Monoclonal antibodies in the study of architecture of plant viruses

Monoclonal antibodies have been used as probes to study the architecture of several plant viruses over the past decade. These studies complement the information obtained through X-ray crystallography and help in delineating epitopes on the surface of the virus. The monoclonal antibodies that recognize distinct epitopes also aid in unravelling the mechanisms of assembly/disassembly of virus particles. Group-specific and strain-specific monoclonal antibodies are widely used in the classification of viruses. The significant developments made in this emerging area are reviewed here with specific examples.

Antigen detection assay with parasite specific monoclonal antibodies for diagnosis of lymphatic filariasis

Background: Lymphatic filariasis is a painful and profoundly disfiguring disease. Infection is usually acquired in childhood but its visible manifestations occur later in life, causing temporary or permanent disability. The importance of developing effective assays to diagnose, monitor and evaluate human lymphatic filariasis has been emphasized by the WHO. Methods: High-affinity monoclonal antibodies (mAbs) specific for recombinant filarial antigen WbSXP-1 were developed. An ELISA based capture assay using monoclonal and polyclonal antibodies for WbSXP-1 was used for detection of circulating filarial antigen. Results: High-affinity monoclonal antibodies (mAbs) were developed that specifically binds both W. bancrofti and B. malayi mf antigens. Two mAbs (1F6H3 and 2E12E3) of subclass IgG2a and IgM showed high affinity, avidity and reactivity to recombinant and mf native antigen. Both the mAbs were used in combination as capture antibodies and polyclonal as detection antibody to develop the assay. The assay showed very high sensitivity towards W. bancrofti mf positive samples compared to endemic normal samples (P<0.0001). Conclusion: A capture assay using high-affinity monoclonal antibodies for WbSXP-1 was developed for the detection of filarial circulating antigen in clinical samples from bancroftian infection. Besides, this would also help in epidemiological studies in endemic areas of filarial infections. (C) 2011 Elsevier B.V. All rights reserved.

Identification and purification of tRNAs containing N6-(delta 2-isopentenyl) adenosine using antibodies specific for N6-(delta-isopentenyl) adenosine

Antibodies specific for the modified nucleoside N6-(delta 2-isopentenyl) adenosine (i6A) were employed to identify the tRNAs containing i6A from an unfractionated tRNA mixture by a nitrocellulose filter binding assay. When radioactive aminoacyl-tRNAs were incubated with i6A-specific antibodies and filtered through nitrocellulose membrane filters, the tRNAs possessing i6A (tRNAtyr and tRNAser) remained on the filters. tRNAarg and tRNAlys which do not contain i6A showed no binding. This finding will be useful as a very simple and rapid assay of such RNAs under a variety of conditions. Purification of i6A containing tRNAs from an unfractionated tRNA mixture was achieved by affinity chromatography of the tRNAs on an i6A antibody-Sepharose column. Nonspecific binding of tRNAs to the column was avoided by the use of purified antibodies.

Docking and free energy simulations to predict conformational domains involved in hCG-LH receptor interactions using recombinant antibodies

Single chain fragment variables (ScFvs) have been extensively employed in studying the protein-protein interactions. ScFvs derived from phage display libraries have an additional advantage of being generated against a native antigen, circumventing loss of information on conformational epitopes. In the present study, an attempt has been made to elucidate human chorionic gonadotropin (hCG)-luteinizing hormone (LH) receptor interactions by using a neutral and two inhibitory ScFvs against hCG. The objective was to dock a computationally derived model of these ScFvs onto the crystal structure of hCG and understand the differential roles of the mapped epitopes in hCG-LH receptor interactions. An anti-hCG ScFv, whose epitope was mapped previously using biochemical tools, served as the positive control for assessing the quality of docking analysis. To evaluate the role of specific side chains at the hCG-ScFv interface, binding free energy as well as residue interaction energies of complexes in solution were calculated using molecular mechanics Poisson-Boltzmann/surface area method after performing the molecular dynamic simulations on the selected hCG-ScFv models and validated using biochemical and SPR analysis. The robustness of these calculations was demonstrated by comparing the theoretically determined binding energies with the experimentally obtained kinetic parameters for hCG-ScFv complexes. Superimposition of hCG-ScFv model onto a model of hCG complexed with the 51-266 residues of LH receptor revealed importance of the residues previously thought to be unimportant for hormone binding and response. This analysis provides an alternate tool for understanding the structure-function analysis of ligand-receptor interactions. Proteins 2011;79:3108-3122. (C) 2011 Wiley-Liss, Inc.

