抗淋病奈瑟菌外膜蛋白I多克隆抗体的制备及其ELISA双抗夹心法的建立

目的 制备抗淋病奈瑟菌外膜蛋白I多克隆抗体,建立检测外膜蛋白I的双抗夹心法。方法 以淋病奈瑟菌及其外膜蛋白I作为抗原,分别免疫健康性家兔及雌性BALB/C小鼠,制备兔抗淋病奈瑟菌和鼠抗外膜蛋白I多克隆抗体,并采用ELISA法检测抗体效价。结果与讨论 经ELISA证明兔抗淋病奈瑟菌多抗可与淋病奈瑟菌外膜蛋白I呈高效价反应,以此建立的ELISA双抗夹心法检测外膜蛋白I可获满意效果。     

抗淋病奈瑟菌外膜蛋白Ⅰ多克隆抗体的制备及其ELISA双抗夹心法的建立

目的制备抗淋病奈瑟菌外膜蛋白Ⅰ多克隆抗体,建立检测外膜蛋白Ⅰ的双抗夹心法.方法以淋病奈瑟菌及其外膜蛋白Ⅰ作为抗原,分别免疫健康雄性家兔及雌性BALB/C小鼠,制备兔抗淋病奈瑟菌和鼠抗外膜蛋白Ⅰ多克隆抗体,并采用ELISA法检测抗体效价.结果与讨论经ELISA证明兔抗淋病奈瑟菌多抗可与淋病奈瑟菌外膜蛋白Ⅰ呈高效价反应,以此建立的ELISA双抗夹心法检测外膜蛋白Ⅰ可获满意结果.     

淋病奈瑟菌候选外膜蛋白的免疫原性及膜定位的初步研究北大核心CSCD

目的对淋病奈瑟菌FA1090全基因组外膜蛋白进行免疫原性和膜定位的初步分析,以评价其作为候选疫苗的潜力。方法采用基因合成的方法构建重组质粒,转化感受态细胞,利用大肠杆菌表达系统对重组蛋白进行诱导表达,通过镍柱层析法纯化重组蛋白;用重组蛋白免疫BALB/c小鼠,制备免疫血清,通过间接ELISA检测抗体效价初步评价蛋白的免疫原性及其在外膜的定位。结果构建了15个重组质粒,获得了相应的纯化蛋白,通过免疫小鼠获得了免疫血清。免疫原性分析蛋白WP_003689500.1诱导小鼠产生相应IgG抗体的能力较强,膜定位分析显示有4个蛋白在外膜表达的可能性较高。结论蛋白WP_003689500.1诱导产生的抗体效价较高,与全细胞抗原的结合能力较强,可作为淋病奈瑟菌候选疫苗。     

淋病奈瑟菌的分型方法及应用

淋病是我国发病率较高的性传播疾病之一,是引起盆腔炎性疾病的主要病原体。常导致不良后果,如输卵管性不孕、宫外孕和慢性盆腔疼痛。此外,流行病学和生物学的研究均证明,淋球菌的感染可促进感染艾滋病病毒的传播。淋球菌分型有助于对淋病传播以及耐药菌株流行状况的了解,为性传播疾病预防和控制措施的制定提供依据。传统的分型方法如营养分型和血清分型曾用于淋病的流行病学研究,但由于这两种分型方法存在其内在缺陷,如费时、费力、分型能力有限等,这些缺点限制了这些分型方法的进一步运用。近年来,基于基因水平的分型方法不断涌现,这些方法不仅简单易行,而且有着比传统方法更强的分型能力。文章对相关研究进展进行综述。     

