静注巨细胞病毒人免疫球蛋白原料血浆的筛选及制备

目的筛选获得高效价血浆并制备静注巨细胞病毒人免疫球蛋白(intravenous immunogloblin against cytomegalovirus,CMV-IVIG)。方法分别使用酶联免疫吸附(enzyme linked immunosorbent assay,ELISA)法及微量细胞中和法对113份人血浆巨细胞病毒(cytomegalovirus,CMV)IgG抗体进行结合效价及中和效价的检测,将两种方法获得的高效价血浆分别制备CMV-IVIG,采用常规微量细胞中和法检测抗CMV中和效价,并与IVIG中和效价进行比较,确定符合要求的免疫球蛋白成品,确立血浆筛选方法。结果对113份血浆进行效价检测,将结合效价≥20 IU/mL以及中和效价≥1∶32的血浆分别收集并制备5%CMV-IVIG,成品批号分别20190401和20190402;20190401批CMV-IVIG成品中和效价为498.28 IU/mL,20190402批CMV-IVIG成品中和效价为2 357.56 IU/mL,而5%IVIG中和效价为259.53 IU/mL。结论 ELISA法筛选获得的血浆制备的CMV-IVIG中和效价为普通IVIG的1.92倍,不符合特异性免疫球蛋白不低于普通免疫球蛋白4倍的效价判定标准,微量细胞中和法筛选获得的血浆制备CMVIVIG中和效价为普通IVIG的9.08倍,符合效价判定标准,表明微量细胞中和法可用于CMV高中和效价血浆的筛选,为CMV-IVIG的制备提供合格的血浆原料。     

静注人免疫球蛋白(pH 4)中人肠道病毒71型中和抗体效价分析

目的对目前市售静注人免疫球蛋白(p H 4)(human intravenous immunoglobulin,IVIG)中的人肠道病毒71型(enterovirus 71,EV71)中和抗体效价进行筛查,为EV71相关疾病的被动免疫治疗提供参考。方法采用微量细胞病变法检测24批IVIG制品中的EV71中和抗体效价,并分组进行比较。结果 24批IVIG制品中的EV71中和抗体效价为841.6~1 024.3 U/m L,比活为16 832~20 486 U/g Ig G。4个季度生产的IVIG中EV71中和抗体效价差异无统计学意义(P0.05),病毒高发季与非高发季采浆生产的IVIG中EV71中和抗体效价差异也无统计学意义(P0.05)。结论 IVIG制品中EV71中和抗体效价可达50 000 U/瓶(蛋白浓度5%,50 m L/瓶),本研究为EV71相关疾病的被动治疗提供了临床参考依据。     

细胞核染色法检测人免疫球蛋白类制品巨细胞病毒中和抗体效价

目的检测静注免疫球蛋白(intravenous immunoglobulin,IVIG)(p H 4)及巨细胞病毒(cytomegalovirus,CMV)免疫球蛋白(IG)中的CMV中和抗体效价。方法采用基于微量细胞病变的细胞核染色法检测39批国产IVIG、8批注册检验巨细胞病毒免疫球蛋白(CMV-IG)以及2批国外CMV-IG制品的CMV中和抗体效价。结果 39批IVIG的CMV中和抗体效价几何平均值为361 U/ml,最低效价为234 U/ml,最高效价为502 U/ml;8批注册检验CMV-IG的CMV中和抗体效价几何平均值为1 113 U/ml,最低效价为923 U/ml,最高效价为1 553 U/ml;2批国外CMVIG的CMV抗体效价分别为579 U/ml和163 U/m。8批注册检验CMV-IG的CMV中和抗体效价显著高于39批IVIG的CMV中和抗体效价(P0.001)。结论国产39批IVIG的CMV中和抗体效价在234~502 U/ml之间,经血浆筛查后投浆制备的CMV-IG显著高于普通IVIG的CMV中和抗体效价。     

