Morphological analysis of the effects of ions and ultraviolet light on colloidal monolayers at the air-water interface.

The formation of fractal aggregates under the external influence of ions or ultraviolet light has been observed in colloidal monolayers trapped at the air-water interface. A morphological analysis of this is reported in this paper. These fractals have many characteristics similar to those observed in other experiments where electrolyte was added to the bulk. However, they exhibit behavior, such as restructuring, particular to the external influence. In most cases the aggregating system displayed scaling analogous to spinodal decomposition, but with the measured fractal dimension (which depends slightly on the external influence) entering in place of the usual spatial dimension.

Pattern Formation in Colloidal Monolayers at the Air-Water Interface.

Using video-microscopic techniques, single layers (monolayers) of charged colloidal particles of 1- and 3-&mgr;m diameter were studied at an air-water interface. The particles normally arranged themselves in an ordered lattice-like array. However, occasionally, the monolayer formed loosely bonded crystallite clusters, here called mesostructures. The monolayer was exposed to a number of external influences: ions, electric current, and ultraviolet light. Depending on the perturbation applied, the lattice-like structure formed fractal aggregates, clusters (mesostructures), and two other particular structures, here referred to as striations and loops. The mechanisms of formation and implications of the different patterns are discussed. Copyright 2001 Academic Press.

Signal amplification in flow cytometry using biotin tyramine.

BACKGROUND: Catalysed reporter deposition (CARD) has been successfully used as a means of signal amplification in solid-phase immunoassays. The procedure relies on the use of horseradish peroxidase (HRP)-conjugated reagents--normally antibodies-in conjunction with substituted phenolic compounds such as biotin tyramine. The HRP catalyses deposition of biotin tyramine around the site of enzyme activity, and streptavidin-HRP can then be added to generate an amplified HRP signal. The possibility of using this technique for solution-phase amplifications has been suggested but not yet demonstrated. METHODS: This paper describes the application of CARD to signal enhancement in flow cytometry. The specific examples described here are those of anti-human CD4 and anti-human CD36 antibodies binding to either human lymphocytes or mixed mononuclear cells. RESULTS: Optimum biotin tyramine concentrations were evaluated, and a fivefold increase in signal was observed over standard detection of the anti-human CD4 antibody with anti-mouse-fluorescein isothiocyanate (FITC). In the example using the anti-CD36 antibody, the biotin tyramine treatment was repeated, resulting in an additional 2.5-fold signal amplification. CONCLUSIONS: The technique described in this report provides a method of amplifying the signals achieved by standard flow cytometry detection reagents.

Directed selection of MIP-1 alpha neutralizing CCR5 antibodies from a phage display human antibody library.

The seven trans-membrane chemokine receptor CCR-5 is a coreceptor for macrophage tropic HIV-1 strains. CCR-5 responds to a number of chemokines, including macrophage inflammatory protein (MIP)-1 alpha. We describe the use of MIP-1 alpha in a biotin tyramine-mediated proximity selection to guide the selection of CCR-5-specific phage antibodies from a large phage display human library. Proximity based selections resulted in a population of antibodies recognizing CCR-5 on primary CD4+ lymphocytes, none of which blocked MIP-1 alpha binding to cells. The selected population of phage antibodies were subsequently used as guide molecules for a second phase of selection that was carried out in the absence of MIP-1 alpha. This generated a panel of CCR-5-binding antibodies, of which around 20% inhibited MIP-1 alpha binding to CD4+. The single chain Fvs (scFv) generated by this step-back selection procedure also inhibited MIP-1 alpha-mediated calcium signaling.

Surface light scattering: a methodological review.

The methods used in the application of light scattering to the study of thermally excited capillary waves on liquid surface or interfaces are reviewed. The focus is on the use of photon correlation to determine the spectroscopic information carried by the scattered light. Particular attention is directed to extraction of the maximum amount of information from the data, including surface viscoelastic parameters and intensities.

Directing phage selections towards specific epitopes.

It is possible to direct selections from antibody repertoires displayed on filamentous phage towards unique epitopes on protein antigens by competing with related molecules. A phage display repertoire of human single chain Fvs (scFvs) was panned three times against foetal haemoglobin (HbF). The selection was dominated by one clone with a Kd of 10 nM but yielded at least 17 others, all of which bound HbF but crossreacted with adult haemoglobin (HbA). To direct selection towards HbF-specific epitopes, the repertoire was preincubated with HbA in solution before each panning. Crossreactive scFvs can form complexes with the soluble HbA and thereby be prevented from binding the immobilized HbF. Four clones with preferential binding to HbF emerged under these conditions. One of these (Hb-1), with a Kd of 6 microM, had exquisite specificity for HbF and could distinguish cells expressing HbF from those expressing HbA by immunocytochemistry and flow cytometry. This antibody has an affinity that is 600-fold lower than the dominant crossreactive clone, and so only emerged under conditions of 'competitive deselection'. Thus, competitive deselection is a viable means for directing selections towards useful epitopes. It permits a more effective 'search' of phage display repertoires and allows the emergence of lower affinity clones with useful specificities. These clones may be useful in themselves or may serve as leads for in vitro affinity maturation.

