共查询到20条相似文献,搜索用时 31 毫秒
1.
Nedonchelle E Pitiot O Vijayalakshmi MA 《Applied biochemistry and biotechnology》2000,83(1-3):287-295
Catalytic autoimmune antibodies from the sera of lupus patients were purified using histidyl-aminohexyl-Sepharose gel and
compared with the antibodies purified with protein A and protein G affinity chromatography. The IgG preparations from the
histidine affinity column had a much higher catalytic activity in hydrolyzing the peptide substrate Pro-Phe-Arg-methyl-coumarinamide
compared to the antibodies obtained by the conventional protein A/G method. This preservation of catalytic activity is attributed
to the gentle buffer conditions used in the histidine ligand method that allowed the integrity of three-dimensional structure
of purified catalytic antibodies. Thus, histidine affinity offer a superior method for isolating autoimmune catalytic antibodies. 相似文献
2.
Singh KV Kaur J Raje M Varshney GC Suri CR 《Analytical and bioanalytical chemistry》2003,377(1):220-224
The correct choice of the elution conditions to break an affinity interaction is important for the successful purification of biomolecules. The optimal elution buffer liberates the bound substance in a minimum volume and maintains the activity of the purified material. The present study demonstrates an enzyme-linked immunosorbent assay (ELISA)-based approach for selection of specific elution conditions for eluting antibodies against a small molecule (atrazine) from pooled sera. Six different elution conditions were tried for the removal of antibodies from the complex. Large-scale purification of anti-atrazine antibodies from the sera was done with a hapten-specific column using an amino-terminal crosslinked agarose gel. Efficacy in terms of total amount of recovery and binding affinities of eluted antibodies from the column were further investigated by ELISA. Results indicate that the ELISA-based elution approach is ideal for the selection of suitable elution buffer that can subsequently be utilized for affinity purification applications. 相似文献
3.
Saccodossi N Cabrera RB Oliver F Gelmi L Magglio DG Fernández-Lahore HM Leoni J Stumpo RR 《Journal of separation science》2004,27(7-8):589-594
The antigenic protein Ro52 was expressed in the E. coli system harboring a 6 x His tag in the form of insoluble inclusion bodies. Direct chemical extraction of the product using 6-8 M urea proved to be effective. Furthermore, the tagged protein was recovered by direct adsorption on Ni2+-loaded commercial adsorbents derivatized with iminodiacetic acid. Screening experiments in small packed columns revealed that selective binding and elution were possible using a denaturing buffer at pH 4.5. The hydrodynamic evaluation of scaled-up fluidized systems showed values for the phi (dynamic zone) parameter in the range 0.95-1.00 for fluidization in buffer and in the range 0.70-0.85 for the biomass-containing feedstock. Removal of macromolecular DNA released by the disrupted biomass was mandatory. Under optimized process conditions good recovery (60-70%) was achieved and a highly purified (95%) product obtained. The purified Ro52 retained its immunoreactivity against sera of patients with systemic lupus erythematosus (SLE) and Sjogren's syndrome-related disorders. The production and application of new recombinant antigens may contribute to increasing the sensitivity and specificity of the detection of anti-Ro antibodies in these autoimmune diseases. 相似文献
4.
尿毒症患者血清中中分子物质的提取及性质的初步表征 总被引:3,自引:0,他引:3
从尿毒症患者血清及腹透液中得到的中分子量物质,具有相同的凝胶色谱行为及紫外、红外吸收行为,它们在206和235nm处,有特征紫外吸收峰;在波数为3246,1516,1122和613cm~(-1)处,有特征红外吸收峰。中分子物经离子交换色谱进一步分离,发现起决定紫外及红外吸收行为的成分集中于第3子峰;其自由氨基酸和肽类物质含量均低于3%。 相似文献
5.
