Preparation of a purified adenovirus diagnostic antigen by gel filtration

1969 ◽  
Vol 15 (10) ◽  
pp. 1167-1171 ◽  
Author(s):  
John R. Polley ◽  
T. S. Webb
Keyword(s):  
Tissue Culture ◽  
Gamma Irradiation ◽  
Gel Filtration ◽  
Culture Fluid ◽  
Pressure Filtration ◽  
Routine Production ◽  
Diagnostic Antigen ◽  
Sepharose 4B ◽  
Molecular Sieving

Gel filtration has been investigated as a method for purifying adenovirus antigens in tissue culture fluid. It was found that by molecular sieving through the agarose Sepharose 4B, most of the infectivity, hemagglutinins, and nonspecific complement-fixing (CF) activity could be separated from a second fraction which contained some hemagglutinin but only a low titer of viable virus and from a third fraction which contained the group-specific complement-fixing antigen but was relatively free of hemagglutinin, viable virus, and nonspecific reactants.For the routine production of a noninfective, purified group-specific diagnostic antigen for adenovirus, the bulk infected fluid is inactivated by heating at 56 °C for 3 hours at pH 5.5 or by gamma irradiation with 2.5 megarads, concentrated about 20-fold by pervaporation or by pressure filtration, purified by gel filtration using Sepharose 4B, concentrated to desired potency, and then lyophilized for stable storage.

PREPARATION OF A STABLE NONINFECTIVE GROUP-SPECIFIC ADENOVIRUS DIAGNOSTIC ANTIGEN

1964 ◽  
Vol 10 (5) ◽  
pp. 747-751
Author(s):  
John R. Polley ◽  
Muriel M. Guerin
Keyword(s):  
Tissue Culture ◽  
Culture Fluid ◽  
Copper Hydroxide ◽  
Diagnostic Antigen ◽  
Sintered Glass ◽  
Antigen Present ◽  
Glass Filter ◽  
Sintered Glass Filter ◽  
Serological Reactivity ◽  
Infected Cells

In the preparation of a group-specific diagnostic antigen for adenoviruses, various procedures for the concentration and purification of the antigen present in the tissue culture fluid of specifically infected cells were investigated. It was found possible to prepare an antigen of increased potency and specificity by passage of the fluid through an ultrafine sintered glass filter, followed by coprecipitation of the antigen with copper hydroxide. This antigen was rendered noninfective by treatment with 0.01% formaldehyde at pH 7 at 37 °C for 4 days or by heating at 56 °C at pH 5.5 for 3 hours. After destruction of the infectivity the antigen was stabilized for storage by lyophilization. This antigen, after such treatment, still retained its serological reactivity.

scholarly journals HUMAN AMYLOID PROTEIN: CHEMICAL VARIABILITY AND HOMOGENEITY

1971 ◽  
Vol 19 (1) ◽  
pp. 1-15 ◽  
Author(s):  
M. HARADA ◽  
C. ISERSKY ◽  
P. CUATRECASAS ◽  
D. PAGE ◽  
H. A. BLADEN ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amyloid Fibril ◽  
Gel Filtration ◽  
Protein S ◽  
Amyloid Protein ◽  
X Ray Diffraction ◽  
Amyloid Proteins ◽  
X Ray ◽  
Chemical Variability ◽  
Sepharose 4B

The morphology of the fibril of amyloid derived from different individuals is similar, but occasionally significant differences are noted. All human amyloid filaments have a "β-pleated sheet" conformation as revealed by x-ray diffraction, and those examined after orientation show a "cross-β" pattern. All amyloid fibril concentrates studied so far can be fractionated to obtain the major amyloid protein component(s) by sequential gel filtration with 5 M guanidine-HCl in 1 N acetic acid on Sepharose 4B and Sephadex G-100 or G-75 columns with the removal of over 28% of proteins representing minor constituents. The major amyloid protein(s) obtained from the spleen and/or liver of six patients is found to contain tryptophan, to be deficient in hydroxylysine and hydroxyproline and usually at least one commonly occurring amino acid and to have a high content of dicarboxylic acid and short chain amino acids and unreactive (blocked) NH2-terminal groups or aspartic acid-asparagine (Asx). However, the amyloid protein(s) from each individual differs from that of the others in molecular weight, in amino acid composition and in the presence or absence of specific tryptic peptides. Amyloid protein(s) from the liver and spleen of the same individual is identical. No chemical characteristics distinguish amyloid proteins derived from cases classified clinically as "primary" from those classified as "secondary." There is a striking chemical similarity between amyloid proteins and the NH2-terminal variable fragment of the light and heavy chain of immumoglobulin proteins.