静注甲型H1N1流感人免疫球蛋白的研制

目的研制高效价静注甲型H1N1流感人免疫球蛋白。方法用甲型H1N1流感疫苗(简称甲流)对固定供血浆者按疫苗说明书免疫后2周开始采集原料血浆,采用血凝抑制法检测原料血浆和制品的甲流抗体效价;采用柱层析法从高效价甲流免疫血浆中提取富含IgM免疫球蛋白的样品,模拟低温乙醇蛋白分离法工艺从小量混合血浆分离IgG样品,测定血浆和提取样品的甲流抗体效价,分析甲流中和抗体效价与IgG、IgM含量的对应关系,判断甲流中和抗体的免疫球蛋白类型;按本公司静脉注射人免疫球蛋白(Intravenous immunoglobulin,IVIG)生产工艺制备静注甲型H1N1流感人免疫球蛋白,并与普通IVIG及相应合并血浆的甲流抗体效价进行比较。结果筛选到甲流抗体效价≥320 HU/ml的合格血浆9 866袋,合格率达33.5%,其中抗体效价≥640 HU/ml的血浆占合格血浆的49%,血浆质量符合《中国药典》三部(2010版)"血液制品生产用人血浆规程"要求;甲流抗体效价与IgG含量呈相应比例关系,而与IgM含量无关,甲流中和抗体的免疫球蛋白类型以IgG为主;制备的甲流人免疫球蛋白制品的甲流中和抗体效价达2 560 HU/ml,为普通IVIG的197倍,其他质量指标符合《中国药典》三部(2010版)"静注人免疫球蛋白制检规程"要求。结论成功研制了静注甲型H1N1流感人免疫球蛋白,在甲流疫情得到有效控制的情况下,仍具有战略储备意义。     

高效价巨细胞病毒人免疫球蛋白原料血浆的筛查

目的分析普通健康供浆员中人巨细胞病毒(human cytomegalovirus,HCMV)免疫球蛋白(IgG)抗体效价分布情况,筛查富含高效价HCMV IgG抗体的原料血浆。方法采用HCMV IgG定量检测试剂,检测本公司下属广西地区五个单采血浆站采集的血浆样本的HCMV IgG抗体效价,检测抗体效价15 PEI-U/ml的血浆样本混合后的HCMV IgG抗体效价,并对血浆中富含HCMV抗体的供浆员血浆进行采集,分析效价分布变化情况。结果健康供浆员28 607份血浆样本的HCMV IgG抗体阳性率为85.2%,其中效价分布在1~10、10~15、15 PEI-U/ml的各占64.5%、8.7%和11.9%;1 318名血浆中富含HCMV抗体的供浆员8 426份血浆样本HCMV IgG抗体阳性率为98.1%,抗体效价15 PEI-U/ml占43.8%,约为普通健康人血浆样本的3.7倍,3个月内平均抗体效价可维持在15 PEI-U/ml以上的有586人,占44.5%;不同浆站抗体效价15 PEI-U/ml的血浆样本混和后的平均效价为33.72 PEI-U/ml。结论健康供浆员中高效价HCMV IgG抗体的比例较高,部分供浆员HCMV IgG抗体高效价水平可稳定维持3个月,从健康供浆员中采集含高效价HCMV IgG抗体的原料血浆,用于制备巨细胞病毒人免疫球蛋白具有可行性。     

静注人免疫球蛋白在治疗自身免疫疾病和炎症所引发皮肤病的应用

静注人免疫球蛋白(intravenous immunoglobulin,IVIG)是由多种蛋白组成,包括IgG、IgM、IgA、IgE抗体及少量的白蛋白,还包括氯化钠、麦芽糖等物质。由于IVIG从健康人血浆中提取,因此含有多种人体所需要的自然抗体,一直以来被用作治疗传染性疾病和免疫缺陷疾病。近些年发现IVIG可以治疗多种临床疾病,包括自身免疫和炎症疾病及其所引起的多种皮肤科相关疾病。本文针对IVIG治疗因自身免疫系统紊乱或炎症引发的皮肤疾病的应用作一综述。     

