Intact natalizumab pharmacokinetics is impacted by endogenous IgG4 concentration.

BACKGROUND: Natalizumab (NAT) pharmacokinetics and pharmacodynamics are complicated by arm exchange with endogenous IgG4, resulting in a mixture of a more potent intact, bivalent form and a less potent, functionally monovalent form. Total NAT and endogenous IgG4 concentrations vary considerably across patients. This study assessed the concentration of intact NAT, and how it relates to total NAT and endogenous IgG4 levels in blood and saliva. METHODS: Paired serum and saliva samples from a small cohort of relapsing-remitting multiple sclerosis patients were measured for levels of intact NAT, total NAT, IgG and IgG4. RESULTS: Intact NAT concentration was dependent on both total NAT and endogenous IgG4 levels. Low endogenous IgG4 led to a higher ratio of intact NAT to total NAT, while the opposite was observed in subjects with high endogenous IgG4. Serum and saliva measurements show good concordance. CONCLUSIONS: Intact NAT concentration is influenced by both NAT pharmacokinetics and endogenous IgG4 levels. Patients with low IgG4 levels can have high concentrations of intact NAT even with lower levels of total NAT, which may explain cases of NAT-associated progressive multifocal leukoencephalopathy (PML) in such patients. Monitoring both forms of NAT could better guide dosing, maximizing drug efficacy and safety.

Accurate prediction of serum antibody levels from noninvasive saliva/nasal samples.

The importance of easily accessible, noninvasive samples, such as saliva, to effectively monitor serum antibody levels has been underscored by the SARS-CoV-2 (COVID-19) pandemic. Although a correlation between saliva and serum antibody titers has been observed, the ability to predict serum antibody levels from measurements in saliva is not well established. Herein, the authors demonstrate that measurements of SARS-CoV-2 antibody levels in both saliva and nasal specimens can be used to accurately determine serum levels by utilizing endogenous total IgG as an internal calibrator. They postulate that this method can be extended to the measurement of many different antibody analytes, making it of high interest for antibody therapeutic drug monitoring applications.

Peptide Mimotope-Enabled Quantification of Natalizumab Arm Exchange During Multiple Sclerosis Treatment.

BACKGROUND: Natalizumab, a therapeutic antibody used to treat multiple sclerosis, undergoes in vivo Fab arm exchange to form a monovalent bispecific antibody. Although highly efficacious, the immunosuppressive activity of natalizumab has been associated with JC polyomavirus-driven progressive multifocal leukoencephalopathy (PML). Development of assays that can distinguish between and quantify bivalent (unexchanged) and monovalent (exchanged) forms of natalizumab in clinical samples may be useful for optimizing extended interval dosing and reducing the risk of PML. METHODS: In vitro natalizumab arm exchange was conducted, along with peptide mimotope and anti-idiotype surface capture chemistry, to enable the development of enzyme-linked immunosorbent assays. RESULTS: An assay using a unique peptide Veritope TM was developed, which can exclusively bind to bivalent natalizumab. In combination with enzyme-linked immunosorbent assays that quantifies total natalizumab, the assay system allows quantification of both natalizumab forms. CONCLUSIONS: In this article, a novel assay for the quantification of unexchanged and exchanged natalizumab variants in clinical samples was developed. This assay will enable investigations into the clinical significance of the relationship of PK/PD with the monovalent-to-bivalent ratio, as it relates to the efficacy of the drug and risk of PML.

Sol-gel-derived silver-nanoparticle-embedded thin film for mass spectrometry-based biosensing.

A porous silver-nanoparticle (AgNP)-embedded thin film biosensor was produced by the sol-gel method. The thin films were used as matrix-free laser desorption ionization mass spectrometry (LDI-MS) biosensors applicable to several chemical classes. In these experiments, UV laser irradiation (337 nm) of the AgNP facilitates desorption and ionization of a number of peptides, triglycerides, and phospholipids. Preferential ionization of sterols from vesicles composed of olefinic phosphosphatidylcholines is also demonstrated, offering the possibility for a simplified approach for sterol analysis from complex mixtures. The composition of the nanoparticles was confirmed by X-ray photoelectron spectroscopy (XPS) and UV-vis spectroscopy. XPS data revealed a binding energy of 368.2 eV, consistent with the previous assignment of the binding energy for the Ag 3d(5/2) peak from Ag(0) at 368.1 ± 0.1 eV. The surface morphology of the thin films was studied by field-emission scanning electron microscopy (FE-SEM) and revealed the presence of nanoparticles and the porous nature of the biosensor.

Label-free biosensing with lipid-functionalized gold nanorods.

The fabrication of a label-free mass spectrometry and optical detection-based biosensor platform for the detection of low-abundance lipophilic analytes in complex mixtures is described. The biosensor consists of a lipid layer partially tethered to the surface of a gold nanorod. The effectiveness of the biosensor is demonstrated for the label-free detection of a lipophilic drug in aqueous solution and a lipopeptide in serum.

