Dissociation Of Antibodies Research Articles

Orientation of Antibody Modified and Reacted on Carboxy-Functionalized Polystyrene Particle Revealed by Zeta Potential Measurement.

The comprehensive understanding of the orientation of antibodies on a solid surface is crucial for affinity-based sensing mechanisms. In this study, we demonstrated that the orientation of primary antibodies modified on carboxy-functionalized polystyrene (PS) particles can be analyzed using zeta potential behavior at different pH based on the combined Gouy-Chapman-Stern model and the acid dissociation of carboxy groups and antibodies. We observed that at low surface concentrations of the primary antibody, a side-on orientation was predominant. However, at higher concentrations (approximately 30000 antibodies per PS particle), the orientation shifted to an end-on type due to steric hindrance. Furthermore, the reaction mechanism of the secondary antibody exhibited pH-dependent behavior. At pH > 7, the zeta potential changes were attributed to the antibody-antibody reaction, whereas at pH < 7, adsorption of secondary antibody onto the PS particle was observed, leading to a change in the orientation of the primary antibody modified on the PS particle to an end-on type. The change in zeta potential due to secondary antibody binding indicated a detection limit of 37000 antibodies per PS particle. As a result, we revealed that the analysis of zeta potential behavior enables the evaluation of antibody orientation and the detection of zeptomole order antibodies. This study represents the first demonstration of this capability. We anticipate that the present concept and results will broaden the quantitative application of zeta potential measurements and have significant implications for research areas, including physical chemistry and analytical chemistry.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics.

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide-ribosome-mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

Acid pH promotes bispecific antibody formation by the redox procedure

Bispecific antibodies (BsAbs), are potential theranostics. Chemical procedures of preparation of BsAbs, in which two monospecific antibodies are split into half molecules and heterodimerized, continue to attract attention in view of their simplicity. Poor dissociation of antibodies with reduced inter-heavy chain disulfides into half molecules under neutral conditions however restricts the BsAbs formation. In this study, we report that the heterodimerization of antibodies can be improved leading to over 6-fold increase in the yield of BsAbs, by carrying out the redox procedure at pH 4.0. In view of improvement in heterodimerization, BsAbs could be conveniently prepared starting from partially purified ion-exchange fraction of the antiserum and purified by twin affinity chromatography on antigen supports. The UV, CD, intrinsic and extrinsic fluorescence spectral analysis of BsAbs prepared by the modified redox procedure were comparable with the native IgG, which suggest the absence of significant acid-pH-induced damage. ThT binding studies and native size exclusion chromatography ruled out amyloid fibril formation.

The impact of antibody/epitope affinity strength on the sensitivity of electrochemical immunosensors for detecting small molecules

A displacement immunoassay involves having a labelled analogue of the analyte (the epitope) already bound to the antibody. The presence of the analyte causes a competition for antibodies, and some of the antibodies dissociates from the epitope so that it can bind with the analyte. Herein, the influence of the affinity of the surface-bound epitope for the antibody on the sensitivity and selectivity of a displacement immunosensor is explored both theoretically and experimentally. An electrochemical immunosensor described previously, where the dissociation of antibodies from an electrode surface causes an increase in current from surface-bound ferrocene species, is used for this purpose. As expected, the ease and effectiveness of the bound antibody being displaced is inversely related to the affinity of the antibody to the surface-bound epitope relative to the analyte in solution as expected. However, if the affinity constant is too low, selectivity and/or sensitivity are compromised. Experimental results are qualitatively compared with a simple mass-action model.

Conformational Analysis of Human ATP-binding Cassette Transporter ABCB1 in Lipid Nanodiscs and Inhibition by the Antibodies MRK16 and UIC2