静注人免疫球蛋白管理现状调研

正静注人免疫球蛋白(intravenous immunoglobulin,IVIG)制品主要成分为Ig G,是人血浆中含量最丰富的免疫球蛋白,从大量健康人群混合血浆(原料血浆)中浓缩分离而成~([1])。目前,IVIG临床适应证广泛,需求量较大,作为一种血液制品,其原料来源稀少,价格昂贵,因此如何合理的管理IVIG资源逐渐备受关注。本文从生产、供应、价格、医保等方面调     

巨细胞病毒IgG定量ELISA检测方法的建立及初步应用

目的建立巨细胞病毒(Cytomegalovirus,CMV)IgG定量ELISA检测方法,并进行初步应用。方法用CMV IgG(50 U/ml)标准品建立CMV IgG ELISA定量检测方法,确定该方法的最佳线性范围;制备室内质控血清并进行标定。对该方法进行重复性、中间精密性、准确性、特异性验证,并与Dia.Pro CMV IgG ELISA试剂盒进行比较。用建立的ELISA法检测689人份健康献浆员血浆的CMV IgG抗体效价。结果建立的CMV IgG定量ELISA检测方法的最佳线性范围为0.000 78～0.05 U/ml;制备的室内质控血清的抗体效价为(5.67±0.55)U/ml;重复性试验检测结果的变异系数为4.6%～9.8%,中间精密性试验检测结果的变异系数为9.7%～10.6%;该方法检测3个浓度标准品的回收率为107.01%～112.97%;样品稀释液和血清中的其他一些干扰因素对检测结果影响较小;该方法与Dia.Pro CMV IgG ELISA试剂盒检测血浆样品的相关系数r=0.81。该方法检测689人份健康献浆员血浆样品的CMV IgG抗体效价≥10 U/ml的占30.8%。结论建立的CMV IgG定量ELISA检测方法操作简便,精密性良好,特异性强,可用于人血浆中CMV IgG的定量检测。     

实验设计法优化高浓度静注人免疫球蛋白制剂的处方

目的通过实验设计法优化高浓度(10%)静注人免疫球蛋白(human immunoglobulin for intravenous injection,IVIG)的制剂处方。方法采用单因素试验(包括等电点、二级结构、分子大小分布,转变中点温度4个指标)确定IVIG(10%)的pH范围,同时经国外相关数据和产品质量指标选择IVIG的稳定剂甘氨酸和聚山梨酯80的浓度范围,再采用实验设计法优化IVIG的pH及稳定剂浓度。以转变中点温度为指标,通过单因素试验优化IVIG(10%)的离子强度。结果最佳IVIG(10%)制剂处方为聚山梨酯80:80 mg/L,pH:4. 5,甘氨酸:0. 25 mol/L。最适离子强度为0 mmol/L氯化钠。结论成功优化了IVIG(10%)的制剂处方,可确保制剂的稳定性,提高临床用药安全性。     

国产静脉注射人免疫球蛋白制品中凝血激活物水平分析

目的分析11家中国血液制品企业共计64批次上市后的静脉注射人免疫球蛋白(immunoglobulin for intravenous injection,IVIG)(p H 4)制品中凝血激活物水平,对国内IVIG制品在促凝血方面的安全性作出评估,为提高制品质量标准提供参考。方法利用非活化的部分凝血活酶时间法(non-activated partial thromboplastin time,NAPTT)、凝固法(一期法)及本实验室建立的改良凝血酶生成试验(modified thrombin generation test,M-TGT),分别测定64批次国产和7批次进口IVIG样品的NAPTT、系列凝血因子活性、活化凝血因子Ⅺ(FⅪa)的含量及凝血酶生成含量达到最高值时所需时间(time to peak,TTP),分析IVIG的凝血激活物水平。结果除国内厂家F所生产的2批IVIG中FⅪ活性为0.030~0.036 IU/ml外,其余国产62批次产品中FⅪ活性均低于试剂最低检测限未检出;所有产品中TTP均大于40 min,FⅪa含量均小于0.37 nmol/L,NAPTT均大于203 s。结论所调查的11家共计64批次的国产IVIG制品的凝血激活物水平均较低。今后在我国是否推行IVIG制品的凝血激活物检测尚需进一步研究。     

