用免疫单扩散法测定低pH静注丙球IgG含量及其影响因素初探

本文报道了低ＰＨ值注丙球ＩｇＧ含量测定免疫单扩散法的建立,并对其影响因素进行了初步试验。结果表明,试验方法较为稳定,简单易行。在低ＰＨ静注丙球ＩｇＧ含量检测时,进口Ｓｉｇｍａ琼脂糖效果优于国产琼脂糖（Ｐ〈０．０１）,国产（东海）琼脂糖不适于ＩｇＧ含量的定量检测;应先将静注丙球ＰＨ调至中性后进行ＩｇＧ含量测定（Ｐ〈０．０５）样品中含有的１０％麦芽糖对试验结果无影响（Ｐ〉０．０５）。     

FMMU白化豚鼠免疫学特性研究

目的　通过测定补体含量、免疫球蛋白含量、淋巴细胞的转化率及红细胞免疫功能 ,比较FMMU白化豚鼠和花色豚鼠的免疫学特性。方法　利用自动生化分析仪测定FMMU白化豚鼠和普通花色豚鼠的免疫球蛋白含量 (IgG、IgA、IgM)、C3、C4含量及总补体水平 ;利用淋巴细胞转化实验测定淋巴细胞的转化率 ;利用红细胞免疫复合物 (immunocomplex ,IC)花环形成试验测定两种豚鼠红细胞免疫粘附功能。结果　FMMU白化豚鼠血清中免疫球蛋白含量 (IgG、IgA、IgM)、C3、C4含量及补体总活性均显著低于花色豚鼠 ;FMMU白化豚鼠淋巴细胞转化率比花色豚鼠略低 ;FMMU白化豚鼠红细胞C3bR花环形成率与花色豚鼠差异无显著性 (P >0 0 5 )。而RBC IC花环率显著低于花色豚鼠 (P <0 0 5 )。结论　封闭群FMMU白化豚鼠具有独特的免疫学特性 ,总体免疫学功能低于花色豚鼠。     

小鼠耐力训练后再力竭运动对体内某些生化指标的影响

目的 :通过分析耐力训练后再力竭运动小鼠部分生化指标的变化来探讨耐力训练提高机体抗疲劳能力可能机制及血液再分配的机制。方法 :建立耐力训练后再力竭运动模型 ,测定血清中超氧化物歧化酶 (SOD)活性、过氧化物酶 (POD)活性、过氧化氢酶 (CAT)活性与丙二醛 (MDA )含量及肝、骨骼肌、心肌与血清中一氧化氮 (NO )含量。结果 :运动后即刻 ,非耐力组血清SOD活性及肝NO含量较安静组显著下降 (P <0 .0 5～ 0 .0 1) ,而血清POD与CAT活性及骨骼肌与血清NO含量则显著升高 (P <0 .0 5～ 0 .0 1) ,其余指标无显著变化 (P >0 .0 5)。耐力组CAT活性显著高于非耐力组 (P <0 .0 5) ,血清NO含量显著低于非耐力组 (P <0 .0 1) ,其余指标两组间无显著差异 (P >0 .0 5)。 2 4h恢复后 ,非耐力组血清CAT活性与MDA含量及肝NO较运动后即刻显著升高 (P <0 .0 5～ 0 .0 1) ,血清与骨骼肌NO含量显著下降 (P <0 .0 5) ,而其余指标则无显著变化 (P >0 .0 5)。耐力组血清SOD活性及肝、血清与心肌NO含量较运动后即刻显著升高 (P <0 .0 5) ,而血清CAT活性显著下降 (P <0 .0 5) ,其余指标无显著变化(P >0 .0 5)。耐力组血清CAT活性与MDA含量显著低于非耐力组 (P <0 .0 5) ,而心肌NO含量显著高于非耐力组 (P <0 .0 5) ,其余指标两组间     

