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 Preparation, Characterization and Diagnostic Valuation of Two Novel Anti-HPV16 E7 Oncoprotein Monoclonal Antibodies

Viruses ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 333
Author(s):  
Renjian Hu ◽  
Zhen Dong ◽  
Kui Zhang ◽  
Guangzhao Pan ◽  
Chongyang Li ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Detection System ◽  
Early Stage ◽  
Sandwich Elisa ◽  
Quantitative Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Method ◽  
Cervical Cancers ◽  
E7 Oncoprotein ◽  
Oncogenic Protein

At present, the clinical detection method of human papillomavirus (HPV) is mainly based on the PCR method. However, this method can only be used to detect HPV DNA and HPV types, and cannot be used to accurately predict cervical cancer. HPV16 E7 is an oncoprotein selectively expressed in cervical cancers. In this study, we prepared an HPV16 E7-histidine (HIS) fusion oncoprotein by using a prokaryotic expression and gained several mouse anti-HPV16 E7-HIS fusion oncoprotein monoclonal antibodies (mAbs) by using hybridoma technology. Two mAbs, 69E2 (IgG2a) and 79A11 (IgM), were identified. Immunocytochemistry, immunofluorescence, immunohistochemistry, and Western blot were used to characterize the specificity of these mAbs. The sequences of the nucleotide bases and predicted amino acids of the 69E2 and 79A11 antibodies showed that they were novel antibodies. Indirect enzyme-linked immunosorbent assay (ELISA) with overlapping peptides, indirect competitive ELISA, and 3D structural modeling showed that mAbs 69E2 and 79A11 specifically bound to the three exposed peptides of the HPV16 E7 (HPV16 E749–66, HPV16 E773–85, and HPV16 E791–97). We used these two antibodies (79A11 as a capture antibody and 69E2 as a detection antibody) to establish a double-antibody sandwich ELISA based on a horseradish peroxidase (HRP)-labeled mAb and tetramethylbenzidine (TMB) detection system for quantitative detection of the HPV16 E7-HIS fusion oncoprotein, however, it was not ideal. Then we established a chemiluminescence immunoassay based on a labeled streptavidin-biotin (LSAB)-ELISA method and luminol detection system—this was sufficient for quantitative detection of the HPV16 E7-HIS fusion oncogenic protein in ng levels and was suitable for the detection of HPV16-positive cervical carcinoma tissues. Collectively, we obtained two novel mouse anti-HPV16 E7 oncoprotein mAbs and established an LSAB-lumino-dual-antibody sandwich ELISA method for the detection of the HPV16 E7-HIS fusion oncogenic protein, which might be a promising method for the diagnosis of HPV16-type cervical cancers in the early stage.

scholarly journals Production and Characterization of Monoclonal Antibodies against Vegetative Cells of Bacillus cereus

2000 ◽  
Vol 66 (5) ◽  
pp. 2278-2281 ◽  
Author(s):  
Nadine Charni ◽  
Claude Perissol ◽  
Jean Le Petit ◽  
Nathalie Rugani
Keyword(s):  
Monoclonal Antibodies ◽  
Bacillus Cereus ◽  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Vegetative Cells ◽  
Immunological Method ◽  
Molecular Masses

ABSTRACT Two monoclonal antibodies (MAbs) against Bacillus cereus were produced. The MAbs (8D3 and 9B7) were selected by enzyme-linked immunosorbent assay for their reactivity with B. cereus vegetative cells. They reacted with B. cereusvegetative cells while failing to recognize B. cereusspores. Immunoblotting revealed that MAb 8D3 recognized a 22-kDa antigen, while MAb 9B7 recognized two antigens with molecular masses of approximately 58 and 62 kDa. The use of MAbs 8D3 and 9B7 in combination to develop an immunological method for the detection of B. cereus vegetative cells in foods was investigated.

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.

