Chemical approaches to penicillin allergy—V. Nature of reaction between rabbit antigen (CRP-penicillin conjugate) and rabbit antipenicillin antibody

The availability of an electrophoretically homogeneous rabbit penicillin carrier receptor protein (CRP) and rabbit antipenicillin antibody afforded an idealin vitro system to calculate the thermodynamic parameters of the binding of14C benzyl penicillin CRP conjugate (antigen) to the purified rabbit antipenicillin antibody. The thermodynamic parameters of this antigen-antibody reaction has been studied by radio-active assay method by using millipore filter. Equilibrium constant (K) of this reaction has been found to be 2·853×109M−2 and corresponding free energy (ΔG) at 4°C and 37°C has been calculated to be −12·02 and −13·5 kcal/mole, enthalpy (ΔH) and entropy (ΔS) has been found to be 361 kcal/mole and +30 eu/mole respectively. Competitive binding studies of CRP-analogue conjugates with the divalent rabbit antibody has been carried out in the presence of14C-penicilloyl CRP. It was found that 7-deoxy penicillin-CRP complex and 6-amino penicilloyl CRP conjugate binds to the antibody with energies stronger than that with the14C-penicilloyl CRP. All the other analogue conjugates are much weaker in interfering with the binding of the penicilloyl CRP with the antibody. The conjugate of methicillin,o-nitro benzyl penicillin and ticarcillin with CRP do not materially interfere in the process.

Realtime kinetic analysis of antigen-antibody interaction using solid phase binding: Transformation of hCG-monoclonal antibody complex

Kinetic constants of MAb-hCG interactions have been determined using solid phase binding of I-125[hCG] to immobilized MAb. While association has been shown to follow the expected pattern, dissociation consists of at least two reversible steps, one with a rate constant of 0.0025 min(-1), and a second with a rate constant of 0.00023 min(-1). Validity of affinity constant measurements in the light of the complex reaction kinetics is discussed, A comparison between the method of surface plasmon resonance technology (BIAcore) and solid phase binding (SPB) for determination of kinetic parameters shows that SPB provides not only a cost-effective approach for determination of realtime kinetic parameters of macromolecular ligand-ligate interaction but also a method with several advantages over the BIAcore system in investigating the mechanism of antigen-antibody interaction.

Monoclonal antibody screening of a phage-displayed random peptide library reveals mimotopes of chemokine receptor CCR5: Implications for the tertiary structure of the receptor and for an n-terminal binding site for HIV-1 gp120

The chemokine receptor CCR5 contains seven transmembrane-spanning domains. It binds chemokines and acts as co-receptor for macrophage (m)-tropic (or R5) strains of HIV-1. Monoclonal antibodies (mAb) to CCR5, 3A9 and 5C7, were used for biopanning a nonapeptide cysteine (C)-constrained phage-displayed random peptide library to ascertain contact residues and define tertiary structures of possible epitopes on CCR5. Reactivity of antibodies with phagotopes was established by enzyme-linked immunosorbent assay (ELISA). mAb 3A9 identified a phagotope C-HASIYDFGS-C (3A9/1), and 5C7 most frequently identified C-PHWLRDLRV-C (5C7/1). Corresponding peptides were synthesized. Phagotopes and synthetic peptides reacted in ELISA with corresponding antibodies and synthetic peptides inhibited antibody binding to the phagotopes. Reactivity by immunofluorescence of 3A9 with CCR5 was strongly inhibited by the corresponding peptide. Both mAb 3A9 and 5C7 reacted similarly with phagotopes and the corresponding peptide selected by the alternative mAb. The sequences of peptide inserts of phagotopes could be aligned as mimotopes of the sequence of CCR5. For phage 3A9/1, the motif SIYD aligned to residues at the N terminus and FG to residues on the first extracellular loop; for 5C7/1, residues at the N terminus, first extracellular loop, and possibly the third extracellular loop could be aligned and so would contribute to the mimotope. The synthetic peptides corresponding to the isolated phagotopes showed a CD4-dependent reactivity with gp120 of a primary, m-tropic HIV-1 isolate. Thus reactivity of antibodies raised to CCR5 against phage-displayed peptides defined mimotopes that reflect binding sites for these antibodies and reveal a part of the gp120 binding sites on CCR5.

