辛酸沉淀结合阴离子交换层析纯化马IgG的研究

目的以辛酸沉淀结合离子交换层析纯化破伤风抗毒素马免疫血浆,获得高质量的马IgG,为抗毒素F(ab')2的制备奠定基础。方法通过对辛酸沉淀马血浆过程中的pH、辛酸浓度以及血浆稀释倍数的实验设计(DoE),研究不同条件对IgG纯度、效价、比活性、浊度以及过滤速度的影响,确定各工艺参数的可操作区间,并结合Capto DEAE阴离子交换层析以流穿模式进一步纯化IgG。结果马血浆经一步辛酸沉淀获得的IgG纯度(SDSPAGE)大于92%、分子排阻色谱(SEC)纯度大于95%,比活性较血浆提高2.14±0.29倍;辛酸沉淀后的IgG样品经阴离子交换层析,可有效去除聚合体以及小分子杂质,将纯度提高至95%(SDS-PAGE)和98%(SEC)。结论马血浆经辛酸沉淀和阴离子交换层析可获得高纯度、高比活的IgG。     

IgG4 levels in polyvalent immunoglobulins and immunoglobulins prepared by precipitation with caprylic acid

Levels of IgG4 in immunoglobulin preparations obtained either by conventional ethanol fractionation or by ethanol and caprylic acid fractionation (Allergam) were measured by an enzyme-immunoassay (competitive Elisa). In 9 ethanol preparations, the mean percentage of IgG4 was 2.1% +/- 0.5. In 10 preparations of Allergam, the mean percentage of IgG4 was 4.6% +/- 1. The concentration of IgG4 in Allergam preparations is about twice the concentration found in conventional ethanol preparations.     

DNA-hydrolysing activity of IgG antibodies from the sera of patients with schizophrenia

It is believed that damage to the membranes of brain cells of schizophrenia (SCZ) patients induces the formation of autoantigens and autoantibodies. Nevertheless, the importance of immunological changes leading to the loss of tolerance to self-antigens in the genesis of SCZ has not been established. The MALDI mass spectra of the IgG light chains of 20 healthy donors were relatively homogeneous and characterized by one peak with only one maximum. In contrast to the healthy donors, the MALDI mass spectra of IgG light chains corresponding to 20 SCZ patients demonstrated, similarly to 20 autoimmune systemic lupus erythematosus (SLE) patients, two maxima of a comparable intensity. In addition, the MALDI spectra of the IgG light chains of five SLE and four SCZ patients contained a small additional brightly pronounced peak with remarkably lower molecular mass compared with the main one. DNase autoantibodies (abzymes) can be found in the blood of patients with several autoimmune diseases, while the blood of healthy donors or patients with diseases without a significant disturbance of the immune status does not contain DNase abzymes. Here, we present the first analysis of anti-DNA antibodies and DNase abzymes in the sera of SCZ patients. Several strict criteria have been applied to show that the DNase activity is an intrinsic property of IgGs from the sera of SCZ patients. The sera of approximately 30% of SCZ patients displayed a higher content of antibodies (compared with 37% of SLE) interacting with single- and double-stranded DNA compared with healthy donors. Antibodies with DNase activity were revealed in 80% of the patients. These data indicate that some SCZ patients may show signs of typical autoimmune processes to a certain extent.     

IgG antibodies with peroxidase-like activity from the sera of healthy Wistar rats

Various catalytic antibodies or abzymes (Abzs) have been detected recently in the sera of patients and animals with many autoimmune diseases, where their presence is most probably associated with autoimmunization. Normal humans or animals usually do not contain Abzs. In contrast, polyclonal Abzs from healthy humans and animals have an intrinsic superoxide dismutase activity and catalyze formation of H(2)O(2) (Wentworth et al., 2000, Proc. Natl. Acad. Sci. USA; 2001, Science). Here, we present the first evidence showing that highly purified native IgGs from the sera of healthy Wistar rats interact with H(2)O(2) and possess peroxidase-like activity. Specific peroxidase activity of IgG preparations from the sera of 10 rats varied in the range 1.6-27% as compared with that for horseradish peroxidase (100%). Antioxidant enzymes such as superoxide dismutases, catalases, and glutathione peroxidases are known to represent critical defence mechanisms for preventing oxidative modifications of DNA, proteins, and lipids. Antioxidant peroxidase activity of Abzs can also play an important role in the protection of organisms from oxidative stress as well as in oxidation of toxic compounds.     

