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PubMed1986

An automatic stimulus generation system for electroretinogram capture and processing.

An automated system is presented for on-line capture and processing of the analog signal obtained in response to light or X-ray stimulation of isolated rat retina maintained in survival by perfusion. The most important part of the system is a microcomputer Apple II (48 K Europlus) equipped with interface boards. Basic and assembler programs automatically deliver light or X-ray stimulation every 5 min. Data capture and data processing are carried out following each retinal response. Calculated parameters of the ERG, and 200 values obtained after sampling of an ERG are placed in a data file on a floppy disc. One hundred ERGs can be stored in this way.

Animals↗

PubMed1983

Plasma corticosterone in wild starlings (Sturnus vulgaris) immediately following capture and in relation to body weight during the annual cycle.

Free-living starlings (Sturnus vulgaris) were caught by mist netting. Blood samples were taken from them between 20 sec and 4 min after capture and assayed for corticosterone. Before 1 min corticosterone levels were constant (10.3 nmol litre-1) but after 1 min they increased rapidly (27.0 +/- 3.6 nmol litre-1 min-1). Male and female free-living starlings were caught throughout the year with mist nets, and traps placed in nest boxes. Blood samples were taken within 1 min of capture and body weights recorded. Plasma corticosterone varied between 3 and 13 nmol litre-1. In both sexes the level was high during winter and through the breeding season. It fell to low levels during late summer and increased again during autumn. Body weight in both sexes was highest in midwinter and lowest in midsummer but there was no correlation between body weight and plasma corticosterone concentration.

Animal Population Groups↗

PubMed1977

Three alkylation methods for the determination of indometacin in plasma by electron capture gas chromatography.

The following alkylation methods for the determination of indometacin in plasma by electron capture gas chromatography are compared: 1, alkylation with diazopropane; 2, extractive alkylation; 3, alkylation by a solid-liquid phase transfer catalysed process. The drug in plasma at pH 4.0 is initially extracted with heptane containing 5% n-pentanol. The methyl ester of indometacin is based as internal standard. After alkylation to the propyl ester according to one of the three alkylation methods, indometacin can be determined down to 5 ng per sample by electron capture gas chromatography. The relative standard deviations (n = 10) at the 200 ng level are 5.1% for the alkylation with diazopropane, 7.5% for the extractive alkylation technique and 3.5% for the alkylation by the solid-liquid phase transfer catalysed process. The comparatively low value obtained by the last method indicates that decomposition of indometacin can be avoided under such mild conditions.

Alkylation↗

PubMed1985

Monoclonal capture antibody ELISA for respiratory syncytial virus: detection of individual viral antigens and determination of monoclonal antibody specificities.

An enzyme-linked immunosorbent assay (ELISA) for respiratory syncytial virus (RSV) that employs solid-phase monoclonal antibodies was developed. RSV antigens bound by these monoclonal capture antibodies were detected by addition of a polyclonal bovine antiserum, followed anti-bovine enzyme conjugate and enzyme substrate. The sensitivity and specific of the assay were determined by titrations of the solid-phase antibodies and by antigen titrations with both unpurified RSV-infected cell culture material and purified RSV nucleocapsids. The addition of a competitive binding step prior to the addition of antigen to the solid-phase antibody provides further evidence of the assay's specificity. Furthermore, the competitive binding assay enables the antigen specificity of monoclonal antibodies to be determined or compared without the use of purified antigens. Monoclonal capture ELISA is a convenient, rapid, and sensitive assay that can be used to measure specific RSV antigens in unpurified preparations as well as to determine anti-RSV antibody specificity and should prove useful in examining other complex antigen-antibody systems.

Antibodies, Monoclonal↗

PubMed1988

The immunochemistry of sandwich-ELISAs. IV. The antigen capture capacity of antibody covalently attached to bromoacetyl surface-functionalized polystyrene.

