more sensitive and specific than the conventional Der f 1-capture ELISA in diagnosing dust mite allergy.

Adolescent↗

PubMed2005

Under-notification of giardiasis in Auckland, New Zealand: a capture-recapture estimation.

Estimation of the degree of undercount is important for disease surveillance. Capture-recapture techniques are now being used to evaluate the completeness of disease ascertainment. This study estimated the level of under-notification of giardiasis in the Auckland adult population using a capture-recapture method. Two independent datasets of giardiasis cases > or = 15 years were generated from the 1998-1999 Auckland Giardiasis Study (AGS) case database and cases notified to Auckland Regional Public Health Services (ARPHS) for the same period of time. Cases were matched and under-notification was estimated using a two-sample capture-recapture method. During the 12-month period, 199 cases participated in the AGS and 413 cases were notified to ARPHS. The capture-recapture calculation indicated that only 49% of cases were notified. Under-notification by a factor of 2 obscures the true burden of giardiasis. Socio-economic conditions and water quality may influence disease notification inversely. Capture-recapture techniques are useful in evaluating the completeness of surveillance.

Adolescent↗