百日咳毒素和丝状血凝素的纯制和检测现况

介绍了百日咳毒素和丝状血凝素的分离纯化及纯度、活性检定的方法,对影响分离纯化这两种活性物质的因素也作了一些分析     

响应面法建立层析纯化去除百日咳丝状血凝素中内毒素工艺

【背景】无细胞组分百日咳疫苗在人群中接种后不良反应发生率大大降低,是未来百日咳疫苗的发展方向,但是新的抗原纯化方式需要工艺中加入内毒素的去除。【目的】使用响应面法优化层析纯化法去除无细胞百日咳疫苗中百日咳丝状血凝素(Filamentous hemagglutinin,FHA)中内毒素的工艺。【方法】通过单因素试验,确定响应面设计范围;根据响应面法设计原理,使用Mini TAB软件,以FHA的回收率和收获的FHA蛋白浓度,同时兼内毒素合格为考察指标,对上样样品量、样品p H、样品电导Cond进行优化,最终确定去除FHA内毒素的层析纯化工艺。【结果】使用目前的层析纯化条件获得的Capto adhere去除FHA的内毒素的最佳工艺条件:p H 5.3,Cond 9.6,Mass 3.0。【结论】用响应面法优化了去除百日咳丝状血凝素中内毒素的层析纯化工艺,这种方法效率高、耗时少,为后续生物制品工艺扩大再生产提供参考。     

百日咳丝状血凝素的纯化及初步鉴定

目的从百日咳鲍特菌中提纯百日咳丝状血凝素(filamentous hemagglutinin,FHA),并对其进行初步鉴定。方法采用珍珠岩吸附层析和羟基磷灰石吸附层析相结合的方法,从百日咳鲍特菌培养上清中纯化FHA;SDSPAGE电泳和PAGE电泳分析样品纯度,并经斑点免疫印迹法对其进行鉴定。结果纯化样品的纯度达到95%以上,斑点免疫印迹中和FHA单抗有反应斑点,与百日咳毒素(pertussis toxin,PT)单抗无反应斑点。结论珍珠岩吸附层析和羟基磷灰石吸附层析相结合的方法可作为一种快速有效的纯化手段提取纯度较高的FHA,有望作为参考品用于百日咳FHA抗原含量检测、抗FHA血清抗体滴度检测及抗原纯度检测。     

百日咳杆菌丝状血凝素的纯制及其生物学特性的研究

介绍了两种纯制百日咳杆菌丝状血凝素的方法，并对不同方法纯化的丝状血凝素的产量、纯度、生物学活性等方面进行了比较，从中选择出一种纯度高、活性好、可以大批量提纯的方法。     

百日咳杆菌丝状血凝素

本文扼要介绍了百日咳杆菌丝状血凝素这一有效免疫原的发现过程和提纯方法,并在阐述其物理化学及生物学特性的基础上,就其作为人用疫苗的前景进行了探讨。     

国产层析填料在百日咳毒素和丝状血凝素纯化中的筛选及应用

目的 筛选适合的国产层析填料以满足对组分百日咳疫苗中百日咳毒素(pertussis toxin, PT)与丝状血凝素(filamentous hemagglutinin, FHA)的分离纯化。方法 筛选对PT与FHA结合能力较好且分离度高的国产层析填料,优化纯化工艺,通过3批纯化试验比较国产填料与进口填料的抗原回收率、目的抗原纯度以及对目的抗原的动态结合载量。结果 通过纯化试验筛选到国产填料SP resin-1与MMC resin-1并优化了纯化工艺。3批纯化试验显示,SP resin-1纯化百日咳抗原工艺稳定,FHA的纯度和回收率与Capto SP ImpRes差异无统计学意义(P>0.05);且与Capto SP ImpRes相比,SP resin-1对抗原的动态结合载量更高。3批PT精纯试验显示,国产填料MMC resin-1精纯PT的工艺稳定,PT的纯度和回收率均与Capto MMC差异无统计学意义(P>0.05),并且MMC resin-1对PT的载量高于Capto MMC。结论 试验筛选出纯化PT与FHA的国产填料SP resin-1与MMC resin-1,纯...     