Generation of a panel of related human scFv antibodies with high affinities for human CEA.

BACKGROUND: A human single chain Fv (scFv) specific for human carcinoembryonic antigen (CEA) has been isolated from a 2.0 x 10(9) phage display library from unimmunised human donors. The dissociation constant of the scFv has been measured by surface plasmon resonance (SPR) and found to be 7.7 x 10(-9) M, with an off-rate component of 6.2 x 10(-3) s-1. In order to investigate directly whether increased affinity leads to improved targeting of CEA-positive tumours, this scFv has been affinity matured by both targeted mutagenesis of the CDRs of heavy and light chains, and by light chain shuffling. STUDY DESIGN: A partial randomisation scheme, biased towards amino acids commonly found as somatic mutations of germline antibody sequences, was used for directed diversification of VH and VL CDR3s. Diversification of the entire VL region was also introduced by light chain shuffling of the parental anti-CEA scFv. Selection of the mutagenised repertoires was carried out to enrich for antibodies with a reduced koff. RESULTS: Sequencing the selected clones identified a number of amino acid changes in the VH CDR3, one of which gave a four-fold reduction in koff. Stringent selection of the light chain shuffled library resulted in several clones with a two- to three-fold reduction in koff. It has been possible to combine the selected changes from both mutagenesis approaches by using the mutagenised heavy chain and a light chain derived by shuffling to give a human scFv with a dissociation constant for human CEA of 6.0 x 10(-10) M. CONCLUSION: A panel of human anti-CEA scFvs has been generated with differing dissociation constants for antigen, which will allow the correlation between tumour targeting efficiency in relation to binding affinity to be assessed directly. The scFv panel will be valuable in the optimisation of human antibodies for immunotherapy.

Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library.

To generate a stable resource from which high affinity human antibodies to any given antigen can be rapidly isolated, functional V-gene segments from 43 non-immunized human donors were used to construct a repertoire of 1.4 x 10(10) single-chain Fv (scFv) fragments displayed on the surface of phage. Fragments were cloned in a phagemid vector, enabling both phage displayed and soluble scFv to be produced without subcloning. A hexahistidine tag has been incorporated to allow rapid purification of scFv by nickel chelate chromatography. This library format reduces the time needed to isolate monoclonal antibody fragments to under two weeks. All of the measured binding affinities show a Kd < 10 nM and off-rates of 10(-3) to 10(-4) s-1, properties usually associated with antibodies from a secondary immune response. The best of these scFvs, an anti-fluorescein antibody (0.3 nM) and an antibody directed against the hapten DTPA (0.8 nM), are the first antibodies with subnanomolar binding affinities to be isolated from a naive library. Antibodies to doxorubicin, which is both immunosuppressive and toxic, as well as a high affinity and high specificity antibody to the steroid hormone oestradiol have been isolated. This work shows that conventional hybridoma technology may be superseded by large phage libraries that are proving to be a stable and reliable source of specific, high affinity human monoclonal antibodies.

Novel developmentally regulated exon identified in the rat fast skeletal muscle troponin T gene.

In theory, the rat fast skeletal muscle troponin T gene can generate 64 different isoforms. Here we report the identification of a novel alternative exon (exon y) that increases the potential isoform variation to 128. The inclusion of exon y in fast skeletal muscle troponin T mRNA occurs in perinatal, but not adult, skeletal muscle. Exon y is located between exons 8 and 9. This is the first time that a developmentally regulated exon located amongst a set of alternatively spliced exons has been described. Exon y is included in two mRNA isoforms. The proteins that these mRNAs would encode have molecular masses greater than that of the largest fast skeletal muscle troponin T isoform lacking exon y. These two proteins correlate well in both size and pattern of expression with the two fast skeletal muscle troponin T isoforms expressed in perinatal skeletal muscle. These results indicate that there is coordinated regulation of the splicing of exon y with other alternative exons.

Viscoelastic relaxation of bilayer lipid membranes: II. Temperature dependence of relaxation time.

Recent studies have demonstrated the high frequency exponential relaxation of a viscoelastic modulus of lipid membranes. It is shown that the temperature dependence of the relaxation time can be extracted from published data on the relevant modulus for solvent-free bilayer lipid membranes of glycerol monoöleate. The relaxation is fastest close to the membrane transition temperature, where the time scale is approximately 20 mus. Possible causes of the transitional minimum of this time scale are discussed.

