scholarly journals Mapping Immunodominant Antibody Epitopes of Abrin

Antibodies ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 11
Author(s):  
Ron Alcalay ◽  
Reut Falach ◽  
Yoav Gal ◽  
Anita Sapoznikov ◽  
Tamar Sabo ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Polyclonal Antibodies ◽  
Spatial Location ◽  
B Subunit ◽  
Control And Prevention ◽  
Binding Pockets ◽  
Linear Epitopes ◽  
A Subunit ◽  
Antibody Interactions ◽  
Antibody Epitopes

Abrin, a toxin isolated from the seeds of Abrus precatorius (jequirity pea) is considered a biological threat agent by the Center for Disease Control and Prevention. To date, there is no effective postexposure treatment for abrin poisoning, and efforts are being made to develop an efficient vaccine and measures for postexposure therapy. Epitope mapping is widely applied as an efficient tool for discovering the antigenic moieties of toxins, thus providing invaluable information needed for the development of vaccines and therapies. Aiming to identify the immunodominant epitopes of abrin, several neutralizing antiabrin polyclonal antibodies were screened using a set of 15-mer peptides spanning the amino acid sequence of either the A or B subunits of abrin. Analysis of the antibody-binding pattern revealed 11 linear epitopes for the A subunit and 14 epitopes for the B subunit that are located on the surface of the toxin and thus accessible for antibody interactions. Moreover, the spatial location of several of these epitopes suggests they may block the galactose-binding pockets or the catalytic domain, thus neutralizing the toxin. These findings provide useful information and suggest a possible strategy for the development and design of an improved abrin-based vaccine and therapeutic antibodies.

scholarly journals Progesterone receptor in the chick oviduct: an immunohistochemical study with antibodies to distinct receptor components.

1984 ◽  
Vol 99 (4) ◽  
pp. 1193-1201 ◽  
Author(s):  
J M Gasc ◽  
J M Renoir ◽  
C Radanyi ◽  
I Joab ◽  
P Tuohimaa ◽  
...  
Keyword(s):  
Progesterone Receptor ◽  
Polyclonal Antibodies ◽  
Smooth Muscles ◽  
Immunoperoxidase Technique ◽  
Cell Nuclei ◽  
Receptor Complexes ◽  
B Subunit ◽  
Chicken Oviduct ◽  
A Subunit ◽  
Immunohistochemical Studies

We performed immunohistochemical studies of chicken oviduct after different fixation procedures, by using antibodies against the progesterone receptor: polyclonal antibodies IgG-G3 against the "8S" form (an oligomere containing progesterone-binding and nonprogesterone-binding units), polyclonal antibodies IgG-RB against the progesterone-binding B subunit, and monoclonal BF4 against the non-progesterone-binding 90,000-mol-wt protein component. Chickens were immature animals with or without estrogen priming, and with or without progesterone treatment. The antibodies were revealed by means of an immunoperoxidase technique that used the avidin-biotin-peroxidase complex, and controls were performed by presaturation of antibodies with the purified 8S-progesterone receptor, the B subunit, and 90,000-mol-wt protein. The progesterone receptor was detected not only in well-characterized target tissues, i.e., in glands and luminal epithelium, but also in stromal cells (some displayed the strongest reaction), in mesothelium, and in fibers of smooth muscles. Only in cell nuclei, whether or not the animals received an injection of progesterone was an antigen revealed corresponding to the B subunit (and/or to the A subunit, because there is immunoreactivity of IgG-RB with both hormone-binding subunits A and B). The 90,000-mol-wt protein was revealed in both cytoplasm and nuclei. These immunohistological data suggest that the concept of steroid action that necessarily involves the original formation of the hormone-receptor complexes in the cytoplasm before translocation to the nucleus, may have to be revised.

scholarly journals Pathogenesis of Shigella diarrhea. IX. Simplified high yield purification of Shigella toxin and characterization of subunit composition and function by the use of subunit-specific monoclonal and polyclonal antibodies.

