scholarly journals Immunopurification and characterization of human α-l-iduronidase with the use of monoclonal antibodies

1989 ◽  
Vol 259 (1) ◽  
pp. 199-208 ◽  
Author(s):  
P R Clements ◽  
D A Brooks ◽  
P A G McCourt ◽  
J J Hopwood
Keyword(s):  
Monoclonal Antibodies ◽  
Normal Control ◽  
Heparan Sulphate ◽  
Molecular Size ◽  
Gel Permeation Chromatography ◽  
Immunoaffinity Chromatography ◽  
Chromatographic Behaviour ◽  
Cultured Skin ◽  
Coomassie Blue ◽  
Column Procedure

alpha-L-Iduronidase from human liver was purified by a three-step five-column procedure and by immunoaffinity chromatography with a monoclonal antibody raised against purified enzyme. Seven bands identified by staining with Coomassie Blue had molecular masses of 74, 65, 60, 49, 44, 18 and 13 kDa and were present in both preparations of the liver enzyme. However, relative to the immunopurification procedure, alpha-L-iduronidase purified by the five-column procedure was considerably enriched in the 65 kDa polypeptide band. The seven bands were identified by Western-blot analysis with two different monoclonal antibodies raised against alpha-L-iduronidase. The chromatographic behaviour of alpha-L-iduronidase on the antibody column was dependent upon the quantity of enzyme loaded. Above a particular load concentration a single peak of enzyme activity was eluted, whereas at load concentrations below the critical value alpha-L-iduronidase was eluted in two peaks of activity, designated form I (eluted first) and form II (eluted second). The following properties of the two forms of alpha-L-iduronidase were determined. (1) The two forms from liver were composed of different proportions of the same seven polypeptides. (2) When individually rechromatographed on the antibody column, each form from liver shifted to a more retarded elution position but essentially retained its chromatographic behaviour relative to the other form. (3) Forms I and II of liver alpha-L-iduronidase showed no difference in their activities towards disaccharide substrates derived from two glycosaminoglycan sources, heparan sulphate and dermatan sulphate. (4) The native molecular size of forms I and II of liver alpha-L-iduronidase was 65 kDa as determined by gel-permeation chromatography. (5) Immunoaffinity chromatography of extracts of human lung and kidney resulted in the separation of alpha-L-iduronidase into two forms, each with different proportions of the seven common polypeptide species. (6) Lung forms I and II were taken up readily into cultured skin fibroblasts taken from a patient with alpha-L-iduronidase deficiency. Liver forms I and II were not taken up to any significant extent. Lung form II gave intracellular contents of alpha-L-iduronidase that were more than double those of normal control fibroblasts, whereas lung form I gave contents approximately equal to normal control values. We propose that all seven polypeptides are derived from a single alpha-L-iduronidase gene product, and that different proportions of these polypeptides can function as a single alpha-L-iduronidase entity.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Human liver glucuronate 2-sulphatase. Purification, characterization and catalytic properties

1989 ◽  
Vol 259 (1) ◽  
pp. 209-216 ◽  
Author(s):  
C Freeman ◽  
J J Hopwood
Keyword(s):  
Enzyme Activity ◽  
Molecular Mass ◽  
Glucuronic Acid ◽  
Heparan Sulphate ◽  
Gel Permeation Chromatography ◽  
Electrophoretic Analysis ◽  
Native Molecular Mass ◽  
Phosphate Ions ◽  
Column Procedure ◽  
Inhibited Enzyme

Human glucuronate 2-sulphatase (GAS), which is involved in the degradation of the glycosaminoglycans heparan sulphate and chondroitin 6-sulphate, was purified almost 2,000,000-fold to homogeneity in 8% yield from liver with a four-step six-column procedure, which consists of a concanavalin A-Sepharose/Blue A-agarose coupled step, a DEAE-Sephacel/octyl-Sepharose coupled step, CM-Sepharose chromatography and gel-permeation chromatography. Although more than 90% of GAS activity had a pI of greater than 7.5, other forms with pI values of 5.8, 5.3, 4.7 and less than 4.0 were also present. The pI greater than 7.5 form of GAS had a native molecular mass of 63 kDa. SDS/polyacrylamide-gel-electrophoretic analysis resulted in two polypeptide subunits of molecular mass 47 and 19.5 kDa. GAS was active towards disaccharide substrates derived from heparin [O-(beta-glucuronic acid 2-sulphate)-(1----4)-O-(2,5)-anhydro[1-3H]mannitol 6-sulphate (GSMS)] and chondroitin 6-sulphate [O-(beta-glucuronic acid 2-sulphate-(1----3)-O-(2,5)-anhydro[1-3H]talitol 6-sulphate (GSTS)]. GAS activity towards GSMS and GSTS was at pH optima of 3.2 and 3.0 respectively with apparent Km values of 0.3 and 0.6 microM respectively and corresponding Vmax values of 12.8 and 13.7 mumol/min per mg of protein respectively. Sulphate and phosphate ions are potent inhibitors of enzyme activity. Cu2+ ions stimulated, whereas EDTA inhibited enzyme activity. It was concluded that GAS is required together with a series of other exoenzyme activities in the lysosomal degradation of glycosaminoglycans containing glucuronic acid 2-sulphate residues.