The antigen capture capacity of antibodies covalently immobilized on injection-molded polystyrene beads was evaluated. Bromoacetyl groups on the bead surfaces rendered them reactive to protein nucleophilic groups. The bromoacetyl surface exhibited up to a ten-fold greater capacity for protein compared to unmodified polystyrene, with no detectable dissociation such as occurs with simple adsorption. Biotinylated anti-fluorescein was immobilized on this surface both through direct covalent attachment and indirectly via streptavidin, which was first covalently attached to the bead. Comparisons of the resulting biological activity, normalized to the amount of anti-fluorescein on the bead, were made between the attachment methods and simple passive adsorption. The presence of the streptavidin spacer on the bromoacetyl surfaces improved the antigen capture capacity of antifluorescein, for fluoresyl-albumin by 45% compared to direct covalent linkage of the antibody to modified polystyrene and by 160% relative to antibody adsorbed on unmodified polystyrene.

Acetates↗

PubMed1989

Determination of human antibodies to specific antigens of cytomegalovirus using an antigen capture immunoassay.

The antigen capture immunoassay which is described herein is based on the binding of specific antigens of cytomegalovirus (CMV) by monoclonal antibodies bound to a solid phase. The specificity of the binding was demonstrated by the analysis of antigens labelled with [35S]methionine and captured by the bound monoclonal antibodies. These specific antigens are recognized in turn by specific anti-cytomegalovirus antibodies in human sera. The immunoassay permits quantitation of these specific anti-cytomegalovirus antibodies and should facilitate both qualitative and quantitative comparisons of the antibodies against specific CMV antigens in different individuals.

Antibodies, Monoclonal↗

PubMed1990

Substantially increased sensitivity of the spot-ELISA for the detection of anti-insulin antibody-secreting cells using a capture antibody and enzyme-conjugated insulin.

This paper describes an antibody capture spot-ELISA for the detection of anti-insulin antibody-secreting cells. The assay is based on the binding of secreted antibodies by immobilised isotype-specific capture antibodies and subsequent detection of insulin-specific antibodies with a conjugate of human insulin and alkaline phosphatase (HI-AP). Compared with the conventional approach, using antigen for coating and employing an enzyme-linked detecting antibody, this technique improved the detection of murine cells secreting anti-insulin antibodies of different IgG subclasses.

Animals↗

PubMed1990

A chemiluminescent, microparticle-membrane capture immunoassay for the detection of antibody to hepatitis B core antigen.

A chemiluminescent, microparticle-membrane capture immunoassay (CLIA/MMC) for the detection of antibody to hepatitis B core antigen (anti-HBc) is described. The assay utilizes recombinant hepatitis B core antigen coupled to carboxylated latex microparticles. Human polyclonal IgG anti-HBc labelled with acridinium competes with antibody in the sample for a limited number of binding sites on the solid phase. After a 40 min incubation at 40 degrees C, the reaction mixture is transferred to a glass fiber capture membrane and washed. A chemiluminescent signal is produced by addition of alkaline peroxide and is quantitated on a semi-automated reader as described. The CLIA/MMC assay was compared with standard EIA and RIA procedures (Corzyme and Corab, respectively, Abbott Laboratories, North Chicago, IL). Assay sensitivities were RIA greater than CLIA/MMC greater than EIA. A population of 200 normal blood donors showed nearly identical distributions with the CLIA/MMC and RIA (mean = 11% inhibition, SD = 13% for both), compared with the EIA (mean = 13% inhibition, SD = 15%). With a selected plasma population (n = 307), the CLIA/MMC immunoassay showed an excellent correlation (r = 0.94) with both the EIA and RIA procedures. Association of anti-HBc reactivity near assay cutoffs with antibody to hepatitis B surface antigen suggested relative specificity in the order RIA greater than CLIA/MMC greater than EIA. The CLIA/MMC procedure, which can be readily automated, provides a non-istopic alternative to current EIA testing with performance more nearly equivalent to RIA.

Acridines↗

PubMed1992

An ELISA sandwich capture assay for recombinant fusion proteins containing glutathione-S-transferase.

A sandwich capture ELISA technique is presented for detection of recombinant proteins sharing a common affinity domain or reagents such as antibodies that bind to these proteins. An activated carrier protein (BSA) is modified with reduced glutathione (GT), forming an affinity capture reagent for glutathione-S-transferase (GST) and recombinant fusion proteins bearing the GST moiety. GT-BSA is immobilized on microtiter plates, and a sandwich is formed consisting of the recombinant fusion protein, reactive antibodies, and detection antibodies. An example is given that demonstrates that this format yields equivalent results to a conventional ELISA test with a panel of newly diagnosed diabetic sera reacting with an islet cell autoantigen.

