Immunochemical Quantification of Metallothioneins of a Marine Mollusc

1988 ◽  
Vol 45 (7) ◽  
pp. 1257-1263 ◽  
Author(s):  
G. Roesijadi ◽  
M. E. Unger ◽  
J. E. Morris
Keyword(s):  
Metal Binding ◽  
Binding Proteins ◽  
Polyclonal Antibodies ◽  
Exchange Chromatography ◽  
Enzyme Linked Immunosorbent Assay ◽  
Low Molecular Weight ◽  
Ammonium Sulfate Precipitation ◽  
Marine Mollusc ◽  
Peroxidase Conjugate

Mercury-induced, low molecular weight, metal-binding proteins were isolated from the marine mussel Mytilus edulis and used as antigen in the development of antibodies and an enzyme-linked immunosorbent assay (ELISA) for quantification of the proteins. Partial characterization of the isolated protein indicated that its properties were consistent with those of metallothionein (MT). In contrast with most MTs, this protein occurred as an apparent dimer and contained glycine at high levels. Polyclonal antibodies against this protein were produced in goats and purified to an IgG fraction by ammonium sulfate precipitation and DEAE ion-exchange chromatography. Ouchterlony analysis and ELISA showed that these antibodies were cross-reactive with two other charge variants of mercury-induced, metal-binding proteins of M. edulis, but not with rabbit MT. The ELISA was based on an indirect, competitive procedure utilizing a rabbit anti-goat IgG–horseradish peroxidase conjugate as the second antibody. Application of this assay to cytosolic extracts of mussel gills indicated 0.5 μg metal-binding proteins/g wet tissue weight in gills of control mussels and elevated levels up to 1780 μg/g following exposure to mercury, cadmium, or copper for 28 d. Zinc was not effective as an inducer of these proteins.

Rabbit antibodies against the low molecular weight folate binding protein from human milk. Use for immunological characterization of human folate binding proteins in an enzyme-linked immunosorbent assay (ELISA)

1987 ◽  
Vol 7 (7) ◽  
pp. 553-557 ◽  
Author(s):  
Mimi Høier-Madsen ◽  
Steen Ingemann Hansen ◽  
Jan Holm
Keyword(s):  
Molecular Weight ◽  
Human Milk ◽  
Immunosorbent Assay ◽  
Binding Proteins ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Low Molecular Weight ◽  
Folate Binding Protein ◽  
Folate Binding Proteins

Antibodies to a low molecular weight folate binding protein isolated from human milk were raised in rabbits and used for development of a two-site enzyme-linked immunosorbent assay (ELISA) for immunological characterization of human folate binding proteins (FBPs). The high and low molecular weight FBPs from human milk were immunologically indistinguishable. Furthermore, the FBPs in human urine and cerebrospinal fluid showed a cross-reactivity of 70% and 30%, respectively. No cross-reactivity of the FBP from cow's milk was observed.

Production and Characterization of Antibodies against Fumonisin B1

1996 ◽  
Vol 59 (9) ◽  
pp. 992-997 ◽  
Author(s):  
FENG-YIR YU ◽  
FUN S. CHU
Keyword(s):  
Detection Limit ◽  
Polyclonal Antibodies ◽  
Fumonisin B1 ◽  
Correlation Coefficients ◽  
Keyhole Limpet Hemocyanin ◽  
Hplc Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Indirect Competitive Elisa ◽  
Antibody Titers

Polyclonal antibodies against fumonisin B1 (FmB1) were produced in rabbits after immunizing the animals with either FmBl-keyhole limpet hemocyanin (KLH) or FmB1 bovine serum albumin (BSA). A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) and an indirect competitive ELISA (idc-ELISA) were used for the characterization of the antibodies and for analysis of the toxin in corn samples. The antibody titers in the serum of rabbits immunized with FmBl-KLH were considerably higher than in those immunized with FmBl-BSA. The antibodies from the rabbits immunized with FmBl-KLH were further characterized. The concentrations causing 50% inhibition of binding of FmB1-horseradish peroxidase (HRP) to the antibodies by FmB1, FmB2 and FmB3 in the ELISA were found to be 0.45, 0.72, and 25 ng/ml, respectively. The detection limit of FmBl, based on 95% confidence at 5% of inhibition of binding of FmBl-HRP conjugate, in buffer of the dc-ELISA was found to be 0.05 ng/ml. In the presence of a matrix such as corn, the detection limit was less than 50 ppb. The overall analytical recoveries of FmBl (50 to 1,000 ng/g) added to the ground corn and then extracted with CH3CN/H2O (1/1, vol/vol) with cleanup and without cleanup in the dc ELISA were found to be 70.5 and 85.9%, respectively. A good correlation was found between the FmBl levels in 2 starch and 10 naturally contaminated corn samples analyzed by the dc-ELISA and the high-pressure liquid chromatography (HPLC) method. The correlation coefficients between ELISA and HPLC were found to be 0.955 (y [ELISA] = 1.3 1x [HPLC] + 77 ppb; P < 0.001) and 0.811 (y = 1.13x + 34 ppb; P < 0.01) for the sample without and with cleanup treatment, respectively.

