Comparative study of thiophilic functionalised matrices for polyclonal F(ab')2 purification

Thiophilic adsorbents have been developed using divinyl sulfone or epoxy activated Streamline quartz base matrix. Their capacity and selectivity for binding polyclonal F(ab')2 fragments generated by whole serum proteolysis was tested. Except for epoxy activated guanidine, all the adsorbents displayed high selectivity for F(ab')2 with dynamic binding capacities ranging from 3 to 10 mg/ml of adsorbent. Thiol immobilised ligands adsorbed more F(ab')2 and the recovery was equal to or more than that from amino immobilised ligands. All adsorbents showed good selectivity for IgG and the dynamic binding capacities were better than for F(ab')2.     

Preparation of latex reagents combined with IgM and its F(ab')2 fragment from commercial ABO blood grouping reagent

To achieve a rapid assay for ABO blood grouping using a latex reagent, two latex reagents were produced, one of which combined with mouse monoclonal immunoglobulin M (IgM) isolated from commercial ABO blood grouping reagent, and the other of which combined with its F(ab')2 fragment prepared by cold pepsin digestion. The latex reagent adsorbing the F(ab')2 fragment was able to detect the 1000-fold diluted saliva and provided much better sensitivity than that of IgM. This suggests that the difference in sensitivity between the two latex reagents is responsible for adsorption orientation of the antigen site on the latex particles. The new assay successfully completed the ABO blood grouping of cigarette ends within 30 min.     

Influence of antibody immobilization strategy on molecular recognition force microscopy measurements

A systematic evaluation of the effects of antibody immobilization strategy on the binding efficiency and selectivity (e.g., ability to distinguish between specific and nonspecific interactions) of immunosurfaces prepared with F(ab') antibody fragments of rabbit Immunoglobulin G (IgG) is described. F(ab') was attached to gold surfaces either (1) directly via the formation of a gold-thiolate bond or (2) indirectly through a series of a bifunctional linkers containing an alkane chain or ethylene glycol spacer. Immobilization of F(ab') via the sulfhydryl reactive group located opposite the antigen binding site ensured optimum orientation of the antigen binding site. X-ray photoelectron spectroscopy (XPS) and surface plasmon resonance (SPR) were used to confirm surface modification with the bifunctional linkers and antibody immobilization, respectively. Binding efficiency assays performed with SPR indicated that increasing the length of the linker increased the antigen binding efficiency. Atomic force microscopy (AFM) adhesion force measurements indicated that AFM probes functionalized with directly immobilized F(ab') more effectively discriminated between specific and nonspecific surface-bound proteins than probes modified indirectly via linker-immobilized F(ab'). In addition, a greater number of antibody-antigen binding events were observed with directly immobilized F(ab')-functionalized probes.     

Study of the adsorption of F(ab')2 onto polystyrene latex beads

An experimental investigation on the adsorption of F(ab')₂ from rabbit IgG onto polystyrene (PS) latex beads is described. All adsorption isotherms were of high affinity and showed well-defined plateaus. Maximum protein adsorption was found around the average isoelectric point (IEP) of the dissolved protein. According to the findings, the F(ab')₂ adsorption on the polystyrene surface is strongly irreversible with respect to ionic strength changes. The pH changes, however, exert a certain effect on the adsorption-desorption process of F(ab')₂ on negatively charged polystyrene surfaces. In order to determine the role played by the electrostatic forces in the F(ab')₂ adsorption onto negatively charged latex particles, an electrokinetic study of the protein-latex complexes has also been carried out. The isoelectric pH of the F(ab')₂-PS complexes is always smaller than the IEP of the dissolved F(ab')₂, indicating that the PS surface charge must partly compensate the positive charge on the protein. Finally, a comprehensive study on the colloidal stability of the sensitized latex beads was performed.     

Production and Characterization of F(Ab’)<Subscript>2</Subscript> Fragments Obtained by Enzymatic Digestion from Murine Anti-MRSA PBP2a Monoclonal Antibodies

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a worldwide health problem. In a previous study, a murine monoclonal antibody (mMAB), capable of binding to PBP2a within MRSA strains, was generated. F(ab’)₂ antibody fragments are widely described in the literature as immunochemical tools and reagents for diagnostics and therapeutics, particularly because of their low immunogenicity and rapid pharmacokinetics. In this study, F(ab’)₂ fragments from mMAB were generated by enzymatic digestion, using pepsin. They were purified by affinity chromatography using protein A and concentrated by a MWCO 50 kDa filtration unit. The results indicate that it is possible to obtain F(ab’)₂ fragments by pepsin digestion. ELISA, western blotting, and fluorescence microscopy data demonstrated that F(ab’)₂ affinity for PBP2a is not lost even after the enzymatic digestion process. As expected, in the pharmacokinetics tests, F(ab’)₂ presented a faster elimination (between 12 and 18 h) compared to IgG. These F(ab’)₂ fragments could be used in future immunodiagnostic applications, including in vitro or in situ radiolabeling and in the treatment of infections caused by this important pathogen.     

