免疫球蛋白制品靶抗体谱、效价水平与交叉反应的实验研究

目的：研究免疫球蛋白制品靶抗体谱、效价水平与交叉反应。方法：采用水痘病毒、巨细胞病毒、Ⅰ与Ⅱ单纯疱疹病毒、狂犬病毒、破伤风类毒素IgGELISA试剂盒及乙肝病毒表面抗原RIA试剂盒分别检测水痘、巨细胞、狂犬、乙肝、破伤风5种特免球蛋白及肌注、静注免疫球蛋白中所含靶抗体效价。结果：由疫苗免疫献血员制备的狂犬、破伤风与乙肝人免疫球蛋白有较高的特异性抗体效价,由筛选高效价血浆制备的巨细胞病毒人免疫球蛋白与其它免疫球蛋白比较,其特异性抗体效价差距较小,人群中也普遍存在水痘病毒与单纯疱疹Ⅰ型病毒感染,其强度较巨细胞病毒弱、较单纯疱疹Ⅱ型病毒强。结论：免疫球蛋白受试品均能检测到7种抗原的靶抗体,其抗体效价水平与主动、被动免疫有关,水痘疫苗能诱导出高效价的抗巨细胞病毒交叉反应抗体,肌注与静注IgG之间存在值得注意的抗体效价差别。     

白喉和破伤风类毒素抗原ELISA检测方法的建立

目的建立定量检测白喉和破伤风类毒素抗原的酶联免疫吸附试验(ELISA)方法。方法分别采用白喉、破伤风单抗包被酶标板,兔抗白喉、破伤风多抗作为二抗,辣根过氧化物酶标记的羊抗兔IgG抗体作为酶标抗体,以平行线法建立定量检测白喉和破伤风类毒素抗原含量的夹心ELISA法,并进行方法学验证。结果两种定量检测ELISA方法的验证结果均符合规定。检测白喉类毒素抗原ELISA方法的定量限为8.90×10-4Lf/ml,回收率为98.35%,批内变异系数(CV)≤10.59%,批间CV≤13.51%;检测破伤风类毒素抗原ELISA方法的定量限为2.13×10~(-3)Lf/ml,回收率为107.28%,批内CV≤13.96%,批间CV≤10.06%。结论建立了白喉、破伤风类毒素抗原ELISA检测方法,该法特异性强、准确度高、重复性好,可用于白喉破伤风疫苗产品的质量控制和生产过程控制。     

深圳市宝安区健康人群百日咳、白喉、破伤风抗体水平监测

目的了解宝安区健康人群破伤风、白喉、百日咳三种疫苗血清抗体水平,评价三种疫苗的免疫实施效果。方法2011年随机抽取宝安区7个街道不同年龄段人群,采用试管凝集试验检测血清抗体水平。结果破伤风、白喉、百13咳抗体保护率分别为98．33％,98．06％,98．33％,各年龄组人群均达到90％以上;几何平均滴度（GMT）分别为1：80．63、1：80．48、1：1409．35。均以21～32岁组最低;三种抗体保护率和GMT有随年龄增大而明显降低的趋势。结论宝安区人群破伤风、白喉、百日咳免疫水平都较高,免疫屏障已经形成,短期内暴发流行百日咳、白喉、破伤风的可能性不大。     

酶联免疫吸附试验(ELISA)和动物法测定人血清破伤风抗体的比较

<正> 人血清中破伤风抗体的测定,在临床上和流行病学上都有十分重要的意义。在临床上是判断病人对破伤风是否具有免疫能力的重要依据;在流行病学上是监测人群破伤风免疫状态的重要方法。小白鼠中和试验是检测人血清破伤风抗体水平的经典方法,凡是抗体效价达到0.01~(IU)/ml以上者便具有足够的破伤风免疫力,动物法的结果准确可靠,为     