Cytokinin binding proteins from mammalian sera

A cytokinin-binding protein fraction was isolated from normal rabbit sera by affinity chromatography. The protein fraction bound tritium labelled N⁶- (δ2-risopentenyl) adenosine and the order of inhibition of this binding by competing non-radioactive compounds was, N⁶-(δ²-isopentenyl) adenosine < N⁶-benzyIadenosine < zeatin-riboside > N⁶-(δ²-isopentenyl) adenine < kinetin riboside > adenosine. The protein fraction showed broad specificity, the prefered cytokinin being N⁶-(δ²-isopentenyl) adenosine. This is the first report of the isolation of cytokinin binding proteins from mammalian sources.     

DNA-hydrolyzing activity of IgG antibodies from the sera of patients with tick-borne encephalitis

DNase autoantibodies (Abzs) can be found in the blood of patients with several autoimmune diseases, while the blood of healthy donors or patients with diseases with an insignificant disturbance of the immune status does not contain DNase Abzs. Here we present the first analysis of the DNase Abzs activity in the patients with tick-borne encephalitis (TBE). Several strict criteria have been applied to show that the DNase activity is an intrinsic property of IgGs from the sera of TBE patients but not from healthy donors. The relative activity of IgGs has been shown to vary extensively from patient to patient, but most of the preparations (91%) had detectable levels of the DNase activity. Polyclonal DNase IgGs were not active in the presence of EDTA or after a dialysis against EDTA, but could be activated by several externally added metal ions, with the level of activity decreasing in the order Mn²⁺ + Ca²⁺ ≥ Mn²⁺+ Mg²⁺ ≥ Mn²⁺ ≥ Mg²⁺ + Ca²⁺ ≥ Co²⁺ ≥ Mg²⁺ > Ca²⁺, while K⁺, Na⁺, Ni²⁺, Zn²⁺, and Cu²⁺ did not stimulate DNA hydrolysis. Affinity chromatography on DNA-cellulose separated the DNase IgGs into many subfractions with various affinities for DNA and very different levels of the relative activity. Possible reasons for catalytic diversity of polyclonal human Abzs are discussed.     

IgG abzymes with peroxidase and oxidoreductase activities from the sera of healthy humans

We present the evidence showing that small fractions of electrophoretically homogeneous immunoglobulin G (IgGs) from the sera of healthy humans and their Fab and F(ab)₂ fragments oxidize 3,3′‐diaminobenzidine through a peroxidase activity in the presence of H₂O₂ and through an oxidoreductase activity in the absence of H₂O₂. During purification on protein G‐Sepharose and gel filtration, the polyclonal IgGs partially lose the Me²⁺ ions. After extensive dialysis of purified Abs against agents chelating metal ions, the relative peroxidase activity decreased dependently of IgG analyzed from 100 to ~10–85%, while oxidoreductase activity from 100 to 14–83%. Addition of external metal ions to dialyzed and non‐dialyzed IgGs leads to a significant increase in their activity. Chromatography of the IgGs on Chelex non‐charged with Cu²⁺ ions results in the adsorption of a small IgG fraction bound with metal ions (~5%), while Chelex charged with Cu²⁺ ions bind additionally ~38% of the total IgGs. Separation of Abs on both sorbents results in IgG separation to many different subfractions demonstrating various affinities to the chelating resin and different levels of the specific oxidoreductase and peroxidase activities. In the presence of external Cu²⁺ ions, the specific peroxidase activity of several IgG subfractions achieves 20–27 % as compared with horseradish peroxidase (HRP, taken for 100%). The oxidoreductase activity of these fractions is ~4–6‐fold higher than that for HRP. Antioxidant enzymes such as superoxide dismutases, catalases, and glutathione peroxidases are known to represent critical defence mechanisms for preventing oxidative modifications of DNA, proteins, and lipids. Peroxidase and oxidoreductase activities of human IgGs could also play an important role in the protection of organisms from oxidative stress and toxic compounds. Copyright © 2015 John Wiley & Sons, Ltd.     