Scanning force microscopy and fluorescence microscopy of microcontact printed antibodies and antibody fragments

Unlabeled primary immunoglobulin G (IgG) antibodies and its F(ab')2 and Fc fragments were attached to oxygen-plasma-cleaned glass substrates using either microcontact printing (MCP) or physical adsorption during bath application from dilute solutions. Fluorescently labeled secondary IgGs were then bound to surface-immobilized IgG, and the relative surface coverage was determined by measuring the fluorescence intensity. Results indicated that the surface coverage of IgG increased with increasing protein solution concentration for both MCP and bath-applied IgG and that a greater concentration of IgG was transferred to a glass substrate using MCP than during physisorption during bath applications. Scanning force microscopy (SFM) showed that patterned MCP IgG monolayers were 5 nm in height, indicating that IgG molecules lie flat on the substrate. After incubation with a secondary IgG, the overall line thickness increased to around 15 nm, indicating that the secondary IgG was in a more vertical orientation with respect to the substrate. The surface roughness of these MCP patterned IgG bilayers as measured by SFM was observed to increase with increasing surface coverage. Physisorption of IgG to both unmodified patterned polydimethylsiloxane (PDMS) stamps and plasma-cleaned glass substrates was modeled by Langmuir adsorption kinetics yielding IgG binding constants of K(MCP) = 1.7(2) x 10(7) M(-1) and K(bath) = 7.8(7) x 10(5) M(-1), respectively. MCP experiments involving primary F(ab')2 and Fc fragments incubated in fluorescently labeled fragment-specific secondary IgGs were carried out to test for the function and orientation of IgG. Finally, possible origins of MCP stamping defects such as pits, pull outs, droplets, and reverse protein transfer are discussed.     

Purification of specific heterologous F(ab)2 fragments with DEAE-Zeta-Prep cartridges for ion-exchange chromatography

A simple two-step procedure for purifying F(ab)2 fragments of horse immunoglobulins is described. In the first step, the horse plasma is diluted, made up to 12% (w/v) with ammonium sulphate and digested with pepsin. In the second step, the previously dialyzed solution is chromatographed. Instead of a normal ion-exchange resin, a DEAE-cellulose, covalently linked to a synthetic vinyl polymer, was used (DEAE-Zeta-Prep). With this assembly it is possible to perform chromatography at a high flow-rate without the problems related to the use of large columns. The yield and purity of the final product are satisfactory. This method has been scaled up for industrial application.     

Asymmetric total synthesis of spongistatins 1 and 2

The total synthesis of spongistatin 1 (1) and spongistatin 2 (2) has been achieved through an advanced-stage intermediate. The synthesis is highlighted by a highly convergent assembly of the four key fragments (the C1-C15 AB fragment 2, the C16-C28 CD fragment 3, the C29-C43 EF fragment 4, and the C44-C51 side chain 5) at a very advanced stage of the synthesis with minimal functional group interconversion. The CD fragment 3 functions as the central building block to which the other fragments are attached. The synthesis of the AB and CD spiroketal fragments is accomplished through the addition of a metalated gamma-pyrone to a beta-alkoxy aldehyde followed by spiroketalization. The EF subunit was assembled with high diastereoselectivity relying on asymmetric aldol reactions of chlorotitanium enolates of N-propionyl oxazolidinethiones and a double diastereoselective boron aldol to join the E and F fragments. Wittig coupling of the CD and EF fragments followed by a diastereoselective aldol reaction between the CDEF ketone and an AB aldehyde set the stage for attachment of the C44-C51 side chains and final macrolactonization and deprotection.     

Probing the orientation of surface-immobilized immunoglobulin G by time-of-flight secondary ion mass spectrometry

Static time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a powerful surface analysis technique for the characterization of protein films because of its chemical selectivity and surface sensitivity. In this study, static ToF-SIMS and principal component analysis (PCA), a multivariate data analysis method, were combined to probe the orientation of surface-immobilized immunoglobulin G (IgG). IgG orientation can enhance its ability to detect its antigen in immunoassay techniques. The IgG used in this work is the mouse monoclonal anti-human chorionic gonadotropin (anti-hCG). Anti-hCG films on different well-defined substrates have been studied using its F(ab')2 and Fc fragments as references. Atomic force microscopy was used to characterize these protein films before static ToF-SIMS analysis. The results from PCA of ToF-SIMS spectra were related to the antibody primary amino acid composition and its three-dimensional structure.     

