scholarly journals Selection and Characterization of Murine Monoclonal Antibodies to Staphylococcus aureus Iron-Regulated Surface Determinant B with Functional Activity In Vitro and In Vivo

2009 ◽  
Vol 16 (8) ◽  
pp. 1095-1104 ◽  
Author(s):  
Martha Brown ◽  
Rose Kowalski ◽  
Julie Zorman ◽  
Xin-min Wang ◽  
Victoria Towne ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Monoclonal Antibodies ◽  
Functional Activity ◽  
Indwelling Catheter ◽  
Lethal Challenge ◽  
Western Blots ◽  
Vitro Assay ◽  
Epitope Recognition

ABSTRACT In an effort to characterize important epitopes of Staphylococcus aureus iron-regulated surface determinant B (IsdB), murine IsdB-specific monoclonal antibodies (MAbs) were isolated and characterized. A panel of 12 MAbs was isolated. All 12 MAbs recognized IsdB in enzyme-linked immunosorbent assays and Western blots; 10 recognized native IsdB expressed by S. aureus. The antigen epitope binding of eight of the MAbs was examined further. Three methods were used to assess binding diversity: MAb binding to IsdB muteins, pairwise binding to recombinant IsdB, and pairwise binding to IsdB-expressing bacteria. Data from these analyses indicated that MAbs could be grouped based on distinct or nonoverlapping epitope recognition. Also, MAb binding to recombinant IsdB required a significant portion of intact antigen, implying conformational epitope recognition. Four MAbs with nonoverlapping epitopes were evaluated for in vitro opsonophagocytic killing (OPK) activity and efficacy in murine challenge models. These were isotype switched from immunoglobulin G1 (IgG1) to IgG2b to potentially enhance activity; however, this isotype switch did not appear to enhance functional activity. MAb 2H2 exhibited OPK activity (≥50% killing in the in vitro OPK assay) and was protective in two lethal challenge models and a sublethal indwelling catheter model. MAb 13C7 did not exhibit OPK (<50% killing in the in vitro assay) and was protective in one lethal challenge model. Neither MAb 13G11 nor MAb 1G3 exhibited OPK activity in vitro or was active in a lethal challenge model. The data suggest that several nonoverlapping epitopes are recognized by the IsdB-specific MAbs, but not all of these epitopes induce protective antibodies.

scholarly journals Functional Analysis of Bacillus anthracis Protective Antigen by Using Neutralizing Monoclonal Antibodies

2004 ◽  
Vol 72 (11) ◽  
pp. 6313-6317 ◽  
Author(s):  
Fabien Brossier ◽  
Martine Lévy ◽  
Annie Landier ◽  
Pierre Lafaye ◽  
Michèle Mock
Keyword(s):  
Functional Analysis ◽  
Monoclonal Antibodies ◽  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Cell Receptor ◽  
Lethal Challenge ◽  
Edema Factor

ABSTRACT Protective antigen (PA) is central to the action of the lethal and edema toxins produced by Bacillus anthracis. It is the common cell-binding component, mediating the translocation of the enzymatic moieties (lethal factor [LF] and edema factor) into the cytoplasm of the host cell. Monoclonal antibodies (MAbs) against PA, able to neutralize the activities of the toxins in vitro and in vivo, were screened. Two such MAbs, named 7.5 and 48.3, were purified and further characterized. MAb 7.5 binds to domain 4 of PA and prevents the binding of PA to its cell receptor. MAb 48.3 binds to domain 2 and blocks the cleavage of PA into PA63, a step necessary for the subsequent interaction with the enzymatic moieties. The epitope recognized by this antibody is in a region involved in the oligomerization of PA63; thus, MAb 48.3 does not recognize the oligomer form. MAbs 7.5 and 48.3 neutralize the activities of anthrax toxins produced by B. anthracis in mice. Also, there is an additive effect between the two MAbs against PA and a MAb against LF, in protecting mice against a lethal challenge by the Sterne strain. This work contributes to the functional analysis of PA and offers immunotherapeutic perspectives for the treatment of anthrax disease.

