scholarly journals Human monoclonal antibody recognizing liver-type aldolase B

1986 ◽  
Vol 240 (3) ◽  
pp. 847-856 ◽  
Author(s):  
N Hibi ◽  
S Arii ◽  
T Iizumi ◽  
T Nemoto ◽  
T M Chu
Keyword(s):  
Monoclonal Antibody ◽  
Human Monoclonal Antibody ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Tetrameric Structure ◽  
Liver And Kidney ◽  
Aldolase B ◽  
Antigen Activity

A human hybridoma clone (4E3) has been established by fusing lymphocytes from a lymph node taken from a breast cancer patient and human lymphoblastoid cells, LICR-LON-HMy2, by the poly(ethylene glycol) method. 4E3 has been stabilized and continued to secrete IgMk antibody into culture medium (greater than 10 micrograms/ml) for over 1 year. The following characteristics of the antigen strongly suggested that 4E3 recognizes liver-type aldolase B (EC 4.1.2.13): the Mr of the native molecule is 160,000 and that of the subunit is 40,000, and thus it has a tetrameric structure of identical subunits; the antigen is abundant in the liver and kidney of human, mouse and rabbit, and is localized by immunohistochemical methods in the cytoplasm of hepatocytes and in the proximal tubules of the kidney; the antigen is precipitable by 50-80% saturation with (NH4)2SO4; the antigen shows charge-dependent heterogeneity on DEAE-cellulose chromatography. To confirm this notion, aldolase B was purified to homogeneity from the liver of human, mouse and rabbit by phosphocellulose chromatography. During the chromatographic purification, the antigen activity as assayed by enzyme-linked immunosorbent assay (e.l.i.s.a.) was superimposed on the enzymic activity of aldolase. Furthermore, monoclonal antibody 4E3 strongly reacted with purified aldolase B in SDS/polyacrylamide-gel electrophoresis followed by Western blotting and also in e.l.i.s.a. using microplates coated with purified enzyme. The reaction between aldolase B and 4E3 activated the human complement system as assessed by the attachment of C3 to the immune complex of aldolase B and 4E3.

scholarly journals Modulation of the Lung Inflammatory Response to Serotype 8 Pneumococcal Infection by a Human Immunoglobulin M Monoclonal Antibody to Serotype 8 Capsular Polysaccharide

2005 ◽  
Vol 73 (8) ◽  
pp. 4530-4538 ◽  
Author(s):  
Tamika Burns ◽  
Maria Abadi ◽  
Liise-anne Pirofski
Keyword(s):  
Monoclonal Antibody ◽  
Inflammatory Response ◽  
Capsular Polysaccharide ◽  
Human Monoclonal Antibody ◽  
Pneumococcal Infection ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Necrosis Factor Alpha ◽  
Monocyte Chemoattractant Protein 1

ABSTRACT The human monoclonal antibody to serotype 8 pneumococcal capsular polysaccharide D11 [immunoglobulin M(κ)] protects wild-type and complement component 4 knockout (C4 KO) mice against lethal intratracheal challenge with serotype 8 pneumococcus, but it does not promote polymorphonuclear leukocyte (PMN)-mediated pneumococcal killing in vitro. In this study, we investigated the effect of D11 on the blood and lung bacterial burdens and the serum and lung expression of inflammatory chemokines and cytokines in an intratracheal challenge model with serotype 8 pneumococcus in C4 KO mice. Pneumococcus was not detected in the blood of D11-treated mice, whereas control mice had high-grade bacteremia with >107 CFU. Control mice had a >5-log increase in lung CFU and D11-treated mice manifested a nearly 3-log increase in lung CFU compared to the original inoculum 24 h after infection. Serum and lung levels of soluble macrophage inflammatory protein 2 (MIP-2) and interleulin-6 (IL-6) as measured by an enzyme-linked immunosorbent assay were lower in D11-treated mice than in control mice 24 h after infection. Real-time PCR was performed to examine lung mRNA chemokine and cytokine expression. The results showed that D11-treated mice had significantly less gamma interferon, MIP-2, IL-12, monocyte chemoattractant protein 1/JE, and tumor necrosis factor alpha expression than control mice 24 h after infection. Histopathology and immunohistochemical staining of lung tissues revealed that D11-treated mice had less inflammation, fewer PMNs, and less myeloperoxidase staining than control mice 24 h after infection. These findings suggest that the efficacy of certain serotype-specific antibodies against pneumococcal pneumonia could be associated with modulation of the lung inflammatory response and a reduction in host damage.

