质控血清09CS中13个肺炎球菌荚膜多糖抗体IgG含量检测范围的确定

目的 建立09CS作为质控血清用于13价肺炎球菌多糖蛋白结合疫苗(13PCV)临床血清样本检测中的检测值范围。方法 用WHO推荐的检测人血清中抗肺炎球菌荚膜多糖抗体IgG的定量ELISA,以国际人肺炎球菌标准血清007sp为标准,将09CS作为待测血清,检测其在13个血清型(1、3、4、5、6A、6B、7F、9V、14、18C、19A、19F、23F型)中抗荚膜多糖抗体IgG含量的值。连续检测09CS血清100余次,计算99%置信区间的各血清型几何平均抗体浓度、标准偏差(SD)和变异系数(CV)。结果 检测得到09C中13个血清型抗荚膜多糖IgG抗体含量以及在99%置信区间(CI)下±2.58倍SD的检测值范围;13个血清型检测结果的CV分别为10.86%、12.52%、13.96%、14.98%、28.77%、11.16%、14.96%、9.31%、10.43%、7.28%、10.86%、12.52%、13.96%,除6A型外,各型CV均低于15%,表明试验间精密度良好;检测次数异常率低于10%。结论 09CS可作为质控血清,用于13价肺炎球菌结合疫苗临床血清中抗荚膜多糖抗体IgG含量的ELISA检测。     

多型调理吞噬试验方法的长期稳定性

目的通过对2010—2016年间09CS针对13个型别的肺炎链球菌荚膜多糖特异性抗体滴度的分析,探讨调理吞噬杀菌试验(multiplexed opsonophagocytic killing assays,MOPA)的长期稳定性。方法统计2010—2016年检测人肺炎链球菌质量控制血清(质控血清)09CS中针对1、3、4、5、6A、6B、7F、9V、14、18C、19A、19F、23F共13个血清型的OPKA滴度,分别计算2010、2011、2012、2013、2014—2016年09CS针对各个型别的OPKA滴度的GMT值及其95%可置信区间;统计09CS针对13个血清型的OPKA滴度在质量控制范围之内的比率;计算2010—2016年09CS针对13个血清型各型别的OPKA滴度CV值。结果从OPKA滴度来看,2010年普遍较低,而2011年、2012年、2013年、2014—2016年这几年间的09CS针对13个血清型的滴度结果稳定。2011—2016年期间检测结果在质控血清滴度范围的比率:2010年最低值是4%,均值为30%;2011年最低值是65%,均值为80%;2012年最低值是73%,均值为85%;而2013年最低值为77%,均值为94%;2014—2016年最低值为71%,均值为90%。2010—2016年09CS针对13个血清型的OPKA滴度各型别CV:各年份的平均值分别为:125%、64%、47%、41%和39%。结论充分证明了从2011年起本实验室建立的MOPA稳定性良好。     

18～60岁健康人群肺炎链球菌IgG抗体水平调查

目的了解18~60岁健康人群肺炎链球菌IgG抗体水平,为肺炎链球菌性疾病的预防与控制提供参考依据。方法选取甘肃、宁夏健康献浆员为调查对象,分为18~30、31~40、41~50、51~60岁4个年龄组,共调查347人。采用WHO推荐的标准化酶联免疫吸附试验检测12种肺炎链球菌血清型IgG抗体水平,并计算IgG抗体阳性率和几何平均浓度(Geometric mean concentration,GMC)。结果质控血清QC907的IgG抗体浓度均值与参考值之间的误差百分数均未超过±40%,测定值符合其参考值范围,各血清型抗体浓度检测结果的CV均30%。347份血清样品肺炎链球菌IgG抗体总阳性率为64.8%~96.0%,GMC为0.37~2.24μg/m L。不同血清型间IgG抗体GMC差异具有统计学意义(P0.05)。不同年龄组同种血清型IgG抗体阳性率及GMC均无统计学意义(P0.05)。除19F型外,不同地区同种血清型IgG抗体阳性率均有统计学意义(P0.05);除4、7F、14和18C型外,不同地区同种血清型IgG抗体GMC间差异均有统计学意义(P0.05)。结论 18~60岁健康人群肺炎链球菌IgG抗体水平普遍较高,对肺炎链球菌主要流行血清型均具有一定的保护力。     

