Monoclonal antibodies against plasma protease inhibitors: I. Production and characterization of 23 monoclonal antibodies against human α2

1984 ◽  
Vol 4 (1) ◽  
pp. 39-48 ◽  
Author(s):  
P. Herion ◽  
D. Siberdt ◽  
G. Garduno Soto ◽  
J. Urbain ◽  
A. Bollen
Keyword(s):  
Monoclonal Antibodies ◽  
Protease Inhibitors ◽  
Light Chains ◽  
Enzyme Linked Immunosorbent Assay ◽  
High Avidity ◽  
Fusion Technique ◽  
Epitope Specificity ◽  
Fast Acting ◽  
Inhibition Pattern

23 hybridomas secreting monoclonal antibodies against human α2, the fast-acting inhibitor of plasmin present in plasma, have been produced by the cell-fusion technique. Isotyping of the monoclonal antibodies has revealed that 14 monoclonal antibodies belong to the class IgG1, 6 to the class IgG2a, and 3 to the class tgG2b. All light chains belong to the κ group. The specificity and relative avidity of these monoclonals have been determined Using an indirect enzyme-linked immunosorbent assay. 13 monoclonals exhibit a relatively high avidity for α2, 5 are of intermediate avidity, and 5 of low avidity. The epitope specificity of these 23 rnonoclonal antibodies, originating from a single mouse, have been examined in inhibition experiments. A group of 10 monoclonal antibodies exhibit a very similar inhibition pattern. Partial inhibition effects displayed by 10 other antibodies define partially overlapping antigenic regions. The binding of these antibodies seems to produce a conformational change in the α2 molecule, reducing the binding of two other antibodies. The last antibody defines an independent epitope.

Monoclonal antibodies against plasma protease inhibitors: II. Production and characterization of 25 monoclonal antibodies against human α1-antitrypsin. Correlation between antigenic structure and functional sites

1984 ◽  
Vol 4 (2) ◽  
pp. 139-147 ◽  
Author(s):  
P. Hérion ◽  
D. Siberdt ◽  
M. Francotte ◽  
J. Urbain ◽  
A. Bollen
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Protease Inhibitors ◽  
Antigenic Structure ◽  
Light Chains ◽  
Enzyme Linked Immunosorbent Assay ◽  
Functional Sites ◽  
Heavy Chains ◽  
Low Levels

Twenty-five hybridomas secreting monoclonal antibodies against human α1-antitrypsim have been produced by the cell-fusion techmque (Kóhler and Milstein, 1976). All antibodies are specific for α1-antitrypsim and carry γ1-antitrypsim heavy chains and κ light chains. Inhibition experiments showed that these monoclonal antibodies define three independent antigenic regions on the α1-antitrypsim molecule; one of these domains appears to be involved in the interaction between α1-antitrypsim and trypsin. In addition, one monoclonal antibody, AATY39, was used to develop an enzyme-linked immunosorbent assay capable of detecting low levels of α1-antitrypsim in the range of 1 to 2 ng/ml.

Production and utilization of monoclonal antibodies to human/rat corticotrophin-releasing factor-41

1990 ◽  
Vol 5 (2) ◽  
pp. 159-166 ◽  
Author(s):  
N. G. N. Milton ◽  
E. W. Hillhouse ◽  
S. A. Nicholson ◽  
C. H. Self ◽  
A. M. McGregor
Keyword(s):  
Monoclonal Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Corticotrophin Releasing Factor ◽  
Tissue Extracts ◽  
Rat Pituitary ◽  
Hypothalamic Tissue ◽  
Dose Dependent

ABSTRACT Murine monoclonal antibodies against human/rat corticotrophin-releasing factor-41 (CRF-41) were produced and characterized for use in the immunological and biological characterization of CRF-41. Spleen cells from BALB/c mice immunized with CRF-41 conjugated to bovine γ-globulin were fused with a BALB/c-derived non-secretor X-63 myeloma line. Hybridomas were selected for CRF antibody production by enzyme-linked immunosorbent assay, and positive hybridomas cloned twice. Three monoclonal antibodies were obtained (KCHMB001, KCHMB002 and KCHMB003) and characterized as IgG1, IgG1 and IgG2a isotypes respectively, with affinity constants for rat CRF-41 of 30, 53 and 34 nmol/l respectively. All three monoclonal antibodies recognize an epitope contained between residues 34 and 41 of the human/rat sequence. The antibodies were able to neutralize the ACTH-releasing activity of rat CRF-41, applied to rat pituitary fragments in vitro, in a dose-dependent manner. Isoelectric focusing showed that KCHMB 003 detected bands of synthetic rat CRF-41 and rat [Met(O)21,38]-CRF-41 at pH 7·1 and 6·8 respectively. Use of KCHMB003 in a two-site enzyme-amplified immunoassay showed that this antibody recognizes both synthetic rat CRF-41 and immunoreactive CRF-41 in rat hypothalamic tissue extracts.

