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«Kinetic Investigations of Thiolate Protected Gold Nanoparticles: Protein Interactions, Electron Transfer, and Precursor Formation By Brian N. Turner ...»

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Competitive binding studies of HRSV F using libraries of monoclonal IgG’s have revealed three unique antigenic sites (A, B, C) with various epitopes (including one in a bridge site AB).78 The A and C sites are conserved while the B site is variable.78 Monoclonal antibody resistant mutants (MARMs) in the A site are understood in terms of specific amino acid changes. Coincidentally, a number of MARMs of RSV type A exhibit amino acid changes between residues 258 and 276. 75, 78-85 Specific amino acid changes in the F protein of RSV type A MARMs are shown in Table 1 with reference as to which monoclonal antibody was used to select the MARM.

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Epitopes in antigenic site A are conformational epitopes, and are, therefore, difficult to mimic in such a way that elicits antibody binding similar to the native structure.75, 78 Failure to reproduce to secondary structure is further compounded when the epitope is highly discontinuous.

Synagis, often called by its pharmaceutical name Palivizumab (PZ), is prescribed to prevent HRSV infection in children during the cold season, and is commercially available from MedImmune. This designer monoclonal pharmaceutical IgG is a humanized form of murine monoclonal antibody (mAb) 1129, 78 where certain regions are identical to the murine structure to conserve the structural integrity of the binding sites, and the rest of the mAb is that of a well characterized human mAb MCPC603. 86, 87 Palivizumab was determined to have at least as strong of an affinity for RSV F as chimeric 1129 via ELISA.86 The commercial availability of this mAb and its high affinity for RSV F protein makes it a desirable tool for constructing immunosensors, mapping HRSV F epitope sequences, and screening antigenic mimics (see Chapter III) presenting HRSV F epitope peptides.

Since this study was completed, crystal structures of HRSV F in complex with mAb F101,88 post-fusion HRSV F,89 and the HRSV F motavizumab peptide epitope bound to the antigen-binding fragment (Fab) of motavizumab90 have been published.

Motavizumab is the next generation of PZ, with 10 fold higher activity but the same epitope region.91 These crystal structures should prove instrumental in understanding the structural basis of the antigen-antibody interaction, and will aid future efforts to construct biomimetic agents.

Epitope Mapping In the construction an antigenic mimic, it is useful to understand the specific intermolecular forces that are responsible for binding HRSV F to PZ. Specifically, the goal is to determine which amino acid side chains are involved and how they are spatially organized. Given this information, a nanomaterials scientist can imagine and create a synthetic system which copies the important parameters without the need to use complicated techniques, expensive and fragile biological systems, and exotic reagents.

Epitope mapping describes any technique where antigens or fragments thereof are analyzed for their affinity to an antibody in order to gain information about which parts of the whole are important for the antigen-antibody interaction. Linear epitope mapping refers to techniques which solely interrogate primary structure while conformational epitope mapping considers the effects of secondary and tertiary structure as well. In the current study, a linear epitope mapping approach to investigate synthetic antigenic site A peptides against PZ was used, but information about the secondary structure was inferred from other studies.

The discovery by Rutledge and coworkers of binding “hot spots” in a proposed long Cterminal epitope sequence of Ebola glycoprotein92 lead this research to apply similar methodology to antigenic site A of HRSV F. Briefly, the researchers discovered that there were two discrete regions in a longer peptide sequence which had local maxima in an ELISA assay against the monoclonal antibody 15H10 that appeared to be centered about two spatially distinct three amino acid sequences, as is shown in Figure 9.

Figure 9: ELISA study of synthetic peptides from Rutledge and coworkers demonstrating that two spatially separate three amino acid sequences are likely to provide the strongest interactions between the glycoprotein of Ebola and the antibody 15H10. 92 Copyright 2008, the Materials Research Society, used with permission.

These “hot spots,” TDK and FID, were validated by place exchanging cysteine-PEG terminated versions of the three amino acid sequences onto tiopronin protected gold nanoparticles and detecting them with a home-made Ebola QCM immunosensor presenting 15H10.

