Isolation and characterization of riboflavin carrier protein from human amniotic fluid

A specific protein exhibiting immunological cross-reactivity with chicken riboflavin carrier protein has been purified to homogeneity from human amniotic fluid by use of ion-exchange and affinity chromatography. The protein is similar to its avian counterpart in terms of molecular size, distribution of 125I-labelled tryptic peptides during finger printing, and preferential binding to riboflavin. Immunologically, they are homologous since most of the monoclonal antibodies raised against the avian protein cross-react with the purified human vitamin carrier.

Refolding of riboflavin carrier protein as probed by biochemical and immunological parameters

The unfolding of the chicken egg white riboflavin carrier protein by disulfide reduction with dithiothreitol led to aggregation with concomitant loss of ligand binding characteristics and the capacity to interact with six monoclonal antibodies directed against surface-exposed discontinuous epitopes. The reduced protein could, however, bind to a monoclonal antibody recognizing sequential epitope. Under optimal conditions of protein refolding, the vitamin carrier protein regained its folded structure with high efficiency with simultaneous complete restoration of hydrophobic flavin binding site as well as the epitopic conformations exposed at the surface in a manner comparable to its native form.

Simultaneous purification of biotin-binding proteins-I and -II from chicken egg yolk and their characterization

Chicken egg yolk biotin-binding protein-I (BBP-I) has been purified to homogeneity along with the tetrameric BBP-II by a common protocol. The purification includes delipidation of egg yolk by butanol extraction, DEAE-Sephacel chromatography, treatment with guanidinium chloride and biotin-aminohexyl-Sepharose affinity chromatography. The identity of purified BBP-I was ascertained by its physicochemical properties as well as by its immunological cross-reactivity and precursor-product relationship with BBP-II.

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.