Slow rotational mobilities of antibodies and lipids associated with substrate-supported phospholipid monolayers as measured by polarized fluorescence photobleaching recovery.

Polarized fluorescence photobleaching recovery has been used to monitor slow rotational motions of a fluorescently-labeled anti-dinitrophenyl mouse IgGl monoclonal antibody (ANO2) specifically bound to substrate-supported monolayers composed of a mixture of distearoylphosphatidylcholine (DSPC) and dinitrophenyldioleoylphosphatidylethanolamine (DNP-DOPE). ANO2 antibodies were labeled with a new bifunctional carbocyanine fluorophore that has two amino-reactive groups; steady-state fluorescence anisotropy data confirmed the expected result that the ANO2-conjugated bifunctional probe had less independent flexibility than ANO2-conjugated unifunctional fluorescence labels. Rotational mobilities were also measured for the fluorescent lipid 1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine (dil) in DSPC and in mixed DSPC/DNP-DOPE monolayers in the presence and absence of unlabeled ANO2 antibodies. The apparent rotational correlation time and fractional mobility of ANO2 on supported monolayers were approximately 70 and approximately 0.3 s, respectively. These measured parameters of rotational mobility did not depend on the ANO2 surface density or on kinetic factors, but addition of unlabeled polyclonal anti-(mouse IgG) antibodies significantly decreased the apparent mobile fraction. The measured fluorescence recovery curves for dil were consistent with two fluorophore populations with rotational correlation times of approximately 4 and approximately 100 s and a population of immobile fluorescent lipid. No difference in fluorescence recovery and decay curves was measured for dil in DSPC monolayers, DSPC/DNP-DOPE monolayers, and DSPC/DNP-DOPE monolayers treated with unlabeled ANO2 antibodies.     

Lateral diffusion and fluorescence microscope studies on a monoclonal antibody specifically bound to supported phospholipid bilayers

Supported phospholipid bilayers prepared by Langmuir-Blodgett techniques were introduced recently as a new model membrane system [Tamm, L.K., & McConnell, H.M. (1985) Biophys. J. 47, 105-113]. Here, supported bilayers are applied to study the lateral diffusion and lateral distribution of membrane-bound monoclonal antibodies. A monoclonal anti-trinitrophenol antibody was found to bind strongly and with high specificity to supported phospholipid bilayers containing the lipid hapten (trinitrophenyl)phosphatidylethanolamine at various mole fractions. The lateral distribution of the membrane-bound antibodies was studied by epifluorescence microscopy. The bound antibodies aggregated into patches on a host lipid bilayer of dimyristoylphosphatidylcholine below the lipid chain melting phase transition and redistributed uniformly on fluid-phase supported bilayers. Lateral diffusion coefficients and mobile fractions of fluorescent phospholipid analogues and fluorescein-labeled antibodies were measured by fluorescence recovery after pattern photobleaching. The lateral diffusion coefficients of the membrane-bound antibodies resembled those of the phospholipids but were reduced by a factor of 2 in the fluid phase. The lipid chain melting phase transition was also reflected in the lateral diffusion coefficient of the bound antibody but occurred at a temperature about 3 deg higher than the phase transition in supported bilayers of pure phospholipids. The antibody lateral diffusion coefficients decreased in titration experiments monotonically with increasing antibody surface concentrations by a factor of 2-3. Correspondingly, a relatively small decrease of the antibody lateral diffusion coefficient was observed with increasing mole fractions of lipid haptens in the supported bilayer.     

Inhomogeneous translational diffusion of monoclonal antibodies on phospholipid Langmuir-Blodgett films.

