scholarly journals Production and characterization of monoclonal antibodies to β-galactoside-binding lectin of bovine heart muscle. Direct evidence that haemagglutinating activity is associated with a 13kDa protein

1984 ◽  
Vol 220 (1) ◽  
pp. 253-260 ◽  
Author(s):  
S R Carding ◽  
R Thorpe ◽  
R A Childs ◽  
M Spitz ◽  
T Feizi
Keyword(s):  
Heart Muscle ◽  
Direct Evidence ◽  
Solid Phase ◽  
Binding Activity ◽  
High Titre ◽  
Immunoglobulin M ◽  
Bovine Heart ◽  
Haemagglutinating Activity ◽  
Binding Lectin

With the aim of obtaining monospecific antibodies against the beta-galactoside-binding lectin of bovine heart muscle, spleen cells from Lou rats immunized with lectin were fused with the rat myeloma line Y3.Ag1.2.3. Two immunoglobulin M (IgM)-producing clones, designated NIBy 142-36/8 and NIBy 143-9/5, derived from separate fusions, were used to generate ascites containing high-titre binding activity against the 13kDa component in preparations of lectin. Direct evidence that haemagglutinating activity is associated with the 13kDa protein was obtained by the specific elution of 13kDa polypeptides with haemagglutinating activity from an immobilized antibody adsorbent. Solid-phase radiobinding assays and immunoblotting of isolated lectins and/or muscle homogenates confirmed the earlier indications with conventional antisera that the beta-galactoside-binding lectins of bovine, human and monkey muscle tissue are antigenically related.

scholarly journals Multiple proteins related to the soluble galactose-binding animal lectin revealed by a monoclonal anti-lectin antibody

1985 ◽  
Vol 228 (1) ◽  
pp. 147-153 ◽  
Author(s):  
S R Carding ◽  
R A Childs ◽  
R Thorpe ◽  
M Spitz ◽  
T Feizi
Keyword(s):  
Solid Phase ◽  
Cell Types ◽  
Binding Activity ◽  
Active Components ◽  
Muscle Tissues ◽  
Bovine Tissue ◽  
Tissue Homogenates ◽  
Binding Lectin ◽  
Animal Lectin ◽  
Galactose Binding Lectin

A monoclonal antibody (NIBy 142-36/8) raised against the soluble galactose-binding lectin of bovine heart muscle has been tested by solid-phase vinyl-plate radiobinding and nitrocellulose immunoblotting with homogenates of various bovine tissues, and the muscle tissues of pig, rabbit, chicken and rat. Muscle lectins of chicken, rabbit and rat differed from those of man and pig in their lack of reactivity with the 36/8 antibody. There was a good correlation of haemagglutinating activities and immunoreactivities of the bovine tissue homogenates, suggesting that the soluble galactose-binding protein is a major haemagglutinin in various tissues. Immunoblotting experiments revealed an array of antigenically active components in the homogenates in addition to the 13 and 26kDa proteins that were previously detected in preparations of purified lectin. These were in the range 36kDa to more than 200kDa, and a different spectrum of immunoreactive components was found in various cell types. Galactose-binding activity was demonstrable in 13, 26 and 36kDa components in certain bovine tissues, suggesting that the immunoreactive components of higher Mr may be inactive precursor forms of the lectin.

scholarly journals Purification and characterization of a saliva-interacting cell-wall protein from Streptococcus mutans serotype f by using monoclonal-antibody immunoaffinity chromatography

1985 ◽  
Vol 228 (1) ◽  
pp. 211-217 ◽  
Author(s):  
F Ackermans ◽  
J P Klein ◽  
J Ogier ◽  
H Bazin ◽  
F Cormont ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Wall ◽  
Streptococcus Mutans ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Immunoaffinity Chromatography ◽  
Binding Activity ◽  
Immunoglobulin M ◽  
Single Polypeptide Chain

A rat monoclonal antibody, LO SM2, of the immunoglobulin M class, specific for a saliva receptor (SR) from Streptococcus mutans serotype f, was able to precipitate the SR from crude cell-wall-associated antigens (WEA) of this bacteria in presence of a detergent mixture. We have then used the technique of monoclonal-antibody immunoaffinity chromatography to purify the S. mutans SR. Pure SR was obtained from a crude WEA fraction with a single chromatographic step. The active SR could be eluted from the column in a highly purified form with 0.2 M-glycine/HC1, pH 2.8. The final yield was about 32% in terms of binding activity. Characterization of the SR by crossed immunoelectrophoresis, sodium dodecyl sulphate- or 4-30%-native-gradient-polyacrylamide-gel electrophoresis showed that the receptor is a single polypeptide chain of Mr approx. 74000. Native or denaturated forms of the SR adsorbed on to a solid support, such as nitrocellulose, are recognized by monoclonal antibody LO SM2, and both forms are still able to bind the ligand, saliva.

scholarly journals Isolation and characterization of adult bovine heart muscle chalones.