束缚应激对大鼠心肌细胞凋亡的影响及中药颐心宁的干预作用

目的 :探讨束缚应激对大鼠心肌细胞凋亡的影响及中药颐心宁抗应激性心肌损伤的作用。方法 :采用琼脂糖凝胶电泳和TUNEL检测心肌细胞凋亡。结果 :①束缚 1、2、4周时 ,应激组DNAladder心肌细胞凋亡结果阴性 ,原位细胞凋亡明显增多 ,统计学有非常显著差异 (P <0 .0 1) ;②颐心宁干预组DNAladder结果阴性 ,而原位细胞凋亡检测结果则较单纯应激大鼠显著减轻 (P <0 .0 5 ) ;③蒸馏水干预组与单纯应激大鼠比较则无统计学意义 (P >0 .0 5 )。结论 :束缚应激可诱导大鼠心肌细胞发生凋亡 ,中药颐心宁可抑制心肌细胞凋亡 ,具有抗应激性心肌损伤的作用。     

HPLC法测定国产葡萄酒中白藜芦醇的含量

采用HPLC法测定国产葡萄酒中白藜芦醇的含量。色谱条件为 :Shim packCLC ODS柱 (15 0mm× 6 0mm) ,流动相 :0 2mol/LH3 PO4 CH3 CN(2 0∶80pH 3 0～ 3 5 ) ,在 30 6nm测定。测得 15种国产葡萄酒中通化干红葡萄酒的白藜芦醇含量最高为 5 6 0mg/L。该方法灵敏 ,重现性好     

硒元素对平菇菌丝体GSH-Px、SOD及MDA的影响

于培养基中加入一定量的亚硒酸钠溶液 ,分别测定了 2个品种平菇菌丝体内GSH Px、SOD活性及MDA含量。结果表明 :3 0、60mg/L组菌丝体内GSH Px、SOD活性极显著升高 (P <0 .0 1 )而MDA含量明显降低 (P <0 .0 5 ) ,随着硒水平的升高 ,GSH Px、SOD活性呈下降趋势而MDA含量则显著升高 (P <0 .0 5 )。因此 ,在培养富硒平菇菌丝体时应适当考虑培养基的硒浓度。     

降低静脉注射用人免疫球蛋白抗补体活性的试验观察

采用CH50试验法测定静脉注射用人免疫球蛋白(IVIG)抗补体活性(ACA),在中性pH条件下,比较了不同的Na^ 含量及不同种类的糖对ACA测定结果的影响。结果表明,NaCl含量由0．2％上升至1．0％时,ACA呈逐渐下降趋势;用5％葡萄糖作稳定剂时ACA最低。IVIG在37℃条件下放置一月后,ACA有明显下降趋势。在半成品配制过程中,pH及各种成份的加入顺序对ACA也有一定影响。     

鲤亚急性喹乙醇中毒的血液生化指标研究

在饲料中分别以 10 0 0mg/kg、2 0 0 0mg/kg、30 0 0mg/kg的喹乙醇剂量对鲤进行了亚急性毒性试验 ,并对中毒鱼进行了血液生化指标的研究 ,经 6周的试验 ,各组的发病率分别为 35 %、70 %、92 .5 % ;死亡率为 2 7.5 %、5 7.5 %、82 5 %。中毒鱼表现为特征性的“应激性出血综合症” ,且Hb含量与RBC数量减少 ,表现为贫血 ;红细胞SOD酶活性降低 ;血清AST、ALT活性升高 ,血清K+ 浓度升高 ,Na+ 浓度降低 ,表现为高血钾和低血钠症。试验组血液生化指标的变化与对照组间存在着显著 (P <0 .0 5 )或极显著的差异 (P <0 .0 1)。     