The translational mobility of fluorescent-labeled monoclonal antibodies specifically bound to supported phospholipid bilayers containing hapten-conjugated phospholipids has been measured as a function of the surface concentration of bound antibodies using fluorescence recovery after photobleaching. Fluorescence recovery curves are fit well by a model that assumes the presence of two populations of antibodies with different lateral diffusion coefficients. The larger diffusion coefficient equals 3.5 x 10(-9) cm2/s, the smaller diffusion coefficient ranges from 1.5 x 10(-9) cm2/s to 2.5 x 10(-10) cm2/s, and the fractional fluorescence recovery associated with the smaller coefficient increases from approximately 0 to approximately 0.7 with increasing concentration of bound antibody. These results suggest that complexes of haptenated phospholipids and antibodies in phospholipid Langmuir-Blodgett films form clusters or domains in a concentration-dependent fashion.     

Binding of a monoclonal antibody and its Fab fragment to supported phospholipid monolayers measured by total internal reflection fluorescence microscopy.

The association of an anti-dinitrophenyl monoclonal antibody and its Fab fragment with supported phospholipid monolayers composed of a mixture of dipalmitoylphosphatidylcholine and dinitrophenyl-conjugated dipalmitoylphosphatidylethanolamine has been characterized with total internal reflection fluorescence microscopy. The surface densities of bound antibodies were measured as a function of the antibody and Fab solution concentrations, and as a function of the solution concentration of dinitrophenylglycine. The apparent association constant of Fab fragments with surface-associated haptens was approximately 10-fold lower than the association constant for haptens in solution, and the apparent surface association constant for intact antibodies was only approximately 10-fold higher than the constant for Fab fragments. Data analysis with simple theoretical models indicated that, at most antibody surface densities, 50-90% of membrane-associated intact antibodies were attached to the surface by two antigen binding sites.     

Submicron structure in L-alpha-dipalmitoylphosphatidylcholine monolayers and bilayers probed with confocal, atomic force, and near-field microscopy.

Langmuir-Blodgett (LB) monolayers and bilayers of L-alpha-dipalmitoylphosphatidylcholine (DPPC), fluorescently doped with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diIC18), are studied by confocal microscopy, atomic force microscopy (AFM), and near-field scanning optical microscopy (NSOM). Beyond the resolution limit of confocal microscopy, both AFM and NSOM measurements of mica-supported lipid monolayers reveal small domains on the submicron scale. In the NSOM studies, simultaneous high-resolution fluorescence and topography measurements of these structures confirm that they arise from coexisting liquid condensed (LC) and liquid expanded (LE) lipid phases, and not defects in the monolayer. AFM studies of bilayers formed by a combination of LB dipping and Langmuir-Schaefer monolayer transfer exhibit complex surface topographies that reflect a convolution of the phase structure present in each of the individual monolayers. NSOM fluorescence measurements, however, are able to resolve the underlying lipid domains from each side of the bilayer and show that they are qualitatively similar to those observed in the monolayers. The observation of the small lipid domains in these bilayers is beyond the spatial resolving power of confocal microscopy and is complicated in the topography measurements taken with AFM, illustrating the utility of NSOM for these types of studies. The data suggest that the small LC and LE lipid domains are formed after lipid transfer to the substrate through a dewetting mechanism. The possible extension of these measurements to probing for lipid phase domains in natural biomembranes is discussed.     

Atomic force microscopy of hydrated phosphatidylethanolamine bilayers.

We present images of the polar or headgroup regions of bilayers of dimyristoyl-phosphatidylethanolamine (DMPE), deposited by Langmuir-Blodgett deposition onto mica substrates at high surface pressures and imaged under water at room temperature with the optical lever atomic force microscope. The lattice structure of DMPE is visualized with sufficient resolution that the location of individual headgroups can be determined. The forces are sufficiently small that the same area can be repeatedly imaged with a minimum of damage. The DMPE molecules in the bilayer appear to have relatively good long-range orientational order, but rather short-range and poor positional order. These results are in good agreement with x-ray measurements of unsupported lipid monolayers on the water surface, and with electron diffraction of adsorbed monolayers.     