The human ATP-binding cassette (ABC) transporter, P-glycoprotein (P-gp; ABCB1), mediates the ATP-dependent efflux of a variety of drugs. As a result, P-gp plays a critical role in tumor cell drug resistance and the pharmacokinetic properties of most drugs. P-gp exhibits extraordinary substrate and inhibitor promiscuity, resulting in a wide range of possible drug-drug interactions. Inhibitory antibodies have long been considered as a possible strategy to modulate P-gp-dependent cancer cell drug resistance, and it is widely suggested that the antibodies MRK16 and UIC2 inhibit P-gp by capturing a single isoform and preventing flux through the catalytic cycle. Although the crystal structures of many bacterial whole transporters, as well as isolated nucleotide-binding domains, have been solved, high resolution structural data for mammalian ABC transporters are currently lacking. It has been extremely difficult to determine the detailed mechanism of transport of P-gp, in part because it is difficult to obtain purified protein in well defined lipid systems. Here we exploit surface plasmon resonance (SPR) to probe conformational changes associated with these intermediate states for P-gp in lipid bilayer nanodiscs. The results indicate that P-gp in nanodiscs undergoes functionally relevant ligand-dependent conformational changes and that previously described inhibitory antibodies bind to multiple nucleotide-bound states but not the ADP-VO(4)-trapped state, which mimics the post-hydrolysis state. The results also suggest that the substrate drug vinblastine is released at stages that precede or follow the post-hydrolysis ADP-PO(4)·P-gp complex.

Beyond Photobleaching, Laser Illumination Unbinds Fluorescent Proteins

Confocal and two-photon fluorescence microscopy techniques using genetically encoded fluorescent probes are widely used in cell biology. Beyond the common problems of photobleaching and phototoxicity, we present evidence that photounbinding also has the potential to compromise such methods, especially in quantitative studies. We show that laser intensities within excitation regimes typical for imaging approaches such as as fluorescence recovery after photobleaching (FRAP), photolysis, or fluorescence correlation spectroscopy (FCS) experiments can cause the dissociation of antibodies from their ligands. Indeed, both one- and two-photon excitation of a fluorescent anti-GFP antibody caused its dissociation from immobilized GFP in vitro. Importantly, with two-photon excitation, the laser intensity threshold for photobleaching was the same as for photounbinding. By contrast, with single-photon excitation, we found a range of laser intensities where photobleaching can be separated from photounbinding. This photounbinding effect was visualized and measured by rebinding a second fluorescent anti-GFP (Green Fluorescent Protein) antibody, indicating that the GFP remained functional for reassociation following the photoinduced dissociation. Finally, we show that this unbinding effect occurs only when at least one binding partner carries a fluorescent label. Our results show that this photounbinding effect can readily remain masked or be misinterpreted as photobleaching, which can compromise the quantitative interpretation of binding studies made using fluorescence microscopy.

A novel biochip technology for detection of explosives – TNT: Synthesis, characterisation and application

This contribution describes the synthesis, characterisation and evaluation of a novel biochip technology for the detection of the explosive substance 2,4,6-trinitrotoluene (TNT). Two types of thiols are self-assembled to produce the biochip on gold, namely oligo(ethylene glycol) (OEG)-alkyl thiols terminated with a hydroxyl group and a TNT-analogue (2,4-dinitrobenzene), respectively. Three different TNT-analogues are mixed in various proportions with hydroxyl-terminated OEG-thiols to obtain highly selective and sensitive biochips with a low non-specific binding. The produced self-assembled monolayers (SAMs) are thoroughly characterised with null ellipsometry, contact angle goniometry, infrared reflection absorption spectroscopy (IRAS) and X-ray photoelectron spectroscopy (XPS) and they all meet high standards in terms of molecular conformation, packing and orientation. The biochip is designed to function as a platform for a competitive label-free immunoassay and two real-time transducers – surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) – are used to monitor the dissociation of on-line immobilised monoclonal antibodies produced against TNT. The three TNT-analogues are all potential candidates for the development of a functional biochip, though one of them displayed superior properties in terms of shorter recovery/stabilisation time after antibody immobilisation and a better response/loading capacity ratio. This is particularly evident when using low antigen (TNT-analogue) content in the mixed SAM.