Longitudinal surface plasmon resonance based gold nanorod biosensors for mass spectrometry.

A "strategy" for analyte capture/ionization based on chemical derivatization of gold nanorods and infrared laser desorption ionization (IR-LDI) is described. This is the first example of laser desorption/ionization of biomolecules using gold nanorods irradiated with an IR laser. LDI is performed at wavelengths (1064 nm) that overlap with the longitudinal surface plasmon resonance (LSPR) mode of gold nanorods. The absorbed energy from the laser facilitates desorption and ionization of the analyte. The wavelength of the LSPR band can be tuned by controlling the aspect ratio (length-to-diameter) of the nanorod. For example, the SPR band for Au nanorods having an aspect ratio of 5:1 is centered at approximately 840 nm, and this band overlaps with the 1064 nm output of a Nd:YAG laser. We show that a variety of biomolecules can be efficiently desorbed and ionized by 1064 nm irradiation of nanorods. We also show that analyte capture can be controlled by surface chemistry of the nanorods. The results of these studies are important for designing nanomaterial-based capture assays for mass spectrometry and interfacing nanomaterials with imaging/spatial profiling mass spectrometry experiments.

Benchtop chemistry for the rapid prototyping of label-free biosensors: Transmission localized surface plasmon resonance platforms.

Herein, a simple label-free biosensor fabrication method is demonstrated based on transmission localized surface plasmon resonance (T-LSPR). The platform, which consists of a silver nanoparticle array, can be prepared in just a few minutes using benchtop chemistry. The array was made by a templating technique in conjunction with the photoreduction of Ag ions from solution. This metal surface was functionalized with biotin-linked thiol ligands for binding streptavidin molecules from solution. For an array of 19 nm diameter silver nanoparticles, a redshift in the T-LSPR spectrum of 24 nm was observed upon protein-ligand binding at saturation. The binding constant was found to be 2x10(12) M(-1). Platforms were also fabricated with silver nanoparticles of 34, 55, and 72 nm diameters. The maximum LSPR wavelength shift was nanoparticle size dependent and the maximum sensitivity was obtained with the smaller nanoparticles.

Tailoring nanoparticle surface chemistry to enhance laser desorption ionization of peptides and proteins.

Gold nanoparticles capped with 4-aminothiophenol have been employed for laser desorption ionization mass spectrometry of biomolecules. We demonstrate that the capped nanoparticles increase ion yields, decrease ion fragmentation, and increase the useful analyte mass range when compared to other nanoparticle systems. These results will allow for further development of nanoparticles with both targeting and enhanced ionization abilities to aid in biomarker screening.

Imaging large arrays of supported lipid bilayers with a macroscope.

Herein, the authors present fluorescence resonance energy transfer (FRET) and two-dimensional protein saturation data acquired from spatially addressed arrays of solid supported lipid bilayers (SLBs). The SLB arrays were imaged with an epifluorescencetotal internal reflection macroscope. The macroscope allowed 1x imaging and had a relatively high numerical aperture (0.4). Such powerful light gathering and large field of view capabilities make it possible to simultaneously image dozens of addressed SLBs. Three experiments have been performed. First, a 9x7 array of supported lipid bilayer was fabricated and imaged in which each bilayer element was individually addressed. Second, a FRET assay was developed between Texas Red-DHPE (1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine) and NBD-PE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-n-(7-nitro-2-1,3-benzoxadiazol-4-yl)). The concentration of dye could be varied at each address and the value of the Forster radius (7.3+/-0.6 nm) was easily abstracted. Third, a ligandreceptor recognition assay was designed to show the two-dimensional number density of proteins which can be bound at saturation. It was found for the streptavidinbiotin pair that the protein saturated at the interface above 3 mol % biotin concentration. This corresponded to a two-dimensional footprint of 40 nm(2) for the streptavidin molecule. These results clearly open the door to using individually addressed bilayers for obtaining large amounts of biophysical data at the supported bilayeraqueous interface. Such abilities will be crucial to obtaining sufficient data for determining the interfacial mechanisms for a variety of membraneprotein interactions.

Direct writing of metal nanoparticle films inside sealed microfluidic channels.

Herein we demonstrate the ability to pattern Ag nanoparticle films of arbitrary geometry inside sealed PDMS/TiO2/glass microfluidic devices. The technique can be employed with aqueous solutions at room temperature under mild conditions. A 6 nm TiO2 film is first deposited onto a planar Pyrex or silica substrate, which is subsequently bonded to a PDMS mold. UV light is then exposed through the device to reduce Ag+ from an aqueous solution to create a monolayer-thick film of Ag nanoparticles. We demonstrate that this on-chip deposition method can be exploited in a parallel fashion to synthesize nanoparticles of varying size by independently controlling the solution conditions in each microchannel in which the film is formed. The film morphology was checked by atomic force microscopy, and the results showed that the size of the nanoparticles was sensitive to solution pH. Additionally, we illustrate the ability to biofunctionalize these films with ligands for protein capture. The results indicated that this could be done with good discrimination between addressed locations and background. The technique appears to be quite general, and films of Pd, Cu, and Au could also be patterned.