scholarly journals Human erythrocyte antigens: II. The In(Lu) gene regulates expression of an antigen on an 80-kilodalton protein of human erythrocytes

Blood ◽  
1984 ◽  
Vol 64 (3) ◽  
pp. 599-606 ◽  
Author(s):  
MJ Telen ◽  
TJ Palker ◽  
BF Haynes
Keyword(s):  
Monoclonal Antibody ◽  
Western Blot ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Two Dimensions ◽  
Antigen Activity

Abstract We have previously shown that a murine monoclonal antibody (A3D8) identifies a human erythrocyte protein antigen whose expression is regulated by the Lutheran inhibitor [In(Lu)] gene. In the present study, we demonstrated by immunoprecipitation and Western blot techniques that the antigen defined by A3D8 was on an 80-kD erythrocyte membrane protein. A second 170-kD protein was coprecipitated with the 80-kD protein but failed to show antigen activity by Western blot analysis. The 170-kD protein, when analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis in two dimensions, was composed of 50- and 30-kD disulfide-linked subunits. In(Lu) Lu[a-b-) erythrocytes differed from Lu(a+b+) or Lu(a-b+) erythrocytes in that In(Lu) deoxycholate erythrocyte membrane extracts contained trace amounts of immunoprecipitable 80-kD protein compared with detergent-solubilized erythrocyte membrane extracts prepared from Lu(a+b+) or Lu(a-b+) subjects.

A monoclonal antibody toBabesia divergenswhich inhibits merozoite invasion

Parasitology ◽  
1987 ◽  
Vol 94 (1) ◽  
pp. 17-27 ◽  
Author(s):  
Caroline M. Winger ◽  
Elizabeth U. Canning ◽  
J. D. Culverhouse
Keyword(s):  
Monoclonal Antibody ◽  
Indirect Fluorescent Antibody Test ◽  
Fluorescent Antibody ◽  
Polyacrylamide Gel Electrophoresis ◽  
Antibody Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Merozoite Invasion ◽  
Babesia Divergens ◽  
Sds Page ◽  
Bovine Erythrocytes

SUMMARYA monoclonal antibody-producing hybridoma cell line has been raised against merozoites ofBabesia divergens. This antibody is strongly reactive against merozoites in an enzyme-linked immunosorbent assay (ELISA) and is also positive in an indirect fluorescent antibody test (IFAT) on preparations of live merozoites. The antibody recognizes an antigen, of molecular weight (Mr) 50–60 K by Western blot analysis on SDS–polyacrylamide gel electrophoresis (SDS–PAGE) gels. Merozoite neutralization assays show that the antibody significantly inhibits merozoite invasion of bovine erythrocytes.

scholarly journals Protection against Candidiasis by an Immunoglobulin G3 (IgG3) Monoclonal Antibody Specific for the Same Mannotriose as an IgM Protective Antibody

2000 ◽  
Vol 68 (3) ◽  
pp. 1649-1654 ◽  
Author(s):  
Yongmoon Han ◽  
Marcia H. Riesselman ◽  
Jim E. Cutler
Keyword(s):  
Monoclonal Antibody ◽  
Cell Surface ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Survival Times ◽  
Vaginal Infection ◽  
Igg Antibody ◽  
Disseminated Candidiasis