1984 ◽  
Vol 160 (6) ◽  
pp. 1767-1781 ◽  
Author(s):  
A Donohue-Rolfe ◽  
G T Keusch ◽  
C Edson ◽  
D Thorley-Lawson ◽  
M Jacewicz
Keyword(s):  
Cell Surface ◽  
Hela Cell ◽  
Specific Activity ◽  
Polyclonal Antibodies ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
High Yield ◽  
B Subunit ◽  
A Subunit ◽  
High Yields

A simple purification scheme for shigella cytotoxin was devised, resulting in high yields (approximately 50%) and a 1,300-fold increase in specific activity compared with the initial crude bacterial cell lysate. The purified toxin was enterotoxic in ligated rabbit ileal loops and neurotoxic when injected into the peritoneal cavity of mice. Measurement of specific activity of cytotoxin and enterotoxin demonstrated that these two toxicities copurify during the fractionation procedure. On sodium dodecyl sulfate gel electrophoresis, the toxin migrated as two polypeptide subunits, an A subunit of 32,000 mol wt and a B subunit of 6,500 mol wt. Chemical cross-linking experiments demonstrate that the toxin is a complex consisting of one A and five B subunits with a molecular weight of 64,000. Polyclonal rabbit anti-toxin and anti-subunit B antisera were produced as well as subunit-specific mouse monoclonal antibodies. All antibodies preincubated with toxin neutralized cytotoxic effects in HeLa cell monolayers. In contrast, only A subunit-specific antibodies were able to neutralize toxin prebound to the HeLa cell surface. Antibody to the B subunit also inhibited binding of 125I-labeled toxin to these cells by 94% or more. These data demonstrate that the B subunit is involved in shigella toxin binding to the cell surface.

scholarly journals Major Neutralizing Sites on Vaccinia Virus Glycoprotein B5 Are Exposed Differently on Variola Virus Ortholog B6

2007 ◽  
Vol 81 (15) ◽  
pp. 8131-8139 ◽  
Author(s):  
Lydia Aldaz-Carroll ◽  
Yuhong Xiao ◽  
J. Charles Whitbeck ◽  
Manuel Ponce de Leon ◽  
Huan Lou ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Protective Efficacy ◽  
Polyclonal Antibodies ◽  
Passive Immunization ◽  
Variola Virus ◽  
Subunit Vaccines ◽  
Structural And Functional Properties ◽  
Antigenic Properties ◽  
Control And Prevention ◽  
A Subunit

ABSTRACT Immunization against smallpox (variola virus) with Dryvax, a live vaccinia virus (VV), was effective, but now safety is a major concern. To overcome this issue, subunit vaccines composed of VV envelope proteins from both forms of infectious virions, including the extracellular enveloped virion (EV) protein B5, are being developed. However, since B5 has 23 amino acid differences compared with its B6 variola virus homologue, B6 might be a better choice for such a strategy. Therefore, we compared the properties of both proteins using a panel of monoclonal antibodies (MAbs) to B5 that we had previously characterized and grouped according to structural and functional properties. The B6 gene was obtained from the Centers for Disease Control and Prevention, and the ectodomain was cloned and expressed in baculovirus as previously done with B5, allowing us to compare the antigenic properties of the proteins. Polyclonal antibodies to B5 or B6 cross-reacted with the heterologous protein, and 16 of 26 anti-B5 MAbs cross-reacted with B6. Importantly, 10 anti-B5 MAbs did not cross-react with B6. Of these, three have important anti-VV biologic properties, including their ability to neutralize EV infectivity and block comet formation. Here, we found that one of these three MAbs protected mice from a lethal VV challenge by passive immunization. Thus, epitopes that are present on B5 but not on B6 would generate an antibody response that would not recognize B6. Assuming that B6 contains similar variola virus-specific epitopes, our data suggest that a subunit vaccine using the variola virus homologues might exhibit improved protective efficacy against smallpox.

scholarly journals Characterization and Epitope Mapping of the Polyclonal Antibody Repertoire Elicited by Ricin Holotoxin-Based Vaccination

2014 ◽  
Vol 21 (11) ◽  
pp. 1534-1540 ◽  
Author(s):  
Ofer Cohen ◽  
Adva Mechaly ◽  
Tamar Sabo ◽  
Ron Alcalay ◽  
Ronit Aloni-Grinstein ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Epitope Mapping ◽  
High Avidity ◽  
Random Peptide ◽  
B Subunit ◽  
Time Period ◽  
Control And Prevention ◽  
A Subunit ◽  
Passive Vaccination ◽  
Short Time