scholarly journals Rapid and simultaneous purification of aflatoxin B1, zearalenone and deoxynivalenol using their monoclonal antibodies and magnetic nanoparticles

2021 ◽  
Author(s):  
Hyuk-Mi Lee ◽  
Hwan-Goo Kang
Keyword(s):  
Monoclonal Antibodies ◽  
Magnetic Nanoparticles ◽  
Aflatoxin B1 ◽  
Animal Feed ◽  
Immunoaffinity Chromatography ◽  
The Novel ◽  
Buffer Solution ◽  
Purification Method ◽  
Recovery Rates ◽  
Single Spike

AbstractTo develop a new simple and simultaneous purification method for mycotoxins in feeds and grains, magnetic nanoparticles (MNPs) conjugated with monoclonal antibodies (mAbs) against mycotoxins were used to separate aflatoxin B1 (AFB1), zearalenone (ZEA) and deoxynivalenol (DON). For a single spike of each mycotoxin into the buffer solution (16% MeOH in PBS), mean recoveries were 93.1–95.0% for AFB1 (5–20 ng/mL spiked), 87.2–96.0% for ZEA (125–500 ng/mL spiked) and 75.2–96.9% for DON (250–1,000 ng/mL spiked) by HPLC and ELISA. Recoveries of AFB1 (20 ng/mL) and ZEA (500 ng/mL) simultaneously spiked into the buffer solution were 87.0 and 99.8%, respectively. Recovery rates of AFB1/DON and DON/ZEA spiked simultaneously were 86.2%/76.6% and 92.0%/86.7%, respectively, at concentrations of 20 ng/mL AFB1, 500 ng/mL ZEA, and 1,000 ng/mL DON. Recoveries using the novel mAb–MNP conjugated system in a buffer solution simultaneously spiked with AFB1, ZEA and DON were 82.5, 94.6 and 73.4%, respectively. Recoveries of DON in animal feed were 107.7–132.5% at concentrations of 250–1,000 ng/g spiked in feed. The immunoaffinity chromatography (IAC) clean-up method was compared with the purification method using novel mAb–MNP. After fortification of animal feed with AFB1 (5, 10 and 20 ng/g feed) and ZEA (125, 250 and 500 ng/g feed), AFB1 and ZEA were purified using both the methods. In the case of the novel mAb-MNP conjugated system, mean recoveries for AFB1 were 89.4, 73.1 and 88.3% at concentrations of 5, 10 and 20 ng/g feed, respectively. For ZEA, mean recoveries were 86.7, 85.9 and 79.1% at concentrations of 125, 250 and 500 ng/g, respectively. For IAC purification, recoveries were 42.9–45.1% for AFB1 and 96.8–103.2% for ZEA. In conclusion, the present purification method using monoclonal antibodies conjugated to MNPs can be used for simple and simultaneous purification of mycotoxins from feed and maize.

scholarly journals Correlations between retention volume and molecular size parameters in gel permeation chromatography of small molecules

1975 ◽  
Vol 28 (1) ◽  
pp. 189 ◽  
Author(s):  
RA Shanks
Keyword(s):  
Molecular Weight ◽  
Small Molecules ◽  
Organic Molecules ◽  
Retention Volume ◽  
Molecular Size ◽  
Gel Permeation Chromatography ◽  
Molar Volumes ◽  
Small Organic Molecules ◽  
Molecular Dimension ◽  
Gel Permeation

Gel permeation columns of Bio Beads S-X8 have been used to provide separation of oligomers and other small organic molecules. Results show successful separations up to molecular weight c. 600. The retention times of compounds have been correlated with the largest molecular dimension of the molecules and also with molar volumes.

