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1.
《Biochimica et Biophysica Acta.Protein Structure》1976,420(2):246-257
IgG separated from an antiserum to estradiol was coupled under various experimental conditions to Sepharose activated either with CNBr or by conversion into a long-armed derivative (the N-hydroxysuccinimide ester). The conjugates were characterized by measurement of the binding parameters, in order to evaluate separately the loss of sites and the loss of affinity. The cross-reactivity with estriol and estrone was measured to obtain information on the occurrence of structural alterations of the antibody site.The results show that the loss of immunoreactivity varies in extent (from 95% to less than 10%) and in nature (loss of sites or of affinity or a combination of both effects) depending on the coupling conditions.The use of a hydrocarbon extension to keep the protein distant from the matrix does not prevent the loss of active sites but is effective in safeguarding the affinity of the residual sites. The loss of sites can be substantially reduced by coupling at a pH value around neutrality and by keeping the protein/matrix mass ratio low. At a coupling pH of 6.4 and at a mass ratio of 0.1–0.2 nmol IgG/mg of Sepharose, the antibodies were insolubilized with a negligible loss of sites and affinity; on increasing the mass ratio (up to 10 nmol IgG/mg Sepharose) there is a progressive loss of sites accompanied by a substantial lowering of the affinity of the residual sites.On the basis of the above-mentioned findings, the nature of the effects occurring when antibodies are transferred from solution onto a solid matrix is discussed. 相似文献
2.
A method of purifying the naturally occurring antibody to alpha-galactoside moiety (anti-alpha-Gal) in human plasma by a single-step affinity chromatography on cross-linked guar galactomannan (CLGG) or agarose (Sepharose 4B) is described. IgG nature of the two preparations, as revealed by agar gel diffusion, as well as their preference for alpha-anomer of galactose, as revealed in inhibition of their agglutination of trypsinized rabbit erythrocytes by sugars, identified them with anti-alpha-Gal. The antibody binding capacity of Sepharose 4B was only a third of that of CLGG. Both gels showed similar dependence on ionic strength for binding. The pH optimum for binding of anti-alpha-Gal to CLGG was 8.0. Significantly anti-alpha-Gal binding to Sepharose was unaffected by CNBr activation and ligand coupling to the gel, thus warning that contaminating plasma could introduce artifacts in agarose-based chromatography of human tissue biomolecules. 相似文献
3.
Epoxy Sepharose, an activated affinity matrix which has been used for immobilisation of carbohydrates has been tried for immobilisation
of proteins. Under normal conditions of coupling at neutral or alkaline pH proteins do not couple to epoxy Sepharose. However,
a very high salt concentration during coupling allows the binding of proteins to epoxy Sepharose at a pH as low as 8.5. Increasing
ionic strength and/or pH facilitates the binding. The bioactivity of the proteins is not destroyed by the immobilisation.
This matrix, unlike cyanogen bromide-Sepharose, retains its ability to bind albumin by 80–90% even after 60 days of storage
in aqueous suspension at 4°C. Its capacity to bind proteins is comparable to that of cyanogen bromide-Sepharose. 相似文献
4.
The interaction between immobilized antibodies against human immunoglobulin G (IgG) and the immunoenzyme complex IgG-peroxidase (IgG-P) was studied. The complex was obtained by covalent binding of IgG to peroxidase modified by sodium periodate. Study of the IgG-P binding kinetics and dissociation of the antibody-(IgG-P) complex showed that the antibodies immobilized on Sepharose reversibly interacted with IgG-P, similar to the antigen-antibody reaction in solution. The efficient values of the binding constants for the antibodies binding to Sepharose covalently and through the antigen-antibody bond are (2,2+/-0,5) 10(8) M-1 and (4,2+/-0,2) 10(8) M-1, respectively. The nature of a carrier and the immobilization method used do not significantly affect the rate of the complex binding to the antibodies. The activation energy of the reaction of IgG-P binding to the antibodies immobilized on Sepharose covalently and through the antigen-antibody bond is 7,3 and 4,1 kcal/mole, respectively. A procedure of titration of immobilized antibodies active sites with the antigen-enzyme complex is discussed. 相似文献
5.