破伤风毒素C片段的基因克隆、表达、纯化及免疫原性分析

目的　对破伤风毒素C片段进行基因克隆、重组表达、蛋白纯化和免疫原性分析。方法　应用PCR技术直接从破伤风梭状芽孢杆菌的质粒DNA中扩增出 1370bp的破伤风毒素C片段(简称TTC)基因 ,将此基因片段插入到表达载体pET 2 2b( )中 ,并在大肠杆菌BL2 1(DE3)中表达。用阴离子交换层析和金属离子螯合亲和层析方法进行蛋白纯化后 ,参照《中国生物制品规程 2 0 0 0年版》的免疫攻击实验方法测定其效价。结果　经SDS PAGE分析 ,重组蛋白的表达量占菌体总蛋白的15 %。免疫印迹实验证实该重组蛋白是破伤风毒素C片段抗原。纯化得到纯度为 96 .5 %的重组蛋白 ,纯化回收率达 4 0 %。免疫攻击实验测定其效价为 18.70 6IU/mg,ED50 为 2 0 μg。经加强免疫 ,1μg免疫剂量即能产生足够的抗体保护动物免受破伤风毒素的攻击。结论　所获得的重组蛋白具有良好的免疫原性 ,为开发基因工程疫苗奠定了基础     

定量检测破伤风类毒素酶联免疫吸附法的建立及初步应用

目的：建立定量检测破伤风类毒素（TT）抗原的双抗体夹心ELISA法,并探索其初步应用。方法：以TT抗毒素为包被抗体,大鼠抗TT多抗为检测抗体,采用相应酶标抗体检测TT抗原含量,建立双抗体夹心ELISA法,并进行方法学验证。结果：TT含量在0～0.062 5 Lf/ml范围内线性关系良好（r>0.99）。该方法与白喉类毒素、百日咳类毒素、百日咳丝状血凝素及百日咳黏附素无明显交叉反应,重复性好,特异性较强,精密度及准确度验证均符合常规质控要求。该法准确检测范围为0.000 625～0.040 000 Lf/ml,定量限度为0.000 625 Lf/ml。采用该方法对TT抗原进行吸附率检测,分别采用该法和絮状单位测定法测定6批TT的絮状含量,2种检测方法高度相关（r=0.94）。结论：建立的定量检测TT的双抗体夹心ELISA法为破伤风疫苗生产过程中TT质量控制提供了有效技术手段。     

深圳市1003名外来劳务工破伤风抗体水平的调查

目的利用横断面基线调查法对深圳市1003名工厂劳务工破伤风抗体水平进行调查研究,制定相应的免疫接种措施,加强对外来劳务工的管理。方法采用酶联免疫吸附试验检测破伤风抗体。结果深圳市1003名工厂外来劳务工来自全国28个省市自治区,抗体阳性率较高的人群主要集中在中部、南部和西南部。1003名外来劳务工破伤风抗体的阳性率为18.6%,其中男性为18.7%,女性为18.4%。29岁以下劳务工破伤风抗体的阳性率为15.7%,30～39岁的阳性率为2.1%,40岁以上的阳性率为0.8%。小学文化及以下劳务工破伤风抗体阳性率为14.8%,初中文化为14.7%,高中或中专文化为21.0%,专科为21.6%,大学本科及以上文化为26.5%。结论深圳市工厂劳务工破伤风抗体水平偏低。抗体阳性率在不同年龄组间存在显著差异,29岁以下劳务工破伤风抗体的阳性率较高。建议政府为外来劳务工进行破伤风疫苗的普种,以进一步提高和保持高水平的百白破混合制剂常规免疫接种率,同时加强外来流动人口免疫接种的监测,防止破伤风病的发生。     

检测IgG抗体效价在诊治母儿血型不合中的意义

目的 探讨检测IgG抗体效价在诊治母儿血型不合中的意义。方法 对孕妇为“O”型血而丈夫为其他血型的怀疑有母儿ABO血型不合的产妇，在孕28周以后进行ABO抗体效价检测。结果 595例孕妇接受筛查，抗体效价小于64的352例产妇所生新生儿中有26例发生新生儿高胆，无1例溶血，抗体效价在64～256之间的226例产妇所生新生儿16例发生新生儿高胆，仅1例溶血，抗体效价512的12例产妇所生新生儿有3例为“O”型血，9例为其它血型，有7例发生新生儿高胆，3例溶血，抗体效价为1024的4例产妇所生新生儿2例为其它血型其中1例发生高胆并溶血，另2例新生儿血型为“O”型，抗体效价为2048的1例新生儿其他血型并发生溶血性黄胆。结论 检测抗体效价可以筛查一部份夫妻血型不合的孕妇作为监测对象加以监测，特别是抗体效价高于512时应该重点监测，但高效价并不一定代表母儿血型不合及溶血。     