ABSTRACT We previously reported that a liposome-mannan vaccine (L-mann) ofCandida albicans induces production of mouse antibodies that protect against disseminated candidiasis and vaginal infection. Immunoglobulin M (IgM) monoclonal antibody (MAb) B6.1, specific for aC. albicans cell surface β-1,2-mannotriose, protects mice against both infections. Another IgM MAb, termed B6, which is specific for a different cell surface mannan epitope, does not protect against disseminated candidiasis. The B6.1 epitope is displayed homogeneously over the entire cell surface, compared to a patchy distribution of the B6 epitope. To determine if protection is restricted to an IgM class of antibody, we tested an IgG antibody. MAb C3.1 was obtained from L-mann-immunized mice. By results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, capture enzyme-linked immunosorbent assay, and immunodiffusion tests, MAb C3.1 is an IgG3 isotype. By epitope inhibition assays, we determined that MAb C3.1 is specific for same mannotriose as MAb B6.1. As expected by the results of the inhibition assays, immunofluorescence microscopy showed that the C3.1 epitope is distributed on the yeast cell surface in a pattern identical to that of the B6.1 epitope. Kidney CFU and mean survival times of infected mice pretreated with MAb C3.1 indicated that the antibody enhanced resistance of mice against disseminated candidiasis. Mice in pseudoestrus that were given MAb C3.1 prior to vaginal infection developed fewer vaginal Candida CFU than control animals that received buffered saline instead of the antibody. The finding that an IgG3 antibody is protective is consistent with our hypothesis that epitope specificity and complement activation are related to the ability of an antibody to protect against candidiasis.

scholarly journals Fine Definition of the Epitope on the gp41 Glycoprotein of Human Immunodeficiency Virus Type 1 for the Neutralizing Monoclonal Antibody 2F5

2001 ◽  
Vol 75 (22) ◽  
pp. 10906-10911 ◽  
Author(s):  
Carol E. Parker ◽  
Leesa J. Deterding ◽  
Christine Hager-Braun ◽  
James M. Binley ◽  
Norbert Schülke ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Human Immunodeficiency Virus ◽  
Human Monoclonal Antibody ◽  
Maldi Ms ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ionization Mass ◽  
Protection Assay ◽  
Virus Envelope ◽  
C Terminus ◽  
Immunodeficiency Virus

ABSTRACT Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), in combination with proteolytic protection assays, has been used to identify the functional epitope on human immunodeficiency virus envelope glycoprotein gp41 for the broadly neutralizing anti-gp41 human monoclonal antibody 2F5. In this protection assay-based procedure, a soluble gp140 protein with a stabilizing intermolecular disulfide bond between the gp120 and gp41 subunits (SOS gp140) was affinity bound to immobilized 2F5 under physiological conditions. A combination of proteolytic enzymatic cleavages was then performed to remove unprotected residues. Residues of SOS gp140 protected by their binding to 2F5 were then identified based on their molecular weights as determined by direct MALDI-MS of the immobilized antibody beads. The epitope, NEQELLELDKWASLWN, determined by this MALDI-MS protection assay approach consists of 16 amino acid residues near the C terminus of gp41. It is significantly longer than the ELDKWA core epitope previously determined for 2F5 by peptide enzyme-linked immunosorbent assay. This new knowledge of the structure of the 2F5 epitope may facilitate the design of vaccine antigens intended to induce antibodies with the breadth and potency of action of the 2F5 monoclonal antibody.

scholarly journals Human monoclonal antibody against Rh(D) antigen: partial characterization of the Rh(D) polypeptide from human erythrocytes

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1491-1497 ◽  
Author(s):  
C Bloy ◽  
D Blanchard ◽  
P Lambin ◽  
D Goossens ◽  
P Rouger ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Gel Electrophoresis ◽  
Human Monoclonal Antibody ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Insoluble Residue ◽  
Binding Studies ◽  
Sds Page ◽  
Immune Precipitation ◽  
D Antigen