Study of human cortical bone and demineralized human cortical bone viscoelasticity

The knowledge of human bone viscoelasticity is an important issue for defining semirigid calcified tissues implants. A very sensitive technique was used to investigate bone viscoelasticity: the thermally stimulated creep method. A study of demineralized human bone was performed to determine the molecular origin of bone viscoelasticity. The thermally stimulated creep spectra of bone and demineralized bone, at the hydrated state, present a similar shape with one main retardation mode located at −133 and −120°C, respectively. This mode is shifted toward higher temperatures after dehydration, revealing the existence of another mode at around −155°C. The analysis of elementary spectra of bone and demineralized bone has shown that retardation times follow an Arrhenius equation, and that two compensation phenomena are observed with comparable compensation parameters. The first compensation phenomenon, which corresponds to the main retardation mode, was attributed to motions of water molecules located inside the collagen triple helix. The second compensation phenomenon, which reveals the existence of another relaxation mode at higher temperatures, was assigned to movements of hydrophilic side chains bound to water molecules. As for the mode observed at around −155°C, it was associated with motions of aliphatic side chains. Overall, bone viscoelasticity originates from the organic matrix. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 79: 2527–2533, 2001     

Lipids in human milk

I have reviewed recent (March 1995–December 1997) papers on human milk lipids including many on fatty acid (FA) composition. The effects of maternal diets on the profiles are apparent. However, more data on the composition of milk lipids are needed. It is noteworthy that so few papers on milk FA composition have reported analyses using high-resolution gas-liquid chromatography columns. Two of these were on milk from women in North America. The diets in North America are varied and the number of analyses few. We do not have a reliable data base showing the ranges of biologically important acids. Except for the gangliosides, few new data on the other lipids appeared during this period.     

Color and human response

Color has played an important role in human life and culture since the beginnings of recorded history. Here are random notes drawn from widely different sources.     

Paf-acether in human skin

Paf is a phospholipid mediator present in human skin which induces inflammatory events such as neutrophil infiltration and increased vascular permeability. Recent data suggest that cutaneous cells, such as fibroblasts and keratinocytes, produce paf and that paf is released during allergic cutaneous reactions. It is tempting to speculate that paf may contribute to the development of various skin disorders with acute and chronic skin inflammation. Paf antagonists may help in bringing answers to this hypothesis and may offer new prospects for the treatment of cutaneous inflammatory diseases. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Glycosphingolipids of human thyroid

Glycosphingolipids were isolated from total lipids of female and male human thyroids by alkaline hydrolysis, silicic acid, diethylaminoethyl-celluose and thin layer chromatography and analyzed by gas liquid chromatography. On the basis of their mobility in two dimensions on thin layer chromatography, IR analysis, and of sugar molar ratio, four neutral glycolipids, a sulfatide, and a hematoside fraction were identified. Glucosyl, plus galactosyl ceramide, and trihexosyl ceramide were the major fractions and accounted for 33% and 28% of total neutral glycolipids, respectively. Dihexosyl ceramide was a mixture of lactosyl and digalactosyl ceramide. The acidic lower phase glycolipids comprised ceramide galactosyl sulfate as the major component of male thyroids. Hematoside was identified tentatively as a minor component of the thyroids of both sexes. Major fatty acids of all neutral glycolipid fractions were 20∶0, 22∶0, 24∶0, and 24∶1; 24∶0 and 24∶1 for sulfatides. Low proportions of α-hydroxy fatty acids were identified. Total neutral glycosphingolipids of male thyroids were comparable in quantities with human liver but lower than kidneys, leucocytes, and platelets. Male thyroids comprised higher quantities of neutral glycosphingolipids (4.04±0.32 μmoles/g total lipid) as compared to females (2.34±0.21 μmoles/g total lipid), and much higher sulfatide than the females. These marked differences may suggest that the biosynthesis of the glycosphingolipids in the thyroid gland is under hormonal control. Similarities in glycosphingolipid composition of human thyroid and kidney are discussed in relation to a possible role played by glycolipids in ion transport, which is a common feature of the two organs.     