Yukiharu Sato Akira Sakurai Nobutaka Takahashi Yeong-Man Hong Yasutsugu Shimonishi Chieko Kitada 《Bioscience, biotechnology, and biochemistry》2013,77(6):1531-1533
A fraction containing IgA (IgA-rich fraction) was prepared from bovine colostrum by anion exchange chromatography using DEAE-Sephadex A-50 and gel filtration on Sephadex G-200. A large amount of IgG1-dimer was found in this fraction, which could not be separated from IgA by repeated gel filtration.The Fc fragment of bovine colostral IgG (IgG-Fc) was prepared from papain digestion mixtures. IgG-Fc was found to be heterogeneous on DEAE-cellulose column chromatography. Two IgG-Fc fractions were obtained, but no antigenic difference was found between them. Anti-IgG-Fc antibodies raised in rabbits by injection of these Fc preparations reacted only with IgG1 and IgG2. An immunoadsorbent (anti-IgG-Fc-Sepharose) was prepared by coupling these anti-IgG-Fc antibodies to CNBr-activated Sepharose 4B.IgA was purified from the IgA-rich fraction by affinity chromatography on anti-IgG-Fc-Sepharose adsorbent. IgG1-dimer was effectively removed by this treatment. The purified sample gave only one precipitin arc characteristic of IgA on immunoelectrophoresis with multiple anti-bovine colostral whey antiserum. A small amount of IgA was found to be adsorbed to the affinity column nonspecifically.When a rabbit was immunized with the purified IgA, besides anti-IgA antibodies, antibodies against the secretory component (SC) were found in the antiserum. This finding leads us to expect that the purified IgA is secretory IgA containing SC. 相似文献
6.
筛选一种高效重组金黄色葡萄球菌蛋白A(SpA)用于制备抗体纯化亲和介质。首先通过基因操作获得金黄色葡萄球菌蛋白A(SpA)的Z结构域单体、二串体、三串体、四串体和五串体基因,将目的基因分别克隆至pET-22b表达载体并转化至大肠杆菌BL21(DE3)感受态细胞,获得不同串联个数的Z结构域基因工程菌,经诱导表达和Ni2+亲和层析纯化得到Z结构域单体和二-五串体蛋白。纯化后的目的蛋白偶联至琼脂糖凝胶作为亲和层析介质,对人免疫球蛋白G(IgG)进行分离纯化。分析比较Z结构域串联体蛋白产量及其偶联的亲和介质对抗体吸附载量的差异。结果表明,构建的Z结构域单体、二串体、三串体、四串体和五串体基因工程菌能有效表达目的蛋白,制备的凝胶亲和介质可特异性吸附人IgG。增加Z结构域串联数,重组蛋白产量和单位摩尔数多聚体蛋白吸附载量获得提高,其中,重组四串体蛋白产量大(160 mg/10 g湿菌体),对抗体的吸附载量高(34.4 mg人IgG/mL胶),更适合作为配基用于亲和层析介质的制备。 相似文献
7.
A procedure is described for coupling wheat germ agglutinin to cyanogen bromide-activated Sepharose to yield a lectin affinity column of high capacity. Covalent linkage of the lectin to the insoluble matrix is carried out in the presence of a mixture of β-1,4-linked N-acetylglucosamine oligosaccharides prepared from chitin. The lectin-affinity column specifically recognizes glycoproteins containing N-acetylglucosamine residues with the capacity of binding 0.6–1.0 mg of ovomucoid per milliliter of gel. The affinity column is stable (as determined by ovomucoid binding) and shows little loss in binding capacity or specificity after repeated usage. Important characteristics for the use of this column to purify glycoproteins are described. 相似文献
8.
Y Yae S Inaba H Sato K Okochi F Tokunaga S Iwanaga 《Biochimica et biophysica acta》1991,1078(3):369-376
A novel thermolabile beta-2 macroglycoprotein ('thermolabile substance' (TLS) or 'Hakata antigen' (HA], which was detected by the precipitating (auto) antibodies of patients with systemic lupus erythematosus, was isolated and characterized. The purification procedure entailed the following steps: isoelectric precipitation in the range between pH 5.2-6.1, hydroxyapatite absorption chromatography, 35% saturated ammonium sulfate precipitation, Sephadex G-200 gel filtration, Pevikon block electrophoresis, lentil lectin affinity chromatography and immobilized rabbit anti-human whole serum IgG column chromatography. Utilizing these procedures, 0.1 mg of HA was purified from 3 1 of pooled human serum. The molecular mass of HA was determined as 650 kDa by Sepharose 4B gel filtration. On SDS-PAGE analysis, HA showed a single band at 35 kDa under reduced conditions and numerous ladder bands between 35 kDa to more than 300 kDa under nonreduced conditions. On analytical ultracentrifugation, HA gave a molecular mass of 520 kDa with a single meniscus and a sedimentation constant of 12.0. The amino acid and carbohydrate analysis of reduced and S-pyridylethylated HA revealed that it contained five residues of hydroxyproline and an N-linked type sugar chain. 相似文献
9.