scholarly journals Production and Characterization of Two Monoclonal Antibodies Specific for Plasmopara halstedii

2000 ◽  
Vol 66 (8) ◽  
pp. 3277-3282 ◽  
Author(s):  
S. Bouterige ◽  
R. Robert ◽  
J. P. Bouchara ◽  
A. Marot-Leblond ◽  
V. Molinero ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Detection System ◽  
Sandwich Elisa ◽  
Plasmopara Viticola ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasmopara Halstedii ◽  
Race 1 ◽  
Molecular Masses ◽  
Fungal Antigens

ABSTRACT Sunflower downy mildew, caused by the fungus Plasmopara halstedii, is a potentially devastating disease. We produced two monoclonal antibodies (MAbs) (12C9 and 18E2) by immunizing mice with a partially purified extract of P. halstedii race 1. Both MAbs detected in enzyme-linked immunosorbent assay (ELISA) all races ofP. halstedii present in France. No cross-reactions were observed with Plasmopara viticola or with other fungi commonly associated with sunflowers. Both MAbs recognized the same three fungal antigens with molecular masses of 68, 140, and 192 kDa. However, the epitopes on the fungal antigens were distinct and repetitive. Seed homogenates from infected plants were incubated in wells coated with MAb 18E2. This resulted in the trapping of P. halstedii antigens that were identified with biotinylated MAb 12C9. No reactions were seen with seed homogenates from healthy plants. Thus, our results suggest that these MAbs might be used to develop a sandwich ELISA detection system for P. halstedii in infected seeds.

scholarly journals Production and Characterization of Monoclonal Antibodies to Psittacine Beak and Feather Disease Virus

1992 ◽  
Vol 4 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Branson W. Ritchie ◽  
Frank D. Niagro ◽  
Kenneth S. Latimer ◽  
W. L. Steffens ◽  
Denise Pesti ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Myeloma Cell ◽  
Enzyme Linked Immunosorbent Assay ◽  
Spleen Cells ◽  
Myeloma Cell Line ◽  
Elisa System ◽  
Mouse Myeloma Cell Line ◽  
Feather Disease Virus ◽  
Mouse Myeloma

Monoclonal antibodies specific for the virus that causes psittacine beak and feather disease (PBFD) were produced by fusing spleen cells from mice immunized with purified concentrated PBFD virus with mouse myeloma cell line Sp2/0. The resulting hybridomas were tested for reactivity against whole purified virus by an enzyme-linked immunosorbent assay (ELISA) system. Four clones, designated 15H8, 8E3, 11G12, and 2C3, were subcloned by limiting dilution. Isotyping indicated that clone 15H8 was secreting IgG, whereas the remaining clones secreted IgM. The secreted immunoglobulins were characterized by reactivity against purified PBFD virus using immunoblotting procedures, by immunohistochemical staining of virus-induced lesions in infected tissues, and by inhibition of PBFD virus agglutination of cockatoo erythrocytes. Antibodies secreted by clones 15H8 and 8E3 had the strongest activity against purified whole virus. Only immunoglobulin secreted by the clone 15H8 could be used to detect viral antigen in infected tissues. None of the monoclonal antibodies had hemagglutination-inhibition activity.

scholarly journals Development of Cell-Based Assays for In Vitro Characterization of Hepatitis C Virus NS3/4A Protease Inhibitors

2005 ◽  
Vol 49 (4) ◽  
pp. 1381-1390 ◽  
Author(s):  
Victoria Chung ◽  
Anthony R. Carroll ◽  
Norman M. Gray ◽  
Nigel R. Parry ◽  
Pia A. Thommes ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Vaccinia Virus ◽  
Protease Inhibitors ◽  
Expression System ◽  
Recombinant Vaccinia Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Recombinant Vaccinia