白喉类毒素酶联免疫检测法的建立及初步应用

目的建立测定吸附无细胞百白破联合疫苗中白喉类毒素酶联免疫检测(ELISA)方法,并进行验证及初步应用。方法以高效价兔抗DT多克隆抗体和相应酶标抗体建立双抗体夹心ELISA法,确定线性范围同时验证该方法的重复性和特异性等确定检测限度,并初步应用。结果 DT含量在0~0.0160 Lf/m L范围内反应曲线线性关系良好(r0.99)。该方法与破伤风类毒素(Tetanus toxoid,TT)、百日咳毒素(Pertussis toxin,PT)、百日咳丝状血凝素(Filamantous hemagglutinin,FHA)与黏着素(Pertactin,Prn)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,通过验证确定的准确检测范围为0.000 8~0.016 0 Lf/m L;检测限度为0.000 8 Lf/m L。该方法对DT抗原进行了吸附率的检测,同时检测了10批白喉类毒素原液与《中华人民共和国药典》三部2010年版规定的絮状单位检测方法进行对比,变异系数低于20%。结论建立了白喉类毒素双抗体夹心ELISA检测方法,为吸附无细胞百白破联合疫苗生产过程中白喉类毒素含量的质量控制提供了有效技术手段。     

百日咳疑似病例实验室检测法的比较研究

目的 对2019年河南省监测医院的百日咳疑似病例进行实时荧光聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)和定量ELISA实验室检测比较.方法 4家监测医院采集百日咳疑似病例的鼻咽拭子和血清,通过real-time PCR检测百日 咳鲍特菌(Bord...     

百日咳杆菌有效免疫原的发酵培养

为扩大生产,采用５００立升发酵罐培养无细胞百日咳菌苗,发现随着培养时间的延续,细胞浓度增高,培养液的ｐＨ值上升,ＰＴ、ＦＨＡ活性、血凝效价逐渐增加、Ｏ２溶压下降、ＣＯ２溶压上升。ｐＨ值达８２时,ＰＴ活性最高为３００ＥＵ／ｍｌ,较现用扁瓶培养方法高５倍。ｐＨ值继续上升时,ＰＴ活性开始下降。ＦＨＡ活性及血凝效价具有相似的变化。通过测定培养液ｐＨ值以确定收获时间,可获得富含ＰＴ、ＦＨＡ、且活性均保持较高水平的培养液     

A new culturing method for production of filamentous hemagglutinin of Bordetella pertussis

The addition of cyclodextrins, especially heptakis (2,6-O-dimethyl)β-cyclodextrin (MeβCD), to synthetic medium, such as Stainer-Scholte medium (D.W. Stainer and M.J. Scholte, J. Gen. Microbiol. 63: 211–220) greatly stimulated the production of filamentous hemagglutinin (F-HA) by Bordetella pertussis Tohama phase I under shaking conditions. Purified F-HA from this culturing system was demonstrated immunochemically and morphologically to be identical with that from static culture.     

Detection of Autographa californica and Heliothis zea baculovirus proteins by enzyme-linked immunosorbent assay (ELISA)

The structural proteins of Autographa californica (AcMNPV) and Heliothis zea (HzSNPV) nuclear polyhedrosis viruses were detected by indirect enzyme-linked immunosorbent assay (ELISA). The immunoassay detected less than 1 ng of AcMNPV protein. The extent of immunological relatedness between AcMNPV-occluded virus and AcMNPV polyhedral protein, AcMNPV-nonoccluded virus, Estigmene acrea granulosis virus, Amsacta moorei entomopoxvirus Heliothis zea NPV, and Lymantria dispar NPV was determined. No immunological relatedless was detected between HzSNPV, AcMNPV, and a persistent rod-shaped virus isolated from the Heliothis zea cell line (IMC-Hz-1). The polyhedral proteins of HzSNPV and AcMNPV were found to be immunologically identical.     