Purification Of Vasoactive Peptides From Human Fibrinogen Degraded By Human Leucocyte Elastase

1981 ◽  
Author(s):  
R Wallin ◽  
M Belew ◽  
K Ohlsson ◽  
T Saldeen
Keyword(s):  
Affinity Chromatography ◽  
Vascular Permeability ◽  
Gel Filtration ◽  
Molar Ratio ◽  
Significant Activity ◽  
Small Peptides ◽  
Human Fibrinogen ◽  
Leucocyte Elastase ◽  
Pm 10 ◽  
Sepharose 4B

The presence of leucocytes around extravascular fibrin deposits suggests that the leucocyte elastases might be partly responsible for the extravascular degradation of fibrin. Our previous studies have shown that the degradation of fibrin(ogen) by plasmin leads to the release of 2 small peptides which markedly increase vascular permeability and induce oedema e.g. in the lungs. The results of this investigation show that small peptides released from fibrinogen after degradation by leucocytes elastases also increase vascular permeability.Human fibrinogen (Kabi, Grade L) was made plasminogenfree by affinity chromatography on Lysine-Sepharose 4B prior to use. The human leucocyte elastases were isolated from extracts of lysosome-like granules of human leukaemic myeloid cells by a combination of gel filtration, affinity chromatography and preparative agarose gel electrophoresis. The fibrinogen (0.5 %) and the leucocyte elastases (in a molar ratio of 100:1) were incubated together for 48 h at +37°C and at pH 8.5. The mixture was then cooled to +4°C to stop the lysis and ultrafiltrated on a DIAFLO PM 10 membrane until the retentate was approximately 10 % of the starting volume. The peptides in the diffusate accounted for about 20 % of the starting material as estimated from absorbance measurements at 280 nm. The diffusate was concentrated by lyophilization and fractionated by chromatography on a column of Bio-Gel P-6. At least 8 fractions were obtained of which only two showed a significant activity in their ability to increase vascular permeability in rat skin. The active peptides in these two fractions were further purified to homogeneity by column zone electrophoresis at various pHs and their amino acid compositions established.

IDENTIFICATION AND PURIFICATION OF A NEW PROTEIN FROM WATER-SOLUBLE EXTRACTS OF HUMAN ADRENAL GLAND

1979 ◽  
Vol 82 (3) ◽  
pp. 383-NP ◽  
Author(s):  
M. A. AL-AWQATI ◽  
Y. B. GORDON ◽  
T. CHARD
Keyword(s):  
Molecular Weight ◽  
Adrenal Gland ◽  
Gel Filtration ◽  
Water Soluble ◽  
Double Immunodiffusion ◽  
Molecular Weights ◽  
Physicochemical Methods ◽  
Two Peaks ◽  
Sepharose 4B ◽  
New Protein

An homogenate of human foetal adrenal gland was subjected to negative immunoabsorption by column chromatography using anti-whole human serum coupled to Sepharose 4B. Two peaks were eluted and used to immunize rabbits. The antisera produced were absorbed and tested for specificity by double immunodiffusion. Two antigens, which appeared to be specific to the adrenal gland, were identified having molecular weights of 25 000 and 65 000 as determined by gel filtration. The lower molecular weight antigen was isolated by physicochemical methods and found to be a protein. The amino acid composition is reported.

scholarly journals BIOCHEMICAL STUDY OF CELLULAR ANTIGEN-ANTIBODY REACTION IN TISSUE CULTURE