HL-60分化细胞表面标志、活性及其对肺炎链球菌调理吞噬杀菌能力的动态变化

目的分析HL-60分化细胞的表面标志、活性及其对肺炎球菌调理吞噬杀菌能力的动态变化。方法以流式细胞仪连续监测分化1～7 d的HL-60细胞表面标志CD11b、CD35和CD71的表达以及活细胞、凋亡细胞和死亡细胞的比例,同时用09CS、QC2、B、C和F 5份质控血清以调理吞噬杀菌试验检测肺炎链球菌血清型6B、7F、14和23F的杀菌滴度。结果分化3～6 d的HL-60细胞表面标志、活细胞比例可达到实验室要求,5份质控血清的调理吞噬杀菌滴度稳定而且在质控范围之内。结论分化3～6 d的HL-60细胞可以用于评价肺炎链球菌疫苗免疫血清的调理吞噬杀菌试验,为调理吞噬杀菌试验的建立和标准化提供了依据。     

NIH小鼠模型在评价13价肺炎球菌结合疫苗免疫原性中的作用

目的 研究小鼠模型在评价13价肺炎球菌结合疫苗(pneumococcal conjugate vaccine, PCV)免疫原性中的作用。方法 15批13价PCV免疫NIH小鼠,免疫3针后采血,检测血清抗不同血清型荚膜多糖IgG抗体的含量和调理吞噬杀菌抗体的水平。检测方法采用WHO推荐的ELISA和OPA。系统性比较IgG抗体含量和调理吞噬杀菌滴度之间的相关关系,包括组内相关关系和均值相关关系以及两者之间在统计学上的差异。结果 (1)各型的组内相关性在每个组之间有所不同23F型OPA滴度与IgG抗体水平之间相关系数比较低,而且各组之间的差异均有统计学意义(P均<0.05);3型的相关系数比较高,而且与大部分血清型(如4、6A、6B、9V、14、18C、19F和23F型)之间的差异均有统计学意义(P均<0.05);而7F型和几乎所有的型(23F型除外)之间差异均无统计学意义(P均>0.05)。另外,方差分析结果表明,各型之间总体的差异有统计学意义(P<0.05)。(2)每组有13个血清型,其在各组之间的差异除了G7组与其他12组的差异有统计学意义之外,其他各组之间...     

不同来源HL60细胞用于调理吞噬杀菌试验的比较

目的通过比较不同来源的HL60细胞分化后细胞的表面标志、活性及其对肺炎链球菌调理吞噬杀菌指数的动态变化,评价HL60细胞的来源对肺炎链球菌调理吞噬杀菌能力的影响。方法使用流式细胞仪连续监测来源于美国ATCC和中国科学院上海细胞研究所的HL60细胞在分化后1～7d的表面标志CDllb、CD35和CD71的表达情况,并观察活细胞、凋亡细胞和死亡细胞的比例;同时使用两种不同来源的细胞检测09CS和B两份质控血清对肺炎链球菌血清型6B、7F、14和23F菌株的调理吞噬杀菌指数,同时观察09CS对13价结合疫苗血清型1、3、4、5、6A、6B、7F、9V、14、18C、19A、19F和23F菌株的调理吞噬杀菌指数。结果两种不同来源HL60细胞分化3～6d细胞的表面标志、活细胞比例均可达到国际实验室的要求指标;两份质控血清对检测菌株的调理吞噬杀菌指数均能稳定于质控范围之内;两种细胞检测的09CS对13价肺炎链球菌结合疫苗血清型菌株的调理吞噬杀菌指数具可比性。结论两种来源不同的HL60细胞分化3-6d均能用于评价肺炎链球菌疫苗免疫血清的调理吞噬杀菌试验,这为调理吞噬杀菌试验及其标准化在国内的建立提供了便利。     