The Effect Of Antiporcine Willebrand Factor Monoclonal Antibodies On The Bleeding Time

1981 ◽  
Author(s):  
E J W Bowie ◽  
D N Fass ◽  
J A Katzmann
Keyword(s):  
Monoclonal Antibodies ◽  
Bleeding Time ◽  
Vitro Assay ◽  
Functional Sites ◽  
Antibody Titers ◽  
Vitro Model ◽  
Von Willebrand ◽  
Willebrand Factor

Murine monoclonal antibodies to porcine Willebrand factor were used to study the role of Willebrand factor in hemostatic plug formation in the in vivo and in vitro skin bleeding times. The in vivo assay requires the intravenous injection of antibody-containing mouse ascitic fluids into normal animals with subsequent ear bleeding time tests performed over a 24-hour period. The in vitro assay was carried out with heparinized normal porcine blood flowing through a 0.5 cm incision in an excised piece of porcine skin. The heat inactivated ascitic fluids, containing the antibodies, were used ;Ln these assays at final dilutions of 2 × 102 through 2 × 105 The 7 antibodies used comprised at least 5 groups with differing reactivities based on assays other than bleeding time tests. The antibody titers in a poreineg Willebrand factor-binding radioimmunoassay ranged from 108 through 1012 . Doses of 10HL of ascitic fluid/kg of body weight (approximately 2 × 103 final dilution) were able to transiently prolong the in vivo bleeding time without alteration of other VIII complex values (VIII:C, ristocetin cofactor activity and Vlll-related antigen). Higher doses resulted in bleeding times similar to von Willebrand pigs (>15 min) immediately following infusion, with a decay of the effect over the next 2 hours. At dilutions of 2 × 104 selected monoclonal antibodies interfered with hemostasis in the in vitro model whereas other antibodies inhibited only at 2 × 102 dilution. With one exception, the potencies of the antibodies appeared to be similar in both assays. The titer of the antibodies in the radioimmunoassay does not appear to correlate with prolongation of the in vitro bleeding time, suggesting specific reactivity with functional sites involved in hemostatic plug formatio.

scholarly journals Variable major proteins of Borrellia hermsii.

1982 ◽  
Vol 156 (5) ◽  
pp. 1312-1324 ◽  
Author(s):  
A G Barbour ◽  
S L Tessier ◽  
H G Stoenner
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Antigenic Variation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Major Protein ◽  
Relapsing Fever ◽  
Western Blots

Borrelia hermsii, a relapsing fever agent, manifests antigenic variation in vivo and in vitro. We studied three mouse-passaged serotypes of strain HS1 (7, 14, and 21) and a HS1 derivative obtained after multiple in vitro passages (C serotype). All four serotypes had two major proteins in whole cell lysates fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One major protein species (pII) had the same apparent subunit molecular weight (or approximately 3.9 X 10(4) in all the serotypes. In contrast, the other abundant protein in lysates, pI, had a different apparent molecular weight in each serotype. In one gel the molecular weights of pIc, pI7, pI14, and pI21 were 1.9, 4.2, 4.1, and 4.0 X 10(4), respectively. Serotype-specific mouse antisera bound to both hemologous and heterologous pIIs, to homologous pI, but not to heterologous pI in Western blots. Hybridomas were raised from spleens of mice infected with B. hermsii. Monoclonal antibodies were identified by immunofluorescence assays using whole organisms. Monoclonal antibodies specific for serotype 7 (H1826) or for serotype 21 (H3326) bound only to pI7 or pI21, respectively, in Western blots. The surface location of the pI was suggested not only by the immunofluorescence studies but also by the labeling of pI7 and pI21 when whole cells of serotypes 7 and 21 were incubated with 125I in the presence of Iodogen. Under the same circumstances, pII was relatively poorly labeled. These studies have identified the variable pI proteins of B. hermsii as serotype-specific antigens. A change from one pI to another may be the basis of antigenic variation of Borrelia species during relapsing fever.

scholarly journals Antibodies directed towards neuraminidase N1 control disease in a mouse model of influenza

2017 ◽  
pp. JVI.01584-17 ◽  
Author(s):  
E.R. Job ◽  
M. Schotsaert ◽  
L.I. Ibañez ◽  
A. Smet ◽  
T. Ysenbaert ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Antiviral Activity ◽  
Influenza A ◽  
Conformational Epitope ◽  
Influenza Viruses ◽  
Lethal Challenge ◽  
H5n1 Influenza ◽  
Antigenic Properties

