Ab Complex Research Articles

Circular Dichroism reveals evidence of coupling between immunoglobulin constant and variable region secondary structure

Antibodies (Ab) are bifunctional molecules with two domains, a constant region (C) that confers effector properties and a variable (V) region responsible of antigen (Ag) binding. Historically the C and V regions were considered to be functionally independent, with Ag specificity being solely determined by the V region. However, recent studies suggest that the C region can affect Ab fine specificity. This has led to the proposal that the C H domain influences the structure of the V region, thus affecting Ab affinity and fine specificity. An inference from this proposal is that V region identical monoclonal Abs (mAbs) differing in C region (eg isotype) would manifest different secondary structures arising from isotype-induced variation in the V–C regions after Ag binding. We hypothesized that such effects could translate into differences in Circular Dichroism (CD) upon Ag–Ab complexes formation. Consequently we studied the interaction of a set of V region identical IgG 1, IgG 2a, IgG 2b and IgG 3 mAbs with glucuronoxylomannan (GXM). The native CD spectra of the pairs IgG 1/IgG 2a and IgG 3/IgG 2b were strikingly similar, implying similar secondary structure content. GXM binding by IgG 1, IgG 2a, IgG 2b and IgG 3 produced different CD changes, with the pairs IgG 1/IgG 2a and IgG 3/IgG 2b again manifesting qualitatively similar trends in secondary structure changes. The magnitude of the changes differed among the isotypes with IgG 2a > IgG 3 > IgG 2b > IgG 1. These differences in CD changes were interpreted to reflect differences in V–C secondary structures.

Chemiluminescent immunoassay of thyroxine enhanced by microchip electrophoresis

A homogeneous chemiluminescent immunoassay of thyroxine (T4) enhanced by microchip electrophoresis separation has been developed. The method deployed the competitive immunoreaction of T4 and horseradish peroxidase (HRP)-labeled T4 (HRP–T4) with anti-T4 mouse monoclonal antibody (Ab). HRP–T4 and the HRP–T4–Ab complex were separated and quantified by using microchip electrophoresis (MCE) with chemiluminescence (CL) detection. Highly sensitive CL detection was achieved by means of HPR-catalyzed luminol–H 2O 2 reaction. Due to the effective MCE separation, the CL analytical signal was less prone to sample matrix interference. Under the selected assay conditions, the MCE separation was accomplished within 60 s. The linear range for T4 was 5–250 nM with a detection limit of 2.2 nM (signal/noise ratio = 3). The current method was successfully applied for the quantification of T4 in human serum samples. It was demonstrated that the current MCE–CL-enhanced competitive immunoassay was quick, sensitive, and highly selective. It may serve as a tool for clinical analysis of T4 to assist in the diagnosis of thyroid gland functions.

ADP but Not Pi Dissociation Contributes to Rate Limitation for Escherichia coli Rho

To define the molecular mechanism by which ATP hydrolysis powers the 5'-->3' travel of homohexameric Escherichia coli transcription termination factor Rho along RNA, rates for association and dissociation of non-RNA substrates and products were measured. Rapid mix/chemical quench and stopped-flow spectrofluorometry measurements were carried out with Rho and [gamma-(32)P]ATP, mantADP, or fluorescently tagged E. coli phosphate-binding protein. The results indicate that the P(i) off-rate is not rate limiting, but at approximately 90 s(-1), the ADP dissociation rate is comparable to the 30 s(-1) k(cat). Previous results indicate that the chemistry step of ATP hydrolysis by Rho is at least 10-fold faster than the overall catalytic cycle. The as yet unmeasured RNA dissociation step, which could be associated with a protein conformation change, might also be a rate-limiting factor.