Solid supported lipid bilayers: From biophysical studies to sensor design.

The lipid bilayer is one of the most eloquent and important self-assembled structures in nature. It not only provides a protective container for cells and sub-cellular compartments, but also hosts much of the machinery for cellular communication and transport across the cell membrane. Solid supported lipid bilayers provide an excellent model system for studying the surface chemistry of the cell. Moreover, they are accessible to a wide variety of surface-specific analytical techniques. This makes it possible to investigate processes such as cell signaling, ligand-receptor interactions, enzymatic reactions occurring at the cell surface, as well as pathogen attack. In this review, the following membrane systems are discussed: black lipid membranes, solid supported lipid bilayers, hybrid lipid bilayers, and polymer cushioned lipid bilayers. Examples of how supported lipid membrane technology is interfaced with array based systems by photolithographic patterning, spatial addressing, microcontact printing, and microfluidic patterning are explored. Also, the use of supported lipid bilayers in microfluidic devices for the development of lab-on-a-chip based platforms is examined. Finally, the utility of lipid bilayers in nanotechnology and future directions in this area are discussed.

Fluid and air-stable lipopolymer membranes for biosensor applications.

The behavior of poly(ethylene glycol) (PEG) conjugated lipids was investigated in planar supported egg phosphatidylcholine bilayers as a function of lipopolymer density, chain length of the PEG moiety, and type of alkyl chains on the PEG lipid. Fluorescence recovery after photobleaching measurements verified that dye-labeled lipids in the membrane as well as the lipopolymer itself maintained a substantial degree of fluidity under most conditions that were investigated. PEG densities exceeding the onset of the mushroom-to-brush phase transition were found to confer air stability to the supported membrane. On the other hand, substantial damage or complete delamination of the lipid bilayer was observed at lower polymer densities. The presence of PEG in the membrane did not substantially hinder the binding of streptavidin to biotinylated lipids present in the bilayer. Furthermore, above the onset of the transition into the brush phase, the protein binding properties of these membranes were found to be very resilient upon removal of the system from water, rigorous drying, and rehydration. These results indicate that supported phospholipid bilayers containing lipopolymers show promise as rugged sensor platforms for ligand-receptor binding.

On the mechanism of the hofmeister effect.

Vibrational sum frequency spectroscopy was used to probe fatty amine monolayers spread on various electrolyte solutions. The spectra revealed ion specific changes in both monolayer ordering and water structure with the former following the Hofmeister series. Separate measurements of the surface potential as a function of ion tracked closely to changes in alkyl chain structure, but less closely to changes in water structure. The disruption of the monolayer ordering could be ascribed to the relative ability of the ions to penetrate past the hydrophilic surface of the monolayer's headgroups and into the more hydrophobic portion of the thin film. The corresponding trends observed in the surface water structure showed significant deviations from the Hofmeister series, leading to the conclusion that the changes in surface water structure, often credited with being the origin of Hofmeister effects, are probably not of primary importance. On the other hand, dispersion forces almost certainly play a large role in the order of the Hofmeister series.

Creating fluid and air-stable solid supported lipid bilayers.

Solid supported lipid bilayers are rapidly delaminated when drawn through the air/water interface. We have discovered that a close packed monolayer of specifically bound protein prevents this process. The protection mechanism worked in two ways. First, when protein-protected bilayers were drawn through the air/water interface, a thin bulk water layer was visible over the entire bilayer region, thereby preventing air from contacting the surface. Second, a stream of nitrogen was used to remove all bulk water from a protected bilayer, which remained fully intact as determined by fluorescence microscopy. The condition of this dried bilayer was further probed by fluorescence recovery after photobleaching. It was found that lipids were not two-dimensionally mobile in dry air. However, when the bilayer was placed in a humid environment, 91% of the bleached fluorescence signal was recovered, indicating long-range two-dimensional mobility. The diffusion coefficient of lipids under humid conditions was an order of magnitude slower than the same bilayer under water. Protected bilayers could be rehydrated after drying, and their characteristic diffusion coefficient was reestablished. Insights into the mechanism of bilayer preservation were suggested.

Thermodynamics of phase transitions in langmuir monolayers observed by vibrational sum frequency spectroscopy.

Vibrational sum-frequency spectroscopy (VSFS) was used to study gauche defects in octadecylamine (ODA) monolayers at the air/water interface. The VSFS spectra provide unique insights into phase transitions that occur as a result of changes in the structure of the monolayer's hydrophobic region. These changes can be attributed to the increased presence of gauche conformers in the ODA alkyl chains during the monolayer's transition from the solid to liquid phase. Temperature-dependent spectra from monolayers at several different pressures were used to assign the phase transition temperature based on the observed changes in microscopic structure. Through application of a two-dimensional form of the Clapeyron equation, the first in situ measurements of the entropy and enthalpy changes associated with gauche conformers in a monolayer were made.