There is increasing evidence to suggest that antibodies directed towards influenza A virus (IAV) neuraminidase (NA) are an important correlate of protection against influenza in humans. Moreover, the potential of NA-specific antibodies to provide broader protection than conventional hemagglutinin (HA) antibodies has been recognized. Herein, we describe the isolation of two monoclonal antibodies, N1-7D3 and N1-C4, directed towards the N1 NA. N1-7D3 binds to a conserved linear epitope in the membrane distal, carboxy-terminal part of the NA and reacted with the NA of seasonal H1N1 isolates ranging from 1977 till 2007 the 2009 H1N1pdm virus as well as A/Vietnam/1194/04 (H5N1). However, N1-7D3 lacked NA inhibition (NI) activity and the ability to protect BALB/c mice against a lethal challenge with a range of H1N1 viruses. Conversely, N1-C4 bound to a conformational epitope that is conserved between two influenza subtypes, the 2009 H1N1pdm and H5N1 IAV and displayed potentin vitroantiviral activity mediating both NI and plaque size-reduction. Moreover, N1-C4 could provide heterosubtypic protection in BALB/c mice against a lethal challenge with 2009 H1N1pdm or H5N1 virus. Glutamic acid residue 311 in the NA was found to be critical for the NA binding and antiviral activity of monoclonal antibody N1-C4. Our data provide further evidence on cross-protective epitopes within the N1 subtype and highlight the potential of NA as an important target for vaccine and therapeutic approaches.ImportanceInfluenza remains a world-wide burden to public health. As such the development of new and novel vaccines and therapeutics against influenza virus is crucial. Human challenge studies have recently highlighted the importance of antibodies directed towards the viral neuraminidase (NA) as an important correlate of reduced influenza-associated disease severity. Furthermore, there is evidence that anti-NA antibodies can provide broader protection than antibodies towards the viral hemagglutinin. Here we describe the isolation and detailed characterization of two N1 NA-specific monoclonal antibodies. One of these monoclonal antibodies broadly binds N1 type NAs and the second one displays NAI, in vitro and in vivo anti-viral activity against 2009 H1N1pdm and H5N1 influenza viruses. These two new anti-NA antibodies contribute to our understanding of the antigenic properties and protective potential of the influenza NA antigen.

scholarly journals Immunoglobulin Fc receptors in clinical strains of Staphylococcus aureus do not confer resistance to Phagocytosis in an in vitro assay

1999 ◽  
Vol 41 (3) ◽  
pp. 143-145
Author(s):  
Benito VEGA ◽  
Jorge D. GARCIA ◽  
Francisco HERNÁNDEZ
Keyword(s):  
Staphylococcus Aureus ◽  
Fc Receptors ◽  
Clinical Samples ◽  
Healthy Individuals ◽  
Phagocytic Cells ◽  
Vitro Assay ◽  
Group A ◽  
Live Bacteria

Staphylococcus aureus binds Immunoglobulin G (IgG) on its external surface due to the presence of specific receptors for the Fc domain of this immunoglobulin. This mechanism represents a kind of camouflage against phagocytic cells. In order to confirm that possibility an in vitro evaluation of the phagocytic activity of leukocytes polymorpho-nuclear (PMN) against strains of Staphylococcus aureus was done, comparing 18 strains isolated from clinical samples and 16 from healthy individuals. The presence of Fc receptors was evaluated by haemagglutination (HA) with erythrocytes group A after incubation of the strains with IgG anti blood group A. Phagocytosis of S. aureus was carried out by mixing live bacteria with a suspension of human PMN and incubating at 37 °C for 1 h; survivors were counted as colony forming units by plating. The strains from clinical specimens showed higher HA than those from healthy individuals (p = 0.01); but the former were killed more efficiently than the latter (80-90% and 40%, respectively). It is may be possible that S. aureus showed different behavior in vivo, where could express other virulence factors to prevent the action of phagocytes.

scholarly journals New monoclonal antibodies that recognize an unglycosylated, conserved, extracellular region of CD44 in vitro and in vivo, and can block tumorigenesis

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250175
Author(s):  
Daniel F. Lusche ◽  
Deborah J. Wessels ◽  
Ryan J. Reis ◽  
Cristopher C. Forrest ◽  
Alexis R. Thumann ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Cancer Cell ◽  
Cell Types ◽  
Western Blots ◽  
Cd44 Isoforms ◽  
Cellular Processes ◽  
Transmembrane Glycoprotein ◽  
Extracellular Region

CD44 is a transmembrane glycoprotein that binds to hyaluronic acid, plays roles in a number of cellular processes and is expressed in a variety of cell types. It is up-regulated in stem cells and cancer. Anti-CD44 monoclonal antibodies affect cell motility and aggregation, and repress tumorigenesis and metastasis. Here we describe four new anti-CD44 monoclonal antibodies originating from B cells of a mouse injected with a plasmid expressing CD44 isoform 12. The four monoclonal antibodies bind to the terminal, extracellular, conserved domain of CD44 isoforms. Based on differences in western blot patterns of cancer cell lysates, the four anti-CD44 mAbs separated into three distinct categories that include P4G9, P3D2, and P3A7, and P3G4. Spot assay analysis with peptides generated inEscherichia colisupport the conclusion that the monoclonal antibodies recognize unglycosylated sequences in the N-terminal conserved region between amino acid 21–220, and analyses with a peptide generated in human embryonic kidney 293 cells, demonstrate that these monoclonal antibodies bind to these peptides only after deglycosylation. Western blots with lysates from three cancer cell lines demonstrate that several CD44 isoforms are unglycosylated in the anti-CD44 target regions. The potential utility of the monoclonal antibodies in blocking tumorigenesis was tested by co-injection of cells of the breast cancer-derived tumorigenic cell line MDA-MB-231 with the anti-CD44 monoclonal antibody P3D2 into the mammary fat pads of mice. All five control mice injected with MDA-MB-231 cells plus anti-IgG formed palpable tumors, while only one of the six test mice injected with MDA-MB-231 cells plus P3D2 formed a tiny tumor, while the remaining five were tumor-free, indicating that the four anti-CD44 mAbs may be useful therapeutically.