肺炎链球菌荚膜多糖特异性IgG抗体定量ELISA检测及质量控制要求

检测肺炎链球菌荚膜多糖(PPS)特异性抗体的酶联免疫吸附试验多年来经历了两次主要改进。介绍了WHO推荐的人血清抗肺炎链球菌(Streptococcus pneumoniae)荚膜多糖抗体IgG定量ELISA(Pn PS ELISA)检测方法、关键试剂、局限性以及方法验证等内容。ELISA定量检测法,为预防肺炎链球菌的婴幼儿侵袭性疾病提供准确的抗体水平,并对肺炎链球菌疫苗的临床评价提供血清学证据。人血清中肺炎链球菌荚膜多糖抗体IgG是判断肺炎链球菌疫苗效力的重要指标。     

人血清中肺炎链球菌荚膜多糖IgG抗体定量ELISA的初步验证

目的对肺炎链球菌荚膜多糖Ig G抗体定量ELISA,用于人血清中12F、19A、22F及33F型Ig G抗体的检测进行初步验证。方法以不同生产企业相同型别的12F、19A、22F及33F型荚膜多糖为包被抗原,用肺炎链球菌荚膜多糖Ig G抗体定量ELISA,对人血清中12F、19A、22F及33F型Ig G抗体进行定量检测,并对该方法的线性、检测限、检测范围、准确度、精密度、特异性进行初步验证。结果该方法检测13份质控血清的12F、19A、22F及33F型Ig G抗体的范围分别是0.02~4.38 ng/m L、0.14~34.68 ng/m L、0.10~25.20 ng/m L和0.12~29.78 ng/m L,r2均0.99,最低检测限分别为0.35 ng/m L、0.37 ng/m L、0.44 ng/m L和0.88 ng/m L。准确度为71.15%;试验间CV值均20%;特异性均85%。结论肺炎链球菌荚膜多糖Ig G抗体定量ELISA,用于人血清中12F、19A、22F及33F型Ig G抗体的检测,需对准确度、精密度和特异性进一步验证。     

人血清中Hib荚膜多糖抗体定量的ELISA方法建立及初步验证

目的比较两种不同活化方法制备的酪胺化Hib荚膜多糖(PRP-Ty)的特性,分别作为包被抗原建立测定人血清中Hib荚膜多糖特异抗体含量的ELISA方法。比较并确定包被抗原,对建立的ELISA方法进行初步验证。方法分别用CNBr和CDAP作为活化剂制备PRP-Ty,经Sepharose CL-4B层析分析相对分子质量分布范围,并确定适宜PRP-Ty抗原包被浓度的间接ELISA方法,以人Hib荚膜多糖IgG抗体定量标准品作为阳性标准,通过四参数非线性拟合计算人血清Hib荚膜多糖IgG抗体含量。结果 CNBr活化法制备的酪胺化Hib荚膜多糖(PRP-TyCNBr)较天然Hib荚膜多糖相对分子质量向小相对分子质量方向偏移,而CDAP活化法制备的酪胺化Hib荚膜多糖(PRP-TyC DAP)较天然Hib荚膜多糖相对分子质量分布无显著变化;两种PRP-Ty在0.65～2.00μg/mL的包被浓度范围内均有良好的包被活性,方法的灵敏度均达到0.02μg/mL IgG抗体检测水平。结论 PRP-TyC DAP作为包被物的ELISA测定方法可以更加真实可靠地反映人血清IgG抗体水平。     