Use of phage display technology to investigate allergen-antibody interactions

Phage display is an advanced technology that can be used to characterize the interactions of antibody with antigen at the molecular level. It provides valuable data when applied to the investigation of IgE interaction with allergens. The aim of this rostrum article is to provide an explanation of the potential of phage display for increasing the understanding of allergen- IgE interaction, the discovery of diagnostic reagents, and the development of novel therapeutics for the treatment of allergic disease. The significance of initial studies that have applied phage display technology in allergy research will be highlighted. Phage display has been used to clone human IgE to timothy grass pollen allergen Phl p 5, to characterize the epitopes for murine and human antibodies to a birch pollen allergen Bet v 1, and to elucidate the epitopes of a murine mAb to the house dust mite allergen Der p 1. The technology has identified peptides that functionally mimic sites of human IgE constant domains and that were used to raise antiserum for blocking binding of IgE to the FcεRI on basophils and subsequent release of histamine. Phage display has also been used to characterize novel peanut and fungal allergens. The method has been used to increase our understanding of the molecular basis of allergen-IgE interactions and to develop clinically relevant reagents with the pharmacologic potential to block the effector phase of allergic reactions. Many advances from these early studies are likely as phage display technology evolves and allergists gain expertise in its research applications.

Amplification of antigen-antibody interactions based on biotin labeled protein-streptavidin network complex using impedance spectroscopy

Antibody was covalently immobilized by amine coupling method to gold surfaces modified with a self-assembled monolayer of thioctic acid. The electrochemical measurements of cyclic voltammetry and impedance spectroscopy showed that the hexacyanoferrate redox reactions on the gold surface were blocked due to the procedures of self-assembly of thioctic acid and antibody immobilization. The binding of a specific antigen to antibody recognition layer could be detected by measurements of the impedance change. A new amplification strategy was introduced for improving the sensitivity of impedance measurements using biotin labeled protein- streptavidin network complex. This amplification strategy is based on the construction of a molecular complex between streptavidin and biotin labeled protein. This complex can be formed in a cross-linking network of molecules so that the amplification of response signal will be realized due to the big molecular size of complex. The results show that this amplification strategy causes dramatic improvement of the detection sensitivity of hIgG and has good correlation for detection of hIgG in the range of 2-10 mug/ml. (C) 2001 Elsevier Science B.V. All rights reserved.

Resonant Rayleigh light scattering response of individual Au nanoparticles to antigen--antibody interaction

A proof-of-concept study was reported on analysis of antigen–antibody recognition based on resonant Rayleigh scattering response of single Au nanoparticles in an imaging chamber. As benefited by a traditional dark-field microscope and a spectrograph, individual Au nanoparticles (30 nm) were observed with high signal-to-noise ratio and they were effectively utilized to monitor changes in refractive index induced by specific binding of the adsorbates. Using PSA antigen as a model, a LSPR ?max shift of about 2.85 nm was recorded for a molecular binding corresponding to 0.1 pg ml-1 of the protein biomarker. This result successfully demonstrates a non-labeling detection system for proteins as well as thousands of different chemical or biological species, and it possesses a great potential as a sensitive, on-chip and multiplexing detection.