scholarly journals Effects of anti-CD3 monoclonal antibodies on functional activity of lymphocytes: studies in vivo and in vitro

2008 ◽  
Vol 99 (2) ◽  
pp. 155-159 ◽  
Author(s):  
K.J. PARLEVLIET ◽  
M. E. D. CHAMULEAU ◽  
S.-L. YONG ◽  
M. H. M. RAASVELD ◽  
I. J. M. BERGE ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Functional Activity

A fully human monoclonal antibody to Staphylococcus aureus iron regulated surface determinant B (IsdB) with functional activity in vitro and in vivo

2010 ◽  
Vol 19 (4) ◽  
pp. 113-128 ◽  
Author(s):  
Tim Ebert ◽  
Sharon Smith ◽  
Greg Pancari ◽  
Desmond Clark ◽  
Richard Hampton ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Monoclonal Antibody ◽  
Functional Activity ◽  
Human Monoclonal Antibody

scholarly journals Human monoclonal antibodies against Staphylococcus aureus surface antigens recognize in vitro biofilm and in vivo implant infections

2021 ◽  
Author(s):  
Lisanne de Vor ◽  
Bruce van Dijk ◽  
Kok P.M. van Kessel ◽  
Jeffrey S. Kavanaugh ◽  
Carla J.C. de Haas ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Monoclonal Antibodies ◽  
Biofilm Formation ◽  
Teichoic Acid ◽  
Human Monoclonal Antibodies ◽  
Human Mabs ◽  
Wall Teichoic Acid ◽  
Alternative Approach

AbstractImplant-associated Staphylococcus aureus infections are difficult to treat because of biofilm formation. Bacteria in a biofilm are often insensitive to antibiotics and host immunity. Monoclonal antibodies (mAbs) could provide an alternative approach to improve the diagnosis and/or treatment of biofilm-related infections. Here we show that mAbs targeting common surface components of S. aureus can recognize clinically relevant biofilm types. We identify two groups of antibodies: one group that uniquely binds S. aureus in biofilm state and one that recognizes S. aureus in both biofilm and planktonic state. In a mouse model, we show that mAb 4497 (recognizing wall teichoic acid (WTA)) specifically localizes to biofilm-infected implants. In conclusion, we demonstrate the capacity of several human mAbs to detect S. aureus biofilms in vitro and in vivo. This is an important first step to develop mAbs for imaging or treating S. aureus biofilms.

scholarly journals Human monoclonal antibodies against Staphylococcus aureus surface antigens recognize in vitro and in vivo biofilm

eLife ◽  
2022 ◽  
Vol 11 ◽  
Author(s):  
Lisanne de Vor ◽  
Bruce van Dijk ◽  
Kok van Kessel ◽  
Jeffrey S Kavanaugh ◽  
Carla de Haas ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Monoclonal Antibodies ◽  
Teichoic Acid ◽  
Human Monoclonal Antibodies ◽  
Human Mabs ◽  
Wall Teichoic Acid ◽  
Prosthetic Joint ◽  
Alternative Approach

Implant-associated Staphylococcus aureus infections are difficult to treat because of biofilm formation. Bacteria in a biofilm are often insensitive to antibiotics and host immunity. Monoclonal antibodies (mAbs) could provide an alternative approach to improve the diagnosis and potential treatment of biofilm-related infections. Here, we show that mAbs targeting common surface components of S. aureus can recognize clinically relevant biofilm types. The mAbs were also shown to bind a collection of clinical isolates derived from different biofilm-associated infections (endocarditis, prosthetic joint, catheter). We identify two groups of antibodies: one group that uniquely binds S. aureus in biofilm state and one that recognizes S. aureus in both biofilm and planktonic state. Furthermore, we show that a mAb recognizing wall teichoic acid (clone 4497) specifically localizes to a subcutaneously implanted pre-colonized catheter in mice. In conclusion, we demonstrate the capacity of several human mAbs to detect S. aureus biofilms in vitro and in vivo.

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