检测人血清中肺炎球菌10A型荚膜多糖IgG抗体的包被抗原适用性研究

目的 确定用于23价肺炎多糖疫苗免疫后临床血清样本检测的包被用10A型肺炎球菌荚膜多糖(Pn10A)。方法 根据WHO推荐的检测人血清中肺炎球菌荚膜多糖IgG抗体含量的ELISA(PnPSELISA),包被不同来源(ATCC、A公司、B公司、5个公司混合)的10A多糖[Pn10A(ATCC)、Pn10A(A)、Pn10A(B)、Pn10A(mix)],检测38份血清中Pn10AIgG抗体的几何平均浓度(GMC)和相同样本免疫前、后的阳转率(免疫后/免疫前≥2为阳转),确定用于临床血清检测的包被Pn10A。结果 用Pn10A(ATCC)、Pn10A(A)、Pn10A(B)包被检测38份相同样本免疫前、后血清中Pn10AIgG抗体的GMC和阳转率,Pn10A(A)与Pn10A(ATCC)包被的检测结果差异无统计学意义(P>0.05),数据一致性好(r>0.9);Pn10A(B)与Pn10A(ATCC)包被的检测结果差异有统计学意义(P<0.05),数据一致性差(r<0.8);再以Pn10A(A)、Pn10A(ATCC)、Pn10A(mix)包被检测另46份相同样本免疫前、后血清中Pn10AIgG抗体的GMC和阳转率,Pn10A(A)、Pn10A(mix)与Pn10A(ATCC)包被的检测结果差异无统计学意义(P>0.05),数据一致性好(r>0.9),免疫前、后GMC值相近,阳转率相近,差异无统计学意义(P>0.05)。结论 Pn10A(A)、Pn10A(mix)与Pn10A(ATCC)均可以作为包被多糖用于检测人血清中肺炎球菌Pn10AIgG抗体;但从长久使用相同抗原检测大批量临床样本的需求考虑,Pn10A(mix)更具有足量、经济的优势。     

肺炎球菌1型荚膜多糖多克隆抗体制备与夹心ELISA法建立及其在多糖浓度测定中的应用

目的：利用肺炎球菌1型全菌体制备多克隆抗体,并且利用该抗体建立肺炎1型荚膜多糖夹心酶联免疫吸附分析法（ Enzyme-linked immunosorbent assay ,ELISA）,用于检测发酵和纯化过程中的多糖浓度。方法用灭活的1型肺炎链球菌免疫家兔6周,获得高滴度的抗多糖血清,经过亲和层析纯化,获得高纯度的兔抗肺炎1型多糖抗体IgG。以纯化IgG作为包被抗体,加入多糖样品,再以生物素化的抗体作为检测抗体,建立夹心ELISA法检测肺炎1型多糖浓度。确定标准曲线的最佳线性范围,并对该方法进行特异性、准确性和精密度验证。结果兔免疫血清经过双向免疫扩散检测抗体滴度可达1∶32;该方法的线性检测范围为1．56～50 ng/mL;最低检测限为3．13 ng/mL。在标准品中混入其他型别多糖或培养基,回收率分别为102％和108％;该方法批内精密度和批间精密度分别为6．08％和7．01％。结论建立的夹心ELISA方法,其特异性、准确性和精密度均良好,可以特异地检测肺炎球菌1型多糖浓度。     

Peptide mimotopes as prototypic templates of broad-spectrum surrogates of carbohydrate antigens.

Peptide mimetics of carbohydrate antigens can function as templates to exploit immune mechanisms to augment vaccine design strategies as they are T cell dependent antigens. In this study we evaluate a peptide mimetic (peptide 105) of the Pneumococcal capsular polysaccharide type 14 (Pn14) as a model antigen to explore differences in antigenicity and immunogenicity of peptide mimotopes. The multiple antigenic peptide (MAP) form, by ELISA, competes with native Pn14 in a concentration-dependent manner for binding to an anti-Pn14 monoclonal antibody. It was observed that peptide priming with a conjugated form (105-BSA) and boosting with Pn14 produced higher levels of Pn14-reactive IgG1, IgG2a, IgG2b and IgG3 than priming and boosting with Pn14. This serum also displayed reactivity with multiple serotypes, as assessed by ELISA. However, when compared with serum from humans immunized with the 23-valent pneumococcal vaccines, mimetic-induced mouse serum did not display a significant ability to mediate opsonophagocytic killing of pneumococci. These results suggest the feasibility of designing mimotopes to render effective humoral responses not only to a single serotype of Streptococcus pneumoniae, but to multiple serotypes at once. Such peptides would simplify currently available vaccine approaches, yet highlights the requirement of more extensive polymerization to fully emulate native antigen.     