Measuring antibody affinity and performing immunoassay at the single molecule level

Fluorescence correlation spectroscopy (FCS) enables direct observation of the translational diffusion of single fluorescent molecules in solution. When fluorescent hapten binds to antibody, analysis of FCS data yields the fractional amounts of free and bound hapten, allowing determination of the equilibrium binding constant. Equilibrium dissociation constants of anti-digoxin antibodies and corresponding fluorescein-labeled digoxigenin obtained by FCS and fluorescence polarization measurements are identical. It is also possible to follow a competitive displacement of the tracer from the antibody by unlabeled hapten using FCS in an immunoassay format. The fluorescence polarization immunoassay for vancomycin detection was used to test the FCS approach. Fitting of the FCS data for the molar fractions of free and bound fluorescein-labeled vancomycin yielded a calibration curve which could serve for determination of the vancomycin concentration in biological samples.     

Structural investigation of the covalent and electrostatic binding of yeast cytochrome c to the surface of various ultrathin lipid multilayers using x-ray diffraction.

X-Ray diffraction was used to characterize the profile structures of ultrathin lipid multilayers having a bound surface layer of cytochrome c. The lipid multilayers were formed on an alkylated glass surface, using the Langmuir-Blodgett method. The ultrathin lipid multilayers of this study were: five monolayers of arachidic acid, four monolayers of arachidic acid with a surface monolayer of dimyristoyl phosphatidylserine, and four monolayers of arachidic acid acid with a surface monolayer of thioethyl stearate. Both the phosphatidylserine and the thioethyl stearate surfaces were found previously to covalently bind yeast cytochrome c, while the arachidic acid surface electrostatically binds yeast cytochrome c. Meridional x-ray diffraction data were collected from these lipid multilayer films with and without a bound yeast cytochrome c surface layer. A box refinement technique, previously shown to be effective in deriving the profile structures of ultrathin multilayer lipid films with and without electrostatically bound cytochrome c, was used to determine the multilayer electron density profiles. The surface monolayer of bound cytochrome c was readily apparent upon comparison of the multilayer electron density profiles for the various pairs of ultrathin multilayer films plus/minus cytochrome c for all cases. In addition, cytochrome c binding to the multilayer surface significantly perturbs the underlying lipid monolayers.     

Study of P-glycoprotein effect on the lipid monolayer properties by the Langmuir-Blodgett technique

Expression of the P-glycoprotein (Pgp) is proved to be one of the main reasons for the development of the multidrug resistance (MDR) phenotype by cancer cells. The effect of Pgp on the properties of lipid monolayers was studied using membrane fractions of sensitive and Pgp over-expressing multidrug resistance cancer cells containing 11, 24 or 32% of Pgp relative to the total content of membrane proteins. The effect of the Pgp membrane concentration on the properties of monolayers prepared from the membrane fractions was analyzed by the Langmuir-Blodgett method. The subphase composition was found to play a critical role in the stability of monolayers at any Pgp concentration. The optimal subphase comprised 10 mM tris-HCl buffer, pH 6.5, which made it possible to create very stable monolayer films with the pressure of collapse of about 30-40 mN/m. Monolayers prepared from membrane fractions of sensitive cells and cells containing the maximum (32%) amount of Pgp were found to be much more stable compared with fractions comprising 11 or 24% of Pgp. The analysis of monolayer compression dynamics revealed three distinct stages: (1) the self-organization of lipid molecules, which is characterized by an abrupt change of surface potential; (2) the compression of Pgp molecules at the constant potential of monolayers; and (3) the compression of lipid molecules, which is characterized by a quasilinear increase of both pressure and surface potential. It was shown that the specific surface areas of monolayers formed from sensitive and Pgp-enriched membranes containing 11 or 24% of Pgp are very similar, whereas the surface area of the monolayer formed from membranes containing 32% of Pgp is nearly 1.5-fold greater. This fact may reflect the effect of the threshold rearrangement of the structure of lipid molecules or cooperative modifications of lipid-Pgp interactions induced by the increase in the Pgp content from 24 to 32%. The effect of verapamil, a well-known Pgp modulator, on the properties of monolayers was studied. It was show that verapamil is able to induce changes of the surface of Pgp-containing monolayers, and these modifications are maximal at the Pgp:verapamil 1:1 molar ratio. The data present the first experimental evidence for the possible intervention of Pgp modulator into the processes of lipid-lipid or lipid-Pgp cooperative interactions within Pgp-enriched membranes.     