Procedure for the isolation of a large peptic fragment of human serum albumin

For a thorough investigation of the drug-binding behaviour and other physicochemical properties of human serum albumin, one needs large amounts of specific fragments of albumin. Such fragments were obtained by careful proteolysis of the native protein with pepsin at pH 3.70. The fast protein liquid chromatographic technique was used to find the optimum experimental conditions for the separation of the fragments. By means of anion-exchange chromatography, chromatofocusing and gel permeation, it was possible to obtain a large fragment with a relative molecular mass of 46,000. The fragment could be assigned to segment 1-387 and therefore consists of domains 1 and 2 of the albumin structure. A 1-g amount of albumin produced 50 mg of a fragment that was 98% homogeneous.     

Investigating alternative acidic proteases for H/D exchange coupled to mass spectrometry: Plasmepsin 2 but not plasmepsin 4 is active under quenching conditions

Structural studies of proteins by hydrogen/deuterium exchange coupled to mass spectrometry (DXMS) require the use of proteases working at acidic pH and low temperatures. The spatial resolution of this technique can be improved by combining several acidic proteases, each generating a set of different peptides. Three commercial aspartic proteases are used, namely, pepsin, and proteases XIII and XVIII. However, given their low purity, high enzyme/protein ratios have to be used with proteases XIII and XVIII. In the present work, we investigate the activity of two alternative acidic proteases from Plasmodium falciparum under different pH and temperature conditions. Peptide mapping of four different proteins after digestion with pepsin, plasmepsin 2 (PSM2), and plasmepsin 4 (PSM4) were compared. PSM4 is inactive at pH 2.2 and 0°C, making it unusable for DXMS studies. However, PSM2 showed low but reproducible activity under DXMS conditions. It displayed no substrate specificity and, like pepsin, no strict sequence specificity. Altogether, these results show that PSM2 but not PSM4 is a potential new tool for DXMS studies.     

Monoclonal antibodies as a tool for structure-function studies of the MDR1-P-glycoprotein

P-glycoprotein is considered one of the most important member of the rapidly growing superfamily of integral proteins known as the ATP-binding cassette (ABC) which in human also include several other multidrug resistance membrane proteins (i.e., MRP), the product of the cystic fibrosis gene, the TAP-1/TAP2 peptide transporters encoded by the major histocompatibility complex genes and the gene encoding for breast cancer resistance protein (BCRP) also known as MXR1 (mitoxantrone resistance protein). Many monoclonal antibodies (MAbs) reacting with distinct P-glycoprotein domains have been isolated and used to study the molecular organization and cellular functions of this ABC protein. MAbs have been used for multidrug resistance (mdr) gene cloning, delineation of the secondary and tertiary structure of P-glycoprotein and molecular analysis of the mechanisms involved in substrate recognition and transport. The immunodetection of the distinct products of the mdr gene family in normal and malignant cells and tissues has greatly contributed to the understanding of the physiological role of P-glycoprotein and its possible involvement in the refractory of tumors to chemotherapy. The present article deals with the immunological methods used for the structure-function studies of the P-glycoprotein. After introducing the basic structural features of this ABC transporter, the antibody based-approach is discussed with aiming to furnishing methodological perspectives for further investigations of the physiological role of P-glycoprotein and the multidrug resistance phenomenon.     

Characterization of the Fc receptors of the murine leukemia L1210

A glycoprotein extract prepared from the plasma membranes of L1210 cells was passed over columns of Sepharose 4B to which either heat-aggregated human IgG or F(ab')2 fragments has been coupled. The intact IgG column bound 35.7 percent of the applied counts, whereas the F(ab')2 columns bound 2.8 percent. The bound glycoproteins were eluted with citrate buffer (pH 3.2) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three peaks with apparent molecular weights of 65,000 45,000, and 28,000 daltons were identified and purified by electroelution from polyacrylamide gels. The isolated proteins were able to bind to the same sub-classes of mouse IgG myeloma proteins as the intact L1210 cells, indicating that these molecules are related to L1210 surface Fc receptors. Amino acid analyses of the 3 proteins were markedly similar suggesting that the observed molecular heterogeneity might be due to carbohydrate differences. Neuraminidase digestion of the isolated proteins resulted in mobility shifts on polyacrylamide gel electrophoresis which were consistent with the interpretation that either the isolated proteins have considerably different sialic acid contents, or that removal of the sialic acid results in disaggregation of an Fc receptor molecule.     