scholarly journals Cadmium-binding proteins of rat testes. Characterization of a low-molecular-mass protein that lacks identity with metallothionein

1984 ◽  
Vol 220 (3) ◽  
pp. 811-818 ◽  
Author(s):  
M P Waalkes ◽  
S B Chernoff ◽  
C D Klaassen
Keyword(s):  
Metal Binding ◽  
Gel Electrophoresis ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Or Groups

Cadmium-binding proteins in the cytosol of testes from untreated rats were separated by Sephadex G-75 gel filtration. Three major testicular metal-binding proteins (TMBP), or groups of proteins, with relative elution volumes of approx. 1.0 (TMBP-1), 1.7 (TMBP-2) and 2.4 (TMBP-3) were separated. Elution of Zn-binding proteins exhibited a similar pattern. TMBP-3 has previously been thought to be metallothionein (MT), and hence this protein was further characterized and compared with hepatic MT isolated from Cd-treated rats. Estimation of Mr by gel filtration indicated a slight difference between MT (Mr 10000) and TMBP-3 (Mr 8000). Two major forms of MT (MT-I and MT-II) and TMBP-3 (TMBP-3 form I and TMBP-3 form II) were obtained after DEAE-Sephadex A-25 anion-exchange chromatography, with the corresponding subfractions being eluted at similar conductances. Non-denaturing polyacrylamide-gel electrophoresis on 7% acrylamide gels indicated that the subfractions of TMBP-3 had similar mobilities to those of the corresponding subfractions of MT. However, SDS (sodium dodecyl sulphate)/12% (w/v)-polyacrylamide-gel electrophoresis resulted in marked differences in migration of the two corresponding forms of MT and TMBP-3. Co-electrophoresis of MT-II and TMBP-3 form II by SDS/polyacrylamide-gel electrophoresis revealed two distinct proteins. Amino acid analysis indicated much lower content of cysteine in the testicular than in the hepatic proteins. TMBP-3 also contained significant amounts of tyrosine, phenylalanine and histidine, whereas MT did not. U.v.-spectral analysis of TMBP-3 showed a much lower A250/A280 ratio than for MT. Thus this major metal-binding protein in testes, which has been assumed to be MT is, in fact, a quite different protein.

scholarly journals Nuclear Detection of Cellular Retinoic Acid Binding Proteins I and II with New Antibodies

1998 ◽  
Vol 46 (10) ◽  
pp. 1103-1111 ◽  
Author(s):  
Marie-Pierre Gaub ◽  
Yves Lutz ◽  
Norbert B. Ghyselinck ◽  
Isabelle Scheuer ◽  
Véronique Pfister ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Binding Proteins ◽  
Polyclonal Antibodies ◽  
Amino Acid Sequences ◽  
Mouse Embryos ◽  
Low Molecular Weight ◽  
E Coli ◽  
Nuclear Receptor Family ◽  
Retinoic Acid Binding Proteins ◽  
Receptor Family

Apart from the retinoic acid nuclear receptor family, there are two low molecular weight (15 kD) cellular retinoic acid binding proteins, named CRABPI and II. Mouse monoclonal and rabbit polyclonal antibodies were raised against these proteins by using as antigens either synthetic peptides corresponding to amino acid sequences unique to CRABPI or CRABPII, or purified CRABP proteins expressed in E. coli. Antibodies specific for mouse and/or human CRABPI and CRABPII were obtained and characterized by immunocytochemistry and immunoblotting. They allowed the detection not only of CRABPI but also of CRABPII in both nuclear and cytosolic extracts from transfected COS-1 cells, mouse embryos, and various cell lines.

scholarly journals Purification and partial characterization of 4-aminobutyrate:2-oxoglutarate aminotransferase from sheep brain and locust ganglia

1988 ◽  
Vol 249 (3) ◽  
pp. 795-799 ◽  
Author(s):  
D Jeffery ◽  
D M Rutherford ◽  
P D J Weitzman ◽  
G G Lunt
Keyword(s):  
Hydrophobic Interaction Chromatography ◽  
Competitive Inhibitor ◽  
Exchange Chromatography ◽  
Mammalian Brain ◽  
Enzyme Linked Immunosorbent Assay ◽  
Initial Comparison ◽  
Michaelis Constants ◽  
Sheep Brain ◽  
Suicide Substrate