Mechanism for Amyloid Precursor-like Protein 2 Enhancement of Major Histocompatibility Complex Class I Molecule Degradation

Earlier studies have demonstrated interaction of the murine major histocompatibility complex (MHC) class I molecule K(d) with amyloid precursor-like protein 2 (APLP2), a ubiquitously expressed member of the amyloid precursor protein family. Our current findings indicate that APLP2 is internalized in a clathrin-dependent manner, as shown by utilization of inhibitors of the clathrin pathway. Furthermore, we demonstrated that APLP2 and K(d) bind at the cell surface and are internalized together. The APLP2 cytoplasmic tail contains two overlapping consensus motifs for binding to the adaptor protein-2 complex, and mutation of a tyrosine shared by both motifs severely impaired APLP2 internalization and ability to promote K(d) endocytosis. Upon increased expression of wild type APLP2, K(d) molecules were predominantly directed to the lysosomes rather than recycled to the plasma membrane. These findings suggest a model in which APLP2 binds K(d) at the plasma membrane, facilitates uptake of K(d) in a clathrin-dependent manner, and routes the endocytosed K(d) to the lysosomal degradation pathway. Thus, APLP2 has a multistep trafficking function that influences the expression of major histocompatibility complex class I molecules at the plasma membrane.

Basophils as TH2‐inducing APCs: the dog can sing but is it a diva?

Basophils as T_H2‐inducing APCs: the dog can sing but is it a diva?

Cytokine/Antibody Complexes: An Emerging Class of Immunostimulants

In recent years, complexes formed from a cytokine and antibodies against that respective cytokine (cytokine/Ab complex) have been shown to induce remarkable powerful changes in the immune system. Strong interest exists especially for complexes formed with Interleukin (IL)-2 and anti-IL-2-antibody (IL-2/Ab complex). IL-2/Ab complex activates maturation and proliferation in CD8(+) T cells and natural killer (NK) cells to a much higher degree than conventional IL-2 therapy. In addition, IL-2/Ab complex does not stimulate regulatory T cells as much as IL-2 alone. This suggests the possibility to replace the conventional IL-2 therapy with a therapy using low-dose IL-2/Ab complex. Further synthetic cytokine/Ab complexes are studied currently, including IL-3/Ab complex for its effects on the mast cell population, and IL-4/Ab complex and IL-7/Ab complex for inducing B and T cell expansion and maturation. Cytokine complexes can also be made from a cytokine and its soluble receptor. Pre-association of IL-15 with soluble IL-15 receptor alpha produces a complex with strong agonistic functions that lead to an expansion of CD8(+) T cells and NK cells. However, cytokine/Ab complexes also occur naturally in humans. A multitude of auto-antibodies to cytokines are found in human sera, and many of these auto-antibodies build cytokine/Ab complexes. This review presents naturally occurring auto-antibodies to cytokines and cytokine/Ab complexes in health and disease. It further summarizes recent research on synthetic cytokine/Ab complexes with a focus on the basic mechanisms behind the function of cytokine/Ab complexes.

Requirement of B cells for generating CD4+ T cell memory.

B cells can influence T cell responses by directly presenting Ag or by secreting Ab that binds to Ag to form immunogenic complexes. Conflicting evidence suggests that persisting Ag-Ab complexes propagate long-term T cell memory; yet, other data indicate that memory cells can survive without specific Ag or MHC. In this study, the roles of B cells and Ag-Ab complexes in T cell responses to lymphocytic choriomeningitis virus (LCMV) infection were investigated using B cell-deficient or B cell-competent mice. Despite normal lymphocyte expansion after acute infection, B cell-deficient mice rapidly lost CD4(+) T cell memory, but not CD8(+) T cell memory, during the contraction phase. To determine whether Ag-Ab complexes sustain CD4(+) T cell memory, T cell responses were followed in B cell-transgenic (mIg-Tg) mice that have B cells but neither LCMV-specific Ab nor LCMV-immune complex deposition. In contrast to B cell-deficient mice, mIg-Tg mice retained functional Th cell memory, indicating that B cells selectively preserve CD4(+) T cell memory independently of immune complex formation. An in vivo consequence of losing CD4(+) T cell memory was that B cell-deficient mice were unable to resolve chronic virus infection. These data implicate a B cell function other than Ab production that induces long-term protective immunity.