Chromatography, Affinity↗

PubMed1992

The physical and functional behavior of capture antibodies adsorbed on polystyrene.

Six monoclonal and two polyclonal antibodies to fluorescein (FLU) were affinity purified and immobilized on Immulon 2 polystyrene as capture antibodies (CAbs): (a) by passive adsorption at pH 9.6, (b) via a streptavidin bridge to a biotinylated carrier molecule, and (c) via an antiglobulin which had been previously adsorbed passively to the polystyrene. Data show that less than 3.0% of the binding sites of monoclonal CAbs and approximately 5-10% of those of polyclonal CAbs were capable of capturing antigen (FLU4.2-BSA) after passive adsorption. Immobilization of CAbs via an antiglobulin or a streptavidin bridge, resulted in the preservation of antibody binding sites to greater than 70% for some monoclonals although immobilization via the streptavidin bridge resulted in the highest number of functional sites/well. The data presented are consistent with studies on other adsorbed proteins which demonstrate that passive adsorption on polystyrene results in the loss of protein function. Furthermore, these data show that generally less than half of the binding sites of antibodies available in solution are available after solid-phase immobilization even when non-adsorptive methods are employed. Some polyclonal anti-FLU also have lower average avidity following passive adsorption compared with CAbs immobilization via a streptavidin bridge. Immunochemical studies revealed that adsorbed polyclonal-CAbs performed like monoclonals when tested with multivalent antigens (FLU10-IgA) but in an expected heterogeneous manner in Scatchard plots when tested using univalent FLU-insulin. This observation implied cross-linking of immobilized CAbs by the multivalent antigen. Because only 5-10% of adsorbed polyclonal CAbs are active, the survivors must be non-randomly distributed in clusters to explain the cross-linking. This was confirmed by scanning electron microscopy which gave rise to the hypothesis that antibodies which retain activity after adsorption, are those present in clusters, i.e., the functional adsorbed CAb is an antibody cluster. Data presented in this report on the behavior of adsorbed CAbs, and reviewed from the work of others for various adsorbed proteins, indicate that the method of passive adsorption at pH 9.6, which is widely used in popular microtiter ELISAs, and which has in many ways revolutionized immunoassay, is a method of protein denaturation. Assayists that utilize passive adsorption of proteins on hydrophobic supports as part of their research need to be cognizant of this phenomenon, while inventors of immunoassay should develop alternative methods of immobilization which do not destroy 90% of the functional activity of solid-phase reactant.

Adsorption↗

PubMed1993

Capture assay for specific IgE. An improved quantitative method.

A capture assay for the measurement of specific IgE in the serum of allergic patients is described, using monoclonal anti-human IgE (coated to the wells of a microtiter plate) and biotinylated allergens in solution. In a single incubation, IgE is bound to the solid phase through the Fc fragment and biotinylated allergens react with their specific IgE Fab regions, if present. In a second step, streptavidin-HRP conjugate is added to reveal the amount of biotin fixed on the solid phase. Quantitative determinations are obtained by comparison with a standard curve of total IgE incubated with a biotinylated monoclonal anti-IgE, complementary to the one employed as capture antibody. The assay is unaffected by allergen-specific IgG, shows good intra- and interassay reproducibility and is linear over a wide range of specific IgE concentrations. The method could be used with a wide range of different allergens.

Animals↗

PubMed1995

Biotin-avidin immobilization of platelet glycoproteins (BAIPG): a new capture assay for the detection of anti-platelet antibodies.