ABSTRACTRicin, one of the most potent and lethal toxins known, is classified by the Centers for Disease Control and Prevention (CDC) as a select agent. Currently, there is no available antidote against ricin exposure, and the most promising therapy is based on neutralizing antibodies elicited by active vaccination or that are given passively. The aim of this study was to characterize the repertoire of anti-ricin antibodies generated in rabbits immunized with ricin toxoid. These anti-ricin antibodies exhibit an exceptionally high avidity (thiocyanate-based avidity index, 9 M) toward ricin and an apparent affinity of 1 nM. Utilizing a novel tissue culture-based assay that enables the determination of ricin activity within a short time period, we found that the anti-ricin antibodies also possess a very high neutralizing titer. In line with these findings, these antibodies conferred mice with full protection against pulmonary ricinosis when administered as a passive vaccination. Epitope mapping analysis using phage display random peptide libraries revealed that the polyclonal serum contains four immunodominant epitopes, three of which are located on the A subunit and one on the B subunit of ricin. Only two of the four epitopes were found to have a significant role in ricin neutralization. To the best of our knowledge, this is the first work that characterizes these immunological aspects of the polyclonal response to ricin holotoxin-based vaccination. These findings provide useful information and a possible strategy for the development and design of an improved ricin holotoxin-based vaccine.

scholarly journals Identification of Two Cross-Neutralizing Linear Epitopes within the L1 Major Capsid Protein of Human Papillomaviruses

2002 ◽  
Vol 76 (13) ◽  
pp. 6480-6486 ◽  
Author(s):  
Alba-Lucia Combita ◽  
Antoine Touzé ◽  
Latifa Bousarghin ◽  
Neil D. Christensen ◽  
Pierre Coursaget
Keyword(s):  
Neutralizing Antibodies ◽  
Polyclonal Antibodies ◽  
Human Papillomaviruses ◽  
Hpv 16 ◽  
Human Papillomavirus Type 16 ◽  
Linear Epitopes ◽  
Cross Neutralization ◽  
Definition Of ◽  
L1 Protein

ABSTRACT The neutralizing activities of polyclonal antibodies and monoclonal antibodies (MAbs) obtained by immunization of mice with L1 virus-like particles (VLPs) were investigated by using pseudovirion infectivity assays for human papillomavirus type 16 (HPV-16), HPV-31, HPV-33, HPV-45, HPV-58, and HPV-59 to obtain a better definition of cross-neutralization between high-risk HPVs. In this study, we confirmed and extended previous studies indicating that most genital HPV genotypes represent separate serotypes, and the results suggest that the classification of serotypes is similar to that of genotypes. In addition, three cross-neutralizing MAbs were identified (HPV-16.J4, HPV-16.I23, and HPV-33.E12). MAb HPV-16.J4 recognized a conserved linear epitope located within the FG loop of the L1 protein, and HPV-16.I23 recognized another located within the DE loop. The results suggested that reactivity of MAb HPV-16.I23 to L1 protein is lost when leucine 152 of the HPV-16 L1 protein is replaced by phenylalanine. This confirmed the existence of linear epitopes within the L1 protein that induce neutralizing antibodies, and this is the first evidence that such linear epitopes induce cross-neutralization. However, the cross-neutralization induced by L1 VLPs represents less than 1% of the neutralizing activity induced by the dominant conformational epitopes, and it is questionable whether this is sufficient to offer cross-protection in vivo.

Changed properties of the A subunit in DNA gyrase with a B subunit mutation

1982 ◽  
Vol 186 (4) ◽  
pp. 572-574 ◽  
Author(s):  
E. S. Bogdanova ◽  
S. M. Mirkin ◽  
Zh. G. Shmerling
Keyword(s):  
Dna Gyrase ◽  
B Subunit ◽  
A Subunit

scholarly journals Functional CBS modules make IMPDHs octameric

2014 ◽  
Vol 70 (a1) ◽  
pp. C1283-C1283
Author(s):  
Gilles Labesse ◽  
Thomas Alexandre ◽  
Laurène Vaupré ◽  
Isabelle Salard-Arnaud ◽  
Joséphine Lai Kee Him ◽  
...  
Keyword(s):  
Analytical Ultracentrifugation ◽  
Quaternary Structure ◽  
Catalytic Domain ◽  
Structural Data ◽  
Site Directed Mutagenesis ◽  
X Ray Crystallography ◽  
Binding Pockets ◽  
Essential Enzyme ◽  
Cbs Domains