Identification of immunodominant epitopes in the filamentous hemagglutinin of Bordetella pertussis

The filamentous hemagglutinin (FHA) of Bordetella pertussis is a principal adhesin, which plays a key role in the colonization of the upper respiratory tract. FHA is also a protective antigen, which has been incorporated in the new generation of acellular vaccines against whooping cough. The protein is synthesized as a large 367-kDa precursor, which is then processed into a 220-kDa secreted polypeptide. To optimize the use of this protein for vaccine purposes it would be helpful to define the regions encompassing immunodominant epitopes. Twelve recombinant plasmids have been generated encoding fusion proteins between fragments of the matured-secreted 220-kDa form of FHA and the vector-encoded phage MS2 polymerase. Protein extracts of the resulting recombinant clones have been tested for reactivity with sera from 20 patients convalescent from whooping cough, and two human standard sera. The results indicate the presence of an immunodominant B cell epitope in the polypeptide coded by a 1-kb DNA fragment encompassing positions 5781-6800 of the published sequence. These results suggest that the identified fragment should be conserved in the formulation of vaccines against pertussis.     

Interaction of the Bordetella pertussis filamentous hemagglutinin with heparin

Heparin, a glycosaminoglycan synthesized in connective tissue-mast cells, appeared to inhibit the hemagglutination of rabbit erythrocytes induced by the filamentous hemagglutinin (FHA), a major adhesin of Bordetella pertussis. This inhibition suggested an interaction of heparin with the FHA region responsible for the hemagglutination activity. FHA-heparin interactions may play a role in bacterial attachment and persistence in the lungs during human pertussis. To confirm a direct FHA-heparin interaction, heparin was used as ligand in an affinity chromatography procedure. This technique allowed to purify FHA directly from the bacterial culture medium in a single-step using heparin-Sepharose CL-6B or Zetaffinity heparin 60 disks. The purified FHA was highly immunoreactive with anti-FHA monoclonal antibodies and showed no signs of degradation after 15 successive cycles of freezing-thawing. The described purification method is simple, and suitable for the rapid preparation of FHA.     

A simple sandwich ELISA (WELYSSA) for the detection of lyssavirus nucleocapsid in rabies suspected specimens using mouse monoclonal antibodies

Monoclonal antibody (MAb)-based capture enzyme-linked immunosorbent assays (ELISA) were developed for the diagnosis of rabies-suspect specimens. A combination of four mouse monoclonal antibodies directed against the rabies virus nucleocapsid was selected and used for the detection. The test was optimized and standardized so that maximum concordance could be maintained with the standard procedures of rabies diagnosis recommended by the WHO expert committee. Using prototype viruses from the different genotypes of lyssavirus and from various geographic origins and phylogenetic lineages, this paper presents a reliable, rapid and transferable diagnostic method, named WELYSSA that readily permits the detection of lyssaviruses belonging to the 7 genotypes of lyssavirus circulating in Europe, Africa, Asia and Oceania. The threshold of detection of lyssavirus nucleocapsids is low (0.8 ng/ml). With a panel of 1030 specimens received for rabies diagnostic testing, this test was found to be highly specific (0.999) and sensitive (0.970) when compared to other recommended rabies diagnostic methods.     

Some factors affecting the detection period of aphid remains in predators using ELISA

Quantitative enzyme-linked immunosorbent assay (ELSIA) was used to detect antigens of the aphid Sitobion averae (F.) in the guts of Linyphiidae, Carabidae and Staphylinidae. The effects of temperature, both constant and variable, and size of meal on the detection period and antigen decay rate were studied in the laboratory. Predators fed freshly-killed aphids were subsequently kept at one of several temperature regimes for a period from 0 to 13 days before being assayed for aphid remains. The proportion of prey remaining at intervals after feeding was measured. Curves were fitted to transformed data and the detection period estimated. The rate of decline in detectable remains was temperature-related, with the rate increasing as temperature increased. Prey remains in Staphylinidae declined much faster than in either Carabidae or Linyphiidae. In all but one case the decline was exponential with time. Variable temperature regimes produced results very similar to those obtained under conditions of constant temperature. Meal size produced a considerable difference in the amount of aphid remains detectable but little difference in the rate of decline or the estimated detection period. Data of the above types are a prerequisite for postmortem quantification of predator meals.