云南水牛的同期发情、超数排卵和胚胎移植试验

为探讨水牛胚胎移植的效果 ,于 2 0 0 2年对云南水牛进行了胚胎移植试验 :①用国产氯前列烯醇(PG) 0 6mg/头·次处理供、受体水牛的同期发情率和可用率分别为 4 3 33% (13/ 30 )和 16 6 7% (5 / 30 ) ;同期发情率经产水牛高于青年水牛 (P =0 0 86 ) ,杂交水牛高于德宏水牛 (P =0 15 3) ,体重 4 0 1～ 5 30kg水牛显著高于体重 30 0～ 4 0 0kg水牛 (P <0 0 5 ) ;发情明显水牛的可用率极显著高于发情不明显的水牛 (P <0 0 1)。②选用河流型摩拉水牛与沼泽型德宏水牛的杂交一代 5头为供体 ,分进口激素组 (n =2 )和国产激素组 (n =3)进行超数排卵 ,共有 2头获 9枚胚胎 ;进口激素组供体的平均获胚数和可移植胚数分别为 2 0枚和 1 5枚 ,比国产激素组分别多 0 33枚 (P =0 4 5 4 )和 1 17枚 (P =0 2 88)。③所获 4枚可用鲜胚分别移植 3头受体 ,结果 90d的妊娠率为 33 33% ,但最终无一头产犊。试验结果表明使用进口FSH 2 4mg +PG (Lutalyse○R) 35mg和国产FSH 11mg +PG 0 8mg对水牛超数排卵有效 ,同时提示需要足够数量的受体 ,从中选用发情表现明显、黄体发育良好的进行胚胎移植 ,才能取得良好效果。     

自发性高血压大鼠心肌胶原与血管紧张素Ⅱ的关系

目的　观察成年 (16周龄 )自发性高血压大鼠 (spontaneouslyhypertensiverat,SHR)与同龄对照组 (WKY)大鼠之间细胞外基质成分的差异及血管紧张素Ⅱ (AngiotensinⅡ ,AngⅡ )在SHR大鼠左室肥厚形成过程的作用。方法　用尾袖法间接测定大鼠血压 ;检测左心室组织及血浆中的血管紧张素转化酶 (angiotensinconvertingenzyme ,ACE)活性 (紫外分光光度法 ) ;放免法测定左室心肌AngⅡ含量。免疫组化测定左室心肌胶原含量 ,用3H -Proline掺入量测定体外培养心肌成纤维细胞 (cardiacfibroblast,CFB)胶原的合成率。结果　 (1) 16周龄SHR大鼠血压明显高于对照组 (WKY)大鼠 ,分别为 (2 7.6 3± 2 .6 7)kPa和 (16 39± 0 54)kPa ,P <0 .0 5;(2 )SHR大鼠左室心肌AngⅡ含量明显高于WKY组 ,分别为 (2 6 6± 75)pg/ 10 0mg和 (134± 4 1)pg/ 10 0mg ,P <0 .0 5;(3)左室重量 (Leftventricalarmass,LVM)SHR明显高于WKY组 ,分别为 (10 14.3± 6 2 .1)mg和 (895.7± 86 .4 )mg ,P <0 .0 5;(4 )心体比 (Letventricrlarmass/bodyeight,LVM/BW )SHR明显高于WKY组 ,分别为 (3.4 4± 0 .15)mg/g和 (2 .17± 0 .11)mg/g ,P <0 .0 5;(5)体外细胞培养的心肌成纤维细胞3H -Proline掺入量随着AngⅡ浓度升高而增加 ,1μmol/L的AngⅡ使SH     

冻干低pH静注丙球质量稳定性观察

对１４批静注丙球保留样品进行３年效期内稳定性观察结果表明：出厂后３年内制品的物理检查和热稳定性试验均合格,ｐＨ值没有变化,ＩｇＧ分子仍以单体形式存在,抗补体活性（ＡＣＡ）和ＰＫＡ含量未见升高,白喉抗毒素效价未见下降。表明我所大规模生产的静注丙球质量是稳定可靠的     

l-Proline reduces IgG dimer content and enhances the stability of intravenous immunoglobulin (IVIG) solutions