Abo-incompatible renal transplantation—dissociation of abo antibodies

Abo-incompatible renal transplantation—dissociation of abo antibodies

Dissociation of antibodies bound to surface-immobilized antigen

The dissociation of antibodies bound to surface-immobilized antigen was investigated by the ELISA, using a hapten (TNP) as antigen. Antibody binding was found to be stable, and no half-time of dissociation could be defined within 69 h. The role of the bivalence of antibodies and the difference between a homogenous and a heterogenous reaction was investigated by comparing the dissociation rate of antigen-antibody complexes formed by monovalent Fab fragments from surface-immobilized antigen and the dissociation rate of TNP-lysine from antibodies in a homogenous liquid phase. Fab fragments were found to dissociate with a half-time value of about 16 h, whereas the homogenous binding of TNP-antibody dissociated with a half-time of less than 4 h, indicating that both the bivalence of antibodies and the solid phase contributed to the stability of surface-bound antigen-antibody complexes. Qualitative differences between antibodies produced by different clones in a polyclonal antibody response to TNP was investigated by a spot assay. The results indicated that a minority of the antibodies produced had the capacity of binding practically irreversible to solid-phase-immobilized antigen. The impact of the results on the interpretation of data from solid-phase assays is discussed together with the biological importance of the findings.

Low avidity as a cause of prozone phenomena

The incidence of prozones was studied, under varioys conditions, in a model indirect haemagglutination system and in a radioimmunoassay — both assays depending on the use of anti-immunoglobulin (anti-Ig). It was found that prozones were correlated with the presence of low avidity antibodies, and that these could compete with high avidity antibodies for the limited amount of anti-Ig available. It is proposed that the relatively rapid dissociation of low avidity antibodies allows them to form immune aggregates with the anti-Ig. With increasing size these aggregates would become more susceptible to being washed off. In this way low avidity antibodies could occupy the anti-Ig, and yet be relatively ineffective, either for haemagglutination or for the binding of radioactively labelled (or fluorescein-labelled) anti-Ig to the antigen.

Rapid recovery of antigen-binding receptors on chicken B cells following anti-lg serum treatment

Rosette-forming cells (RFC) from the peripheral blood of sheep red blood cell (SRBC)-immunized chickens were characterized as B cells which manifest antigen-binding receptors but no antibody secretion. Lymphocytes were pretreated at 0 degrees C with anti-Ig serum, washed, incubated further at various temperatures and harvested for the rosette-forming cells (RFC) assay. Anti-Ig treatment blocked all RFC formation and providing that the cells were maintained at less than 10 degrees C regeneration did not occur up to 6 h. Complete recovery of RFC formation occurred within 10 min at 37 degrees C following treatment with low but not with high concentrations of anti-Ig serum. However, recovery from inhibition by high-dose anti-Ig treatment was achieved by including chicken IgG in the incubation medium. The recovery of receptors was reduced following "sandwich" treatment of lymphocytes. When cells were treated with anti-Ig antibody, washed and exposed to various drug inhibitors of "capping" the regeneration of RFC was prevented; in contrast, inhibitors of protein synthesis were ineffective. The results were interpreted in terms of dissociation of anti-Ig antibodies from the Ig receptor at an early stage of the ligand-induced receptor redistribution process.

Utilization of glutaraldehyde cross-linked antibodies in the purification of a plant virus

Gels satisfactory for partial purification of a plant virus were produced by treatment of whole or reconstituted tobacco ringspot virus (TRSV) antisera with aqueous solutions of glutaraldehyde. With partially purified TRSV retention capacities of immunoadsorbents from whole antisera ranged from 50 to 70% while those of immunoadsorbents from reconstituted concentrated antisera ranged from 85 to 96% of the added TRSV. Attempts to dissociate the antibody-antigen complex with high salt concentrations resulted in the dissociation and solubilization of antibodies to TRSV. On the other hand, dissociation with eluents of low pH (0.1 M glycine·HCl buffer, pH 2.8) resulted in the dissociation and subsequent elution of virus antigen from the complex. Between 20 and 25% of the retained virus was dissociated and eluted from the immunoadsorbents using low pH and employing a batch procedure. When immunoadsorbents were used to isolate TRSV from crude sap, the isolated virus fractions showed the typical absorbency of a nucleoprotein at 240 nm and 260 nm, respectively and were between 80 and 85% as infectious as partially purified virus. TRSV particles were present in these fractions as shown by electron microscopy.