The effects of charged residues in peptide antigens on the binding characteristics of polyclonal antipeptide antibodies were studied using immunoadsorbents prepared by coupling the antibodies to CNBr-activated Sepharose 4B. Among the antipeptide antibodies, an antibody to the peptide without charged residues showed the most stable interaction with the peptide to the changes in pH. Conversely, the binding affinity of antibodies to the pep-tides with histidine residues having a unique pKa value of 6.0 decreased steeply with pH at around 6.0. The binding affinity of an antibody to the peptide with many charged residues decreased steeply with an increase in the ionic strength (adjusted by NaCl). Since circular dichroism (CD) spectrum measurements indicate that these peptides show disordered structures in the pH range of adsorption measurement, the dependence of peptide-antibody interaction on environmental conditions is attributed to the characteristics of side chains of the peptides. These results indicate that the dependence of the binding affinity of antipeptide antibodies on pH and the ionic strength is dominantly affected by the number and the pKa values of charged residues in the peptides. 相似文献
10.
Under basic conditions β-ecdysone can be covalently linked to the oxirane residue of epoxy-activated Sepharose 6B. Such an ecdysone-modified column matrix retards antibodies to β-ecdysone while permitting, however, the free passage of other antibodies in rabbit antiserum. The bound anti-ecdysone antibodies can subsequently be eluted by a low pH (3.8), high salt (0.5 m) buffer. The utility of such an affinity column for the isolation of ecdysone receptors is discussed. 相似文献
11.
An improved ELISA method for the detection of Staphylococcal Enterotoxin B (SEB) in protein A preparations is presented. Fab fragments were obtained by digestion with papain of anti-SEB IgG bound to SEB immobilized on Sepharose 4B. Anti-SEB and peroxidase-labeled Fab fragments from secondary antibodies were successfully used in a modified ELISA of SEB in protein A preparations. SEB-Sepharose was used repeatedly for the production of anti-SEB Fab fragments by papain digestion without loss of affinity. In addition, for the purification of SEB from crude culture filtrates, an initial step utilizing a combined heat and pH treatment for the removal of significant amounts of contaminating proteins without losses of toxin activity is presented. This pretreatment step yielded positive effects in further downstream processing considering both shortened time and an increase in total recovery. 相似文献
12.
To procure an affinity gel capable of purifying antibody against the cytochrome P450 component of estrogen synthetase (P450ES), we coupled purified P450ES to agarose supports. OUr purpose was to compare two differently-activated agarose gels (Affi-Gel 15 and Tresyl-activated Sepharose) with the same P450ES preparation to compare the efficiency of coupling and the yield of purified antibody. Using supplier-directed protocols to covalently attach P450ES to each of the supports, and identical procedures to bind and elute anti-P450ES, we reached the following conclusions. Tresyl-activated Sepharose bound greater amounts of antigen than Affi-Gel 15 based on the amount of residual antigen after the coupling procedure and the amount of bound antigen detected by an ELISA-type method. However, both ligand-coupled supports yielded comparable amounts of purified anti-P450ES at a 48-fold purification relative to the starting IgG preparation. 相似文献
13.
The shedding of immunoglobulin-binding factor (IBF) has been studied in peripheral blood lymphocytes during 1 h incubation at 37 degrees C. IBF was detectable in the incubating medium on the basis of their ability to bind immunoglobulin G (IgG) specifically. IBF was affinity purified on Sepharose beads coated with bovine IgG, fluoresceinated and identified by their biological activities, i.e., for binding immunoglobulin-binding factor labeled with fluoresceinizothiocyanate (FITC-IBF) to erythrocytes coated with purified antibodies (EAIgG) by fluorometric binding assay. The effect of various pH, temperatures and proteolytic enzymes on the binding properties of FITC-IBF to EAIgG was also studied. We showed that IBF are sensitive to pronase E, higher temperature and pH. 相似文献
14.