Liquid intravenous immunoglobulin (IVIG) products offer improved convenience in preparation but often lack sufficient stability to allow room temperature storage. Furthermore, clinical tolerability may be affected due to formation of idiotype/anti-idiotype IgG dimers and/or aggregates. Here we report on the development of a 10% IVIG formulation with optimized stability achieved by the use of l-proline. The stability of concentrated liquid IVIG was strongly pH dependent. Aggregate formation, yellowish discoloration of the solution and loss of anti-hepatitis B surface antigen (HBs) antibody activity was minimal at intermediate pH (pH 4.8–5.3). Fragmentation of IgG was highest at low pH (pH 4.1). Idiotype/anti-idiotype IgG dimer formation was highest at neutral pH and was reduced with decreasing pH. The presence of l-proline further improved stability by inhibiting protein aggregation, reducing loss of anti-HBs antibody activity and decreasing coloring, particularly compared with glycine formulations. The IgG dimer content was up to 30% lower in solutions containing l-proline compared with those containing glycine or other stabilizers. In conclusion, a weakly acidic pH of approximately 5 and l-proline as stabilizer are optimal conditions for long-term stability of a liquid IVIG. l-proline, an amphiphilic, naturally occurring amino acid, is superior to glycine in restricting IgG dimer formation.     

采用多种抗原测定静注人免疫球蛋白(pH4)中抗体Fc段生物学活性的初探

目的初步探讨采用多种抗原测定静注人免疫球蛋白(IVIG)(pH4)中抗体的Fc段生物学活性,了解IVIG中抗体的Fc段生物学活性。方法采用补体活化的经典途径中免疫复合物激活补体的方法,将不同浓度的麻疹病毒、风疹病毒、乙肝表面抗原(HBsAg)、破伤风类毒素、脑膜炎球菌P64k外膜蛋白和白喉类毒素6种抗原分别致敏人O型血红细胞形成红细胞-抗原结合物;然后,6种致敏红细胞分别与IVIG孵育,与特异性抗体形成红细胞-抗原-抗体复合物;最后,此复合物与补体反应,在541 nm波长处读取吸光值,并绘制溶血反应动力学曲线,分别计算IVIG中针对上述6种抗原IgG的Fc段生物学活性。采用此方法,用6种抗原致敏的红细胞测定IVIG的Fc段生物学活性10次,验证此方法的重复性。结果麻疹病毒、风疹病毒、HBsAg、破伤风类毒素和脑膜炎球菌P64k外膜蛋白致敏的红细胞分别与供试品和补体反应后,测定的溶血反应动力学曲线较平缓,而白喉类毒素致敏的红细胞与供试品和补体反应后,测定的溶血反应动力学曲线下降明显,呈典型的"S"型曲线。计算结果显示,IVIG中针对此六种抗原的抗体Fc段生物学活性相对于参考品均大于80%。Fc段生物学检测方法重复性较好。结论采用多种抗原分别致敏红细胞,可以用来检测IVIG中多种抗体的Fc段生物学活性,为深入了解IVIG制品中的多种抗体的Fc段生物学活性奠定了基础。     

IL-2 and GM-CSF are regulated by DNA demethylation during activation of T cells, B cells and macrophages

High-dose intravenous immunoglobulin (IVIG) preparations are currently used for the treatment of autoimmune diseases such as immune thrombocytopenic purpura (ITP). Although the mechanisms of IVIG efficacy remain enigmatic, some clinical and laboratory studies suggest that interaction of the Fc domain of IgG, especially the Fc domain of dimeric IgG, with its receptors (Fc gamma receptors; FcγRs) plays an essential role. In this study, IVIG was dimerized with chemical crosslinkers to augment its therapeutic efficacy. Dimerized IVIG was found to have a much higher affinity for FcγRs than monomeric IVIG. In a mouse ITP model, chemically dimerized IVIG abrogated the decrease in platelet numbers in the blood that was caused by an anti-platelet antibody at a dose that was one tenth of the required dose of IVIG. These results suggest that chemical dimerization of IVIG should greatly improve the efficacy of IVIG therapy of ITP.     