Abstract A human monoclonal anti-Rh(D) antibody produced by an Epstein-Barr virus (EBV)-transformed B-cell line (IgG1(lambda), clone H2D5D2) has been purified on protein A-Sepharose column and used for binding studies and immune precipitation of the blood group rhesus (Rh) antigens. Scatchard plot analyses show that the 125I-labeled antibody (iodo-gen procedure), binds to 1.09 X 10(5), 0.43 X 10(5), and 0.32 X 10(5) antigen sites on each D--/D--, R2R2 and R1R1 RBC, respectively, with an association constant of approximately 0.6 X 10(8) mol/L-1. Immune precipitation studies indicate also that the Rh(D) antigen of the Rh(D)-positive RBCs is carried by a 29 kd polypeptide as deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE). No material could be precipitated from Rh(D)-negative or Rhnull RBCs. These results indicate that the monoclonal and the polyclonal human anti-Rh(D) behave similarly. A sample (Blo., presumed genotype R2r or R0R2) showing an increased number of antigen sites (0.76 X 10(5)/cell) and a high binding constant (5.7 X 10(8) mol/L-1) was used, as well as D--/D-- RBCs, for further purification of the 29-kd component. Extraction by Triton X-100 (0.1% to 5%) of the immune complexes formed between the membrane-bound Rh(D) antigens and the monoclonal antibody as well as a direct quantitative estimate of the 29- kd component, suggest that the Rh(D) polypeptide is loosely bound to the skeleton, since less than or equal to 80% can be solubilized from the membrane. In similar conditions, glycophorin A showed a slight association with the Triton-insoluble residue, whereas glycophorin B was easily and completely extracted. In contrast, both the minor RBC sialoglycoproteins, glycophorin C and glycoprotein gamma, remained predominantly bound to the membrane skeleton. The purified Rh(D) polypeptide obtained from Blo. and D--/D-- RBCs by immunoprecipitation and preparative gel electrophoresis was homogenous as judged by SDS- PAGE. Amino acid composition indicated that the Rh(D) protein contained sulfhydryl groups which are essential for biological activity.

Some Anticardiolipin Antibodies Recognize a Combination of Phospholipids With Thrombin-Modified Antithrombin, Complement C4b-Binding Protein, and Lipopolysaccharide Binding Protein

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4248-4255 ◽  
Author(s):  
Josiane Arvieux ◽  
Gilles Pernod ◽  
Véronique Regnault ◽  
Luc Darnige ◽  
Jérôme Garin
Keyword(s):  
Bovine Serum ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Anticardiolipin Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Lipopolysaccharide Binding Protein ◽  
Lipopolysaccharide Binding

Abstract The standard enzyme-linked immunosorbent assay (ELISA) for anticardiolipin antibodies (ACA) detects a heterogenous group of antibodies against cardiolipin on its own, β2-glycoprotein I (β2GPI), and, potentially, other phospholipid-binding plasma proteins from bovine or human origin. In an attempt to identify new proteic targets of ACA, we selected 6 patients who possessed cofactor-dependent ACA but no antibody to human or bovine β2GPI detectable in the β2GPI-ELISA. Three of these samples proved to recognize β2GPI in combination with cardiolipin, but not β2GPI directly immobilized on γ-irradiated polystyrene or agarose beads. In the other cases, the component required for ACA binding was purified from adult bovine serum or plasma by means of ammonium sulfate precipitation and chromatography on Phenyl-Sepharose, diethyl aminoethyl (DEAE)-cellulose, heparin-Ultrogel, and Sephacryl S-300 columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis coupled to N-terminal amino acid microsequencing identified the cofactors of patients no. 4, 5, and 6 ACA as lipopolysaccharide binding protein (LBP), complement C4b-binding protein (C4BP), and the thrombin-antithrombin (AT) complex, respectively. Adsorption of each of these cofactor preparations with cardiolipin liposomes led to suppression of ACA reactivity, concomitant with the loss of bands from SDS gels corresponding to sequenced material. Bacterial lipopolysaccharide (which forms high-affinity complexes with LBP) specifically neutralized the cofactor activity of the LBP preparation in a concentration-dependent manner. Bovine serum and plasma, as well as the C4BP preparation, optimally supported the binding of a rabbit anti-C4BP antiserum to immobilized cardiolipin. The binding of a rabbit anti-AT antiserum to solid-phase cardiolipin was sustained by the thrombin-AT preparation and bovine serum, but neither by bovine plasma nor by native AT, thus reproducing the behavior of patient no. 6 ACA. Taking advantage of the restricted recognition by the latter ACA of a cofactor from bovine origin appearing upon clotting, we studied the generation of such activity in human plasma supplemented with bovine AT or bovine prothrombin before clotting. In these conditions, patient no. 6 antibody binding to cardiolipin required the addition of bovine AT, whereas addition of bovine prothrombin alone was ineffective. We therefore concluded that those ACA targeted bovine AT once it has been modified/cleaved by thrombin. These findings underline the wide heterogeneity of ACA and the links that may exist between various coagulation pathways, inflammation and the complement system.