Isolation of antigen/antibody complexes through organic solvent (ICOS) method

We developed a method termed ICOS (isolation of antigen–antibody complexes through organic solvent) for comprehensive isolation of monoclonal antibodies (mAbs) bound to molecules on the cell surface. By mixing a large number of phage particles of an antibody (Ab) library with living cells, antigen (Ag)–Ab complexes were formed on the cell surface. The mixture was overlaid on organic solution in a tube and subjected to centrifugation. Phages bound to cells were recovered from the precipitate. The phage fraction isolated turned out to contain mAbs that bind to very heterogeneous epitopes and show strong binding activity to Ags. The ICOS method was applied to isolation of human mAbs that may be therapeutic against cancers. Sixty percent of clones isolated by the screening of a phage Ab library against cancer cells turned out to bind to various kinds of tumor-associated Ags. The precise protocol of ICOS method and the rationale of efficient screening were described.

Immunohistochemical Localization of Interleukin-6 in Human Pancreatitis

The aim of the study was to identify immunohistochemically the localization of interleukin (IL)-6 in normal pancreas and in chronic pancreatitis (CP). Samples of tissues of normal pancreas (n=5) and CP (n=16), were verified histopathologically and then IL-6 was localized by immunohistochemical staining using the monoclonal antihuman IL-6 antibody and test LSAB2-HRP to visualize IL-6/Ab complexes. In slices of the pancreas, derived from patients with CP, a much stronger immunohistochemical reaction was noticed as compared with controls specimens. IL-6 was localized in exocrine, islet cells and ducts cells of the pancreas. Interestingly, this cytokine was detected in cytoplasm and very close to nucleus. Moreover, in cases of CP with inflammatory infiltration, there were a markedly stronger IL-6 expression, than that observed in specimens without infiltrate. In conclusion, the results presented herein clearly demonstrated a moderate and strong expression of IL-6 in exocrine and endocrine cells of patients with CP. These observations provide further support for the existence of local immune-pancreatic interactions.

Generation and Characterization of Monoclonal Antibodies against Human Interferon-lambda1

Members belonging to the interferon-lambda (IFN-λ) family exert protective action against viral infection; however, the mechanisms of their action have remained elusive. To study IFN-λ biology, such as endocytosis of IFN-λ, we produced monoclonal antibodies (Abs) against human IFN-λ and examined their usefulness. Methods: We purified recombinant human IFN-λ1 expressed in Escherichia coli by using affinity columns. Then, we generated hybridoma cells by fusing myeloma cells with splenocytes from IFN-λ1-immunized mice. For evaluating the neutralizing activity of the monoclonal Abs against IFN-λ1, we performed RT-PCR for the MxA transcript. In order to study the binding activity of IFN-λ and the monoclonal Ab complex on HepG2 cells, we labeled the monoclonal Ab with rhodamine and determined the fluorescence intensity. Results: Four hybridoma clones secreting Abs specific to IFN-λ1 were generated and designated as HL1, HL2, HL3, and HL4. All the Abs reacted with IFN-λ1 in the denatured form as well as in the native form. Abs produced by HL1, HL3, and HL4 did not neutralize the induction of the MxA gene by IFN-λ1. We also demonstrated the binding of the HL1 monoclonal anbitody and IFN-λ complex on HepG2 cells. Conclusion: Monoclonal Abs against IFN-λ1 were produced. These Abs can be used to study the cellular binding and internalization of IFN-λ.

Biophysical Characterization of Anticoagulant Hemextin AB Complex from the Venom of Snake Hemachatus haemachatus