scholarly journals Investigation of the Microcharacteristics of Asphalt Mastics under Dry–Wet and Freeze–Thaw Cycles in a Coastal Salt Environment

Materials ◽  
2019 ◽  
Vol 12 (16) ◽  
pp. 2627 ◽  
Author(s):  
Qinling Zhang ◽  
Zhiyi Huang
Keyword(s):  
Salt Spray ◽  
Molecular Size ◽  
Gel Permeation Chromatography ◽  
Surface Phenomenon ◽  
High Concentration ◽  
Force Microscopy ◽  
Freeze Thaw ◽  
Two Factors ◽  
Asphalt Mastics ◽  
Styrene Butadiene

In the coastal areas of southeastern China, high temperatures and humidity in the summer and microfreezing in the winter, as well as a high concentration of salt spray in the environment, seriously deteriorate the durability of asphalt mixtures. Therefore, the microcharacteristics of asphalt mastics (asphalt mixed with mineral filler) under the effect of chlorine salt and “dry–wet and freeze–thaw” (DW-FT) cycles were investigated by Fourier-transform infrared (FTIR) spectroscopy, gel permeation chromatography (GPC), and atomic force microscopy (AFM) techniques. Two factors, including asphalt mastic types (base and styrene-butadiene-styrene (SBS)-modified mastics) and numbers of DW-FT cycles, were considered based on the natural environment. Regression functions were established to explore the relationship between the FTIR, GPC, and AFM indexes. The results indicate that there were no chemical reactions between the asphalt and filler because the infrared spectrum of the base and SBS-modified mastics were similar. With the increase of the salt “DW-FT” cycle numbers, the sulfoxide index and large molecular size ratios ( L M S % ) increased, and the surface roughness ( R q and R a ) of the morphology decreased, as illustrated by a flatting mastics surface phenomenon in the AFM test. Regression analysis confirmed that there was a high correlation between the FTIR, GPC, and AFM indexes, and formation of the bee structures was closely related to the long chain index. The SBS-modified mastics had a better antiaging performance with a lower increase in the sulfoxide index after the salt “DW-FT” cycles in the coastal environment.

scholarly journals Fucose content of keratan sulphates from bovine articular cartilage

1991 ◽  
Vol 273 (2) ◽  
pp. 307-310 ◽  
Author(s):  
G H Tai ◽  
G M Brown ◽  
H G Morris ◽  
T N Huckerby ◽  
I A Nieduszynski
Keyword(s):  
Molecular Weight ◽  
Articular Cartilage ◽  
Molecular Size ◽  
Gel Permeation Chromatography ◽  
Repeat Sequence ◽  
Keratan Sulphate ◽  
Bovine Articular Cartilage ◽  
Gel Permeation ◽  
Fucose Content ◽  
Galactose Content

Alkaline-borohydride-reduced keratan sulphate chains were isolated from bovine articular cartilage (6-8-year-old animals). Nine keratan sulphate fractions of increasing molecular weight were prepared by gel-permeation chromatography on a calibrated column of TSK 30 XL. The samples were analysed for fucose and galactose contents (% by wt. of keratan sulphate) and fucose/galactose ratio. The fucose content increased with molecular size, but the galactose content remained constant. It was concluded that the alpha(1→3)-linked fucose [Thornton, Morris, Cockin, Huckerby, Nieduszynski, Carlstedt, Hardingham & Ratcliffe (1989) Biochem. J. 260, 277-282] was located within the poly-N-acetyl-lactosamine repeat sequence of articular-cartilage keratan sulphate.

VON WILLEBRAND FACTOR (VWF) ANTIGENS IN URINE

1987 ◽  
Author(s):  
A M V Silveira ◽  
B Hessel ◽  
B Blombäck
Keyword(s):  
Monoclonal Antibodies ◽  
Von Willebrand Factor ◽  
High Performance ◽  
Molecular Size ◽  
Von Willebrand Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Normal Urine ◽  
Von Willebrand ◽  
Willebrand Factor