1960 ◽  
Vol 112 (2) ◽  
pp. 249-255 ◽  
Author(s):  
Akira Tokuda ◽  
Hideo Hayashi ◽  
Kinishiro Matsuba
Keyword(s):  
Tissue Culture ◽  
Protease Activity ◽  
Biochemical Study ◽  
Culture Fluid ◽  
Partial Purification ◽  
Trichloracetic Acid ◽  
Heat Stable ◽  
Antibody Reaction ◽  
Antigen Antibody ◽  
Cellular Antigen

Decrease in the protease activity of the culture fluid observed at later stages of the antigen-antibody reaction is believed to be due to the release of an inhibitor by the cells. The inhibitor was submitted to partial purification: it is heat-stable, non-precipitated by trichloracetic acid and non-dialyzable. It inhibits certain cellular and tissue proteases and papain but is inactive against trypsin. It is suggested that the balance between protease and anti-protease released may determine the intensity, extent, and duration of certain sensitization phenomena.

scholarly journals Immunosuppressive activity of a subline of the mouse EL-4 lymphoma. Evidence for minute virus of mice causing the inhibition.

1976 ◽  
Vol 143 (1) ◽  
pp. 187-205 ◽  
Author(s):  
G D Bonnard ◽  
E K Manders ◽  
D A Campbell ◽  
R B Herberman ◽  
M J Collins
Keyword(s):  
Tissue Culture ◽  
Culture Fluid ◽  
Inhibitory Factor ◽  
Minute Virus Of Mice ◽  
G Cells ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Lymphocyte Cultures ◽  
Minute Virus ◽  
Kilham Rat Virus

Filtered culture fluids from the early in vitro passages of a subline of the C57BL/6 mouse EL-4 lymphoma, EL-4(G-), were strongly inhibitory for BABL/c vs. C57BL/6 mixed lymphocyte cultures (MLC). The inhibitory activity could be preserved by storage at -75 degrees C or 4 degrees C, thus allowing its further characterization. The inhibitory factor was particulate (nondialyzable, sedimentable at 100,000 g for 1 h), very small (recovered after 0.10 mum filtration), sensitive to UV irradiation, but heat stable (56 degrees C, 1 h) and resistant to chloroform. It was infectious, since later, noninhibitory passages of EL-4(G-) tissue culture cells became strongly inhibitory upon inoculation with the culture fluid. This data was consistent with the inhibitory factor being an infectious virus. Virus analysis by mouse antibody production tests revealed that viruses were indeed present in EL-4(G-) ascites cells and in the culture fluid, and not in a late passage of EL-4(G-) tissue culture cells which were not inhibitory. Neutralization of the inhibitory factor was achieved by pretreatment with ascitic fluid or with the sera raised against those (EL-4(G-)-derived materials which contained viruses. Mouse reference immune sera against minute virus of mice (MVM) completely neutralized the inhibitory factor in the culture fluid or in EL-4(G-) ascites cells. Two prototype MVM strains, and one Kilham rat virus preparation, did not inhibit the mouse MLC. Thus, the possibility exists that a variant of MVM, or an unidentified virus, has been grown and selected for in EL-4(G-) cells and recognized, due to its immunosuppressive characteristics. In any event, immunosuppression by EL-4(G-) cells was not mediated by the tumor cells, their metabolic products, or associated endogenous type C viruses, but by an exogenous virus, most likely a variant MVM with immunosuppressive characteristics. This adds weight to a parallel observation from our laboratory on the immunosuppressive effects of Kilham rat virus in rat lymphocyte cultures.

scholarly journals Extra Purified Exosomes from Human Placenta Contain an Unpredictable Small Number of Different Major Proteins