Hemextin AB complex from the venom of Hemachatus haemachatus is the first known natural anticoagulant that specifically inhibits the enzymatic activity of blood coagulation factor VIIa in the absence of factor Xa. It is also the only known heterotetrameric complex of two three-finger toxins. Individually only hemextin A has mild anticoagulant activity, whereas hemextin B is inactive. However, hemextin B synergistically enhances the anticoagulant activity of hemextin A and their complex exhibits potent anticoagulant activity. In this study we characterized the nature of molecular interactions leading to the complex formation. Circular dichroism studies indicate the stabilization of β-sheet in the complex. Hemextin AB complex has an increased apparent molecular diameter in both gas and liquid phase techniques. The complex formation is enthalpically favorable and entropically unfavorable with a negative change in the heat capacity. Thus, the anticoagulant complex shows less structural flexibility than individual subunits. Both electrostatic and hydrophobic interactions are important for the complexation; the former driving the process and the latter helping in the stabilization of the tetramer. The tetramer dissociates into dimers and monomers with the increase in the ionic strength of the solution and also with increase in the glycerol concentration in the buffer. The two dimers formed under each of these conditions display distinct differences in their apparent molecular diameters and anticoagulant properties. Based on these results, we have proposed a model for this unique anticoagulant complex.

Internalizing Antibody-Based Targeted Gene Delivery for Human Cancer Cells

Gene therapy, a potential solution to hereditary and nonhereditary diseases, faces the challenges of safe and specific gene delivery. Cationic carrier molecules (e.g., liposome and polymers) that form noncovalent complexes with negatively charged DNA have been in use as nonviral gene delivery vectors. Although they tend to be relatively less efficient than viral systems, they have inherent advantages of flexibility and safety. Their derivatives, in conjugation with functional molecules such as peptides, proteins, growth factors, and antibodies, have been focused on to generate nanocarriers with low toxicity, high stability, high efficiency, and cell-specific targeting features. Here we describe internalizing polyclonal and monoclonal antibodies against a stress chaperone, mortalin/mtHsp70. We demonstrate that these internalizing anti-mortalin antibodies (i-mot Ab) could be employed for (1) internalization of nanoparticles (quantum dots, Qdots) and the generation of illuminating cells and (2) gene delivery. By using cancer and normal human cells in parallel, we further demonstrate that gene delivery can be specifically enhanced in human cancer cells if cationic polymer polyethylenimine (PEI) and i-mot Ab complex are used and may provide a novel cancer-targeting nanocarrier.

Stochastic innovation as a mechanism by which catalysts might self-assemble into chemical reaction networks.

We develop a computer model for how two different chemical catalysts in solution, A and B, could be driven to form AB complexes, based on the concentration gradients of a substrate or product that they share in common. If A's product is B's substrate, B will be attracted to A, mediated by a common resource that is not otherwise plentiful in the environment. By this simple physicochemical mechanism, chemical reactions could spontaneously associate to become chained together in solution. According to the model, such catalyst self-association processes may resemble other processes of "stochastic innovation," such as Darwinian evolution in biology, that involve a search among options, a selection among those options, and then a lock-in of that selection. Like Darwinian processes, this simple chemical process exhibits cooperation, competition, innovation, and a preference for consistency. This model may be useful for understanding organizational processes in prebiotic chemistry and for developing new kinds of self-organization in chemically reacting systems.

Protective role of unconjugated bilirubin on complement-mediated hepatocytolysis

Hyperbilirubinemia and complement-mediated immune attack on hepatocyte membrane are common features of certain hepatic diseases. To assess whether unconjugated bilirubin (UB) counteracts complement-mediated hepatocytolysis, we first generated a rabbit polyclonal antibody (Ab) against rat hepatocyte plasma membrane (RHPM). An assay performed with isolated rat hepatocytes in the presence of the polyclonal Ab and rat serum as complement donor demonstrated that UB inhibits cell lysis, as lactate dehydrogenase release into the medium was inhibited by the pigment in a dose-dependent manner. Immunofluorescence microscopy studies showed that UB significantly attenuates the binding of C3 to the hepatocyte–Ab complex. Further enzyme immunoassay studies showed that UB interferes the binding of C1q to purified anti-RHPM IgG, also in a dose-dependent manner. A dot-blot assay showed that [ 14C]-UB binds to C1q and human serum albumin (HSA) to a similar extent. A differential spectrum analysis of UB in the presence of C1q further confirmed that the pigment interacts with this protein. In conclusion, we demonstrated an inhibitory action of UB on complement-mediated Ab-induced hepatocytolysis, this action being evidenced at pathophysiological pigment concentrations (171 μM and higher). A direct binding of the pigment to C1q is likely involved.