Monoclonal antibodies specific for prothrombin fragment 1.2 and their use in a quantitative enzyme-linked immunosorbent assay

1993 ◽  
Vol 39 (4) ◽  
pp. 583-591 ◽  
Author(s):  
M J Hursting ◽  
B T Butman ◽  
J P Steiner ◽  
B M Moore ◽  
M C Plank ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Human Plasma ◽  
Sandwich Elisa ◽  
Peptide Sequence ◽  
Carboxyl Terminus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Thrombotic Risk ◽  
Prothrombin Fragment ◽  
Amino Terminal

Abstract Prothrombin fragment 1.2 (F1.2) is an activation peptide generated during a critical event of blood coagulation, the conversion of prothrombin to thrombin. As a marker of thrombin generation, F1.2 has clinical potential in assessing thrombotic risk and monitoring anticoagulant therapy. In developing a highly specific, monoclonal antibody-based immunoassay of human plasma F1.2, we generated six murine anti-F1.2 monoclonal antibodies, using as immunogen a synthetic peptide (sequence: CGSD-RAIEGR) similar to the unique carboxyl terminus of F1.2. Each antibody bound F1.2 but not prothrombin. Epitope mapping studies with one antibody (5-3B) showed that optimum binding required six to eight amino acids plus a terminal arginine to emulate the F1.2 carboxyl terminus. A quantitative sandwich ELISA for human plasma F1.2 was configured with monoclonal antibody 5-3B as the capture antibody and peroxidase-labeled polyclonal antibodies to the F1.2 amino-terminal region as detector antibodies. Calibrators were prepared by adding purified F1.2, 0-10 nmol/L, to F1.2-depleted plasma. Assay characteristics included the following: mean (+/- SD) analytical recovery of 98% +/- 13%; no interference from lipemia, hemolysis, icterus, or thrombolytic agents; 0.08 nmol/L sensitivity; and mean intra- and interassay imprecision (three lots) < 12% at both low and high concentrations of F1.2.

Rapid, sensitive two-site ELISA for detection of cows' milk in goats' or ewes' milk using monoclonal antibodies

1994 ◽  
Vol 61 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Didier Levieux ◽  
Annie Venien
Keyword(s):  
Monoclonal Antibodies ◽  
Sandwich Elisa ◽  
High Specificity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Factors Affecting ◽  
Assay Performance ◽  
Capture Antibody ◽  
Constant Quality ◽  
Species Specific ◽  
Β Lactoglobulin

SummaryA sandwich ELISA (enzyme-linked immunosorbent assay) of the two-site type has been successfully developed for the detection of cows' milk in goats' or ewes' milk. The assay uses two monoclonal antibodies (MAb) raised in mice against cows' β-lactoglobulin (β-lg). These MAb recognize different epitopes of the β-lg, which are sufficiently distinct to allow simultaneous binding of the corresponding antibodies. One of the MAb recognizes a species-specific epitope of the bovine β-lg and was adsorbed to a plastic microtitration plate (capture antibody). The second MAb was labelled with peroxidase and used to detect the captured cows' β-lg. Factors affecting assay performance were investigated. The optimized assay is highly specific, reproducible (intra- and inter-assay CV were 8 and 13% respectively) and sensitive: as little as 5 ng β-lg/ml or 1 part cows' milk per 100000 parts goats' or ewes' milk can be detected. The technique is robust, cheap, rapid, reliable and suitable for high sample throughput, semi-automation and screening surveys. The MAb used guarantee the high specificity of the assay and indefinite reagent supply of constant quality once approved by collaborative national or international trials.

Characterization of rotavirus subgroup-specific monoclonal antibodies and use in single-sandwich ELISA systems for rapid subgrouping of human strains

1989 ◽  
Vol 107 (3-4) ◽  
pp. 315-322 ◽  
Author(s):  
G. Gerna ◽  
Antonella Sarasini ◽  
Maria Torsellini ◽  
Angela di Matteo ◽  
F. Baldanti ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Sandwich Elisa

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.

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