2019 ◽  
Vol 20 (10) ◽  
pp. 2434 ◽  
Author(s):  
Evgeniya E. Burkova ◽  
Alina E. Grigor’eva ◽  
Dmitrii V. Bulgakov ◽  
Pavel S. Dmitrenok ◽  
Valentin V. Vlassov ◽  
...  
Keyword(s):  
Interleukin 1 ◽  
Gel Filtration ◽  
Normal Pregnancy ◽  
Maldi Mass Spectrometry ◽  
2D Electrophoresis ◽  
Cytoplasmic Actin ◽  
Sds Page ◽  
Two Peaks ◽  
Sepharose 4B ◽  
Different Sources

Exosomes are nanovesicles (30–100 nm) containing various RNAs and different proteins. Exosomes are important in intracellular communication, immune function, etc. Exosomes from different sources including placenta were mainly obtained by different types of centrifugation and ultracentrifugations and were reported to contain from a few dozen to thousands of different proteins. First crude exosome preparations from four placentas (normal pregnancy) were obtained here using several standard centrifugations but then were additionally purified by gel filtration on Sepharose 4B. Individual preparations demonstrated different gel filtration profiles showing good or bad separation of exosome peaks from two peaks of impurity proteins and their complexes. According to electron microscopy, exosomes before gel filtration contain vesicles of different size, ring-shaped structures forming by ferritin and clusters of aggregated proteins and their complexes. After filtration through 220 nm filters and gel filtration exosomes display typically for exosome morphology and size (30–100 nm) and do not contain visible protein admixtures. Identification of exosome proteins was carried out by MS and MS/MS MALDI mass spectrometry of proteins’ tryptic hydrolyzates after their SDS-PAGE and 2D electrophoresis. We have obtained unexpected results. Good, purified exosomes contained only 11–13 different proteins: CD9, CD81, CD-63, hemoglobin subunits, interleukin-1 receptor, annexin A1, annexin A2, annexin A5, cytoplasmic actin, alkaline phosphatase, serotransferin, and probably human serum albumin and immunoglobulins. We assume that a possible number of exosome proteins found previously using crude preparations may be very much overestimated. Our data may be important for study of biological functions of pure exosomes.

Plasma protein recovery from spent tissue culture fluid

1991 ◽  
Vol 13 (4) ◽  
pp. 261-264 ◽  
Author(s):  
P. K. Ng ◽  
C. Figueroa ◽  
G. Mitra
Keyword(s):  
Tissue Culture ◽  
Plasma Protein ◽  
Culture Fluid ◽  
Protein Recovery

scholarly journals Structural studies on the glycoproteins from bovine cervical mucus

1978 ◽  
Vol 173 (3) ◽  
pp. 941-947 ◽  
Author(s):  
G P Roberts
Keyword(s):  
Physical Properties ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Cervical Mucus ◽  
Cysteine Residues ◽  
Structural Studies ◽  
Disulphide Bonds ◽  
Structural Differences ◽  
Sepharose 4B

The depolymerization of bovine cervical glycoprotein resulting from cleavage of disulphide bonds. Pronase digestion and both procedures sequentially was assessed by using gel filtration. Cleavage of disulphide bonds followed by Pronase digestion produced more extensive depolymerization than did either treatment alone, and gel filtration of the products resulted in two major peaks of glycosylated material on Sepharose CL-2B and Sepharose 4B. The glycopolypeptides in both peaks had similar sugar and sulphate compositions, but they migrated to different extents on gel electrophoresis. Electrophoretic studies indicated that both glycopolypeptides were derived from the same glycoprotein molecule and not from a mixture of two similar glycoproteins. Pronase digestion of glycoproteins in which the disulphide bonds had been labelled with iodo-[1-14C]acetamide revealed that most of the cysteine residues were situated in regions susceptible to Pronase. The results show the presence of two types of structural regions in bovine cervical glycoprotein, namely ‘naked’ peptide or non-glycosylated regions and glycopolypeptide subunit regions in which glycopolypeptides of two different sizes predominate. Comparison of the cervical glycoproteins isolated from mucus secreted during oestrus and pregnancy, by the methods outlined above, did not reveal any structural differences in the glycoproteins to explain the different physical properties of the mucus secreted under these conditions.

Sign in / Sign up

Export Citation Format

Share Document