Hydrolytic cleavage of ammonia-borane complex for hydrogen production

A new process for generating hydrogen via near room temperature hydrolysis of AB complex using small amounts of platinum group metal catalyst has been studied. Using in situ 11B NMR spectroscopy, the overall rate of K 2Cl 6Pt catalyzed hydrolysis of AB complex was calculated to be third-order. The pre-exponential factor ( A) and the activation energy ( E a) of Arrhenius equation, ln k = ln A − E a/ RT, were determined to be: A = 1.6 × 10 11 L mol −1 s −1 and E a = 86.6 kJ mol −1 for temperature range of (25–35 °C). X-ray photoelectron spectroscopy of the residue suggested that the platinum salt was reduced from Pt 4+ to Pt 0 within the course of the reaction and X-ray diffraction analysis pattern for the residue showed crystallized single-phase boric acid.

Intramolecular domain–domain association/dissociation and phosphoryl transfer in the mannitol transporter of Escherichia coli are not coupled

The Escherichia coli mannitol transporter (II(Mtl)) comprises three domains connected by flexible linkers: a transmembrane domain (C) and two cytoplasmic domains (A and B). II(Mtl) catalyzes three successive phosphoryl-transfer reactions: one intermolecular (from histidine phosphocarrier protein to the A domain) and two intramolecular (from the A to the B domain and from the B domain to the incoming sugar bound to the C domain). A key functional requirement of II(Mtl) is that the A and B cytoplasmic domains be able to rapidly associate and dissociate while maintaining reasonably high occupancy of an active stereospecific AB complex to ensure effective phosphoryl transfer along the pathway. We have investigated the rate of intramolecular domain-domain association and dissociation in IIBA(Mtl) by using (1)H relaxation dispersion spectroscopy in the rotating frame. The open, dissociated state (comprising an ensemble of states) and the closed, associated state (comprising the stereospecific complex) are approximately equally populated. The first-order rate constants for intramolecular association and dissociation are 1.7 (+/-0.3) x 10(4) and 1.8 (+/-0.4) x 10(4) s(-1), respectively. These values compare to rate constants of approximately 500 s(-1) for A --> B and B --> A phosphoryl transfer, derived from qualitative line-shape analysis of (1)H-(15)N correlation spectra taken during the course of active catalysis. Thus, on average, approximately 80 association/dissociation events are required to effect a single phosphoryl-transfer reaction. We conclude that intramolecular phosphoryl transfer between the A and B domains of II(Mtl) is rate-limited by chemistry and not by the rate of formation or dissociation of a stereospecific complex in which the active sites are optimally apposed.

Separation method based on affinity reaction between magnetic and nonmagnetic particles for the analysis of particles and biomolecules

A separation method is reported for particle and biochemical analysis based on affinity interactions between particle surfaces under magnetic field. In this method, magnetic particles with immunoglobulin G (IgG) or streptavidin on the surface are flowed through a separation channel to form a deposition matrix for selectively capturing nonmagnetic analytes with protein A or biotin on the surface due to specific antigen (Ag)–antibody (Ab) interactions. This separation method was demonstrated using model reactions of IgG–protein A and streptavidin–biotin on particle surface. The features of this new separation method are (1) the deposited Ag–Ab complex can be examined and further analyzed under the microscope, (2) a kinetic study of complex binding is possible, and (3) the predeposited matrix can be formed selectively and changed easily. The detection limits were about 10 −11 g. The running time was less than 10 min. The selectivities of studied particles were 94% higher than those of label-controlled particles. This method extends the applications of analytical magnetapheresis to nonmagnetic particles. Preliminary study shows that this separation method has a great potential to provide a simple, fast, and selective analysis for particles, blood cells, and immunoassay related applications.