Alice Schlichtiger Claudia Baier Meng-Xin Yin Andrew B. Holmes Makiko Maruyama Ralf Strasser Ulrich Rant Markus Thaler Peter B. Luppa 《Analytical and bioanalytical chemistry》2013,405(1):275-285
Antiphospholipid antibodies (aPL) are a relevant serological indicator of antiphospholipid syndrome (APS). A solid-state surface with covalently bound ω-amine-functionalized cardiolipin was established and the binding of β2-glycoprotein I (β2-GPI) was investigated either by use of surface plasmon resonance (SPR) biosensor, by electrically switchable DNA interfaces (switchSENSE) and by scanning tunneling microscopy (STM). STM could clearly visualize the attachment of β2-GPI to the cardiolipin surface. Using the switchSENSE sensor, β2-GPI as specific ligand could be identified by increased hydrodynamic friction. The binding of anti-cardiolipin antibodies (aCL) was detected against the ω-amine-functionalized cardiolipin-modified SPR biosensor (aCL biosensor) using sera from healthy donors, APS patients and syphilis patients. Our results showed that the aCL biosensor is a much more sensitive diagnostic device for APS patients compared to previous methods. The specificity between β2-GPI-dependent autoimmune- and β2-GPI-independent infection-associated types of aPLs was also studied and they can be distinguished by the different binding kinetics and patterns. 相似文献
6.
Kozyr AV Kolesnikov AV Zelenova NA Sashchenko LP Mikhalap SV Bulina ME Ignatova AN Favorov PV Gabibov AG 《Applied biochemistry and biotechnology》2000,83(1-3):255-68; discussion 268-9, 297-313
The cytotoxicity of DNA-specific autoantibodies from sera of patients with systemic lupus erythematosis (SLE) and with lymphoproliferative diseases, and from blood of healthy donors was examined on tumor-cell lines L929 and HL-60. DNA-binding IgG fractions from SLE and chronic lymphocytic leukemia (CLL) sera were cytotoxic at concentrations of up to 10(-10) M. No detectable changes in cell viability were observed after incubation with antibodies devoid of DNA-binding activity and DNA-specific antibodies isolated from blood of healthy donors and patients with T-cell lymphoma, B-cell lymphosarcoma, and acute B-cell leukemia. There was good correlation between the cytotoxic activity and DNA-hydrolyzing activity of anti-DNA antibodies. The cytotoxic effect of DNA-binding antibodies presumably was complement-independent, because it was attributed only to the Fab fragment. The cytotoxic effect was completely inhibited by preincubation with double-stranded DNA (dsDNA). Both the cytotoxic effect and the DNA-hydrolyzing activity of anti-DNA antibodies were significantly increased in the antibody fraction that displayed cross-reactivity with nuclear matrix proteins. Possible mechanisms for the formation and pathogenicity of cytotoxic anti-DNA antibodies are discussed in this article. 相似文献
7.
A. V. Kozyr A. V. Kolesnikov N. A. Zelenova L. P. Sashchenko S. V. Mikhalap M. E. Bulina A. N. Ignatova P. V. Favorov A. G. Gabibov 《Applied biochemistry and biotechnology》2000,83(1-3):255-269
Thecytotoxicity of DNA-specific autoantibodies from sera of patients with systemic lupus erythematosis (SLE) and with lymphoproliferative
diseases, and from blood of healthy donors was examined on tumor-cell lines L929 and HL-60. DNA-binding IgG fractions from
SLE and chronic lymphocytic leukemia (CLL) sera were cytotoxic at concentrations of up to 10−10 M. No detectable changes in cell viability were observed after incubation with antibodies devoid of DNA-binding activity
and DNA-specific antibodies isolated from blood of healthy donors and patients with T-cell lymphoma, B-cell lymphosarcoma,
and acute B-cell leukemia. There was good correlation between the cytotoxic activity and DNA-hydrolyzing activity of anti-DNA
antibodies. The cytotoxic effect of DNA-binding antibodies presumably was complement-independent, because it was attributed
only to the Fab fragment. The cytotoxic effect was completely inhibited by preincubation with double-stranded DNA (dsDNA).
Both the cytotoxic effect and the DNA-hydrolyzing activity of anti-DNA antibodies were significantly increased in the antibody
fraction that displayed cross-reactivity with nuclear matrix proteins. Possible mechanisms for the formation and pathogenicity
of cytotoxic anti-DNA antibodies are discussed in this article. 相似文献
8.