Quantitative recovery, selective removal and one-step purification of human parotid and leukemic lysozymes by immunoadsorption 总被引:5,自引:0,他引:5
B J MacKay V J Iacono J M Zuckerman E F Osserman J J Pollock 《European journal of biochemistry》1982,129(1):93-98
Immunoadsorption affinity chromatography was used to isolate and purify human lysozyme. The immunoadsorbent was prepared by coupling sheep anti-(human leukemic lysozyme) IgG to epoxy-activated Sepharose 6B. Lyophilized parotid saliva (21) was resuspended in distilled water (325 ml, 50 mg/ml, w/v) and applied to a column which had a capacity to bind 4.25 mg human enzyme. Non-adsorbed material did not contain lysozyme, as determined by enzymatic and immunological analyses. All lysozyme activity present in the applied sample (1.97 mg) bound to and was desorbed from the column by elution with 0.2 M sodium acetate HCl buffer, pH 1.8. The isolated material was homogeneous as determined by cationic and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, ultracentrifugation, amino acid and amino-terminal analyses, and immunoelectrophoretic analysis. The one-step purification procedure yielded a 1370-fold increase in specific activity. Human lysozyme was also selectively purified by this method from an ammonium sulfate precipitate of the urine of a patient with chronic monocytic leukemia. Amino acid and polyacrylamide gel electrophoretic analyses indicated that the purified enzyme was identical to human lysozyme isolated from leukemic urine by classical biochemical techniques. 相似文献
15.
The antibodies were prepared from antisera of rabbits immunized with bovine serum albumin containing covalently bound p-aminophenyl-beta-D-galactopyranoside (APG) and purified by affinity chromatography on APG-containing ovalbumin immobilized by BrCN-activated Sepharose 4B. The antibodies possessed a selective specificity for APG and interacted with different APG-containing proteins, including APG-containing lysosomal alpha-glucosidase. The purified antibodies are immunoglobulins of G type as was determined from the molecular weights of native and dissociated antibodies and from the immunochemical assays with antibodies against rabbit IgG. 相似文献
16.
C R Mendelson E E Wright C T Evans J C Porter E R Simpson 《Archives of biochemistry and biophysics》1985,243(2):480-491
Aromatase cytochrome P-450 (P-450AROM) was partially purified from human placental microsomes by hydrophobic affinity chromatography using Phenyl-Sepharose and ion-exchange chromatography on DEAE-cellulose. The resulting preparation had a specific activity of 2 nmol/mg protein with respect to cytochrome P-450 content and displayed a type I difference spectrum upon addition of the substrate androstenedione. When the cytochrome P-450-enriched fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue, there was an enrichment of two proteins having apparent molecular weights of 50,000 and 55,000. The bands containing these proteins were removed from unstained polyacrylamide gels and injected separately or together into three rabbits. An aliquot of the serum or an immunoglobulin (IgG) fraction prepared from the serum of the rabbit injected with the 55-kDa band or with both the 50- and 55-kDa bands inhibited aromatase activity of human placental microsomes by 80%; this IgG had no effect on 17 alpha-hydroxylase or 21-hydroxylase activities of human fetal adrenal microsomes. In contrast, the serum of the rabbit injected with the 50-kDa band had little capacity to inhibit placental aromatase activity. By immunoblot analysis, it was found that the IgG from the serum of the rabbit immunized with the 55-kDa protein bound specifically to a protein of 55 kDa in human placental microsomes. Monoclonal antibodies were prepared from a hybridoma cell line derived from the spleen cells of mice immunized against the 55-kDa protein. The monoclonal IgG was covalently linked to a Sepharose 4B column and was used for immunoaffinity chromatography of cytochrome P-450AROM. The finding that cytochrome P-450 and the 55-kDa protein were selectively retained by the affinity column and eluted with NaCl (2 M) and glycine (0.2 M, pH 3.0) and that this fraction contained aromatase activity upon reconstitution with purified NADPH-cytochrome P-450 reductase and phospholipid, is indicative that the 55-kDa protein is indeed cytochrome P-450AROM. These findings are also indicative that both the monoclonal and polyclonal IgGs are specific for human cytochrome P-450AROM. 相似文献
17.