Analysis and functional consequences of increased Fab-sialylation of intravenous immunoglobulin (IVIG) after lectin fractionation

It has been proposed that the anti-inflammatory effects of intravenous immunoglobulin (IVIG) might be due to the small fraction of Fc-sialylated IgG. In this study we biochemically and functionally characterized sialic acid-enriched IgG obtained by Sambucus nigra agglutinin (SNA) lectin fractionation. Two main IgG fractions isolated by elution with lactose (E1) or acidified lactose (E2) were analyzed for total IgG, F(ab')(2) and Fc-specific sialic acid content, their pattern of specific antibodies and anti-inflammatory potential in a human in vitro inflammation system based on LPS- or PHA-stimulated whole blood. HPLC and LC-MS testing revealed an increase of sialylated IgG in E1 and more substantially in the E2 fraction. Significantly, the increased amount of sialic acid residues was primarily found in the Fab region whereas only a minor increase was observed in the Fc region. This indicates preferential binding of the Fab sialic acid to SNA. ELISA analyses of a representative range of pathogen and auto-antigens indicated a skewed antibody pattern of the sialylated IVIG fractions. Finally, the E2 fraction exerted a more profound anti-inflammatory effect compared to E1 or IVIG, evidenced by reduced CD54 expression on monocytes and reduced secretion of MCP-1 (CCL2); again these effects were Fab- but not Fc-dependent. Our results show that SNA fractionation of IVIG yields a minor fraction (approx. 10%) of highly sialylated IgG, wherein the sialic acid is mainly found in the Fab region. The tested anti-inflammatory activity was associated with Fab not Fc sialylation.     

Different IVIG Glycoforms Affect In Vitro Inhibition of Anti-Ganglioside Antibody-Mediated Complement Deposition

Intravenous immunoglobulin (IVIG) is the first line treatment for Guillain–Barré syndrome and multifocal motor neuropathy, which are caused by anti-ganglioside antibody-mediated complement-dependent cytotoxicity. IVIG has many potential mechanisms of action, and sialylation of the IgG Fc portion reportedly has an anti-inflammatory effect in antibody-dependent cell-mediated cytotoxicity models. We investigated the effects of different IVIG glycoforms on the inhibition of antibody-mediated complement-dependent cytotoxicity. Deglycosylated, degalactosylated, galactosylated and sialylated IgG were prepared from IVIG following treatment with glycosidases and glycosyltransferases. Sera from patients with Guillain–Barré syndrome, Miller Fisher syndrome and multifocal motor neuropathy associated with anti-ganglioside antibodies were used. Inhibition of complement deposition subsequent to IgG or IgM autoantibody binding to ganglioside, GM1 or GQ1b was assessed on microtiter plates. Sialylated and galactosylated IVIGs more effectively inhibited C3 deposition than original IVIG or enzyme-treated IVIGs (agalactosylated and deglycosylated IVIGs). Therefore, sialylated and galactosylated IVIGs may be more effective than conventional IVIG in the treatment of complement-dependent autoimmune diseases.     

Anti-A and anti-B activity in batches of different intravenous immunoglobulin products determined using a direct haemagglutination method.

The European Pharmacopoeia requires that manufacturers assess intravenous immunoglobulin (IVIG) products for antibodies against blood groups A and B using an indirect anti-globulin test (AGT). However, this method suffers from the disadvantage that the anti-globulin reagent may be neutralised by excess IgG and invalidate the data generated. In view of this, we have used a direct microtitre-based haemagglutination method to screen batches of IVIG products from five manufacturers for anti-A and anti-B, and compared the titres with those reported by the manufacturers. The range of reported titres varied 32-fold across the different products, whereas virtually all the direct method titres fell within a 4-fold range for each specificity. This indicated that the discrepancies in reported titres were due to inconsistencies in manufacturers' testing methodology and/or interpretation of results. Our finding that the anti-globulin reagent used to bring about agglutination of anti-A- or anti-B-sensitised erythrocytes in the AGT was neutralised by excess IgG at least down to a 1 in 8 dilution of IVIG (from 5% (w/v) IgG) casts serious doubts on the suitability of the AGT for testing high immunoglobulin concentration products.     

The effect of solvent environment on the conformation and stability of human polyclonal IgG in solution.