依据单胺氧化酶B(monoamine oxidase B, MAOB)的疏水特性,建立了一种从猪肝中分离纯化MAOB的新方法。用含有1% Triton X-100的膜蛋白裂解液制备粗酶,以饱和度为20%~50%的硫酸铵反抽提进行粗提,再利用自制的配基密度为75.7 μmol/mL的苯基疏水色谱及Sepharose Q High Performance离子交换色谱进一步分离纯化,得到纯化倍数为18.2、酶比活为135 U/mg的MAOB。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析显示为相对分子质量约60 000的单一蛋白质带。采用高效液相色谱-电喷雾串联质谱对该酶进行鉴定,证实为MAOB。本研究所用分离纯化方法可以有效纯化MAOB, 为MAOB的深入研究提供技术支撑。 相似文献
9.
The polyclonal antibodies to juveniles of Paragoniums westermani (PwJ-PcAbs) from sera of Wistar rats infected with Paragoniums westerrnani (P. w.)were purified by Sephadex G 200 chromatography. Next the shared serological antigens of P. w. metacercaria and juveniles (PwMJ-SAg) from the crude antigens of the metacercariae (M-NS-Ag) were purified with immuno-affinity chromatography on cyanogen bromide-activated cross-linked Sepharose 4B beads coupled withPwJ-PcAbs. PwMJ-SAg, agroup of glycoprotein molecules shown by the staining test, were specific serological antigens of P. w. metacercariae and juveniles, identified by the immunoabsorb test and immunoelectrophoresis. By SDSPAGE, PwMJ-SAg were fractionated to seven bands, including major bands A(27.SK) and Bi(19.5 K), the two major serological antigen molecules. 20 sera samples from the patients with the nonpulmonary type of P. w. paragonimiasis were detected using PwMJ-SAg and M-NS-Ag by Dot-ELISA, and the difference of sensitivity between two antigens 相似文献
10.
Z J Jiang Y P Shen W X Zhao 《Science in China. Series B, Chemistry, life sciences & earth sciences》1990,33(2):178-187
The polyclonal antibodies to juveniles of Paragoniums westermani (PwJ-PcAbs) from sera of Wistar rats infected with Paragoniums westermani (P.w.) were purified by Sephadex G 200 chromatography. Next the shared serological antigens of P.w. metacercaria and juveniles (PwMJ-SAg) from the crude antigens of the metacercariae (M-NS-Ag) were purified with immuno-affinity chromatography on cyanogen bromide-activated cross-linked Sepharose 4B beads coupled with PwJ-PcAbs. PwMJ-SAg, a group of glycoprotein molecules shown by the staining test, were specific serological antigens of P.w. metacercariae and juveniles, identified by the immunoabsorb test and immunoelectrophoresis. By SDS-PAGE, PwMJ-SAg were fractionated to seven bands, including major bands A (27.5 K) and Bi (19.5 K), the two major serological antigen molecules. 20 sera samples from the patients with the nonpulmonary type of P. w. paragonimiasis were detected using PwMJ-SAg and M-NS-Ag by Dot-ELISA, and the difference of sensitivity between two antigens was highly statistically significant (P less than 0.001). BALB/c mice, in the early stage of infection with P. w. metacercaria, were immunized with PwMJ-SAg. The spleen cells of the mice were isolated and fused with SP2/o, a murine myeloma cell line. After three subclonal cultures, eight cell lines secreting monoclonal antibodies (McAbs) to PwMJ-SAg were prepared from 384 wells of hybridoma cells. All McAbs were IgG1 subclass. 相似文献
11.
12.