纯化血清不同倍比稀释法检测孕妇IgG型抗A或抗B抗体效价的比较

目的探讨纯化后的血清不同倍比稀释法检测孕妇ABO血型IgG型抗A抗体效价、抗B抗体效价的可行性。方法采用1∶16、1∶32、1∶4倍比稀释法对同一份效价为512的IgG型抗A抗体的孕妇血清进行检测,再采用以上倍比稀释法检测200例孕妇IgG型抗A抗体效价、抗B抗体效价。结果三种倍比稀释法对同一份效价为512的IgG型抗A抗体孕妇血清检测的凝集强度与效价积分为45、47、45分;再采用三种倍比稀释法检测200例孕妇IgG型抗A抗体效价、抗B抗体效价,异常率为35.5%、36.0%、35.0%,三种方法检测的结果无显著性差异。结论采用更高倍比稀释纯化后血清检测孕妇血清中IgG型抗A抗体或抗B抗体效价,具有敏感性适当、缩短批量检测时间、节约试剂耗材等优点,适合对大批量血标本进行检测,具有一定实际应用价值。     

获得诺贝尔奖金的免疫学工作者

1901 1901年,第一个医学诺贝尔奖金获得者为Emil von Beheing(1854～1917)。他是在柏林klch’s研究所Robert koch的指导下学习的。继1883年Loffler分离出白喉杆菌及1888年Roux和Yersin鉴定出白喉外毒素后,Von Beheing及其同事Kitasato等人(1890～1892)指出,白喉及破伤风的免疫性是由于存在循环抗毒素,并提出被动     

Antibodies against vaccine-preventable diseases in pregnant women and their offspring in the eastern part of Germany

Maternal and cord blood samples of 290 pregnant women in the eastern part of Germany with a mean age of 28 years (16-41 years) were analyzed for antibodies to vaccine-preventable diseases. Both mothers and infants had detectable levels of antibodies to mumps in 96% and to tetanus in 93% of cases. Detectable levels to poliomyelitis, diphtheria, measles and rubella varied from 55% to 91%. Cord blood samples had a significantly higher prevalence of antibodies to pertussis (61%) and diphtheria (81%) in comparison to maternal samples (pertussis 37%, diphtheria 70%) as well as significantly enhanced antibody concentrations to diphtheria. In conclusion, the prevalence of antibodies to pertussis (61%), diphtheria (81 %), poliomyelitis (55-59%) and measles (85%) is suggested to be insufficient in newborn infants to protect them against these infectious diseases.     

Serological response to filamentous hemagglutinin and lymphocytosis-promoting toxin of Bordetella pertussis.

Serum antibody responses to the filamentous hemagglutinin and the lymphocytosis-promoting toxin of Bordetella pertussis after vaccination with diphtheria and tetanus toxoids and pertussis vaccine, adsorbed, were assayed by using the enzyme-linked immunosorbent assay. The effect of early immunization, during the first week of life, on the antibody response also was determined. After vaccination, immunoglobulin G (IgG) and IgM directed against both the filamentous hemagglutinin and the lymphocytosis-promoting toxin were detected. Generally, antibody titers increased with subsequent injections and the age of the children. Maternal antibodies against filamentous hemagglutinin and lymphocytosis-promoting toxin were detected in cord blood. The ability of an infant to produce serum IgG anti-lymphocytosis-promoting toxin after vaccination with pertussis vaccine was inversely related to the cord blood serum IgG anti-lymphocytosis-promoting toxin titer at birth. A good antibody response was observed in infants with low cord blood titers, and a poor antibody response was seen in infants with high cord blood values. The IgM anti-lymphocytosis-promoting toxin response was good in groups with both low and high cord blood titer, with no significant difference observed between the two groups. No IgA anti-lymphocytosis-promoting toxin or IgA anti-filamentous hemagglutinin titers were observed in vaccines. IgA antibodies were observed in convalescent sera from two adults and may be presumptive evidence of infection with B. pertussis.     

Comparative safety and immunogenicity of an acellular versus whole-cell pertussis component of diphtheria-tetanus-pertussis vaccines in Senegalese infants

A diphtheria and tetanus toxoid two-component acellular pertussis vaccine (DTaP), consisting of 25 g glutaraldehyde-detoxified pertussis toxin (PT) and 25 g native filamentous hemagglutinin (FHA), was compared with diphtheria and tetanus toxoid whole-cell pertussis vaccine (DTwP) in a randomized, double-blind manner in 286 Senegalese infants inoculated at two, four, and six months of age. In infants receiving DTaP a significantly lower rate of local reactions, crying and fever was observed than in infants receiving DTwP. One month after the third dose, the geometric mean titres for FHA antibodies were higher in the DTaP group, whereas increases in PT antibody titres were higher in the DTwP group. More than 90% of the infants had a fourfold or more increase in antibodies to both PT and FHA with either vaccine. Diphtheria, tetanus, and polio antibody responses were also measured and found to be comparable between the two groups. The results of this pilot study support the implementation of a field trial to compare the protective efficacy of these vaccines against pertussis in the same setting.     

