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Beaded agarose affinity chromatography of bovine fibroblast interferon
1988
Allen, G.K. | Rosenquist, B.D.
Bovine fibroblast interferon (BoF-IFN), produced in bovine embryonic kidney cell cultures by priming and infection with bluetongue virus, was partially purified by controlled pore glass chromatography. The partially purified B0F-IFN then was subjected to beaded agarose affinity chromatography. The IFN eluted by affinity chromatography in 2 distinct fractions-1 after the addition of 1M NaCl and the other one after the addition of 1.5M NaCl containing 50% ethylene glycol. Analysis of fractions by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis revealed a broad molecular weight range (14,900 to 27,900) for the IFN eluted by 1M NaCl, and 2 discrete molecular weight ranges (16,000 to 19,500 and 28,300 to 34,000) for IFN eluted by 1.5M NaCl containing 50% ethylene glycol. The specific activity of the IFN eluted with 1.5M NaCl containing ethylene glycol was 2.85 X 10(6) U/mg of protein, compared with 5.7 X 10(5) U/mg of protein in the controlled pore glass-purified IFN.
اظهر المزيد [+] اقل [-]Partial purification and characterization of bovine fibroblast interferon
1988
Allen, G.K. | Grothaus, G.D. | Rosenquist, B.D.
Bovine fibroblast interferon (BoF-IFN), produced in primary bovine embryonic kidney cell cultures after priming and infection with bluetongue virus, was purified by controlled pore glass (CPG) chromatography to a specific activity of 10(6) U/mg of protein, with 40% recovery of the original activity. The crude IFN was concentrated more than sevenfold during purification. This proved to be a relatively simple, practical method of obtaining sufficient quantities of partially purified natural BoF-IFN for further studies. The CPG-purified BoF-IFN was further concentrated by sequential ultrafiltration and was analyzed by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis (SDS-PAGE). Interferon, recovered from denaturing conditions either by dialysis against phosphate-buffered saline solution or by dilution in cell culture medium containing 10% fetal bovine serum, migrated as a single stainable protein with molecular weight of 21,000 on analytic SDS-PAGE gels. Recovered IFN activity from preparative SDS-PAGE totalled 8.7% of that applied. Attempts to further pruify CPG-purified BoF-IFN by zinc chelate affinity chromatography were unsuccessful.
اظهر المزيد [+] اقل [-]Isolation and characterization of epizootic hemorrhagic disease virus from sheep and cattle in Colorado
1988
Thompson, L.H. | Mecham, J.O. | Holbrook, F.R.
Epizootic hemorrhagic disease virus was isolated from cattle and sheep in northeastern Colorado during July and August 1984. The isolates were identified as serotype 2 by plaque-inhibition serotyping, genome electropherotyping, and protein analysis.
اظهر المزيد [+] اقل [-]Quality control criteria for quantitative enzyme-linked immunosorbent assay of porcine immunoglobulins A and M
1988
Kelley, K.W. | Kleiss, A.J. | Brief, S.
Using radioimmunoassay methods, quality control criteria were applied to monoclonal antibodies produced to measure porcine immunoglobulins by quantitative ELISA. Porcine IgM and IgA were purified to homogeneity and were used to produce murine hybridomas that secreted antibodies against IgM, IgA, and immunoglobulin light chains. A competitive ELISA was developed to measure IgM, and a sandwich ELISA was to quantify IgA in serum and colostrum. Both ELISA were tested for specificity, accuracy, sensitivity, and precision. Monoclonal antibodies were specific for porcine IgM or IgA in serum and colostrum, and competitive and sandwich ELISA fulfilled all validation criteria.
اظهر المزيد [+] اقل [-]Identification of viral antigens that induce antibody responses on exposure to coronaviruses
1988
Ingersoll, J.D. | Wylie, D.E.
Various techniques were used to look for protective, non-cross-reactive antibodies in the sera of cats exposed to virulent feline infectious peritonitis virus (FIPV). Antibodies reactive with feline enteric coronavirus (FECV) from FIPV-exposed cats were adsorbed by several passages over an FECV-Sepharose column. In an ELISA against FECV and FIPV, the activity against both viruses was removed at the same rate; thus, no FIPV-specific antibodies could be identified. By gel electrophoresis-derived ELISA, the responses of cats surviving FIPV exposure were compared with those of cats succumbing to FIPV exposure to determine whether survival could be correlated with an antibody response against a particular virus protein. Results indicated that both groups responded in the same way to the matrix envelope protein and nucleocapsid proteins. Even though the response to peplomer in each group was weak, the survivor group responded better to this protein. Furthermore, the response of this group to the peplomer protein had the highest correlation with virus neutralization titer.
اظهر المزيد [+] اقل [-]Dermal dysplasia characterized by collagen disorder-related skin fragility in a cow
1988
Kawaguchi, T. | Fukazawa, H. | Naito, Y. | Okada, K.
Holstein cow 1 was examined because of skin fragility and delayed healing of skin wounds, which were markedly exacerbated around the time of parturition. A skin biopsy sample was obtained, and light microscopy revealed irregular deposition of thin collagen fibers in a dermal matrix. Although diffuse inflammation did not occur, the number of plump fibroblasts was increased. Electron microscopy revealed poor construction of collagen fibrils in the dermal matrix. Biochemical analysis of the dermis revealed a normal amount of collagen and uronic acid, but sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveled an increased proportion of soluble alpha-, beta-, and gamma-collagen chains of normal molecular weights. Neither procollagen nor its intermediates devoid of amino- or carboxy-terminal extension peptide were observed. Dermal collagen from cow 1 was more soluble in a neutral salt solvent, 0.5M acetic acid, and the acid containing pepsin than was dermal collagen from healthy cow 2. The peptic digestion profile of dermis from cow 1 revealed a lowered degree of intermolecular cross-linking and destabilization of helical structure in the dermis collagen. The extrahelical peptic cleavage of collagen before cyanogen bromide digestion resulted in release of more fragments derived from carboxy-terminal part of alpha1 chains in dermis of cow 1 than in dermis of healthy cow 2.
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