Use of monoclonal antibodies to detect a phytotoxic glycopeptide produced by Ophiostoma ulmi, the Dutch elm disease pathogen

1985 ◽  
Vol 63 (7) ◽  
pp. 1177-1184 ◽  
Author(s):  
Nicole Benhamou ◽  
G. B. Ouellette ◽  
J. G. Lafontaine ◽  
J. R. Joly
Keyword(s):  
Monoclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Myeloma Cell ◽  
Dutch Elm Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ophiostoma Ulmi ◽  
Mouse Myeloma Cell Line ◽  
Immunocytochemical Techniques ◽  
Host Tissues

Two hybridomas that secrete antibodies specific for a phytotoxic glycopeptide from Ophiostoma ulmi (Buism.) Nannf. were produced by fusing spleen cells of mice immunized with the purified toxin and the Sp2-0/Ag14 mouse myeloma cell line. Specificity of these antibodies was first demonstrated by an enzyme-linked immunosorbent assay (ELISA), then by immunoblotting on nitrocellulose membrane after sodium dodecyl sulfate – polyacrylamide gel electrophoresis of the glycopeptide. Both clones produced antibodies of IgM class as determined by immunodiffusion. These monoclonal antibodies were utilized to detect and localize the toxic glycopeptide in pathogen cells and infected host tissues by immunohistochemical and immunocytochemical techniques.

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 Role of interleukin-15 and interleukin-18 in the secretion of sIL-6R and sgp130 by human neutrophils

2003 ◽  
Vol 12 (3) ◽  
pp. 179-183 ◽  
Author(s):  
E. Jablonska ◽  
M. Marcinczyk
Keyword(s):  
Monoclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Neutrophils ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cytoplasmic Proteins ◽  
Wide Range ◽  
Interleukin 15 ◽  
Culture Supernatants

Background:Available data indicate that neutrophils (PMN) produce a wide range of cytokines with the potential to modulate immune response. Recent investigation have shown that interleukin (IL)-15 and IL-18 potentiated several functions of normal neutrophils. It has been reported that IL-18-induced cytokine production may be significantly enhanced by coincident addition of IL-15.Aims:In the present study we compared the effect of recombinant human (rh)IL-15 and rhIL-18 as well as effect of a rhIL-15 and rhIL-18 combination on the induction secretion of sIL-6Rα and sgp130 by human neutrophils. Methods: PMN were isolated from heparinized whole blood of healthy persons. The PMN were cultured for 18 h at 37°C in a humidified incubator with 5% CO2. rhIL-15 and/or rhIL-18 and lipopolysaccharide were tested to PMN stimulation. The culture supernatants of PMN were removed and examined for the presence of sIL-6R and sgp130 by human enzyme-linked immunosorbent assay kits. Cytoplasmic protein fractions of PMN were analysed for the presence of sIL-6R and sgp130 by western blotting using monoclonal antibodies capable of detecting these proteins. Cells were lysed and cytoplasmic proteins were electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The resolved proteins were transferred onto nitrocellulose and incubated with the primary monoclonal antibodies anti-sIL-6R and anti-sgp130. The membranes were incubated at room temperature with alkaline phosphatase anti-mouse immunoglobulin G. Immunoreactive protein bans were visualized by an AP Conjugate Substrate Kit.Results and conclusion:The results of our investigation revealed that IL-15 alone, similarly to IL-18, has no significant ability for the regulation of both soluble IL-6 receptors, sIL-6R and sgp130, released by human neutrophils. It is interesting to note that the secretion of sgp130 was changed after PMN stimulation with rhIL-15 in the presence of rhIL-18. The combination of rhIL-15 and rhIL-18 was shown to induce PMN to secretion relatively higher amounts of sgp130 compared with the stimulation of PMN with rhIL-15 alone and rhIL-18 alone. The results obtained suggest that IL-15 and IL-18, belonging to the inflammatory cytokines, through the regulation of sgp130 secretion must be also considered as anti-inflammatory mediators that may influence the balance reactions mediated by the IL-6 cytokine family.