Effect of bovine prothrombin fragment 1 on the translational diffusion of phospholipids in Langmuir-Blodgett monolayers.

Previous work has shown that bovine prothrombin fragment 1 binds to supported planar membranes composed of phosphatidylcholine and phosphatidylserine in a Ca(2+)-specific manner (Tendian et al. (1991) Biochemistry 30, 10991; Pearce et al. (1992) Biochemistry 31, 5983-5995). In the present work, fluorescence pattern photobleaching recovery has been used to examine the effect of membrane-bound fragment 1 on the translational diffusion coefficients of two fluorescent phospholipids in fluid-like phosphatidylserine/phosphatidylcholine Langmuir-Blodgett monolayers. The results show that saturating concentrations of fragment 1, in the presence of Ca2+, reduce the diffusion coefficient of nitrobenzoxadiazolyl-conjugated phosphatidylserine (NBD-PS) and nitrobenzoxadiazolyl-conjugated phosphatidylcholine (NBD-PC) by factors of approximately four and two, respectively. Ca2+ or fragment 1 alone do not have a statistically significant effect on NBD-PS or NBD-PC diffusion. In addition, a nonspecific protein (ovalbumin) does not change the diffusion coefficients of the fluorescent phospholipids either in the absence or presence of Ca2+. The fractions of the fluorescent phospholipids that are laterally mobile are approximately 0.9 for all samples. These results are interpreted with several models for possible mechanisms by which extrinsically bound proteins might retard phospholipid diffusion in membranes.     

Binding of antibiotic amphotericin B to lipid membranes: monomolecular layer technique and linear dichroism-FTIR studies

Amphotericin B (AmB) is one of the main antibiotics applied in treatment of deep-seated mycotic infections. Tensiometric technique has been applied to monitor binding of AmB, from the water subphase, to the lipid monomolecular layers, formed with dipalmitoylphosphatidylcholine at the air-water interface. Time dependencies of surface pressure in the monolayers demonstrate strong enhancement of AmB binding to monolayers brought about by sterols present in the membranes. The monolayers have been deposited to a solid support and examined by means of FTIR spectroscopy. FTIR measurements show that majority of the AmB molecules which bind to the membranes are localized in the polar headgroup region. The results of the linear dichroism-FTIR measurements are consistent with the microscopic picture according to which the molecules of the membrane-bound AmB are distributed among two orientational fractions: one horizontal and one vertical with respect to the plane of the membrane (59% versus 41% respectively, in the case of the membrane formed with the pure lipid without sterols). The presence of cholesterol in the membranes (50 mol% with respect to lipid) slightly affects such a distribution (53% horizontal versus 47% vertical) but the presence of ergosterol has a pronounced effect in the increase in population of the fraction of horizontally bound AmB (85% horizontal vs. 15% vertical). The results of the measurements indicate that mode of action of the AmB consists in disruption of the polar headgroup region of biomembranes, brought about by the AmB molecules bound horizontally with respect to the plane of the membrane.     

Binding of antibiotic amphotericin B to lipid membranes: monomolecular layer technique and linear dichroism-FTIR studies

Amphotericin B (AmB) is one of the main antibiotics applied in treatment of deep-seated mycotic infections. Tensiometric technique has been applied to monitor binding of AmB, from the water subphase, to the lipid monomolecular layers, formed with dipalmitoylphosphatidylcholine at the air-water interface. Time dependencies of surface pressure in the monolayers demonstrate strong enhancement of AmB binding to monolayers brought about by sterols present in the membranes. The monolayers have been deposited to a solid support and examined by means of FTIR spectroscopy. FTIR measurements show that majority of the AmB molecules which bind to the membranes are localized in the polar headgroup region. The results of the linear dichroism-FTIR measurements are consistent with the microscopic picture according to which the molecules of the membrane-bound AmB are distributed among two orientational fractions: one horizontal and one vertical with respect to the plane of the membrane (59% versus 41% respectively, in the case of the membrane formed with the pure lipid without sterols). The presence of cholesterol in the membranes (50 mol% with respect to lipid) slightly affects such a distribution (53% horizontal versus 47% vertical) but the presence of ergosterol has a pronounced effect in the increase in population of the fraction of horizontally bound AmB (85% horizontal vs. 15% vertical). The results of the measurements indicate that mode of action of the AmB consists in disruption of the polar headgroup region of biomembranes, brought about by the AmB molecules bound horizontally with respect to the plane of the membrane.     