High-performance affinity chromatography with immobilization of protein A and L-histidine on molded monolith 总被引:2,自引:0,他引:2
Reactive monoliths of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) have been prepared by "in-situ" copolymerization of the monomers in the presence of porogenic diluents. Protein A and L-histidine were immobilized on the monoliths directly or through a spacer arm, respectively. The properties of these two kinds of affinity columns were characterized, and the results showed that the columns with coupling of ligands by a spacer arm have some extent of non-specific adsorption for bovine serum albumin. The affinity column based on the monolithic polymer support provided us with good hydrodynamic characteristic, low flow resistance, and easy preparation. These two affinity columns were used for the purification of immunoglobulin G from human serum. The purity of the purified IgG was detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The stability of the protein A affinity column was investigated, and its performance remained invariable after half a year. The effects of the nature and the pH of the buffer system on the adsorption capacity of human IgG on histidyl affinity column were also investigated. The protein A affinity column is favorable for rapid analysis of human IgG samples. In contrast, the advantages of mild elution conditions, high stability, as well as low cost provide the histidyl column further potential possibility for fast removal of IgG from human plasma in clinical applications. 相似文献
18.
Trichokirin-S1,a small ribosome-inactivating peptide recently purified from the seeds ofTrichosanthes kirilowii,has potential clinical applications because of its small molecular mass.Two stablestrains of hybridomas (1F11 and 2A5) that can secrete highly specific monoclonal antibodies (mAbs) againstTrichokirin-S1 have been developed using the hybridoma technique.The isotypes of these two mAbs,1F11and 2A5,were determined to be IgG_(2a) and IgG_1,respectively.The affinity constants,which were measuredby non-competitive ELISA,were found to be 2.3×10~8 M~(-1) and 2.8×10~8 M~(-1),respectively.An immunoaffinitymethod using 2A5-coupled Sepharose 4B was successfully developed to purify Trichokirin-S1.These twoantibodies have also been used to detect Trichokirin-S1 in Western blot. 相似文献
19.
The possibility to purify glucose oxidase from Penicillium vitale on immunosorbent containing specific antibodies to the enzyme covalently bound with Sepharose 4B is studied. The method of affinity chromatography was applied, beside routine methods of fractionating blood serum proteins, to isolate specific antibodies from antiserum of rabbits immunized with glucose oxidase. Immobilized on Sepharose glucose oxidase was used as biospecific sorbent. Specific antibodies to the enzyme were isolated using chromatograpy of gamma-globulins mixture followed by protein desorption from the column with 1 M NaC1 and 3% glucose. Antibodies were immobilized by their covalent binding to activated Sepharose. The immunosorbent obtained was used to purify low active preparation of glucose oxidase by means of affinity chromatography under conditions worked out for the antibodies isolation. The enzyme was eluted from the column with 1 M NaC1 (pH 3.0) containing 3% glucose. 5-Fold purified enzyme preparation was isolated. 相似文献
20.
Ca2+ binding to chromaffin vesicle matrix proteins: effect of pH, Mg2+, and ionic strength 总被引:5,自引:0,他引:5
Recently we found that Ca2+ within chromaffin vesicles is largely bound [Bulenda, D., & Gratzl, M. (1985) Biochemistry 24, 7760-7765]. In order to explore the nature of these bonds, we analyzed the binding of Ca2+ to the vesicle matrix proteins as well as to ATP, the main nucleotide present in these vesicles. The dissociation constant at pH 7 is 50 microM (number of binding sites, n = 180 nmol/mg of protein) for Ca2+-protein bonds and 15 microM (n = 0.8 mumol/mumol) for Ca2+-ATP bonds. When the pH is decreased to more physiological values (pH 6), the number of binding sites remains the same. However, the affinity of Ca2+ for the proteins decreases much less than its affinity for ATP (dissociation constant of 90 vs. 70 microM). At pH 6 monovalent cations (30-50 mM) as well as Mg2+ (0.1-0.5 mM), which are also present within chromaffin vesicles, do not affect the number of binding sites for Ca2+ but cause a decrease in the affinity of Ca2+ for both proteins and ATP. For Ca2+ binding to ATP in the presence of 0.5 mM Mg2+ we found a dissociation constant of 340 microM and after addition of 35 mM K+ a dissociation constant of 170 microM. Ca2+ binding to the chromaffin vesicle matrix proteins in the presence of 0.5 mM Mg2+ is characterized by a Kd of 240 microM and after addition of 15 mM Na+ by a Kd of 340 microM.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献