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Influence de certains facteurs sur la détection par ELISA de vestiges de pucerons à l'intérieur des prédateurs

Résumé La recherche d'antigènes du puceron Sitobion avenae F. a été effectuée dans les tractus digestifs de Linyphiidae, Carabidae et Staphylinidae par ELISA (adsorption des antigènes sur l'anticorps fixé et dosage par l'anticorps enzymatiquement marqué). Les influences de la température, —soit constante, soit périodique —, de l'importance du repas, du moment de la détection et de la vitesse de disparition des antigènes ont été examinées au laboratoire. Des prédateurs alimentés en pucerons tués depuis peu ont été conservés de un à 13 jours à defférents régimes de températures avant la recherche de vestiges de pucerons. Une courbe d'atalonnage a été établie pour permettre la conversion de la valeur de la densité optique observée en mg de vestiges de pucerons en fonction du temps écoulé depuis le repas. Les données ont été soumises à une transformation angulaire et les courbes ajustées pour estimer la période de détection.La vitesse de disparition de vestiges détectables a augmenté avec la température. Les vestiges ont disparu beaucoup plus vite dans les Staphylinidae que dans les Carabidae ou les Linyphiidae. A l'exception d'un cas, la disparition était une expontentielle du temps. Les thermopériodes ont produit les mêmes effets que les températures constantes. La taille du repas a provoqué des différences considérables sur la quantité de pucerons décelable, mais peu sur la vitesse de la disparition ou la durée de la période où la détection est possible. L'obtention d'informations de ce type est une condition préalable à toute quantification post-mortem des repas de prédateurs.

     

双抗体夹心法检测3-硝基酪氨酸方法的建立及应用

目的：建立一种准确、快速的双抗体夹心酶免疫吸附的方法,以定量检测组织中过氧亚硝基阴离子的水平。方法：分别以小鼠源性抗3-硝基酪氨酸单克隆IgG抗体和兔源性抗3-硝基酪氨酸IgG抗体为包被抗体和检测抗体,采用正交设计方法摸索以上各抗体的浓度,建立定量检测3-硝基酪氨酸（3-NT）的双抗体夹心ELISA法。同时,测定心肌缺血再灌注大鼠心肌组织3-NT的含量。结果：本研究建立的双抗体夹心ELISA法检测3-NT的最低检测下限为0．10ng·ml^-1,具有良好线性关系的检测范围是（0．15-7．50）ng·ml^-1（r^2=0．995）;心肌缺血再灌注组大鼠的心肌组织中的3-NT水平为（1022．42±97．35）ng·mg pro^-1,明显高于假手术组（246．58±56．52ng·mg pro^-1,P〈0．01）。结论：本研究建立的定量检测3-NT的双抗体夹心ELISA法能够方便、准确、快速地检测组织中3-NT的含量,为定量检测组织中ONOO-的水平提供了新的方法。     

无细胞百日咳菌苗纯度和生物学特性研究

本文对无细胞百日咳菌苗的纯度、免疫力和毒性进行了研究。实验证明菌苗中不仅含有百日咳毒素（ＰＴ）和丝状血凝素（ＦＨＡ）,而且还含有百日咳凝集素和百日咳粘着素。菌苗的纯度为５０％－９５．７％,菌苗中ＰＴ和ＦＨＡ的比例为１：１－１：１８。     

肺炎克雷伯氏菌特异性抗原的筛选与鉴定

目的寻找肺炎克雷伯氏菌（Klebsiella pneumoniae）血清学检测用特异性抗原。方法双向电泳分离K.pneumoniae总蛋白,通过免疫印迹（Western blotting）与常见病原菌的多抗反应筛选特异性抗原蛋白,原核表达该蛋白并用ELISA法验证。结果获得K.pneumoniae的双向电泳图谱。寻找Western blotting中与K.pneumoniae自身多抗反应而不与与其它病原菌多抗反应的蛋白,质谱鉴定为酸性磷酸酶（Acid phosphatase,GI：238894261）。经表达纯化并以酶联免疫吸附试验（ELISA）验证,证明该蛋白作为包被抗原的灵敏度高,特异性强。结论 K.pneumoniae酸性磷酸酶是适用于该菌感染检测特异性抗原蛋白。