Insights into differential modulation of receptor function by hinge region using novel agonistic lutropin receptor and inverse agonistic thyrotropin receptor antibodies

We report two antibodies, scFv 13B1 and MAb PD1.37, against the hinge regions of LHR and TSHR, respectively, which have similar epitopes but different effects on receptor function. While neither of them affected hormone binding, with marginal effects on hormone response, scFv 13B1 stimulated LHR in a dose-dependent manner, whereas MAb PD1.37 acted as an inverse agonist of TSHR. Moreover, PD1.37 could decrease the basal activity of hinge region CAMs, but had varied effects on those present in ECLs, whereas 13B1 was refractory to any CAMs in LHR. Using truncation mutants and peptide phage display, we compared the differential roles of the hinge region cysteine box-2/3 as well as the exoloops in the activation of these two homologus receptors. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The antibodies against the computationally designed mimic of the glycoprotein hormone receptor transmembrane domain provide insights into receptor activation and suppress the constitutively activated receptor Mutants

The exoloops of glycoprotein hormone receptors (GpHRs) transduce the signal generated by the ligand-ectodomain interactions to the transmembrane helices either through direct hormonal contact and/or by modulating the interdomain interactions between the hinge region (HinR) and the transmembrane domain (TMD). The ligand-induced conformational alterations in the HinRs and the interhelical loops of luteinizing hormone receptor/follicle stimulating hormone receptor/thyroid stimulating hormone receptor were mapped using exoloop-specific antibodies generated against a mini-TMD protein designed to mimic the native exoloop conformations that were created by joining the thyroid stimulating hormone receptor exoloops constrained through helical tethers and library-derived linkers. The antibody against the mini-TMD specifically recognized all three GpHRs and inhibited the basal and hormone-stimulated cAMP production without affecting hormone binding. Interestingly, binding of the antibody to all three receptors was abolished by prior incubation of the receptors with the respective hormones, suggesting that the exoloops are buried in the hormone-receptor complexes. The antibody also suppressed the high basal activities of gain-of-function mutations in the HinRs, exoloops, and TMDs such as those involved in precocious puberty and thyroid toxic adenomas. Using the antibody and point/deletion/chimeric receptor mutants, we demonstrate that changes in the HinR-exoloop interactions play an important role in receptor activation. Computational analysis suggests that the mini-TMD antibodies act by conformationally locking the transmembrane helices by means of restraining the exoloops and the juxta-membrane regions. Using GpHRs as a model, we describe a novel computational approach of generating soluble TMD mimics that can be used to explain the role of exoloops during receptor activation and their interplay with TMDs.

Antibodies against the extracellular domain of human Notch1 receptor reveal the critical role of epidermal-growth-factor-like repeats 25-26 in ligand binding and receptor activation

The Notch signalling pathway is implicated in a wide variety of cellular processes throughout metazoan development. Although the downstream mechanism of Notch signalling has been extensively studied, the details of its ligand-mediated receptor activation are not clearly understood. Although the role of Notch ELRs EGF (epidermal growth factor)-like-repeats] 11-12 in ligand binding is known, recent studies have suggested interactions within different ELRs of the Notch receptor whose significance remains to be understood. Here, we report critical inter-domain interactions between human Notch1 ELRs 21-30 and the ELRs 11-15 that are modulated by calcium. Surface plasmon resonance analysis revealed that the interaction between ELRs 21-30 and ELRs 11-15 is similar to 10-fold stronger than that between ELRs 11-15 and the ligands. Although there was no interaction between Notch 1 ELRs 21-30 and the ligands in vitro, addition of pre-clustered Jagged1Fc resulted in the dissociation of the preformed complex between ELRs 21-30 and 11-15, suggesting that inter-domain interactions compete for ligand binding. Furthermore, the antibodies against ELRs 21-30 inhibited ligand binding to the full-length Notch1 and subsequent receptor activation, with the antibodies against ELRs 25-26 being the most effective. These results suggest that the ELRs 25-26 represent a cryptic ligand-binding site which becomes exposed only upon the presence of the ligand. Thus, using specific antibodies against various domains of the Notch1 receptor, we demonstrate that, although ELRs 11-12 are the principal ligand-binding site, the ELRs 25-26 serve as a secondary binding site and play an important role in receptor activation.