Orientation and lateral mobility of cytochrome c on the surface of ultrathin lipid multilayer films.

We have previously shown that cytochrome c can be electrostatically bound to an ultrathin multilayer film having a negatively charged hydrophilic surface; furthermore, x-ray diffraction and absorption spectroscopy techniques indicated that the cytochrome c was bound to the surface of these ultrathin multilayer films as a molecular monolayer. The ultrathin fatty acid multilayers were formed on alkylated glass, using the Langmuir-Blodgett method. In this study, optical linear dichroism was used to determine the average orientation of the heme group within cytochrome c relative to the multilayer surface plane. The cytochrome c was either electrostatically or covalently bound to the surface of an ultrathin multilayer film. Horse heart cytochrome c was electrostatically bound to the hydrophilic surface of fatty acid multilayer films having an odd number of monolayers. Ultrathin multilayer films having an even number of monolayers would not bind cytochrome c, as expected for such hydrophobic surfaces. Yeast cytochrome c was covalently bound to the surface of a multilayer film having an even number of fatty acid monolayers plus a surface monolayer of thioethyl stearate. After washing extensively with buffer, the multilayer films with either electrostatically or covalently bound cytochrome c were analyzed for bound protein by optical absorption spectroscopy; the orientation of the cytochrome c heme was then investigated via optical linear dichroism. Polarized optical absorption spectra were measured from 450 to 600 nm at angles of 0 degrees, 30 degrees, and 45 degrees between the incident light beam and the normal to the surface plane of the multilayer. The dichroic ratio for the heme alpha-band at 550 nm as a function of incidence angle indicated that the heme of the electrostatically-bound monolayer of cytochrome c lies, on average, nearly parallel to the surface plane of the ultrathin multilayer. Similar results were obtained for the covalently-bound yeast cytochrome c. Furthermore, fluorescence recovery after photobleaching (FRAP) was used to characterize the lateral mobility of the electrostatically bound cytochrome c over the monolayer plane. The optical linear dichroism and these initial FRAP studies have indicated that cytochrome c electrostatically bound to a lipid surface maintains a well-defined orientation relative to the membrane surface while exhibiting measurable, but highly restricted, lateral motion in the plane of the surface.     

Translational diffusion of bovine prothrombin fragment 1 weakly bound to supported planar membranes: measurement by total internal reflection with fluorescence pattern photobleaching recovery.

Previous work has shown that bovine prothrombin fragment 1 binds to substrate-supported planar membranes composed of phosphatidylcholine (PC) and phosphatidylserine (PS) in a Ca(2+)-specific manner. The apparent equilibrium dissociation constant is 1-15 microM, and the average membrane residency time is approximately 0.25 s-1. In the present work, fluorescence pattern photobleaching recovery with evanescent interference patterns (TIR-FPPR) has been used to measure the translational diffusion coefficients of the weakly bound fragment 1. The results show that the translational diffusion coefficients on fluid-like PS/PC planar membranes are on the order of 10(-9) cm2/s and are reduced when the fragment 1 surface density is increased. Control measurements were carried out for fragment 1 on solid-like PS/PC planar membranes. The dissociation kinetics were similar to those on fluid-like membranes, but protein translational mobility was not detected. TIR-FPPR was also used to measure the diffusion coefficient of the fluorescent lipid NBD-PC in fluid-like PS/PC planar membranes. In these measurements, the diffusion coefficient was approximately 10(-8) cm2/s, which is consistent with that measured by conventional fluorescence pattern photobleaching recovery. This work represents the first measurement of a translational diffusion coefficient for a protein weakly bound to a membrane surface.     