候选无细胞百白破-Sabin株灭活脊髓灰质炎联合疫苗在大鼠上的免疫保护效果研究

目的观察候选无细胞百白破-Sabin株灭活脊髓灰质炎联合疫苗(DTaP-sIPV)在大鼠中的免疫保护效果,为疫苗临床前研究提供依据。方法将候选疫苗DTaP-sIPV、无细胞百白破-灭活脊髓灰质炎-b型流感嗜血杆菌联合疫苗(DTaP-IPV/Hib)、吸附无细胞百白破-b型流感嗜血杆菌联合疫苗(DTaP/Hib)、百日咳疫苗效力参考品(全细胞疫苗,wP)按0、30、60 d 3剂免疫程序免疫Wistar大鼠,检测各组大鼠每剂免疫后的血清中各组分抗体水平。在免疫完成后3周,用百日咳18323株通过气雾攻击的方式感染大鼠。在感染后的第3、7、14、21和28天检测各组白细胞数、肺部菌落克隆形成数以及百日咳疫苗组分抗体变化水平。结果候选疫苗组3剂次免疫完成后PT抗体几何平均滴度(GMT,log2)为16.74,FHA抗体GMT为18.44,PRN抗体GMT为10.75,DT抗体GMT为17.34,TT抗体GMT为17.84,针对3种Sabin脊髓灰质炎病毒株(Ⅰ、Ⅱ和Ⅲ型)的抗体的GMT分别为7.57、8.41和9.70,均达到100%阳转。候选疫苗抗原组分抗体除了PRN和I型IPV外,其他组分抗体水平均与疫苗对照组相比无显著性差异。在基础免疫完成后3周对大鼠进行百日咳杆菌气雾攻击,各疫苗组均表现较好的保护效果,白细胞水平都呈现平稳状态,虽然在肺部也检测到少量细菌定植,但各疫苗组间差异不明显,且在感染后第28天都清除至检测限;而空白对照组在肺部则检测到了大量细菌定植,且在感染后第28天都并未清除至检测限,百日咳特异性的FHA和PRN抗体在感染后的第14天也出现了相应的升高。结论候选疫苗在Wistar大鼠模型上具有较好的免疫保护效果。     

Assessment of serologic immunity to diphtheria-tetanus-pertussis after treatment of Korean pediatric hematology and oncology patients

The aim of this study was to investigate the diphtheria-tetanus-pertussis antibody titers after antineoplastic treatment and to suggest an appropriate vaccination approach for pediatric hemato-oncologic patients. A total of 146 children with either malignancy in remission after cessation of therapy or bone marrow failure were recruited. All children had received routine immunization including diphtheria-tetanus-acellular pertussis vaccination before diagnosis of cancer. The serologic immunity to diphtheria, tetanus and pertussis was classified as: completely protective, partially protective, or non-protective. Non-protective serum antibody titer for diphtheria, tetanus and pertussis was detected in 6.2%, 11.6%, and 62.3% of patients, respectively, and partial protective serum antibody titer for diphtheria, tetanus and pertussis was seen in 37%, 28.1%, and 8.9% of patients. There was no significant correlation between the severity of immune defect and age, gender or underlying disease. Revaccination after antineoplastic therapy showed significantly higher levels of antibody for each vaccine antigen. Our data indicates that a large proportion of children lacked protective serum concentrations of antibodies against diphtheria, tetanus, and pertussis. This suggests that reimmunization of these patients is necessary after completion of antineoplastic treatment. Also, prospective studies should be undertaken with the aim of devising a common strategy of revaccination.     