Inosine-5'-monophosphate dehydrogenase (1, 2) (IMPDH) is a major target for antiviral, antiparasitic, antileukemic and immunosuppressive therapies. It is an ubiquitous and essential enzyme for the biosynthesis of guanosine nucleotides. Up to now, IMPDHs have been reported as tetrameric enzymes harbouring a catalytic domain and a tandem of cystathionine-ß-synthase (CBS) modules. The latter had no precise function assigned despite their nearly absolute conservation among IMPDHs and consistent indication of their importance in vivo. The aim of our study was to provide evidence for the role of the CBS modules on the quaternary structure and on the regulation of IMPDHs. A multidisciplinary approach involving enzymology, site-directed mutagenesis, analytical ultracentrifugation, X-ray crystallography, SAXS, cryo-electron microscopy and molecular modelling allowed us to demonstrate that the Pseudomonas aeruginosa IMPDH is functionally active as an octamer and allosterically regulated by MgATP via each CBS module. Revisiting deposited structural data, we found this newly discovered octameric organization conserved in other IMPDH structures. Meanwhile, we demonstrated that the human IMPDH1 formed two distinct octamers that can pile up into isolated fibres in the presence of MgATP while its pathogenic mutant D226N, localised into the CBS domains, appeared to form massively aggregating fibres. The dramatic impact of this mutation could explain the severe retinopathy adRP10. Our data (3) revealed for the first time that IMPDH has an octameric architecture modulated by MgATP binding to the CBS modules, inducing large structural rearrangements. Thus, the regulatory CBS modules in IMPDHs are functional and they can either modulate catalysis or/and macromolecular assembly. Targeting the conserved effector binding pockets identified within the CBS modules might be promising to develop antibacterial compounds or drugs to counter retinopathy onset.

Control of protein phosphatase 2A by simian virus 40 small-t antigen

1991 ◽  
Vol 11 (4) ◽  
pp. 1988-1995
Author(s):  
S I Yang ◽  
R L Lickteig ◽  
R Estes ◽  
K Rundell ◽  
G Walter ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Simian Virus 40 ◽  
Histone H1 ◽  
Simian Virus ◽  
T Antigen ◽  
Light Chains ◽  
B Subunit ◽  
Phosphatase 2A ◽  
A Subunit ◽  
Small T Antigen

Soluble, monomeric simian virus 40 (SV40) small-t antigen (small-t) was purified from bacteria and assayed for its ability to form complexes with protein phosphatase 2A (PP2A) and to modify its catalytic activity. Different forms of purified PP2A, composed of combinations of regulatory subunits (A and B) with a common catalytic subunit (C), were used. The forms used included free A and C subunits and AC and ABC complexes. Small-t associated with both the free A subunit and the AC form of PP2A, resulting in a shift in mobility during nondenaturing polyacrylamide gel electrophoresis. Small-t did not interact with the free C subunit or the ABC form. These data demonstrate that the primary interaction is between small-t and the A subunit and that the B subunit of PP2A blocks interaction of small-t with the AC form. The effect of small-t on phosphatase activity was determined by using several exogenous substrates, including myosin light chains phosphorylated by myosin light-chain kinase, myelin basic protein phosphorylated by microtubule-associated protein 2 kinase/ERK1, and histone H1 phosphorylated by protein kinase C. With the exception of histone H1, small-t inhibited the dephosphorylation of these substrates by the AC complex. With histone H1, a small stimulation of dephosphorylation by AC was observed. Small-t had no effect on the activities of free C or the ABC complex. A maximum of 50 to 75% inhibition was obtained, with half-maximal inhibition occurring at 10 to 20 nM small-t. The specific activity of the small-t/AC complex was similar to that of the ABC form of PP2A with myosin light chains or histone H1 as the substrate. These results suggested that small-t and the B subunit have similar qualitative and quantitative effects on PP2A enzyme activity. These data show that SV40 small-antigen binds to purified PP2A in vitro, through interaction with the A subunit, and that this interaction inhibits enzyme activity.

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