Several 'capture' assays are currently employed to identify specific platelet antibodies, but all require the use of murine monoclonal antibodies (MoAbs) against the antigen of interest. We have developed a new antigen capture assay for the detection of platelet reactive antibodies, based on platelet surface sialoglycoprotein labelling with biotin hydrazide, and a following immobilization of the biotinylated platelet proteins to microtiter wells that had been coated with streptavidin. The resulting solid phase can then be used in a simple ELISA to detect serum and platelet associated antibodies. We describe here two versions of this biotin-avidin immobilization of platelet glycoproteins (BAIPG) assay. In BAIPG assay type I, the test sera are directly incubated in microtiter wells previously coated with streptavidin plus biotinylated platelet proteins. The BAIPG type II procedure involves the incubation of sera with biotinylated platelets before platelet solubilization, and, after platelet lysis, the immobilization of the immune complexes to streptavidin-coated wells. In both cases, the bound antibodies are determined by alkaline phosphatase conjugated anti-human IgG. Using BAIPG type I, positive results were obtained in 7/33 patients with idiopathic thrombocytopenic purpura (ITP), 1/10 patients with secondary immune thrombocytopenia (SIT) and 4/17 with non-immune thrombocytopenia (NIT). The BAIPG type II test was positive in 13 out of 33 patients with ITP, in six out of ten patients with SIT, and in three out of the 17 patients with NIT. A comparison between BAIPG and monoclonal antibody immobilization of platelet antigens (MAIPA) assays showed a high degree of correlation between the two methods. These results suggest that the BAIPG assay is a valuable new tool for the detection of anti-platelet antibodies.

Autoantibodies↗

PubMed1996

Autoantibody-mediated capture and presentation of autoantigen to T cells via the Fc epsilon receptor by a recombinant human autoantibody Fab converted to IgE.

Fc epsilon receptor (CD23)-mediated capture of IgE-antigen complexes by B cells provides a powerful antigen presenting system. Our goal was to develop a system using high affinity, human, organ-specific monoclonal autoantibodies for antigen capture by B cells. For this purpose, we converted a recombinant human autoantibody to TPO from a Fab (SP1.4) to an IgE molecule. Sera from all patients with autoimmune thyroid disease contain autoantibodies with the same epitope as SP1.4. The SP1.4 H and L chain V region genes were spliced by overlap PCR to a mammalian, non-immunoglobulin signal peptide and transferred to expression vectors for human IgG1 and kappa, respectively. After inserting the IgE constant region genes into the H chain vector, the kappa and IgE H chain vectors were expressed in SP2/0 cells. SP1.4-IgE retains its high affinity (Kd) for TPO (approximately 2 x 10(-10) M), recognizes the same epitope as Fab SP1.4 and, importantly binds to a different epitope than does Fab TR1.9. Binding of preformed complexes of SP1.4-IgE and biotinylated TPO to EB virus transformed B cells (EBVL) was weakly detectable by flow cytometry and was displaced by unlabeled TPO. SP1.4-IgE/125I-TPO complex binding to EBVL was much more clearly evident, was also inhibited by the addition of unlabeled TPO, and was greatly reduced by preincubation of the EBVL with anti-CD23. Further, autologous EBVL preincubated with SP1.4-IgE/TPO complexes stimulated proliferation of TPO-specific T cells. IgE autoantibody-mediated antigen focusing to B cells is unlikely to operate in vivo but is, instead, a powerful investigative tool. In conclusion, SP1.4-IgE is the first monoclonal human autoantibody to be developed for IgE-mediated antigen presentation to T cells by EBVL. Recombinant human autoantibodies converted to IgE, possibly in combinations if their epitopes permit simultaneous binding to the same molecule, provide a unique system to generate human T cell lines and clones specific for peptides naturally processed from internalized high affinity autoantibody/autoantigen complexes.

Antigen Presentation↗

PubMedDate not supplied

The effect of transiency on perceived velocity of visual patterns: a case of "temporal capture".

We measured the points of subjective equality of velocity for dynamic unidirectionally moving random-dot patterns with different amounts of transiency. The transiency was changed by varying the time a dot would move before being randomly replotted within the stimulus. The perceived velocity of patterns moving at intermediate velocities (4 or 6 deg/sec) was increased by decreasing the point lifetime while no speedup was observed at high velocities (12 deg/sec). A speedup was also observed when a few stationary points of short lifetime were introduced into a stimulus. The non-directional transiency generated by these flickering points seems to be captured by the moving pattern and biases the velocity estimate. We term this phenomenon "temporal capture". The results are in agreement with models that determine velocity by comparing the activity in lower and higher temporal frequency channels. Our stimuli would selectively increase activity in high temporal frequency channels and thus lead to an increase in perceived velocity.