固相萃取-亲水作用色谱法测定废水中四环素类抗生素 总被引:1,自引:0,他引:1
建立了固相萃取(SPE)-亲水作用色谱法(HILIC)测定废水中金霉素(CTC)、强力霉素(DC)、四环素(TC)和土霉素(OTC) 4种四环素类抗生素(TCs)残留的新方法.水样经Oasis HLB固相萃取柱净化富集后, 采用以氨基色谱柱及高极性有机溶剂-水相缓冲溶液为流动相的亲水作用色谱法(HILIC)进行分析. 对流动相中缓冲溶液的类型和pH值、离子强度、 有机溶剂的浓度以及流速进行了优化, 确定了以V(乙腈)∶V(6.7 mmol/L柠檬酸铵缓冲溶液, pH 4.0)=85∶ 15混合液为流动相的最佳条件.本方法具有良好的线性关系(r> 0.999)和重现性(峰面积RSD<1.0%), 最低检出限(S/N=3)为12~30 μg/L, 4种四环素类药物添加水平在0.5~10 μg/L范围内的标准加入回收率为 90.6%~106.5%; 相对标准偏差为 2.5%~6.2%.本方法简便、准确、流动相离子强度低,适合于与质谱联用,用于屠宰场污水及医院污水等实际样品检测,结果满意. 相似文献
13.
动物血清中免疫球蛋白和白蛋白的等电点分别约为7.8和4.8,根据它们等电点的较大差别,利用Q SepharoseTM-XL强阴离子交换色谱结合分子排阻色谱同时分离纯化这2种蛋白。以0.02 mol/L pH 8.0的Tris-HCl缓冲液平衡离子交换色谱柱并将已稀释10倍的高免疫的兔血清上样,采用pH分段洗脱。在pH 6.0时以0.3 mL/min低流速洗脱得到高纯度的免疫球蛋白,继续在pH 4.0时洗脱,再辅以Sephadex G-75分子排阻色谱可获得纯度大于95%的白蛋白。对纯化后的蛋白进行活性检测,证明所纯化的免疫球蛋白和白蛋白都保持正常的生物活性。蛋白质含量测定说明免疫球蛋白的纯化回收率达到95%以上,而白蛋白的纯化回收率大于90%。该法简便快速,可同时从动物血清中纯化出保持生物活性的免疫球蛋白和白蛋白,纯化效率高。 相似文献
14.
15.
Glutathione reductase was purified from chicken liver and some characteristics of the enzyme were investigated. The purification
procedure was composed of four steps: preparation of homogenate, ammonium sulfate precipitation, 2′,5′-ADP Sepharose 4B affinity
chromatography, and Sephadex G-200 gel filtration chromatography. Owing to the four consecutive procedures, the enzyme was
purified 1714-fold, with a yield of 38%. Specific activity at the final step was 120 enzyme unit (EU)/mg of protein. The purified
enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight
of the enzyme was found to be 100 kDa by Sephadex G-200 gel filtration chromatography, and the subunit molecular weight was
found to be 43 kDa by SDS-PAGE. Optimum pH, stable pH, optimum ionic strength, and optimum temperature were 7.0, 7.4, 0.75
M Tris-HCl buffer including 1 mM EDTA, and 50°C, respectively. K
M and V
max values for NADPH and glutathione disulfide (GSSG) substrates were also determined for the enzyme. 相似文献
16.
Sunil Chhatre Daniel G. BracewellNigel J. Titchener-Hooker 《Journal of chromatography. A》2009,1216(45):7806-7815
A microscale approach is described which screens conditions for recovering polyclonal antibodies from ovine sera by mixed-mode cation-exchange chromatography. The impact of pH and loading buffer salt concentration were assessed using robotically operated 20 μL packed pipette tips. Low salt concentrations delivered capacities up to 41 mg/mL, while only half this level was obtained at high salt concentrations. Two of the screened conditions were then tested in a 10 mL packed bed and overall trends in capacity, yield and purity were found to be retained. Microscale pipette tips thus provided a useful basis for the rapid, approximate definition of a chromatography design space. 相似文献
17.