METHOD OF DETECTING BIOPRODUCTS USING LOCALIZED SURFACE PLASMON RESONANCE SENSOR OF GOLD NANOPARTICLES

(EN)Disclosed is a method of detecting bioproducts using Localized Surface Plasmon Resonance (LSPR) of gold nanoparticles, which can diagnose bioproducts based on changes in the maximum wavelength occurred by an antigen-antibody reaction after immobilization of the gold nanoparticles onto a glass panel. A sensor using such method exhibits high sensitivity, is low in price, and makes quick diagnosis possible, thereby being applicable to various biological fields associated with environmental contaminants, pathogens and the like, as well as diagnosis of diseases. Further, it provides a technology for manufacturing a sensor having higher sensitivity, low price and quick performance, as compared to conventional methods using SPR.

Characteristics of the histamine release from hamster cheek pouch mast cells stimulated by lectins from Brazilian beans and concanavalin A

We have characterized the histamine releasing effects of lectins extracted from Brazilian beans, in comparison to concanavalin A, in hamster cheek pouch cell suspensions containing mast cells. The lectins from Dioclea virgata, Canavalia brasiliensis, and Dioclea rostrata induce histamine release in a similar manner to concanavalin A, but appear to differ in potency and efficacy. The effects depended on the temperature, pH, and metabolic energy, demonstrating the non-cytotoxic nature of the histamine release. It is suggested that the lectins studied act by the same mechanism as concanavalin A (interacting with sugars in the antibodies bound to the mast cells), since high concentrations of glucose inhibit the histamine release. The lectins at high concentrations quench the histamine release. This suppression is reversed by increasing calcium concentration, suggesting that the lectins bind to the calcium that is essential for the secretion, thereby confirming and extending our previous data using the lectin from Dioclea virgata in rat peritoneal mast cells.

Clonagem e expressão da glicoproteína transmembrana do vírus linfotrópico de células T humanas em sistema procarioto

HTLV-1 is the virus that causes T cell lymphoma/leukemia in adults and a neurological disorder known as HTLV-associated myelopathy or tropical spastic paraparesis. One of the transmission means is through contaminated blood and its byproducts. Because of the risk of HTLV-associated infections, screening for HTLV was introduced for Brazilian blood donors in 1993. Most of the diagnostic kits used in the national blood banks are bought from foreign companies. Brazil does not have the technology to produce this material and there is a need to produce diagnostic systems with national technology. In this study, we show the expression of gp21/HTLV-1 in Escherichia coli and its reactivity towards monoclonal antibodies and the antibodies of infected patients. Expressing these proteins is the first step towards obtaining diagnostic kits with Brazilian biotechnology.

Characterization of yolk sac proteins of Bos indicus cattle embryos

The yolk sac is an embryonic membrane that is essential for the embryo's initial survival in many mammals. It also plays an important role in the production of proteins necessary for development. We studied proteins of the yolk sac in bovine embryos at up to 40 days of gestation. We examined the yolk sac of 17 bovine embryos at different gestational periods, measuring a-fetoprotein, alpha-1-antitrypsin, and transferrin. This experiment was carried out by Western blot technique, associated with electrophoresis on a 6% sodium dodecyl sulfate polyacrylamide gel. Mouse monoclonal antibody anti-human-alpha-fetoprotein, mouse antibody anti-human-transferrin and rabbit polyclonal anti-human-alpha-1-antitrypsin were used as primary antibodies, and conjugated peroxidase as a secondary antibody. We detected the three proteins in some of the yolk sac samples; however, the bands in some specimens (samples) were weak, maybe a result of poor antigen-antibody reaction, since the antibodies used in this study were not specific to bovine proteins. The fact that weak bands appeared might be due to a weak cross-reaction.