Humans↗

PubMed1996

Two contributions of motion induction: a preattentive effect and facilitation due to attentional capture.

By combining the paradigms of motion induction (presentation of an inducing stimulus, followed after a short delay by the presentation of an elongated bar next to it) and visual search (many-item displays with or without a pop-out target), it was possible to demonstrate the existence of two separate contributions to the motion induction effect. Illusory motion in the test bar could be produced either preattentively or by facilitation due to attentional capture. The former effect is fast, independent of the delay between the inducers and the test bar and operating simultaneously at all locations across the visual display, the latter is slower (full strength in 200-300 msec) and confined to the vicinity of the pop-out inducer. The two possibly also differ in their spatial extent, the attentional capture effect extending over a larger area around the inducer. We conclude that the motion induction effect can be used to show the existence of several effects due to the sudden presentation of a visual stimulus.

Attention↗

PubMed1981

Preparation of biological fluids for simultaneous analysis of prostaglandin cyclo-oxygenase synthesized compounds by gas chromatography with electron capture detection.

A method for isolating climax products of the arachidonic acid cascade from biological fluids is described which allows simultaneous measurement of PGs (PGE2, PGD2, 6-keto-PGF1 alpha, TXB2, 6-keto-PGE1, 6, 15-diketo-13,14-dihydro-PGF1 alpha) by electron capture detection of pentafluorobenzyloxime methyl ester trimethylsilyl (TMS) ether derivatives. PGs are adsorbed onto Amberlite XAD-2 from acidified solution and nonadhering substances removed by sequential administration of water, then petroleum ether. PGs are extracted into methanol. Following evaporation and reconstitution in water, the PGs are extracted into ethyl acetate at pH 3 and the ethyl acetate extracts are purified by lipidex column chromatography. Derivatization to pentafluorobenzyloxime methyl ester TMS ethers of PGs in the sample is followed by separation on either glass packed-columns or SCOT capillary columns, and detection by an electron capture detector. PGA2, added to the unpurified sample, is used as an internal standard for quantification. The methods have performed well on all biological fluids thus far examined. Examples of chromatographs from urine, Krebs-perfused lung effluents, and blood are shown.

Animals↗

PubMed1986

A model study of the use of monoclonal antibodies in capture enzyme immunoassays for antigen quantification exploiting the epitope map of tick-borne encephalitis virus.

On the basis of an epitope model, capture enzyme immunoassay systems using monoclonal antibodies have been devised for the detection and quantification of Tick-borne encephalitis virus and compared with a reference system employing polyclonal sera. Monoclonal antibodies were used both as capture and detector antibodies, their suitability depending primarily on their avidity and intrinsic background activity. A considerable increase in sensitivity was achieved by combining antibodies to different non-overlapping epitopes. Biotinylation of the detector antibodies allowed the construction of multiple site simultaneous binding assays. Furthermore the use of monoclonal antibodies of defined serological specificity made virus type identification possible. This assay can therefore be used as a rapid 'test of identity' as required during the manufacture of viral vaccines.

Animals↗

PubMed1993

Capture immunoassay for ruminant tumor necrosis factor-alpha: comparison with bioassay.

Monoclonal antibodies and IgG purified from rabbit polyclonal antiserum, raised against recombinant bovine tumor necrosis factor-alpha (TNF-alpha), have been employed in ELISA procedures to quantitate bovine TNF-alpha. These antibodies were potent in neutralizing the biological activity of recombinant as well as natural bovine TNF-alpha. The monoclonal antibodies were used as capture antibodies and were either passively adsorbed or covalently linked to ELISA plates. Polyclonal rabbit anti-TNF IgG was used as the detecting antibody in combination with a biotinylated anti-rabbit serum and a streptavidin-horseradish peroxidase conjugate. The detection limit for recombinant TNF-alpha medium was 10 pg ml-1 and in bovine or ovine serum was 35 pg ml-1. A good correlation was found between the ELISA and the WEHI-164 Clone 13 biologic assay when TNF-alpha was measured in medium containing serum or in serum. This capture ELISA was also capable of detecting ovine, but not porcine. TNF in supernatants from cultures of lipopolysaccharide-stimulated pulmonary alveolar macrophages.

Animals↗