Lipids and human milk

To support the growth and development of the breast‐fed infant, human milk provides the dietary essential fatty acids, linoleic acid (LA; 18:2n‐6), α‐linolenic acid (ALA, 18:3n‐3), as well as longer‐chain polyunsaturated fatty acids including arachidonic acid (20:4n‐6) and docosahexanoic (DHA 22:6n‐3). The linoleic acid, alpha‐linolenic acid, DHA and arachidonic acid concentration of pasteurized and unpasteurized human milk remains stable during the first month of storage at –20°C and –80°C. However after the first month, a slow decrease in concentration progresses until the end of 6 months of storage at both temperatures. The levels of n‐6 and n‐3 fatty acids, particularly linoleic acid, alpha‐linolenic acid and DHA, in human milk vary widely within and among different populations, and are readily changed by maternal dietary intake of the respective fatty acid. The present paper reviews recent understanding from key researchers of maternal diet and human milk fat composition and form our work the effect of milk fat composition on storage conditions. It is important to understand that maternal diet can affect human milk fat composition and subsequently infant development and growth.     

Lipids of human myocardium

The major lipid classes, including some phospholipids, and their fatty acid profiles have been measured in portions of left ventricular muscle and psoas muscle obtained at autopsy. Atrial appendages and ventricular muscle removed during cardiac surgery were examined also. The proportions of the individual phospholipids were the same in all the muscles, having an excess of phosphatidyl ethanolamine and phosphatidyl serine compared with the serum. Their fatty acid profiles resembled those obtained from other locations. The triglyceride content of the myocardium was relatively constant (except in the atrial appendage) but did rise slightly with increasing obesity. The free fatty acid concentration in the myocardium was relatively high and had a variable fatty acid profile.     

Fats in human nutrition

Summary Dietary fats represent the most compact chemical energy available to man. They contain twice the caloric value of an equivalent weight of sugar. However dietary fats should not be thought of solely as providers of unwanted calories as fats are as vital to cell structure and biological function as protein. If an individual consumes food items of high fat content, an adequate protein and vitamin intake should be assured in order to provide the lipotropic factors necessary for normal fat metabolism. It may be more judicious to control the total caloric intake under such circumstances rather than to resort to periods of semi-starvation or to drastically decrease the dietary fat intake which could result in an increase in hunger pangs and an actual increase in total caloric intake. If the excess calories furnished by carbohydrates are converted to fatin vivo, the problem of obesity could not be solved under conditions of increased total caloric intake. The problem could be solved by a curtailed intake of a diet which includes meat, milk, eggs, vegetables, fruits, and sufficient cereals and bread to provide for an adequate protein, vitamin, and caloric intake. Dietary fats provide the essential linoleic acid which seems to have both a structural and functional role in animal tissue. Although the optimum total intake of linoleic acid by man has not been established, it is evident that the level of intake in the American dietary pattern could be increased. However the indiscriminate substitution of soft for hard fats seems undesirable as an excess consumption of highly unsaturated fatty acids may change the functional value of the triglycerides in the depot fats and may put an undue stress on the antioxidant supply availablein vivo.     

Desmosterol in human milk

Milk samples were collected from mothers at 2,6, 12 and 16 weeks postpartum. Desmosterol was found to be present in all the milk samples. Identification of desmosterol was based on retention times with two gas liquid chromatography (GLC) columns and verified by GC-m ass spectrometry. The concentration of desmosterol in breast milk increased significantly (P<0.5) from 0.6 mg/100 ml at 2 weeks to 1.3 mg/100 ml at 16 weeks postpartum. Desmosterol was not significantly correlated with total lipid, total cholesterol or free cholesterol in the milk. Scientific Contribution No. 960, Storrs Agricultural Experiment Station, University of Connecticut, Storrs, CT 06268.