Isolation and characterization of monoclonal antibodies to proline dehydrogenase from Escherichia coli K-12

1987 ◽  
Vol 65 (6) ◽  
pp. 507-513 ◽  
Author(s):  
Janet M. Wood ◽  
Kimberley A. C. C. Taylor ◽  
Denise J. McClellan ◽  
G. Gregg Lawrie ◽  
Richard L. Krogsrud ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tween 20 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Proline Dehydrogenase ◽  
Western Blots ◽  
Isolation And Characterization ◽  
K 12

The PutA protein of Escherichia coli K-12 serves as both proline dehydrogenase and the repressor controlling the expression of genes putP and putA. Thirty-eight hybridoma cell lines were isolated using mice immunized with proline dehydrogenase purified from a bacterial membrane extract. The monoclonal antibodies secreted by those cells showed varying affinities for proline dehydrogenase by enzyme-linked immunosorbent assay (ELISA). Nine antibodies labelled the PutA protein in Western blots after sodium dodecyl sulfate – Polyacrylamide gel electrophoresis and two of the five tested also labelled the undenatured PutA protein. Three antibodies bound proteins present in a peripheral membrane protein fraction from both putA+ bacteria and a putA::Tn5 mutant strain. Urea denaturation eliminated the proline: 2,6-dichloroindophenol (DCIP) oxidoreductase activity, but did not alter the immunoreactivity of the PutA protein. Tween 20, which caused 1.8-fold increases in Km (proline) and Vmax for proline:DCIP oxidoreductase, increased the avidity of the antibody from hybridoma line 2.1C10.3 fivefold. The antibodies from hybridoma lines 2.1C10.2, 1.2C10.3, and 1.1B07.1 were shown by electron microscopy of immunogold-labelled preparations or by ELISA to bind the membrane-associated PutA protein, whereas those from hybridoma lines 2.1A08.2 and 1.4C09.1 failed to recognize that antigen form. These antibodies will serve as probes of the relationships among protein domain, conformation, and function for the PutA protein.

scholarly journals TechLab and Alexon Giardia Enzyme-Linked Immunosorbent Assay Kits Detect Cyst Wall Protein 1

1999 ◽  
Vol 37 (3) ◽  
pp. 611-614 ◽  
Author(s):  
James H. Boone ◽  
Tracy D. Wilkins ◽  
Theodore E. Nash ◽  
Jill E. Brandon ◽  
Elizabeth A. Macias ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Cyst Wall ◽  
Fluorescent Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Sequences ◽  
Enzyme Linked Immunosorbent Assay ◽  
Stool Specimens ◽  
Human Stool ◽  
Immunofluorescence Antibody

A Giardia lamblia antigen detected by the TechLabGiardia Test (TechLab, Inc., Blacksburg, Va.) and the Alexon ProSpecT Giardia microplate assay (Alexon, Inc., Sunnyvale, Calif.) was purified by immunoaffinity chromatography from supernatant fluids of encystment cultures. Two major proteins (M r 22,000 and 26,000) were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie staining that did not resemble the GSA65 antigen reportedly detected by the Alexon test. These proteins reacted intensely with the monoclonal antibodies used in both commercial enzyme-linked immunosorbent assays (ELISAs). Both proteins had identical N-terminal amino acid sequences and were identified as cyst wall protein 1 (CWP1). The 26-kDa form appeared early during encystment followed by the appearance of the 22-kDa form. Recombinant CWP1 (M r 26,000) was strongly positive in both commercial tests. CWP1 was stable in human stool specimens, resistant to degradation by proteases andN- and O-glycanases, and unaffected by oxidation with sodium periodate. Two minor proteins withM rs of 32,000 and 39,000 were detected in CWP1 preparations by using a sensitive fluorescent protein stain. Both were identified as CWP2, and neither reacted with the monoclonal antibodies from the commercial tests. We analyzed 535 stool specimens for CWP1 by using both commercial ELISAs and resolved discrepant results by using routine ova and parasite examination (O&P) and on immunofluorescence antibody assay. The presence of CWP1 correlated well between both ELISAs (98.7% correlation). Our results demonstrate that both commercial ELISAs detect CWP1, which is a useful diagnostic marker because it is highly stable, is secreted in large amounts by encysting trophozoites, and correlates well with O&P.

scholarly journals Development, Characterization, and Diagnostic Applications of Monoclonal Antibodies against Bovine Rotavirus

2000 ◽  
Vol 7 (2) ◽  
pp. 288-292 ◽  
Author(s):  
Yousif Al-Yousif ◽  
Fahad Al-Majhdi ◽  
Cindy Chard-Bergstrom ◽  
Joe Anderson ◽  
Sanjay Kapil
Keyword(s):  
Monoclonal Antibodies ◽  
Fluorescent Antibody ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Western Blot Assay ◽  
Bovine Rotavirus ◽  
Group A ◽  
Diagnostic Applications ◽  
Subtype A