The Effect of Fluorophore Conjugation on Antibody Affinity and the Photophysical Properties of Dyes

Because the degree of labeling (DOL) of cell-bound antibodies, often required in quantitative fluorescence measurements, is largely unknown, we investigated the effect of labeling with two different fluorophores (AlexaFluor546, AlexaFluor647) in a systematic way using antibody stock solutions with different DOLs. Here, we show that the mean DOL of the cell-bound antibody fraction is lower than that of the stock using single molecule fluorescence measurements. The effect is so pronounced that the mean DOL levels off at approximately two fluorophores/IgG for some antibodies. We developed a method for comparing the average DOL of antibody stocks to that of the isolated, cell-bound fraction based on fluorescence anisotropy measurements confirming the aforementioned conclusions. We created a model in which individual antibody species with different DOLs, present in an antibody stock solution, were assumed to have distinct affinities and quantum yields. The model calculations confirmed that a calibration curve constructed from the anisotropy of antibody stocks can be used for determining the DOL of the bound fraction. The fluorescence intensity of the cell-bound antibody fractions and of the antibody stocks exhibited distinctly different dependence on the DOL. The behavior of the two dyes was systematically different in this respect. Fitting of the model to these data revealed that labeling with each dye affects quantum yield and antibody affinity differentially. These measurements also implied that fluorophores in multiply labeled antibodies exhibit self-quenching and lead to decreased antibody affinity, conclusions directly confirmed by steady-state intensity measurements and competitive binding assays. Although the fluorescence lifetime of antibodies labeled with multiple fluorophores decreased, the magnitude of this change was not sufficient to account for self-quenching indicating that both dynamic and static quenching processes occur involving H-aggregate formation. Our results reveal multiple effects of fluorophore conjugation, which must not be overlooked in quantitative cell biological measurements.     

Interactions of the human calcitonin fragment 9-32 with phospholipids: a monolayer study

Human calcitonin and its C-terminal fragment 9-32 (hCT(9-32)) administered in a spray translocate into respiratory nasal epithelium with an effect similar to intravenous injection. hCT(9-32) is an efficient carrier to transfer the green fluorescent protein into excised bovine nasal mucosa. To understand the translocation of hCT(9-32) across plasma membranes, we investigated its interactions with phospholipids and its interfacial structure using model lipid monolayers. A combination of physicochemical methods was applied including surface tension measurements on adsorbed and spread monolayers at the air-water interface, Fourier transform infrared, circular dichroism, and atomic force microscopy on Langmuir-Blodgett monolayers. The results disclose that hCT(9-32) preferentially interacts with negatively charged phospholipids and does not insert spontaneously into lipid monolayers. This supports a nonreceptor-mediated endocytic internalization pathway as previously suggested. Structural studies revealed a random coil conformation of hCT(9-32) in solution, transforming to alpha-helices when the peptide is localized at lipid-free or lipid-containing air-water interfaces. Atomic force microscopy studies of monolayers of the peptide alone or mixed with dioleoylphosphatidylcholine revealed that hCT(9-32) forms filaments rolled into spirals. In contrast, when interacting with dioleoylphosphatidylglycerol, hCT(9-32) does not adopt filamentous structures. A molecular model and packing is proposed for the spiral-forming hCT(9-32).     

The interaction of an anti-lipid antibody (TEPC 15) with a model biomembrane system (monolayer)

The interaction which occurs between an anti-lipid antibody (TEPC 15) and two phospholipids, phosphatidylcholine and phosphatidylethanolamine, when they are arranged in a lipid monolayer system has been studied. It is shown that the antibody is stabilised under the influence of a high lateral pressure when it is mixed with a lipid monolayer and that the behaviour of the antibody depends upon the lipid used. Measurements of the surface pressure and surface potential parameters of the lipid monolayers indicate that the antibody interacts differently with phosphatidylcholine compared with phosphatidylethanolamine. The antibody also exhibits a different interaction when it is pretreated with phosphorylcholine prior to being spread with a phosphatidylcholine monolayer. The interaction of the antibody with phosphatidylcholine-cholesterol monolayers has also been studied.     