Analysis of human chorionic gonadotropin-monoclonal antibody interaction in BIAcore

Kinetic studies of macromolecular ligand-ligate interaction have generated ample interest since the advent of plasmon resonance based instruments like BIAcore. Most of the studies reported in literature assume a simple 1 : 1 Langmuir binding and complete reversibility of the system. However we observed that in a high affinity antigen-antibody system [human chorionic gonadotropin-monoclonal antibody (hCG-mAb)] dissociation is insignificant and the sensogram data cannot be used to measure the equilibrium and kinetic parameters. At low concentrations of mAb the complete sensogram could be fitted to a single exponential. Interestingly we found that at higher mAb concentrations, the binding data did not conform to a simple bimolecular model. Instead, the data fitted a two-step model, which may be because of surface heterogeneity of affinity sites. In this paper, we report on the global fit of the sensograms. We have developed a method by which a single two-minute sensogram can be used in high affinity systems to measure the association rate constant of the reaction and the functional capacity of the ligand (hCG) immobilized on the chip. We provide a rational explanation for the discrepancies generally observed in most of the BIAcore sensograms

Study of Interaction Force between Antigen and Antibody Using Flow Chamber Method

A parallel plate flow chamber was used to study the interaction force between human IgG (immobilized on a chip surface as ligand) and goat anti-human IgG (immobilized on microspheres surface as receptor). First, it was demonstrated that the binding force between the microspheres and the chip surface came from the bio-specific interaction between the antigen and the antibody. Secondly, it was obtained that the critical shear rate to detach microspheres from the chip surface increases with the ligand surface concentration. Finally, two models to estimate the antigen-antibody bond strength considering bonds' positions were proposed and analyzed.

Visualization of molecule interaction between antigen and antibody - One of ellipsometric imaging applications

It is to investigate molecule interactions between antigen and antibody with ellipsometric imaging technique and demonstrate some features and possibilities offered by applications of the technique. Molecule interaction is an important interest for molecule biologist and immunologist. They have used some established methods such as immufluorcence, radioimmunoassay and surface plasma resonance, etc, to study the molecule interaction. At the same time, experimentalists hope to use some updated technique with more direct visual results. Ellipsometric imaging is non-destructive and exhibits a high sensitivity to phase transitions with thin layers. It is capable of imaging local variations in the optical properties such as thickness due to the presence of different surface concentration of molecule or different deposited molecules. If a molecular mono-layer (such as antigen) with bio-activity were deposited on a surface to form a sensing surface and then incubated in a solution with other molecules (such as antibody), a variation of the layer thickness when the molecules on the sensing surface reacted with the others in the solution could be observed with ellipsometric imaging. Every point on the surface was measured at the same time with a high sensitivity to distinguish the variation between mono-layer and molecular complexes. Ellipsometric imaging is based on conventional ellipsometry with charge coupled device (CCD) as detector and images are caught with computer with image processing technique. It has advantages of high sensitivity to thickness variation (resolution in the order of angstrom), big field of view (in square centimeter), high sampling speed (a picture taken within one second), and high lateral resolution (in the order of micrometer). Here it has just shown one application in study of antigen-antibody interaction, and it is possible to observe molecule interaction process with an in-situ technique.

Application of impedance spectroscopy for monitoring colloid Au-enhanced antibody immobilization and antibody-antigen reactions

We used colloidal An to enhance the amount of antibody immobilized on a gold electrode and ultimately monitored the interaction of antigen-antibody by impedance measurement. Self-assembly of 6 nm (diameter) colloidal An onto the self-assembled monolayers (SAMs) of 4-aminothiophenol modified gold electrode resulted in an easier attachment of antibody. The redox reactions of [Fe(CN)(6)](4-)/[Fe(CN)(6)](3-) on the gold surface were blocked due to the procedures of self-assembly of 4-aminothiophenol and antibody immobilization, which were investigated by cyclic voltammetry and impedance spectroscopy. The interaction of antigen with grafted antibody recognition layers was carried out by soaking the modified electrode into a phosphate buffer at pH 7.4 with various concentrations of antigen at 37 degreesC for 30 min. The antibody recognition layers and their interactions with various concentrations of antigen could be detected by measurements of the impedance change. The results show that this method has good correlation for detection of Hepatitis B virus surface antigen in the range of 0.5-200 mug/l and a detection limit of about 50 ng/l.