ABSTRACT Hybridomas secreting monoclonal antibodies (MAbs) against the Nebraska calf diarrhea strain of bovine rotavirus (BRV) were characterized. Indirect fluorescent-antibody assay, immunodot assay, and immunoprecipitation were used to select hybridomas that produced anti-BRV MAbs. Seven of the MAbs were shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot assay to be reactive with the BRV outer capsid protein, VP7, which has a molecular mass of 37.5 kDa. None of the seven MAbs were reactive with canine rotavirus, bovine coronavirus, or uninfected Madin-Darby bovine kidney cells. Two clones, 8B4 (immunoglobulin G2a [IgG2a]) and 2B11 (IgG1), were found suitable for use in an antigen capture enzyme-linked immunosorbent assay for detecting BRV in bovine fecal samples. Both were subtype A specific (G6 subtype) but did not react with all isolates of BRV group A.

scholarly journals Use of Monoclonal Antibodies in Detection and Serological Classification of Peanut Stripe Virus1

1989 ◽  
Vol 16 (2) ◽  
pp. 63-66 ◽  
Author(s):  
J. N. Culver ◽  
J. L. Sherwood ◽  
M. R. Sanborn
Keyword(s):  
Monoclonal Antibodies ◽  
Western Blotting ◽  
Polyclonal Antibodies ◽  
Myeloma Cell ◽  
Plant Viruses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Peanut Stripe Virus ◽  
Mouse Myeloma Cell Line ◽  
Related Plant

Abstract Monoclonal antibodies (MABs) to peanut stripe virus (PStV) were obtained by fusing spleen cells from mice immunized with PStV to a mouse myeloma cell line. Two IgG2a subclass MABs, each binding with different antigenic sites, and rabbit polyclonal antibodies (PABs) were compared for their reaction to PStV and other serologically related plant viruses. One MAB reacted to PStV but not to other serologically related viruses. The other MAB reaction of the MABs and PABs to PStV in peanut leaf and seed tissues were compared by enzyme-linked immunosorbent assay (ELISA), dot-immunobinding assay, and Western blotting. MABs were found to be equivalent to or better than PABs in detecting PStV by ELISA and dot-immunobinding assay, but one MAB was not suitable for Western blotting.

Production and Characterization of Monoclonal Antibodies To Detect Vibrio cholerae Serogroup O1 in a Rapid Enzyme-Linked Immunosorbent Assay

1996 ◽  
Vol 59 (11) ◽  
pp. 1153-1157 ◽  
Author(s):  
CHARLES W. NOAH ◽  
SHERILYN S. POTEET ◽  
MILDRED M. LISTER ◽  
CHARLES N. RODERICK ◽  
DON B. SMITH ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Vibrio Cholerae ◽  
Immunosorbent Assay ◽  
Bacterial Species ◽  
Myeloma Cell ◽  
Saline Control ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mouse Myeloma Cell Line ◽  
Vibrio Spp

Monoclonal antibodies (MAbs) were developed for a rapid and efficient screening procedure to detect cultures of Vibrio cholerae serogroup O1. Spleen cells of BALB/c mice previously immunized with an attenuated control strain of V. cholerae were fused with mouse myeloma cell line SP2/0. An enzyme-linked immunosorbent assay (ELISA) was used to test cultural hybridoma secretions of two MAbs against 120 strains of V. cholerae O1, 38 strains of V. cholerae non-O1, 15 strains of other Vibrio spp., and 20 strains of other bacterial species. Results of tests using both MAbs were identical. The MAbs successfully detected all of the confirmed serotype O1 strains. Three additional V. cholerae strains that agglutinated antisera and the saline control were considered serologically inconclusive. Of these, one was detected as positive for V. cholerae by both MAbs. The MAbs gave no false-positive reactions when tested against the confirmed non-O1 strains, other Vibrio spp., and other bacterial species. Use of this ELISA will enhance the speed and accuracy needed for detecting V. cholerae O1 cultures.

scholarly journals Structural and functional analysis of the complement component factor H with the use of different enzymes and monoclonal antibodies to factor H

1985 ◽  
Vol 232 (3) ◽  
pp. 841-850 ◽  
Author(s):  
J Alsenz ◽  
T F Schulz ◽  
J D Lambris ◽  
R B Sim ◽  
M P Dierich
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Sites ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Factor H ◽  
Enzyme Linked Immunosorbent Assay ◽  
Terminal Sequence ◽  
Tryptic Fragment ◽  
Cofactor Activity ◽  
Third Component Of Complement