Immune Hemolysis: Serial Studies on the Dissociability of IgG and IgM Anti-Forssman Antibodies Using the C′la Fixation and Transfer Technique

Summary The C′1a fixation and transfer test was found to be a useful procedure for measuring the dissociation of rabbit IgG and IgM anti-Forssman antibodies from sensitized sheep erythrocytes. During the first few weeks after the start of immunization, IgG antibodies were found to be of such low avidity that much antibody could be removed from sensitized erythrocytes simply by washing at 25°C to 30°C. Nearly all antibody activity could be retained on the cells by washing at 0°C with low ionic strength buffer. With IgM fractions from the same sera, avidity was much higher. Only in preimmune sera were low-avidity IgM antibodies observed. Both classes of antibody showed a progressive increase in avidity with time after the start of immunization, although the major increase was much earlier for the IgM than for the IgG class.

EXPERIMENTAL GLOMERULONEPHRITIS. V. STUDIES ON THE INTERACTION OF NEPHROTOXIC ANTIBODIES WITH TISSUE OF THE RAT.

A direct correlation between the amount of kidney-fixing antibody and the degree of associated renal injury was demonstrated for rabbit and duck nephrotoxic antibodies. No evidence for a qualitative difference among nephrotoxic antibodies of a given type was obtained. It appeared that duck nephrotoxic antibody was directed against a wider spectrum of rat renal antigens than was rabbit nephrotoxic antibody. In order to produce immediate proteinuria an amount of rabbit or duck gamma-2 kidney-fixing antibody capable of occupying approximately 45 per cent or more of the capillary filtration surface was needed. For immediate proteinuria an amount of rabbit gamma-2 kidney-fixing antibody capable of reacting with more than one-half of the available antigenic sites was needed. Less than twice that amount of rabbit antibody completely saturated available antigenic sites in the kidney. Virtually all nephrotoxic antibodies in hyperimmune rabbit nephrotoxic sera were of the gamma-2 variety while nephrotoxic antibodies in comparable duck nephrotoxic sera were found in gamma-2 (with 5.8 and 7.4S sedimentation coefficients) and gamma-1M fractions. Gamma-1M duck nephrotoxic antibody was 60 times more potent a nephritogen than gamma-2 duck nephrotoxic antibody on a molecular basis. Mercaptoethanol abolished the nephrotoxicity of gamma-1M duck antibody and reduced that of gamma-2 duck antibodies but had no effect on rabbit gamma-2. In no case did mercaptoethanol treatment have an effect on the kidney-fixing properties of the antibodies involved. After injection of nephrotoxic antibodies there appeared to be a prompt fixation of a majority of the antibody to tissue antigens primarily in the kidney. However, a small amount of potentially kidney-fixing antibody remained in the circulation for a considerable period apparently reflecting a dissociation of less avid antibodies with an equilibrium between fixed and free antibody. The role of this more easily dissociable antibody in the progression of nephrotoxic nephritis is not certain but it is possible that it could play a role in the early progression of the disease.

INSULIN ANTIBODY VARIATIONS IN RABBITS AND GUINEA PIGS AND MULTIPLE ANTIGENIC DETERMINANTS ON INSULIN

1. A method is presented for measuring the degree to which insulin antibodies in one antiserum react with an insoluble insulin complex saturated with antibodies from a different antiserum. 2. Many rabbits produce antibodies which bind to portions of the insulin molecule to which antibodies from guinea pigs or other rabbits cannot bind. 3. Occasional guinea pigs produce antibodies which bind to portions of the insulin molecule to which antibodies from rabbits or other guinea pigs cannot bind. 4. Studies with labeled antisera and repeated incubations of test antisera with antibody insulin complexes demonstrate the individual antibody variations to be due to antibodies directed to different determinants and not to dissociation of antibodies from the same determinant on the insulin molecule. 5. More than one antibody molecule can simultaneously bind to an insulin molecule. 6. Insulin has a multiplicity of antigenic determinants. 7. The relationship between antigenic determinants, insulin antibodies, and neutralization of insulin by antisera is discussed. 8. The determinants to which insulin antibodies are directed appear to be characteristic for the individual rabbit or guinea pig immunized. It is postulated therefore that genetic factors direct antibody production toward specific determinants when insulin is the antigen.