Preparation and mass spectrometric study of egg yolk antibody (IgY) against rabies virus

Rabies virus was used as the antigen to immunize laying chickens. Anti-rabies virus immunoglobulin Y(IgY) was isolated from yolks of the eggs laid by these chickens using a two-step salt precipitation and one-step gel filtration protocol. The purified IgY was reduced with dithiothreitol, and heavy chains (HC) and light chains (LC) were obtained. In addition, the purified IgY was digested with pepsin and the fragment with specific antigen binding properties (Fab) was produced. Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOFMS), the average molecular weights of IgY, HC, LC, and Fab were determined as 167 250, 65 105, 18 660, and 45,359 Da, respectively. IgY has two structural differences compared with mammalian IgGs. First, the molecular weight of the heavy chain of IgY is larger than that of its mammalian counterpart, while the molecular weight of the light chain of IgY is smaller. Second, upon pepsin digestion, anti-rabies virus IgY is degraded into Fab, in contrast to mammalian IgG, which has been reported to be degraded into F(ab')(2) under the same conditions.     

Atmospheric chemistry of n-C(x)F(2)(x)(+1)CHO (x = 1, 2, 3, 4): fate of n-C(x)F(2)(x)(+1)C(O) radicals

Smog chamber/FTIR techniques were used to study the atmospheric fate of n-C(x)F(2)(x)(+1)C(O) (x = 1, 2, 3, 4) radicals in 700 Torr O(2)/N(2) diluent at 298 +/- 3 K. A competition is observed between reaction with O(2) to form n-C(x)()F(2)(x)()(+1)C(O)O(2) radicals and decomposition to form n-C(x)F(2)(x)(+1) radicals and CO. In 700 Torr O(2)/N(2) diluent at 298 +/- 3 K, the rate constant ratio, k(n-C(x)F(2)(x)(+1)C(O) + O(2) --> n-C(x)F(2)(x)(+1)C(O)O(2))/k(n-C(x)F(2)(x)(+1)C(O) --> n-C(x)F(2)(x)(+1) + CO) = (1.30 +/- 0.05) x 10(-17), (1.90 +/- 0.17) x 10(-19), (5.04 +/- 0.40) x 10(-20), and (2.67 +/- 0.42) x 10(-20) cm(3) molecule(-1) for x = 1, 2, 3, 4, respectively. In one atmosphere of air at 298 K, reaction with O(2) accounts for 99%, 50%, 21%, and 12% of the loss of n-C(x)F(2)(x)(+1)C(O) radicals for x = 1, 2, 3, 4, respectively. Results are discussed with respect to the atmospheric chemistry of n-C(x)F(2)(x)(+1)C(O) radicals and their possible role in contributing to the formation of perfluorocarboxylic acids in the environment.     

Determination of the rate constants for the NH2(X2B1) + NH2(X2B1) and NH2(X2B1) + H Recombination reactions with collision partners CH4, C2H6, CO2, CF4, and SF6 at low pressures and 296 K. Part 2

The recombination rate constants for the reactions NH2(X2B1) + NH2(X2B1) + M → N2H4 + M and NH2(X2B1) + H + M → NH3 + M, where M was CH4, C2H6, CO2, CF4, or SF6, were measured in the same experiment over presseure ranges of 1-20 and 7-20 Torr, respectively, at 296 ± 2 K. The NH2 radical was produced by the 193 nm laser photolysis of NH3. Both NH2 and NH3 were monitored simultaneously following the photolysis laser pulse. High-resolution time-resolved absorption spectroscopy was used to monitor the temporal dependence of both species: NH2 on the (1)2(21) ← (1)3(31) rotational transition of the (0,7,0)A2A1 ← (0,0,0)X2B1 electronic transition near 675 nm and NH3 in the IR on either of the inversion doublets of the qQ3(3) rotational transition of the ν1 fundamental near 2999 nm. The NH2 self-recombination clearly exhibited falloff behavior for the third-body collision partners used in this work. The pressure dependences of the NH2 self-recombination rate constants were fit using Troe’s parametrization scheme, k(inf), k(0), and F(cent), with k(inf) = 7.9 × 10(-11) cm3 molecule(-1) s(-1), the theoretical value calculated by Klippenstein et al. (J. Phys. Chem. A113, 113, 10241). The individual Troe parameters were CH4, k(0)(CH4) = 9.4 × 10(-29) and F(cent)(CH4) = 0.61; C2H6, k(0)(C2H6) = 1.5 × 10(-28) and F(cent)(C2H6) = 0.80; CO2, k(0)(CO2) = 8.6 × 10(-29) and F(cent)(CO2) = 0.66; CF4, k(0)(CF4) = 1.1 × 10(-28) and F(cent)(CF4) = 0.55; and SF6, k(0)(SF6) = 1.9 × 10(-28) and F(cent)(SF6) = 0.52, where the units of k0 are cm6 molecule(-2) s(-1). The NH2 + H + M reaction rate constant was assumed to be in the three-body pressure regime, and the association rate constants were CH4, (6.0 ± 1.8) × 10(-30); C2H6, (1.1 ± 0.41) × 10(-29); CO2, (6.5 ± 1.8) × 10(-30); CF4, (8.3 ± 1.7) × 10(-30); and SF6, (1.4 ± 0.30) × 10(-29), with units cm6 molecule(-1) s,(-1) and the systematic and experimental errors are given at the 2σ confidence level.     