1981 ◽  
Vol 78 (7) ◽  
pp. 4161-4164 ◽  
Author(s):  
J. A. Kriek ◽  
B. J. van der Walt ◽  
A. J. Bester
Keyword(s):  
Heart Muscle ◽  
Bovine Heart ◽  
Isolation And Characterization

scholarly journals Chemical modification studies on Abrus agglutinin. Involvement of tryptophan residues in sugar binding

1984 ◽  
Vol 217 (3) ◽  
pp. 773-781 ◽  
Author(s):  
S R Patanjali ◽  
M J Swamy ◽  
V Anantharam ◽  
M I Khan ◽  
A Surolia
Keyword(s):  
Binding Activity ◽  
Carbohydrate Binding ◽  
Amino Acid Residues ◽  
Partial Protection ◽  
Abrus Precatorius ◽  
Biphasic Pattern ◽  
Saccharide Binding ◽  
Tryptophan Residues ◽  
Haemagglutinating Activity ◽  
Binding Lectin

The galactose-binding lectin from the seeds of the jequirity plant (Abrus precatorius) was subjected to various chemical modifications in order to detect the amino acid residues involved in its binding activity. Modification of lysine, tyrosine, arginine, histidine, glutamic acid and aspartic acid residues did not affect the carbohydrate-binding activity of the agglutinin. However, modification of tryptophan residues carried out in native and denaturing conditions with N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide led to a complete loss of its carbohydrate-binding activity. Under denaturing conditions 30 tryptophan residues/molecule were modified by both reagents, whereas only 16 and 18 residues/molecule were available for modification by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide respectively under native conditions. The relative loss in haemagglutinating activity after the modification of tryptophan residues indicates that two residues/molecule are required for the carbohydrate-binding activity of the agglutinin. A partial protection was observed in the presence of saturating concentrations of lactose (0.15 M). The decrease in fluorescence intensity of Abrus agglutinin on modification of tryptophan residues is linear in the absence of lactose and shows a biphasic pattern in the presence of lactose, indicating that tryptophan residues go from a similar to a different molecular environment on saccharide binding. The secondary structure of the protein remains practically unchanged upon modification of tryptophan residues, as indicated by c.d. and immunodiffusion studies, confirming that the loss in activity is due to modification only.

The Immuno-Electron Microscopic Localization of the Mo-Fe Protein Component of Nitrogenase in Cells of Azotobacter Vinelandii

1973 ◽  
Vol 31 ◽  
pp. 574-575
Author(s):  
J. T. Stasny ◽  
R. C. Burns ◽  
R. W. F. Hardy
Keyword(s):  
Cellular Localization ◽  
Direct Evidence ◽  
Azotobacter Vinelandii ◽  
Intracellular Localization ◽  
Protein Component ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Fe Protein ◽  
Intracytoplasmic Membranes

Structure-functlon studies of biological N2-fixation have correlated the presence of the enzyme nitrogenase with increased numbers of intracytoplasmic membranes in Azotobacter. However no direct evidence has been provided for the internal cellular localization of any nitrogenase. Recent advances concerned with the crystallizatiorTand the electron microscopic characterization of the Mo-Fe protein component of Azotobacter nitrogenase, prompted the use of this purified protein to obtain antibodies (Ab) to be conjugated to electron dense markers for the intracellular localization of the protein by electron microscopy. The present study describes the use of ferritin conjugated to goat antitMo-Fe protein immunoglobulin (IgG) and the observations following its topical application to thin sections of N2-grown Azotobacter.

Application of solid-phase immune electron microscopy to study physical and stability characteristics of enterically transmitted non-a, non-b hepatitis virus

1988 ◽  
Vol 46 ◽  
pp. 80-81
Author(s):  
Charles D. Humphrey ◽  
E. H. Cook ◽  
Karen A. McCaustland ◽  
Daniel W. Bradley
Keyword(s):  
Electron Microscopy ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Solid Phase ◽  
Etiologic Agent ◽  
World Health ◽  
Health Concern ◽  
Morphological Identification ◽  
Immune Electron Microscopy

Enterically transmitted non-A, non-B hepatitis (ET-NANBH) is a type of hepatitis which is increasingly becoming a significant world health concern. As with hepatitis A virus (HAV), spread is by the fecal-oral mode of transmission. Until recently, the etiologic agent had not been isolated and identified. We have succeeded in the isolation and preliminary characterization of this virus and demonstrating that this agent can cause hepatic disease and seroconversion in experimental primates. Our characterization of this virus was facilitated by immune (IEM) and solid phase immune electron microscopic (SPIEM) methodologies.Many immune electron microscopy methodologies have been used for morphological identification and characterization of viruses. We have previously reported a highly effective solid phase immune electron microscopy procedure which facilitated identification of hepatitis A virus (HAV) in crude cell culture extracts. More recently we have reported utilization of the method for identification of an etiologic agent responsible for (ET-NANBH).