Bordetella pertussis infection in mice: correlation of specific antibodies against two antigens, pertussis toxin, and filamentous hemagglutinin with mouse protectivity in an intracerebral or aerosol challenge system

The correlation of titers of specific serum immunoglobulin G antibodies against two antigens, pertussis toxin (PT), and filamentous hemagglutinin (FHA), which are the main components of pertussis vaccine in Japan, with mouse protectivity was examined by both intracerebral and aerosol challenge systems with virulent Bordetella pertussis cells. Titer of the antibodies was calculated from the enzyme-linked immunosorbent assay (ELISA) unit given arbitrarily to reference antibodies. PT antibody titer which protected 50% of mice was indistinguishable in both active immunization followed by intracerebral challenge and passive immunization followed by aerosol challenge. The 50% effective dose was 23 ELISA U/ml in the former mice and 24 ELISA U/mouse in the latter. In the intracerebral challenge system, FHA did not elicit a protective response but was very helpful for PT as an immunizing antigen. When anti-FHA immunoglobulin G coexisted with anti-PT immunoglobulin G in mice, the 50% effective dose of PT antibody was 4.4 or 10 ELISA U/mouse in intracerebral or aerosol challenge systems, respectively. In this active immunization system, pertussis toxoid of 1 micrograms or 0.1 microgram/mouse produced PT antibody of ca. 20 or 5 ELISA U/ml, respectively. It was concluded that pertussis toxoid or its antibody was much more potent than Formalin-treated FHA or its antibody; Formalin-treated FHA or its antibody was helpful when it was administered with pertussis toxoid toxoid or its antibody, however.     

中华仓鼠卵巢细胞在检测百日咳菌免疫原中的应用

采用中华仓鼠卵巢(CHO)细胞检测百日咳菌主要免疫原—百日咳毒素(LPF)和丝状血凝素(FHA),仅发现LPF使CHO细胞呈现明显的特异性簇聚反应,敏感性高,能检测出100pg/ml蛋白氮抗原量。与小鼠白细胞增多促进试验相比,CHO细胞法具有简便、经济、省时等优点,可作为一种检测百日咳毒素免疫原的实用辅助方法。     

Immunization of teenagers with a fifth dose of reduced DTaP-IPV induces high levels of pertussis antibodies with a significant increase in opsonophagocytic activity

Waning vaccine-induced immunity against Bordetella pertussis is observed among adolescents and adults. A high incidence of pertussis has been reported in this population, which serves as a reservoir for B. pertussis. A fifth dose of reduced antigen of diphtheria-tetanus-acellular-pertussis and inactivated polio vaccine was given as a booster dose to healthy teenagers. The antibody activity against B. pertussis antigens was measured prior to and 4 to 8 weeks after the booster by different assays: enzyme-linked immunosorbent assays (ELISAs) of IgG and IgA against pertussis toxin (PT) and filamentous hemagglutinin (FHA), IgG against pertactin (PRN), opsonophagocytic activity (OPA), and IgG binding to live B. pertussis. There was a significant increase in the IgG activity against PT, FHA, and PRN following the booster immunization (P < 0.001). The prebooster sera showed a geometric mean OPA titer of 65.1 and IgG binding to live bacteria at a geometric mean concentration of 164.9 arbitrary units (AU)/ml. Following the fifth dose, the OPA increased to a titer of 360.4, and the IgG concentration against live bacteria increased to 833.4 AU/ml (P < 0.001 for both). The correlation analyses between the different assays suggest that antibodies against FHA and PRN contribute the most to the OPA and IgG binding.     

Impaired humoral response to vaccines among HIV-exposed uninfected infants

Little is known about the vaccine protective response for infants born from HIV-infected mothers. We evaluated the antibody response to hepatitis B, tetanus, and diphtheria vaccine in vertically HIV-exposed uninfected infants and compared them to those of control infants not exposed to the virus. The quantitative determination of specific neutralizing antibodies against hepatitis B, diphtheria, and tetanus were performed blindly on serum samples. The results showed that 6.7% of the HIV-exposed uninfected individuals were nonresponders to hepatitis B vaccine (anti-HBs titer, <10 mIU/ml), and 64.4% were very good responders (anti-HBs titer, ≥1,000 mIU/ml), whereas only 3.6% of the nonexposed infants were nonresponders (χ(2)=10.93; 1 df). The HIV-exposed uninfected infants showed protective titers for diphtheria and tetanus but lower geometric mean anti-tetanus titers compared to those of the HIV-unexposed infants. Our data point to the necessity of evaluating vaccine immune responses in these children and reinforced that alterations in lymphocyte numbers and functions reported for newborns from HIV-infected mothers interfere with the vaccine response.