We report here the first purification to homogeneity of 4-aminobutyrate: 2-oxoglutarate aminotransferase (EC 2.6.1.19) (GABA-T) from an invertebrate source (locust) and its initial comparison with that of GABA-T from mammalian brain (sheep). The enzyme from both organisms was found to be a dimer of similar-sized subunits, with a native Mr of approx. 97,000. The pI of GABA-T from the locust was 6.7 and that of the sheep enzyme was 5.5. Michaelis constants for 4-aminobutyric acid (GABA) and 2-oxoglutarate were respectively 0.79 +/- 0.16 mM and 0.27 +/- 0.08 mM for the locust enzyme and 2.2 +/- 0.24 mM and 0.22 +/- 0.11 mM for the sheep enzyme. 5-(Aminomethyl)-3-isoxazolol (muscimol) was a competitive inhibitor of both enzymes, whereas 5-amino-1,3-cyclohexadienylcarboxylic acid (gabaculine) acted as a potent suicide substrate. However, 3-aminopropane-1-sulphonic acid, diaminobutyric acid, 1,2,3,4-tetrahydro-1-methyl-3-pyridinecarboxylic acid (isoguvacine), beta-(aminomethyl)-4-chlorobenzenepropanoic acid (baclofen), bicuculline and picrotoxin did not inhibit either enzyme at concentrations below 100 mM. Polyclonal antisera raised against GABA-T from the sheep failed to cross-react with the enzyme from locust in either an Ouchterlony immunodiffusion plate or a competitive enzyme-linked immunosorbent assay. The purification procedures differed considerably. Ion-exchange chromatography, which was found suitable for the purification of GABA-T from the sheep, was ineffective with locust enzyme, which was finally purified by hydrophobic-interaction chromatography and chromatofocusing.

Purification and partial characterization of a plasma inhibitor of tonin

1981 ◽  
Vol 59 (4) ◽  
pp. 256-261 ◽  
Author(s):  
J. Tremblay ◽  
G. Thibault ◽  
J. Gutkowska ◽  
R. Boucher ◽  
J. Genest
Keyword(s):  
Molecular Weight ◽  
Plasma Proteins ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sedimentation Velocity ◽  
Exchange Chromatography ◽  
Ammonium Sulfate Precipitation ◽  
Angiotensin I ◽  
Stokes Radius

A plasma inhibitor of tonin activity in the rat, was purified by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. Its purity was investigated by analytical electrophoresis on polyacrylamide gel and by ultracentrifugation sedimentation velocity. The molecular weight (360 000) of the purified inhibitor was determined by sodium dodecyl sulfate electrophoresis and its isoelectric point (4.5) by gel isoelectrofocusing. The Stokes radius (640 nm) was evaluated by gel filtration studies and a frictional ratio (f/f0) of 1.95 was calculated from the molecular weight and Stokes radius.Kinetic studies using angiotensin I as substrate showed that the inhibition of tonin by the purified inhibitor was noncompetitive and does not exceed 70%. Electrophoresis showed the same mobility for [125I]tonin bound to plasma proteins and for [125I]tonin bound to the purified inhibitor. The inhibitor may be a protein resembling half of the dimeric protease inhibitor rat α1-macroglobulin or human α2-macroglobulin

scholarly journals Production of Monoclonal Antibodies Specific for the i and 1,2 Flagellar Antigens of Salmonella typhimurium and Characterization of Their Respective Epitopes

1998 ◽  
Vol 64 (12) ◽  
pp. 5033-5038 ◽  
Author(s):  
N. de Vries ◽  
K. A. Zwaagstra ◽  
J. H. J. Huis in’t Veld ◽  
F. van Knapen ◽  
F. G. van Zijderveld ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Salmonella Typhimurium ◽  
Immunosorbent Assay ◽  
Specific Antibody ◽  
Polyclonal Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Antibodies ◽  
Minimal Area

ABSTRACT Salmonella typhimurium expresses two antigenically distinct flagellins, each containing a different H antigen (i and 1,2), the combination of which is highly specific for this serotype. In this study, overlapping recombinant flagellin fragments were constructed from the fliC(H:i) and fljB (H:1,2) flagellin genes, and the expression products were tested for binding to H antigen-specific monoclonal and polyclonal antibodies. A minimal area, 86 amino acids for H:i and 102 amino acids for H:1,2, located in the central variable domain of each flagellin was required for the binding of serotype-specific antibodies, providing further evidence for the presence of a discontinuous H epitope. Two peptides comprising these areas were shown to be highly suitable for application as antigens in an enzyme-linked immunosorbent assay detecting S. typhimurium-specific antibody.

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