Hemextin AB Complex – A Snake Venom Anticoagulant Protein Complex That Inhibits Factor VIIa Activity

Snake venom is a veritable gold mine of bioactive molecules, capable of binding to a wide variety of pharmacological targets, including the blood coagulation cascade. Here, we report the isolation and characterization of two synergistically acting anticoagulant three-finger proteins, hemextin A and hemextin B, from the venom of Hemachatus haemachatus (African Ringhals cobra). Hemextin A but not hemextin B exhibits mild anticoagulant activity. However, hemextin B interacts with hemextin A and forms a complex (hemextin AB complex), and synergistically enhances its anticoagulant potency. Prothrombin time assay showed that these two proteins form a 1:1 complex. Using a ‘a dissection approach’, we found that hemextins A and AB complex prolong clotting by inhibiting extrinsic tenase activity. Further studies showed that hemextin AB complex potently inhibits the proteolytic activity of factor VIIa (FVIIa) and its complexes. Kinetic studies showed that hemextin AB complex is a non-competitive inhibitor of FVIIa-soluble tissue factor proteolytic activity with a K_i of 25 nM. Hemextin AB complex is the first reported natural inhibitor of FVIIa that does not require either tissue factor or factor Xa scaffold to mediate its inhibitory activity. Molecular interactions of hemextin AB complex with FVIIa/tissue factor-FVIIa may provide a new paradigm in the search for anticoagulants inhibiting the initiation of blood coagulation.

Homopolyvalent antibody–antigen interaction kinetic studies with use of a dual-polarization interferometric biosensor

We used dual-polarization interferometry (DPI) to study the interaction kinetics between a 'homopolyvalent' antigen (Ag) and a monoclonal antibody (Ab). A model system, which uses a monoclonal Ab against a homopentameric Ag, C-reactive protein (CRP), is presented with principle and experiments for the study of the interactions between an Ab and an Ag that has multiple identical epitopes. This allows evaluation of the dissociation constant (K(D)) and of the binding stoichiometry by DPI based on measurements of phase changes of Ab-Ag complexes in the transverse magnetic (TM) and transverse electric (TE) polarization modes. The average experimental value of K(D) found by the DPI technique for anti-CRP Ab was shown to be in close agreement with the value obtained by an indirect competition-enzyme-linked immunosorbent assay (ELISA). Moreover, the total number of Ab combining sites on the DPI sensor chip was calculated, and the binding stoichiometry of the surface Ag-Ab complex was obtained. This study illustrates the advantages of the DPI method in biosensing in its capacity for simultaneous evaluation of the thickness and refractive index (density, mass) of adsorbed layers. This allowed a comprehensive analysis of affinity reactions between an Ab having two binding sites and a multi-sited Ag.

Regional and segmental flexibility of antibodies in interaction with antigens of different size

The interaction of antibodies (Abs) with protein antigens (Ags) of different size, such as hen egg white lysozyme, ovalbumin, and bovine serum albumin, was examined using analytical ultracentrifugation, electrospray ionization time-of-flight mass spectrometry, and surface plasmon resonance in order to estimate regional and segmental Ab flexibility. When both Abs and Ags were free in solution, sedimentation equilibrium and surface plasmon resonance analyses showed the formation of an Ag(2)Ab(1) complexes regardless of Ag size, suggesting that the Fab arms were able to move to avoid interference between Ags bound to Ab combining sites. The Ag(2)Ab(1) complex, as well as the Ag(1)Ab(1) complex, was observed by MS. However, when Abs were immobilized on the surface of a sensor chip through the Fc region, the stoichiometry of the Ag-Ab complex was dependent on the Ag size; Ag(2)Ab(1) forming with hen egg white lysozyme and Ag(1)Ab(1) with ovalbumin and bovine serum albumin. These results indicated that immobilization of the Fc region reduces the dynamic range of the Fab arms and results in interference from the first Ag bound to either combining site, which in turn prevents the binding of the second Ag to the other combining site. Our results allow us to propose that the Fab arms of B-cell receptors whose Fc regions are immobilized on cell surface have a reduced dynamic range.