The action of six different enzymes on the function and structure of Factor H was investigated by use of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, haemagglutination, two enzyme-linked immunosorbent assay systems and an assay for Factor I cofactor activity. Six monoclonal antibodies directed against the 38 kDa tryptic fragment of Factor H [which contains the binding site for C3b (a 180 kDa fragment of the third component of complement) and the cofactor activity] were also used to detect cleavage products derived from the same fragment. Elastase, chymotrypsin A4 or trypsin first cleaved Factor H to 36-38 kDa fragments carrying all six monoclonal anti-(Factor H)-binding sites. In parallel, the interaction of Factor H with surface-bound C3b was lost, whereas the cofactor function was preserved. Further cleavage of the 36-38 kDa fragments into two 13-19 kDa fragments (one carrying the MAH4 and MRC OX 24 epitopes, the other the MAH1, MAH2, MAH3 and MRC OX 23 epitopes) destroyed cofactor activity. Pepsin, bromelain or papain rapidly split off a 13-15 kDa fragment of Factor H carrying the MAH1, MAH2, MAH3 and MRC OX 23 epitopes and destroyed all tested functions of Factor H. Ficin cleaved Factor H into disulphide-linked fragments smaller than 25 kDa, but did not affect the functions of the Factor H molecule. The 38 kDa tryptic fragment of Factor H is the N-terminal end of the Factor H molecule, as determined by N-terminal sequence analysis. A model is presented of the substructure of Factor H.

Screening of Single-Chain Variable Fragments against TSP50 from a Phage Display Antibody Library and Their Expression as Soluble Proteins

2006 ◽  
Vol 11 (5) ◽  
pp. 546-552 ◽  
Author(s):  
Jingyan Wei ◽  
Yang Liu ◽  
Songchuan Yang ◽  
Junjie Xu ◽  
Hangtian Kong ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Phage Display ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sandwich Elisa ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibody Library ◽  
Single Chain ◽  
Single Chain Variable Fragments ◽  
Phage Display Antibody

A novel gene, testes-specific protease 50 ( TSP50), is abnormally activated and differentially expressed in most patients with breast cancer, suggesting it as a novel biomarker for this disease. The possibility that TSP50 may be an oncogene is presently under investigation. In this study, the single-chain variable fragments (scFvs) against TSP50 were panned from a phage display antibody library using TSP50-specific peptide, pep-50, as a target antigen. After 4 rounds of panning, 3 clones (A1, A11, and C8) from the library were verified to show strong binding affinities for TSP50 by enzyme-linked immunosorbent assay (ELISA) and to contain the variable region genes of the light and heavy chains of scFv antibodies but different complementary determining regions by sequencing. The genes of scFv-A1 and scFv-A11 were cloned into expression vector pPELB and successfully expressed as a soluble protein in Escherichia coli Rosetta. The yields of expressions were about 4.0 to 5.0 mg of protein from 1 L of culture. The expressed proteins were purified by a 2-step procedure consisting of ion-exchange chromatography, followed by immobilized metal affinity chromatography. The purified proteins were shown a single band at the position of 31 KDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Sandwich ELISA demonstrated that the expressed scFv proteins were able to specifically react with pep-50, laying a foundation for the investigation of the function of TSP50 in the development and treatment of breast cancer.

scholarly journals Production and characterisation of monoclonal antibodies against immunoglobulins of Cirrhinus mrigala (Hamilton 1822)

2020 ◽  
Vol 67 (2) ◽  
Author(s):  
Snatashree Mohanty ◽  
M. Makesh ◽  
K. V. Rajendran ◽  
P. P. Suresh Babu ◽  
Deepika Anand ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Catla Catla ◽  
Cirrhinus Mrigala ◽  
Indian Major Carps ◽  
Sds Page ◽  
Igg1 Isotype ◽  
Major Carps

Serum immunoglobulins (Ig) of mrigal Cirrhinus mrigala (Hamilton 1822) immunised with bovine serum albumin (BSA), were purified by affinity chromatography using BSA-CL agarose column. The purified mrigal Ig (m-Ig) was characterised under reducing condition by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) which revealed two bands of 85 and 26 kDa corresponding to heavy and light chain, respectively. Following fusion of splenocytes from Balb/c mice immunised with purified m-Ig with myeloma cells, three hybridomas showing reactivity with m-Ig were cloned by limiting dilution. The monoclonal antibodies (MAbs) generated by these clones were designated as 3B2-E12, 3B2-F9 and 4C3-B2 and characterised by western blotting and isotyping. Western blot analysis of the supernatant from the three clones with purified m-Ig indicated that, all the three MAbs were specific to heavy chain. Isotyping revealed that 3B2-E12 MAb was of IgG1 isotype whereas the other two MAbs were of IgG2a isotype. Cross reactivity of anti-mrigal Ig MAb (3B2-E12) was observed with serum Ig of Catla catla and Labeo rohita indicating semi-conserved nature of Ig in Indian major carps.

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