scholarly journals A Recombinant Human Monoclonal Antibody to Human Metapneumovirus Fusion Protein That Neutralizes Virus In Vitro and Is Effective Therapeutically In Vivo

2007 ◽  
Vol 81 (15) ◽  
pp. 8315-8324 ◽  
Author(s):  
John V. Williams ◽  
Zhifeng Chen ◽  
Gabriella Cseke ◽  
David W. Wright ◽  
Christopher J. Keefer ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Human Monoclonal Antibody ◽  
Virus Titer ◽  
Human Metapneumovirus ◽  
Enzyme Linked Immunosorbent Assay ◽  
F Protein ◽  
Fold Reduction ◽  
Hmpv Infection

ABSTRACT Human metapneumovirus (hMPV) is a recently discovered paramyxovirus that is a major cause of lower-respiratory-tract disease. hMPV is associated with more severe disease in infants and persons with underlying medical conditions. Animal studies have shown that the hMPV fusion (F) protein alone is capable of inducing protective immunity. Here, we report the use of phage display technology to generate a fully human monoclonal antibody fragment (Fab) with biological activity against hMPV. Phage antibody libraries prepared from human donor tissues were selected against recombinant hMPV F protein with multiple rounds of panning. Recombinant Fabs then were expressed in bacteria, and supernatants were screened by enzyme-linked immunosorbent assay and immunofluorescent assays. A number of Fabs that bound to hMPV F were isolated, and several of these exhibited neutralizing activity in vitro. Fab DS7 neutralized the parent strain of hMPV with a 60% plaque reduction activity of 1.1 μg/ml and bound to hMPV F with an affinity of 9.8 ×10−10 M, as measured by surface plasmon resonance. To test the in vivo activity of Fab DS7, groups of cotton rats were infected with hMPV and given Fab intranasally 3 days after infection. Nasal turbinates and lungs were harvested on day 4 postinfection and virus titers determined. Animals treated with Fab DS7 exhibited a >1,500-fold reduction in viral titer in the lungs, with a modest 4-fold reduction in the nasal tissues. There was a dose-response relationship between the dose of DS7 and virus titer. Human Fab DS7 may have prophylactic or therapeutic potential against severe hMPV infection.

scholarly journals Human erythrocyte antigens: II. The In(Lu) gene regulates expression of an antigen on an 80-kilodalton protein of human erythrocytes

Blood ◽  
1984 ◽  
Vol 64 (3) ◽  
pp. 599-606
Author(s):  
MJ Telen ◽  
TJ Palker ◽  
BF Haynes
Keyword(s):  
Monoclonal Antibody ◽  
Western Blot ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Two Dimensions ◽  
Antigen Activity

We have previously shown that a murine monoclonal antibody (A3D8) identifies a human erythrocyte protein antigen whose expression is regulated by the Lutheran inhibitor [In(Lu)] gene. In the present study, we demonstrated by immunoprecipitation and Western blot techniques that the antigen defined by A3D8 was on an 80-kD erythrocyte membrane protein. A second 170-kD protein was coprecipitated with the 80-kD protein but failed to show antigen activity by Western blot analysis. The 170-kD protein, when analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis in two dimensions, was composed of 50- and 30-kD disulfide-linked subunits. In(Lu) Lu[a-b-) erythrocytes differed from Lu(a+b+) or Lu(a-b+) erythrocytes in that In(Lu) deoxycholate erythrocyte membrane extracts contained trace amounts of immunoprecipitable 80-kD protein compared with detergent-solubilized erythrocyte membrane extracts prepared from Lu(a+b+) or Lu(a-b+) subjects.

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