Purification of capsular polysaccharide produced by Streptococcus pneumoniae serotype 19A

Streptococcus pneumoniae is a major cause of invasive infection in young infants and older adults. There are currently 90 capsular serotypes identified and 23 serotypes (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F, and 33F) are responsible for about 90% of invasive disease. Among the more than 90 different S. pneumoniae serotypes, serotype 19A is globally very prevalent. A simplified purification procedure including adjustment of cell lysate pH to 4.5, fractionation with 50- 80% ethanol, and dialysis rendered capsular polysaccharide (CPS) in a yield of 31.32 +/- 3.11 mg from 1 l culture (75% recovery after lyses). The product contained only 69.6 microng of protein (99.78% purity) and 0.8 mg (sum of the precipitants from 50~60%, 60~70%, and 70~80%) of nucleic acid (97.45% purity). The purified CPS was conjugated with bovine serum albumin; the product size ranged from 100 to 180 kDa.     

鼠疫菌F1抗体/抗原酶联免疫诊断试剂盒的应用评价

目的对兰州生物制品研究所有限责任公司研制的鼠疫菌F1抗体酶联免疫诊断试剂盒和鼠疫菌F1抗原酶联免疫诊断试剂盒进行临床应用评价。方法采用双抗原/抗体夹心酶联免疫吸附试验(ELISA)、间接血球凝集试验(IHA)、胶体金免疫层析试验(GICA)3种方法的诊断试剂对比检测云南省地方病防治所中心实验室保藏的和现场采集的血清样品和脏器样品,对血清样品做鼠疫菌F1抗体检测,对脏器样品做鼠疫菌F1抗原检测。结果在358份血清样品中,ELISA试剂检出F1抗体阳性52份(14.52%),IHA试剂检出阳性37份(10.34%),GICA试剂检出阳性45份(12.57%)。ELISA与IHA试剂的符合率为95.23%,与GICA试剂的符合率为96.92%。经统计学χ2检验,ELISA试剂检出F1抗体阳性率高于IHA试剂(χ2=11.53,P=0.000 7),与GICA试剂检出的差异无统计学意义(χ2=3.27,P=0.070 4)。进一步分析滴度差值频数,ELISA试剂检测人血清的敏感性高于IHA试剂的样品占87.5%。在117份脏器样品中,3种试剂均检出F1抗原阳性15份(12.82%),符合率100%。滴度差值频数比较,ELISA试剂检测敏感性高于反向间接血球凝集试验(RIHA)试剂的样品为78.57%。结论兰州生物制品研究所有限责任公司研制的鼠疫菌F1抗体酶联免疫诊断试剂盒和鼠疫菌F1抗原酶联免疫诊断试剂盒性质特异,其敏感性优于IHA试剂盒和GICA试剂条,值得在鼠疫的监测和快速诊断中推广应用。     

The indices of antipneumococcal immunity in children with chronic pneumonia

In 76 children with chronic pneumonia the levels of serum antibodies to pneumococcal capsular polysaccharide antigens (serotypes 1, 3, 6B, 8, 9N, 15F, 23F), O-polysaccharide and pneumococcal protein somatic antigens were determined by ELISA techniques. The study showed that in sick children the content of antipneumococcal antibodies in the blood increased with age. No correlation between the content of total immunoglobulins and that of antipneumococcal antibodies in the blood of the patients was established, but a sharp decrease in the concentration of antibodies was registered in a child with hypoglobulinemia. No increase in the level of antibodies to pneumococcal antigens was observed in cases of the exacerbation of Pneumococcus-induced inflammatory process in the lungs.     