ABSTRACT A recombinant vaccinia virus, expressing the NS3-to-NS5 region of the N clone of hepatitis C virus (HCV), was generated and utilized both in a gel-based assay and in an enzyme-linked immunosorbent assay (ELISA) to evaluate the pyrrolidine-5,5-trans-lactams, a series of inhibitors of the HCV NS3/4A protease. The absolute levels of processed, mature HCV nonstructural proteins in this system were found to decrease in the presence of the trans-lactams. Monitoring of this reduction enabled end points and 50% inhibitory concentrations to be calculated in order to rank the active compounds according to potency. These compounds had no effect on the transcription or translation of the NS3-5 polyprotein at concentrations shown to inhibit NS3/4A protease, and they were shown to be specific inhibitors of this protease. The ELISA, originally developed using the vaccinia virus expression system, was modified to utilize Huh-7 cells containing an HCV replicon. Results with this assay correlated well with those obtained with the recombinant vaccinia virus assays. These results demonstrate the utility of these assays for the characterization of NS3/4A protease inhibitors. In addition, inhibitors of other viral targets, such as polymerase and helicase, can be evaluated in the context of the replicon ELISA.

Production and characterization of monoclonal antibodies to the sapstaining fungus Ophiostoma piceae

1994 ◽  
Vol 40 (1) ◽  
pp. 35-44 ◽  
Author(s):  
Srabani Banerjee ◽  
Judy Little ◽  
Maria Chan ◽  
Brian T. Luck ◽  
Colette Breuil ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Wall ◽  
Monoclonal Antibodies ◽  
Light And Electron Microscopy ◽  
Cross Reactivity ◽  
Cell Wall Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Enzymatic Modification ◽  
Ophiostoma Piceae

A sensitive immunological tool has been developed to detect the sapstaining fungus Ophiostoma piceae 3871, which plagues the wood industry. Monoclonal antibodies (1F3(1), 4G3(14), 4G2(4), and 2B6(24)) produced against cell wall protein extracts of this fungus were specific. Specificity was estimated by enzyme linked immunosorbent assay, western blotting, and light and electron microscopy using the immunogold technique. Electron microscopy revealed gold particles localized on the outer surface of the cell wall. When screened against 24 biological control fungi the antibodies showed pratically no cross-reactivity (< 4%). When tested against 19 other staining fungi, the antibodies recognized three strains of Ophiostoma piceae, 1F3(1) recognized Phialophora botulispora, and the antibodies showed less than 5% reactivity with the other fungi. Chemical and enzymatic modification of the antigen revealed that the epitopes recognized by the monoclonal antibodies were glycospecific. Although the antibodies were produced against the cell wall protein extracts of the fungus grown in liquid culture, they also recognized the fungus growing in wood and therefore can be employed to investigate wood colonization by this fungus.Key words: Ophiostoma piceae, monoclonal antibodies, glycoprotein.

Production and characterization of monoclonal antibodies specific for the functional domains of poly(ADP-ribose) polymerase

1986 ◽  
Vol 64 (4) ◽  
pp. 368-376 ◽  
Author(s):  
Daniel Lamarre ◽  
Brian Talbot ◽  
Yvan Leduc ◽  
Sylviane Muller ◽  
Guy Poirier
Keyword(s):  
Monoclonal Antibodies ◽  
Dna Binding ◽  
Binding Site ◽  
Limited Proteolysis ◽  
Antigenic Site ◽  
Light Chains ◽  
Immunoglobulin Light Chains ◽  
Substrate Binding Site ◽  
Dna Binding Site

Monoclonal antibodies were developed against poly(ADP-ribose) polymerase and analyzed for their reactivity against the NAD+- and DNA-binding fragments. Two fusions were performed to obtain hybridomas and the resulting anti-poly(ADP-ribose) polymerase antibodies were further screened by characterization of their immunoglobulin light chains. Five different hybridomas were isolated which produced different immunoglobulin light chains, all of which were specific for poly(ADP-ribose) polymerase. The specificities of these antibodies were determined by immunoblotting against the purified poly(ADP-ribose) polymerase, its autodegradation fragments, and the fragments prepared by limited proteolysis with chymotrypsin and papain. These fragments have been suggested to contain the NAD+-binding site, the DNA-binding site, and the automodification site, respectively. All the monoclonal antibodies reacted with the 116 kdalton (kDa) band corresponding to the purified enzyme. Four antibodies reacted exclusively with antigenic site(s) on the 46-kDa fragment which contains the DNA-binding site. A fifth antibody reacted exclusively with a clearly different antigenic site on the 74- and 54-kDa fragments which possess the NAD+ (substrate) binding site. The immunoreactivity with the major autodegradation products (69- and 46-kDa fragments) of the purified enzyme confirms this difference between the two groups of antibodies. The 22-kDa fragment corresponding to the automodification site does not show any immunoreactivity with the antibodies.

Sign in / Sign up

Export Citation Format

Share Document