TEM characterization of Au/Polysilicon interactions

1990 ◽  
Vol 48 (4) ◽  
pp. 650-651
Author(s):  
N. David Theodore ◽  
Leslie H. Allen ◽  
C. Barry Carter ◽  
James W. Mayer
Keyword(s):  
Solid Phase ◽  
Single Crystal Silicon ◽  
Chemical Vapor ◽  
Electrical Contacts ◽  
Silicon Substrates ◽  
Crystal Silicon ◽  
Transmission Electron ◽  
Polysilicon Films ◽  
The Stability

Metal/polysilicon investigations contribute to an understanding of issues relevant to the stability of electrical contacts in semiconductor devices. These investigations also contribute to an understanding of Si lateral solid-phase epitactic growth. Metals such as Au, Al and Ag form eutectics with Si. reactions in these metal/polysilicon systems lead to the formation of large-grain silicon. Of these systems, the Al/polysilicon system has been most extensively studied. In this study, the behavior upon thermal annealing of Au/polysilicon bilayers is investigated using cross-section transmission electron microscopy (XTEM). The unique feature of this system is that silicon grain-growth occurs at particularly low temperatures ∽300°C).Gold/polysilicon bilayers were fabricated on thermally oxidized single-crystal silicon substrates. Lowpressure chemical vapor deposition (LPCVD) at 620°C was used to obtain 100 to 400 nm polysilicon films. The surface of the polysilicon was cleaned with a buffered hydrofluoric acid solution. Gold was then thermally evaporated onto the samples.

Characterization of Monoclonal Anti-human Factor VII Antibody (B101/B1) that Recognized Three-dimensional Structures near Arg at Position 79 in the First EGF-like Domain

1998 ◽  
Vol 79 (01) ◽  
pp. 104-109 ◽  
Author(s):  
Osamu Takamiya
Keyword(s):  
Monoclonal Antibodies ◽  
Tissue Factor ◽  
Human Factor ◽  
Solid Phase ◽  
Three Dimensional ◽  
Coagulation Factors ◽  
Factor Vii ◽  
Dimensional Structure ◽  
Epidermal Growth

SummaryMurine monoclonal antibodies (designated hVII-B101/B1, hVIIDC2/D4 and hVII-DC6/3D8) directed against human factor VII (FVII) were prepared and characterized, with more extensive characterization of hVII-B101/B1 that did not bind reduced FVIIa. The immunoglobulin of the three monoclonal antibodies consisted of IgG1. These antibodies did not inhibit procoagulant activities of other vitamin K-dependent coagulation factors except FVII and did not cross-react with proteins in the immunoblotting test. hVII-DC2/D4 recognized the light chain after reduction of FVIIa with 2-mercaptoethanol, and hVIIDC6/3D8 the heavy chain. hVII-B101/B1 bound FVII without Ca2+, and possessed stronger affinity for FVII in the presence of Ca2+. The Kd for hVII-B101/B1 to FVII was 1.75 x 10–10 M in the presence of 5 mM CaCl2. The antibody inhibited the binding of FVII to tissue factor in the presence of Ca2+. hVII-B101/B1 also inhibited the activation of FX by the complex of FVIIa and tissue factor in the presence of Ca2+. Furthermore, immunoblotting revealed that hVII-B101/B1 reacted with non-reduced γ-carboxyglutaminic acid (Gla)-domainless-FVII and/or FVIIa. hVII-B101/B1 showed a similar pattern to that of non-reduced proteolytic fragments of FVII by trypsin with hVII-DC2/D4 on immunoblotting test. hVII-B101/B1 reacted differently with the FVII from the dysfunctional FVII variant, FVII Shinjo, which has a substitution of Gln for Arg at residue 79 in the first epidermal growth factor (1st EGF)-like domain (Takamiya O, et al. Haemosta 25, 89-97,1995) compared with normal FVII, when used as a solid phase-antibody for ELISA by the sandwich method. hVII-B101/B1 did not react with a series of short peptide sequences near position 79 in the first EGF-like domain on the solid-phase support for epitope scanning. These results suggested that the specific epitope of the antibody, hVII-B101/B1, was located in the three-dimensional structure near position 79 in the first EGF-like domain of human FVII.

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