Human urine was analyzed using a sensitive enzyme linked immunosorbent assay (ELISA) for von Willebrand factor (VWF) antigen. Urine of healthy persons contained VWF immunoreactivity. In the urine of a patient with severe von Willebrand disease, the VWF antigen was only detectable after intravenous infusion of VWF-Factor VIII concentrate. The VWF antigen in normal urine was analyzed by gel permeation high performance liquid chromatography (HPLC) and gel electrophoresis combined with immunoblotting. These analyses revealed three immunoreactive components of Mr 350 kDa, 60 kDa, and 20 kDa, respectively, the 60 kDa being the major component. Monoclonal antibodies of known specificity to VWF molecule were used in ELISA and immunoblotting to analyze urinary VWF. The three components reacted with an antibody to the central part of VWF, which is called fragment I, and contains the binding site for collagen. No significant immunoreac-tion was observed with monoclonal antibodies to the Nor C-terminal portions of VWF.VWF derivatives of molecular size similar to the largest urinary antigens were also observed in normal plasma. However, there is not an obvious relationship between these plasma forms and the products in urine since reduction of plasma and urine yields different products.These results indicate that VWF antigens excreted in normal urine are most likely fragments of VWF produced by limited degradation in vivo. This degradation preserves the central part of VWF molecule, the one which reacts with the antibody that blocks the binding of VWF to collagen.

scholarly journals Genome Structure ofBacillus cereustsu1 and Genes Involved in Cellulose Degradation and Poly-3-Hydroxybutyrate Synthesis

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Hui Li ◽  
Suping Zhou ◽  
Terrance Johnson ◽  
Koen Vercruysse ◽  
Ouyang Lizhi ◽  
...  
Keyword(s):  
High Performance ◽  
Peptide Mapping ◽  
Cellulose Degradation ◽  
Genome Structure ◽  
Molecular Size ◽  
Gel Permeation Chromatography ◽  
Mass Spectrometry Analysis ◽  
Cellulolytic Activity ◽  
Spectrometry Analysis ◽  
Genes Encoding

In previous work, we reported on the isolation and genome sequence analysis ofBacillus cereusstrain tsu1 NCBI accession number JPYN00000000. The 36 scaffolds in the assembled tsu1 genome were all aligned withB. cereusB4264 genome with variations. Genes encoding for xylanase and cellulase and the cluster of genes in the poly-3-hydroxybutyrate (PHB) biosynthesis pathway were identified in tsu1 genome. The PHB accumulation inB. cereustsu1 was initially identified using Sudan Black staining and then confirmed using high-performance liquid chromatography. Physical properties of these PHB extracts, when analyzed with Raman spectra and Fourier transform infrared spectroscopy, were found to be comparable to the standard compound. The five PHB genes in tsu1(phaA,phaB,phaR,phaC,andphaP)were cloned and expressed with TOPO cloning, and the recombinant proteins were validated using peptide mapping of in-gel trypsin digestion followed by mass spectrometry analysis. The recombinantE. coliBL21 (DE3) (over)expressingphaCwas found to accumulate PHB particles. The cellulolytic activity of tsu1 was detected using carboxymethylcellulose (CMC) plate Congo red assay and the shift towards low-molecular size forms of CMC revealed by gel permeation chromatography in CMC liquid culture and the identification of a cellulase in the secreted proteome.

Immunoaffinity chromatography of bovine FSH using monoclonal antibodies

1987 ◽  
Vol 115 (2) ◽  
pp. 283-288 ◽  
Author(s):  
K. F. Miller ◽  
R. A. Goldsby ◽  
D. J. Bolt
Keyword(s):  
Monoclonal Antibodies ◽  
Pituitary Hormones ◽  
Immunoaffinity Chromatography ◽  
Cross Reactivity ◽  
Scatchard Analysis ◽  
Spleen Cells ◽  
Chromatographic Techniques ◽  
One Step ◽  
Kappa Light Chains ◽  
Pituitary Homogenate

ABSTRACT Bovine FSH (bFSH) was used to immunize BALB/c mice. Spleen cells were fused to the SP 2/0 cell line to produce hybridomas that secreted monoclonal antibodies to bFSH. One of these antibodies (USDA-bFSH-MC28) was extensively characterized and found to be a gamma 1 with kappa light chains, having extremely low cross-reactivity with other bovine pituitary hormones and with ovine and porcine FSH. The dissociation constant as measured by Scatchard analysis was 4·3 nmol/l, and proved to be in a very useful range for affinity chromatography. In an essentially one-step immunoaffinity chromatography procedure, bFSH was easily isolated in a single chromatographic step from crude anterior pituitary homogenate with better yield and with the same purity as classical chromatographic techniques. J. Endocr. (1987) 115, 283–288

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