Quartz Crystal Microbalance (QCM) The QCM consists of a piezoelectric quartz resonator with metal electrodes on either side. The combination of electrodes and resonator is attached to an oscillator circuit.

QCM has become a useful analytical technique due to the linear relationship between mass deposited on the crystal and the frequency of the crystal resonance.93 Oscillator circuits that permit liquid measurements have made the technique useful in the area of bioanalytical chemistry.94, 95 The ideal oscillator for this technology is quartz because it has desirable mechanical, electrical, chemical, and thermal properties. 94 Specifically, ATcut quartz crystals have a temperature coefficient that is nearly zero between 0 and 50 ºC, making them ideal for QCM measurements at ambient temperatures without sophisticated temperature control.96 Electrode wrapped quartz crystals manufactured by Maxtek Inc. resonate at different frequencies depending upon their thickness. Conventionally, quartz crystals used in QCM measurements are identified on the basis of this resonant frequency. For example, a 5 MHz QCM crystal is 333 µm thick and a 9 MHz QCM crystal is 185 µm thick. The crystals are 1” in diameter, the oversized front electrode is ½” in diameter and the concentric electrode located on the reverse side (contact electrode) is ¼” in diameter. 97 The overlapping area of the contact electrode and front electrode is 0.3419 cm2. The overlapping region has been shown to be the sensitive area by scanning electrochemical mass sensitivity experiments.98 The consequences of this are that deposited mass (mass added to the crystal by covalent, electrostatic, physical etc. interactions) has a maximum effect on resonant frequency in the overlapping region and a small effect outside of the overlapping region (known as field fringing). Such an electrode geometry sacrifices some sensitivity in order to negate fringing field effects from non-specific binding on the quartz itself.99 Fringing field effects refer to mass loading that is outside of the fully overlapping area between the two electrodes that still affects the electric field and thus elicits a small, but significant effect on crystal frequency. The manual for the Maxtek RQCM cites that the particular electrode geometry (small contact electrode with an oversized sensing electrode) was chosen to ensure a “more consistent deposition across the active area of the crystal.”97 In order to calculate mass loading using the QCM, one takes advantage of the following relationship between mass and frequency shift, proposed by Sauerbrey and henceforth





named the Sauerbrey equation:

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where f0 is the first harmonic resonant frequency of the unloaded crystal, A is the active  (sensitive) area [m2] of the QCM crystal, ρq is the density of quartz (2.648 kg/m3) and μq is the shear modulus of AT-cut quartz (2.95 x 1011 N/cm2).93, 97 The sensitive area, again, is the overlap area of the large front electrode with the small rear electrode. Cf for our crystals are 0.0566 Hz cm2/g for a 5 MHz crystal and 0.1834 Hz cm2/g for a 9 MHz crystal. When calculating mass, it is important to recognize that frequency is not dependent upon mass loading alone in a liquid system. Viscosity has a major effect on

the frequency, as shown by the following equation:

–  –  –

where ηL [N s m-2] is the viscosity of the liquid in contact with the crystal, and ρL is the  density of the liquid in contact with the crystal. 100 This specific component of the total frequency change is directly related to a change in resistance, given by the following

equation:

–  –  –

where ΔR [Ω] is the change in resistance, and e26 [kg V s-2] is the piezoelectric constant of  quartz.100, 101 Therefore, frequency shift due to viscosity can be calibrated to separate frequency due to mass loading by use of solutions of a non-loading analyte, such as sucrose, leading to a concentration dependent decrease in frequency, which results from viscosity. The slope of a plot of Δf versus ΔR is used as a correction factor to subtract out viscosity induced frequency shift.

The practical equation to be used for data analysis in the current studies involves first

rearranging equation 2-1:

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where c is the calibration factor in units of Hz/Ω attained from the plot of frequency  versus resistance for solutions of varying viscosity. The value for c should be independently determined for each crystal used. Typical c values for the Maxtek crystals used in this research are 2.095 Hz/Ω for a 5 MHz crystal and 15.3 Hz/Ω for a 9 MHz crystal.