Monoclonal antibodies that inhibit IgE binding

Four monoclonal antibodies were produced that inhibit IgE binding to the high affinity IgE receptor (Fc epsilon R) on rat basophilic leukemia cells. The four monoclonal antibodies (mAb) fall into two groups. The first group was comprised of 3 antibodies (mAb BC4, mAb CD3, and mAb CA5) that reacted with the Fc epsilon R at epitopes close or identical to the IgE-binding site. With 125I-labeled antibodies there was reciprocal cross-inhibition between the antibodies and IgE. The antibodies activated both RBL-2H3 cells and normal rat mast cells for histamine release. The 3 antibodies immunoprecipitated the previously described alpha, beta, and gamma components of the receptor. The number of radiolabeled Fab fragments of 2 of these antibodies bound per cell was similar or equal to the number of IgE receptors. In contrast, the mAb BC4 Fab bound to 2.1 +/- 0.4 times the number of IgE receptor sites. Therefore, the portion of the Fc epsilon R exposed on the cell surface must have two identical epitopes and an axis of symmetry. These 3 monoclonal antibodies recognize different but closely related epitopes in the IgE-binding region of the Fc epsilon R. The fourth monoclonal antibody (mAb AA4) had different characteristics. In cross-inhibition studies, IgE and the other 3 monoclonals did not inhibit the binding of this 125I-labeled monoclonal antibody. The number of molecules of this antibody bound per cell was approximately 14-fold greater than the Fc epsilon R number. This monoclonal antibody caused the inhibition of histamine release and it appears to bind to several cell components.     

Analysis of a common-antigen lipopolysaccharide from Pseudomonas aeruginosa.

Lipopolysaccharide isolated from Pseudomonas aeruginosa PAO1 (O5 serotype) was separated into two antigenically distinct fractions. A minor fraction, containing shorter polysaccharide chains, reacted with a monoclonal antibody to a P. aeruginosa common antigen but did not react with antibodies specific to O5-serotype lipopolysaccharide. In contrast, fractions containing long polysaccharide chains reacted only with the O5-specific monoclonal antibodies. The shorter, common-antigen fraction lacked phosphate and contained stoichiometric amounts of sulfate, and the fatty acid composition of this fraction was similar to that of the O-antigen-specific fraction. The lipid A derived from the serotype-specific lipopolysaccharide cross-reacted with monoclonal antibodies against lipid A from Escherichia coli, while the lipid A derived from the common antigen did not react. We propose that many serotypes of P. aeruginosa produce two chemically and antigenically distinct lipopolysaccharide molecules, one of which is a common antigen with a short polysaccharide and a unique core-lipid A structure.     

Structured thin films as functional components within biosensors

This review provides an introduction to the field of thin films formed by Langmuir-Blodgett or self-assembly techniques and discusses applications in the field of biosensors. The review commences with an overview of thin films and methods of construction. Methods covered will include Langmuir-Blodgett film formation, formation of self-assembled monolayers such as gold-thiol monolayers and the formation of multilayers by the self-assembly of polyelectrolytes. The structure and forces governing the formation of the materials will also be discussed. The next section focussed on methods for interrogating these films to determine their selectivity and activity. Interrogation methods to be covered will include electrochemical measurements, optical measurements, quartz crystal microbalance, surface plasmon resonance and other techniques. The final section is dedicated to the functionality of these films, incorporation of biomolecules within these films and their effect on film structure. Species for incorporation will include antibodies, enzymes, proteins and DNA. Discussions on the location, availability, activity and stability of the included species are included. The review finishes with a short consideration of future research possibilities and applications of these films.