Factors affecting the specific activity of immobilized antibodies and their biologically active fragments.

Factors affecting the specific activity of immobilized antibodies and their biologically active fragments were studied with goat anti-mouse and goat anti-human immunoglobulin G. Antibodies were immobilized on HW 65 polymeric support matrix activated with carbonyldiimidazole, hydrazide and iodoacetic acid. The most significant factors influencing the specific activity of stochastic coupling of antibodies are multi-site attachment, multiple orientations and steric hindrance imposed by crowding of antibody and the size of the antigen. In oriented immobilization the specific activity is affected only by steric hindrance. The specific activity of immunosorbents prepared by immobilization of F(ab') fragments can be improved to almost 100% by limiting the amount of protein immobilization and the size of the antigen. The present study shows the protocols for optimizing immobilized antibody performance.     

Hydrogen/deuterium exchange on yeast ATPase supramolecular protein complex analyzed at high sensitivity by MALDI mass spectrometry

To evaluate the ability of hydrogen/deuterium exchange of amide protons followed by mass spectrometry (HXMS) to yield topological information about supramolecular protein complexes, this approach has been tested with the 370 kDa hetero-oligomeric complex of yeast F1-ATPase. The study was focused on the epsilon subunit (6612 Da) of the complex. Deuterium back exchange due to the chromatographic isolation step of this subunit was strongly reduced by means of fast micro-chromatography, and MALDI-MS was used to analyze either the intact subunit or peptide mixtures resulting from its proteolytic cleavage. A deuterium labeling kinetic study was conducted with epsilon subunit being a part of the F1 native complex. The effect of a secondary structure was also investigated by means of HXMS on the isolated epsilon subunit. Finally, to determine which regions of epsilon subunit are accessible to solvent in F1-ATPase during exchange, the complex was submitted to hydrogen/deuterium exchange, the epsilon subunit was purified by micro-chromatography, digested by pepsin, and resulting peptide fragments were analyzed by MALDI-MS. The combination of hydrogen/deuterium exchange, fast micro-chromatography and MALDI-MS was shown to be a fast and efficient way to obtain detailed topological information for the epsilon subunit when it is engaged in the ATPase complex.     

Investigating the structural properties of amyloid-like fibrils formed in vitro from beta2-microglobulin using limited proteolysis and electrospray ionisation mass spectrometry

The protein beta(2)-microglobulin (beta(2)m) aggregates to form classical amyloid fibrils in patients undergoing long-term haemodialysis. Amyloid-like fibrils with a cross-beta fold can also be formed from wild-type beta(2)m under acidic conditions in vitro. The morphology of such fibrils depends critically on the conditions used: incubation of beta(2)m in low ionic strength buffers at pH 2.5 results in the formation of long (microm), straight fibrils while, at pH 3.6, short (<500 nm) fibrils form. At higher ionic strengths (0.2-0.4 M) at pH 1.5-3.6, the fibrils have a distinct curved and nodular morphology. To determine the conformational properties of beta(2)m within in vitro fibrils of different morphologies, limited proteolysis of each fibril type using pepsin was performed and the resulting peptide fragments identified by tandem mass spectrometry. For comparison, the proteolytic degradation patterns of monomeric beta(2)m and seven synthetic peptides spanning the entire sequence of the intact protein were similarly analysed. The results show that fibrils with different morphologies result in distinct digestion patterns. While the curved, worm-like fibrils are relatively weakly protected from proteolysis, the long, straight fibrils formed at pH 2.5 at low ionic strength show only a single cut-site at Val9, demonstrating that substantial refolding of the initially acid-denatured and unprotected state of beta(2)m occurs during assembly. The data demonstrate that the organisation of the polypeptide chain in fibrils with different morphological features differs considerably, despite the fact that the fibrils possess a common cross-beta architecture.