Elucidation of structural and antigenic properties of pneumococcal serotype 11A, 11B, 11C, and 11F polysaccharide capsules

Despite the emerging impact of serogroup 11 serotypes in Streptococcus pneumoniae epidemiology, the structures of serogroup 11 capsule types have not been fully elucidated, particularly the locations of O-acetyl substitutions. Here, we report the complete structures of the serotype 11B, 11C, and 11F polysaccharides and a revision to the serotype 11A capsular polysaccharide using nuclear magnetic resonance (NMR). All structures shared a linear, tetrasaccharide backbone with a pendant phosphopolyalcohol. Three of four saccharides are conserved in all serotypes. The individual serotype capsules differed in the identity of one saccharide, the pendant phosphopolyalcohol, and the O-acetylation pattern. Though the assigned locations of O-acetate substitutions in this study differed from those of previous reports, our findings were corroborated with strong correlations to serology and genetics. We examined the binding of serotyping sera to serogroup 11 polysaccharides by using flow cytometry and an inhibition-type enzyme-linked immunosorbent assay (ELISA) and found that de-O-acetylation of capsular polysaccharides by mild hydrolysis decreases its immunoreactivity, supporting the crucial role of O-acetylation in the antigenicity of these polysaccharides. Due to strong correlations between polysaccharide structures and capsule biosynthesis genes, we were able to assign target substrates for the O-acetyltransferases encoded by wcwC, wcwR, wcwT, and wcjE. We identified antigenic determinants for serogroup 11 serotyping sera and highlight the idea that conventional serotyping methods are not capable of recognizing all putative variants of S. pneumoniae serogroup 11.     

Evaluation of a multiplex PCR for detection of serotypes K1, K2 and K5 in Klebsiella sp. and comparison of isolates within these serotypes

A multiplex PCR using targets within the serotype-specific region of the capsular polysaccharide synthesis gene cluster of serotypes K1, K2 and K5 was evaluated using the 77 reference serotype strains of Klebsiella, and a panel of clinical isolates subjected previously to conventional serotyping. The PCR was highly specific for these serotypes, which are those most associated with virulence in humans and horses. PCR confirmed that isolates of the K5 serotype had cross-reacted with antiserum for other serotypes, particularly for K7. K5 isolates received by our laboratory were almost exclusively from thoroughbred horses, and were submitted for screening prior to breeding programmes. Most, including a reference strain isolated in 1955, belonged to a cluster of genetically similar isolates of sequence type (ST) 60. K1 isolates, all from humans, belonged to a previously identified cluster of ST 23.     

Characterization of the lipopolysaccharide O antigens of Actinobacillus pleuropneumoniae serotypes 9 and 11: antigenic relationships among serotypes 9, 11, and 1.

The antigenic lipopolysaccharide O polysaccharides of capsular serotypes 9 and 11 were examined by chemical, immunological, and nuclear magnetic resonance methods. Immunodiffusion tests carried out on these O antigens indicated that both contained common epitopes which were also shared by Actinobacillus pleuropneumoniae serotype 1. Chemical analysis and high-field nuclear magnetic resonance spectroscopy showed that the O antigens of serotypes 9 and 11 were high-molecular-weight polymers consisting of a backbone of repeating trisaccharide units composed of alpha-L-rhamnopyranosyl and alpha-D-glucopyranosyl residues (2:1). One of the alpha-L-rhamnose units forms a branch point and is stoichiometrically substituted with terminal 2-acetamido-2-deoxy-beta-D-glucose residues in the serotype 11 O polysaccharide, but only to the extent of 25% in the serotype 9 O polysaccharide. Thus, the serotype 9 O polysaccharide contains two different repeating units: a tetrasaccharide unit with the same structure as that of the serotype 11 O polysaccharide and a trisaccharide unit: [formula: see text] where R = beta-D-GlcpNAc for serotype 1 and 11 O polysaccharides, and R = H (75%) and R = beta-D-GlcpNAc (25%) for serotype 9. The structure of the previously determined serotype 1 O polysaccharide (E. Altman, J.-R. Brisson, and M. B. Perry, Biochem. Cell. Biol. 64:17-25, 1986) is identical to that of the serotype 11 O polysaccharide. We propose a more complete serotyping scheme for A. pleuropneumoniae which includes designation of both the capsular (K) and O antigens.