Asymmetrically glycosylated IgG isolated from non-immune human sera

When human IgG or its F(ab')2 fragment purified from a pool of non-immune sera was passed through a Con A-Sepharose column, 12% of the molecules bound to concanavalin A. While 44% of Fab' and 72% of Fd' fragments obtained from F(ab')2 retained by concanavalin A and eluted with methyl alpha-D-mannoside bound to concanavalin A, the Fab' and Fd' fragments obtained from non-retained F(ab')2 and the L chains and Fc fragments did not interact with the lectin. Only Fd' fragment obtained from the F(ab')2 retained by concanavalin A inhibited the fixation of guinea-pig erythrocytes to concanavalin A. These results are similar to those previously observed for IgG antibodies of different animal species and indicate that partial asymmetric glycosylation is a general phenomenon that is not restricted exclusively to IgG molecules with known specificity.     

Rapid isolation of total RNA from mammalian tissues

A rapid procedure for the isolation of total RNA from small amounts of mammalian tissue (35 to 150 mg) is described. Tissues were homogenized in the presence of RNase inhibitors but in the absence of strong detergents. Contaminants were removed by phenol/chloroform extraction and Sephadex column chromatography. Total RNAs were precipitated with ethanol and sodium acetate. The RNAs isolated were intact and suitable for mRNA quantitation via Northern blot or slot-blot analyses. This procedure isolates total RNAs in high yield and purity, without CsCl ultracentrifugation, and is especially useful when mRNAs must be quantitated from many samples.     

Properties of immobilized lipase from Bacillus stearothermophilus MC7. Acidolysis of triolein with caprylic acid

Extracellular bacterial lipases are promising biocatalysts for industry, because they are stable and active enzymes from easily available sources. A lipase from Bacillus stearothermophilus MC7 was immobilized on four polymer carriers by physical adsorption: chitosan, DEAE-cellulose, polypropylene, and polyurethane. The four biocatalysts differ in their hydrolytic activity against long-chain and short-chain triglycerides. Lipase MC7 immobilized on polypropylene (PP-MC7) stands out with its high activity against tributirin. According to the preliminary data, all four preparations were suitable for application in the test reaction of acidolysis of triolein with caprylic acid. The highest degree of conversion of the initial triolein was achieved in the presence of PP-MC7 preparation—50%. But variation of the reaction conditions did not lead to synthesis of the target di-substituted product (dicapryloyl-oleoylglycerol, COC). Reaction proceeds as a selective mono-substitution in the glycerol backbone.     

炭疽灭活和裂解芽胞抗原免疫家兔后血清中的IgG抗体应答

炭疽杆菌芽胞在炭疽免疫中发挥基本作用。实验中以炭疽活芽胞疫苗为原形,建立了制备灭活和裂解炭疽芽胞的方法,研究了各种灭活和裂解炭疽芽胞疫苗不同浓度、不同剂次免疫家兔的抗芽胞和毒素IgG应答,总结分析了各种灭活和裂解炭疽芽胞疫苗用于新疫苗成分之一的可能性。甲醛灭活炭疽芽胞疫苗设芽胞浓度2.5×108剂量组、5×108剂量组、1×109剂量组,于0、4、8周时3次免疫。在3剂免疫后血清抗炭疽芽胞IgG水平持续升高,首次免疫后4、8、12周时家兔血清中抗芽胞IgG几何平均滴度可达到600～16000。裂解炭疽芽胞疫苗的制备和动物免疫中,只采取了2.5×108芽胞浓度,两剂免疫,免疫时间为0、4周。在首次免疫后4、8、12周时家兔血清中抗芽胞IgG几何平均滴度分别为362、776和388。各时间点采集的家兔血清未能测出或只测出极微量的抗炭疽毒素IgG。通过上述研究认为,以裂解炭疽芽胞抗原作为炭疽疫苗成分之一,其抗原性和免疫原性是适宜的;免疫剂量可以设定为2.5×108芽胞浓度上下;免疫次数可定为2剂间隔1个月。