QCM data can also be used to extract thermodynamic parameters for the systems being studied. For the generic system Ag + Ab ⇄ AgAb where Ag is an antigen and Ab is an

antibody, the equilibrium constant, K, is defined by:

–  –  –

where C is the adsorbate concentration, and mmax is the maximum mass that can be  adsorbed by the monolayer, and m/mmax = , the degree of surface coverage.102 Determination of K from either of the above equations (8 or 9), allows calculation of the

Gibb’s free energy of adsorption, Gads using the following equation:

–  –  –

where R is the gas constant and T is the temperature of the system. Studying the variation  of the system with temperature, the enthalpy of adsorption, Hads, can be determined

using the following equation:

–  –  –

The full process from data collection to thermodynamic quantities is rigorous, but allows  for determination of whether various biological processes are entropy or enthalpy driven.

Thermodynamic understanding, in turn, allows for better understanding of the structurefunction relationship in biological molecules. From an evolutionary perspective, this allows researchers to understand how a protein used to be structured, what modifications it made to get where it is, and exactly how those changes made the protein function better than its predecessor.

The ability to determine real time kinetic and thermodynamic parameters is only one aspect that makes the construction of QCM based immunosensors desirable. QCM also had the advantages of low cost, portability, and a number of analytical advantages. A good case for comparison is surface plasmon resonance (SPR). Whereas QCM uses acoustic waves and measures frequency, SPR uses optical waves and measures reflection minimum. Either instrument provides data that can be used to study protein interactions in real time. SPR performs at least as well as QCM in terms of detection limit, sensitivity, and detection time;103, 104 however, QCM instrumentation is less expensive (about $5,000 compared to about $100,000 for the lowest specifications of either instrument) and, reportedly, easier to use, and can make measurements on thicker layers without loss of sensitivity (200 nm for SPR versus 400 nm for QCM). 105-107 Either technique measures up well in terms of analytical figures of merit when compared to many techniques employed in the field. In terms of time consumption, culture of a nasal swab takes about four days for detection and polymerase chain reaction (PCR) takes one to two days for detection. Rapid ELISA techniques can be employed with total analysis time of 30 minutes to 1 hour, but the cost to the patient is about $300.00 per test, and the pieces, such as antibody solutions and control samples, are not reusable.108 The cost could be lowered by a platform like QCM or SPR because of their amenability to reusability, as solid-phase stabilized biomolecules have longer shelf lives. Additionally, all of the above techniques are largely qualitative or semi-quantitative at best, and therefore give less information about total viral load. The dominant RSV immunoassay in the clinical environment, direct fluorescent antibody (DFA), takes about 2 hours.109 A QCM immunosensor that is reusable should be much quicker and cheaper than this technique, while providing more information about the disease.

–  –  –

Instrumentation Solution phase resistance and frequency measurements to characterize biological interactions were made using a Maxtek, Inc. 3-channel Research QCM operating at a driving voltage of 125 mV RMS and using Maxtek 5 or 9 MHz AT-cut quartz crystals with gold electrodes. Flow cells were also purchased from Maxtek. Solutions were delivered through the flow cells using a Cole Parmer Masterflex console drive peristaltic pump.

Materials Reagent and optima grade solvents, N-2-mercaptopropionylglycine (tiopronin), bovine serum albumin (fraction V, 96%), tween-20 (molecular biology grade), α-cyano-4hydroxycinnamic acid, 3,3',5,5'-Tetramethylbenzidine (TMB, liquid substrate, supersensitive, for ELISA), thioanisole, and anisole were purchased from Sigma-Aldrich.

Common laboratory salts and concnetrated ammonium hydroxide were reagent grade and purchased from Fisher scientific. Concentrated sulfuric acid was purchased from EMD.

Absolute ethanol was purchased from Pharmco-AAPER. Immulon 2HB well plates, maleimide coated well plates, and cysteine-HCl were purchased from Thermo scientific.



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