Novel instrumental effects in surface fluctuation spectroscopy: lens aberrations.

The performance of a heterodyne spectrometer used for quasielastic light scattering studies of the fluctuations of liquid surfaces was investigated. Previously unreported instrumental effects were found, which appear to arise from the optical arrangement. It is shown that these effects are due to one particular lens aberration, coma. It is further shown that the spectrometer remains usable even in the presence of such aberrations, provided that care is taken in its calibration.

Viscoelastic relaxation of bilayer lipid membranes. Frequency-dependent tension and membrane viscosity.

Photon correlation spectroscopy has been used to study capillary waves on black lipid membranes of glycerol monooleate at temperatures above the lipid transition. For the first time the tension and viscosity of solvent-free bilayers have been observed to display a frequency dependence. The variations of both parameters can be accounted for by a Maxwell viscoelastic fluid model having a relaxation time of 37 microseconds. The equilibrium (omega = 0) tension is compatible with literature values. The present results do not suffice to precisely define the specific molecular processes involved, but relaxation times similar to the present are associated with certain phenomena in phospholipid vesicles. Bilayers containing hydrocarbon solvent do not show such relaxation, presumably due to their weaker intermolecular interactions.

Phase transitions in monoglyceride bilayers. A light scattering study.

Thermotropic phase transitions in single planar bilayers of glycerol mono-oleate have been investigated using quasi-elastic light scattering from thermally excited membrane fluctuations. In certain cases both spectroscopic and intensity information were derived from the observations. For solvent-free bilayers transitional changes were observed in several membrane parameters: in tension, viscosity and thickness, in a combination of lipid orientational order parameter and dielectric anisotropy, and in the lateral compression modulus. These changes, particularly those in membrane thickness and in the anisotropy/order combination, were clearly indicative of a chain-melting transition in the lipid molecules. The chain-melting transition temperature was identified as 16.6 +/- 0.03 degrees C (delta T 1/2 = 1.5 degrees C). The other changes tended to cluster around 12.5 and 16.6 degrees C, suggesting that a two-stage transition was involved. Analysis of pretransitional fluctuations in membrane viscosity, based on a Landau approach, suggested that at the transition the membrane was close to a critical point (T = 12.7 degrees C). Less information was accessible for membranes containing n-decane within their structure. In this case, the change in membrane tension was much smaller than in the solvent-free case and the transition was considerably broadened. These effects accord with an increase in 'interactive volume' within the bilayer due to solvent inclusion.

Photon correlation spectroscopy as a probe of planar lipid bilayer phase transitions.

Photon correlation spectroscopy has been applied to study phase transitions of planar bilayer membranes. The membrane tension and one specific membrane viscosity are probed. Difficulties arising in the measurement of the temperature dependence of these properties are discussed and a servo-control system to overcome them is described. Typical data are presented for monoglyceride bilayers. Membranes incorporating cholesterol display effects below the lipid transition temperature which are interpreted in terms of separation within the membrane into cholesterol-rich fluid regions and regions of lipid in the gel phase. Some of the cholesterol-rich regions are apparently of macroscopic extent.

Photon correlation spectroscopy of bilayer lipid membranes.

Light scattering by thermal fluctuations on simple monoglyceride bilayer membranes has been used to investigate the viscoelastic properties of these structures. Spectroscopic analysis of these fluctuations (capillary waves) permits the nonperturbative measurement of the interfacial tension and a shear interfacial viscosity acting normal to the membrane plane. The methods were established by studies of solvent and nonsolvent bilayers of glycerol monooleate (GMO). Changes in the tension of GMO/n-decane membranes induced by altering the composition of the parent solution were detected and quantified. In a test of the reliability of the technique controlled variations of the viscosity of the aqueous bathing solution were accurately monitored. The technique was applied to solvent-free bilayers formed from dispersions of GMO in squalane. The lower tensions observed attested to the comparative absence of solvent in such bilayers. In contrast to the solvent case, the solvent-free membranes exhibited a significant transverse shear viscosity, indicative of the enhanced intermolecular interactions within the bilayer.

Cholesterol-induced effects on the viscoelasticity of monoglyceride bilayers.

Changes in the viscoelastic properties of glycerol monooleate bilayers resulting from the incorporation of cholesterol into the membranes have been measured. The interface tension increases with the cholesterol concentration, reaching saturation for a 4.2:1 mole ratio of cholesterol:lipid in the film-forming solution. Incorporation of cholesterol in the membrane causes the appearance of a large intrinsic viscosity; this also increases with the sterol content of the membrane. Molecular models of lipid-sterol interactions and packing are considered to explain both the observed changes in membrane properties and similarities with comparable lipid systems.