de Pinho SC Zollner RL De Cuyper M Santana MH 《Colloids and surfaces. B, Biointerfaces》2008,63(2):249-253
Phosphatidylcholine-based magnetoliposomes containing specific ligands for biological molecules, so-called affinity magnetoliposomes (AML), may prove to be useful as adsorbents in applications such as diagnosis or anchoring and delivery of drugs at specific sites in the human body. In the present study, the performance of affinity magnetoliposomes to adsorb anticardiolipin antibodies (aCL) from a previously characterized pool of patients with autoimmune diseases is described. The magnetic vesicles were prepared by enrobing nanometer-sized colloidal magnetite particles with a phospholipid bilayer composed of dimyristoylphosphatidylcholine (DMPC) and the affinity lipid ligand cardiolipin (CL). Adsorption of antibodies onto the affinity magnetoliposomes assayed using a high-gradient magnetophoresis (HGM) system, in which the magnetoliposomes were first magnetically captured on stainless steel fibers, and which were subsequently overflowed either with a pool of sera from autoimmune patients or sera of healthy individuals as a control. The spectrophotometric assay showed stronger changes in absorbance spectra when the affinity magnetoliposomes containing cardiolipin were added to sera of autoimmune patients than when they were added to sera of healthy individuals. The breakthrough curves obtained from a frontal analyses of the adsorption in the magnetophoresis system showed a 10% difference for total adsorbed IgG when sera of autoimmune and healthy individuals were assayed on magnetoliposomes containing cardiolipin. 相似文献
18.
Alexander G. Gabibov Gennady V. Gololobov Oxana I. Makarevich Dmitry V. Schourov Elena A. Chernova Rana P. Yadav 《Applied biochemistry and biotechnology》1994,47(2-3):293-303
Catalysis by antibodies could be a frequent phenomenon if the immune system generates a sufficiently diverse number of antibodyactive
sites, some of which may possess catalytic activity. A catalytic antibody can be expected to do more damage than one that
simply binds antigen. The best biochemical marker of systemic lupus erythematosus (SLE) is presence of autoantibodies to DNA.
In the present article, we describe the DNA-hydrolyzing activity of DNA-binding autoantibodies purified from SLE patients.
The substrates employed were supercoiled plasmid, radiolabeled plasmid fragments, and oligonucleotides. Hydrolysis of DNA
by the antibodies was indicated by the appearance of fragments visualized by ethidium bromide staining of agarose gels or
autoradiography of polyacrylamide gels. Changes in linear dichroism values were also indicative of DNA hydrolysis. The antibody
activity was purified by protein A-sepharose chromatography, high-performance liquid chromatography gel filtration, and DNA-affinity
chromatography. Scrupulous control studies were done to demonstrate that DNA-hydrolyzing activity really belongs to the antibodies.
Purified Fab fragments showed hydrolyzing activity, whereas the Fc fragment was inactive. The specificity of DNA cleavage
was investigated, and the rate parameters of hydrolysis by antibodies and conventional nucleases were compared. 相似文献
19.
20.
Electrophoretic studies of antimitochondrial antibodies: identification of mitochondrial antigens specific to primary biliary cirrhosis 总被引:1,自引:0,他引:1
Mitochondrial inner membrane proteins extracted from beef heart tissue were examined for reactivity to antimitochondrial antibody (AMA) present in sera of patients with primary biliary cirrhosis (PBC) by an immunoblotting technique. Four proteins, which reacted with AMA, had molecular masses of 70 kDa, 50 kDa, 47 kDa and 40 kDa, as defined by their relative mobility (Rf) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All sera of 114 PBC patients were positive with at least one and as many as four of the mitochondrial proteins. The major antigenic proteins of mitochondrial inner membrane to which AMA reacts were the 70 kDa and 47 kDa proteins. All PBC sera containing antibodies to the 50 kDa and/or 40 kDa proteins reacted with 70 kDa as well. The isolation of antigen reacting with AMA of PBC is important to warrant further study of AMA and the cause of the disease. The isolation of responsible antigens had been difficult because the four antigens were insoluble. However, the antigen newly found by us, the 36 kDa fragment, obtained by partial trypsin digestion, is soluble. Using several procedures, the antigenic protein target of AMA was purified from mitochondria for the first time. We determined the N-terminal sequence of the soluble 36 kDa fragment, 25 residues in length. Until now the N-terminal sequence of the 36 kDa protein has not shown significant homology with any known protein. The present results of antigen purification would contribute to the elucidation of the epitopes of AMA antigen. 相似文献