Stability of therapeutic IgG preparations is an important issue as adequate efficacy and safety has to be ensured throughout a long shelf life. To this end, denaturation and aggregation have to be avoided. In many cases sugars are applied for stabilizing IgG in relatively high concentration (5-10%). However, certain sugars (sucrose, maltose) are responsible for adverse effects including renal failure. In this work we reassessed the effect of pH and stabilizers to optimize the solvent environment and minimize the amount of additives without endangering quality and stability. Since both biological function and aggregation depend on the conformational properties of individual IgG molecules, two sensitive and rapid physical methods were introduced to assess conformational changes and structural stability as a function of pH and addition of standard stabilizers. It was observed that the conformational stability decreases with decreasing pH, while the resistance against aggregation improves. The optimum pH range for storage is 5.0-6.0, as a compromise between conformational stability and the tendency for oligomerization. Intriguingly, additives in physiologically acceptable concentration have no effect on the thermal stability of IgG. On the other hand, glucose or sorbitol, even at a concentration as low as 1%, have significant effect on the tertiary structure as revealed by near-UV-CD spectroscopy, reflecting changes in the environment of aromatic side-chains. Although, 0.3% leucine does not increase conformational stability, it decreases the aggregation tendency even more efficiently than 1% glucose or sorbitol. Both pH and storage temperature are decisive factors for the long-term stability of IgG solutions. An increase in the dimer content was observed upon storage at 5 degrees C which was partly reverted upon incubation at 37 degrees C. Storage at temperatures higher than 5 degrees C may help to maintain an optimal proportion of dimers. Regarding the known side effects, and their limited stabilizing capacity at low concentration, it is advisable to omit sugars at intravenous immunoglobulin (IVIG) formulation. Hydrophobic amino acids give promising alternatives.     

冻干静脉注射人血丙种球蛋白的研制

作者参照瑞典ＫａｂｉＶｉｔｒｕｂ药厂的“Ｇａｍｍｏｎａｔｉｖ”的制备方法,以低温乙醇法的ＦⅡ沉淀为原料,经ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０吸附,加入人血白蛋白作为稳定剂制备静脉注射人血丙种球蛋白（ＩＶＩＧ）。该制剂的γ－球蛋白纯度＞９４％,ＩｇＧ单体二聚体含量＞９７％,ＩｇＧ各亚类均存在且构成比与正常人血浆相似。抗补体活性（ＡＣＡ）和前激肽释放酶激活剂活性（ＰＫＡ）符合规程要求,安全性和稳定性好。该制剂为冻干剂型,便于运输和保存。     

Intravenous immunoglobulin treatment of four patients with juvenile polyarticular arthritis associated with persistent parvovirus B19 infection and antiphospholipid antibodies

Children with rheumatic oligoarthritis and polyarthritis frequently establish persistent parvovirus B19 infections that may be associated with the production of antiphospholipid antibodies (anti-PL IgG). In this study we analysed the influence of high-dose intravenous immunoglobulin (IVIG) therapy on virus load, on the level of anti-PL IgG and its potential capacity to improve the patients' clinical status. Four juvenile patients with long-lasting polyarticular rheumatic diseases and persistent parvovirus B19 infection, associated in three cases with the presence of antibodies against beta2-glycoprotein I (anti-beta2GPI IgG), were treated with two cycles of IVIG on five successive days (0.4 g/kg per day). Clinical parameters including scores of disease activity, virus load and anti-PL IgG levels were determined before, during and after treatment. Two patients showed a complete remission that has lasted 15 months. During that period they showed neither clinical nor laboratory signs of inflammation. Viral DNA was not detectable in serum, and a decrease in anti-beta2GPI IgG was observed. As assessed by the Childhood Health Assessment Questionnaire and the Health-related Quality of Life Questionnaire for Children, both patients were no longer restricted in their activities of daily living and no impact on the health-related quality of life was observed. In one patient the therapy failed: there was no improvement of symptoms and no decrease in virus load or inflammatory parameters. In the fourth patient, clinical and laboratory parameters did not improve despite a decrease in both viral load and anti-PL IgG. Our results show that the use of IVIG to treat parvovirus B19-triggered polyarticular rheumatic disease of childhood might offer an opportunity to improve this disabling condition.