肺炎克雷伯菌FimA的表达纯化及多克隆抗体的制备

目的表达及纯化肺炎克雷伯菌(Klebsiella pneumoniae)菌毛FimA蛋白并制备多克隆抗体。方法在Clustal Omega网站上比对不同细菌FimA蛋白的同源性。设计特异引物,PCR扩增肺炎克雷伯菌FimA基因,双酶切后将其连接到表达质粒pET28a,构建重组质粒pET28a-FimA。将pET28a-FimA转化表达菌BL21 star(DE3)后培养,使用IPTG诱导FimA蛋白表达。使用IEDG在线网站分析FimA蛋白的B细胞表位,然后用层析纯化的重组FimA蛋白免疫小鼠,获得免疫血清,ELISA检测IgG、IgG1、IgG2a和IgA.结果肺炎克雷伯菌的FimA与其他FimA蛋白同源性较低,为23.6%?30.6%。构建的重组质粒PET28a-FimA在大肠埃希菌中能表达21.6×10^3的重组FimA蛋白,经亲和层析后得到单一电泳条带的高纯度目的蛋白。用FimA蛋白免疫小鼠,获得高滴度的IgG和IgA,且IgG2a的A450值高于IgG1.结论成功构建了表达质粒pET28a-FimA,获得高纯度的重组蛋白FimA。FimA具有免疫原性,用该蛋白免疫小鼠获得高滴度的多克隆抗体。     

淋病奈瑟菌常见抗生素耐药机制的研究进展

淋病奈瑟菌是淋病的病原菌。淋病目前在我国性传播疾病中感染率较高,有效的抗生素治疗是防治淋病的主要方法,但淋病奈瑟菌可通过改变作用靶点、改变对细胞膜的通透性、产生抗生素灭活酶以及药物外排机制等方式降低对抗生素的易感性。为了更好地指导临床用药、对淋病进行预防和防治,需要严密监测淋病奈瑟菌的耐药情况。本文针对淋病奈瑟菌常见抗生素的耐药机制进行综述。     

用羊种布鲁氏菌外膜蛋白作抗原的ELISA调查血清抗体的初步结果

酶联免疫吸附试验(ELISA),因其敏感性较高已广泛地应用于布病的检测。使用的抗原多为光滑型牛种布鲁氏菌1生物型如104M的培养物或是其脂多糖(LPS)。其和光滑型布鲁氏菌产生的血清抗体可呈现阳性反应,但对布鲁氏菌粗糙性抗原产生的抗体则不能发生可见的阳性结果,而布鲁氏菌属各菌种     

牛呼吸道合胞体病毒双抗体夹心ELISA检测方法的建立

目的建立牛呼吸道合胞体病毒(BRSV)双抗体夹心ELISA(DAS-ELISA)检测方法。方法以重组纯化的BRSV-G蛋白免疫家兔和小鼠,制备抗G蛋白的多克隆抗体(PcAb)和单克隆抗体(MAb),方阵滴定法优化DAS-ELISA方法抗体工作浓度及反应条件,并对其敏感性、特异性、符合率进行验证。结果获得5株稳定分泌抗G蛋白MAb的杂交瘤细胞株,分泌抗体亚类均为IgG1型κ链,Western blot、IFA试验表明PcAb和MAb均与G蛋白和BRSV发生特异性反应,MAb作为捕获抗体的最佳包被浓度为2.5μg/mL,PcAb作为检测抗体最佳工作浓度为5μg/mL,判定临界值为0.22,最低检测限为1.43μg/mL,批内和批间重复性试验变异系数均小于10%,与常引起牛呼吸道疾病的几种病原均无交叉反应,与RT-PCR的敏感性、特异性、符合率分别为92.0%、100%、95.6%。结论建立的检测BRSV的DAS-ELISA方法可用于临床样品的大规模检测,为BRSV疫情的监测和快速诊断奠定了基础。     

淋病奈瑟氏菌的耐药性及耐药质粒的研究现状

淋病是我国目前最常见的性传播疾病之一。随着抗生素的广泛使用,淋病奈瑟氏菌的耐药性也越来越严重。该文就淋病奈瑟氏菌的耐药现状、耐药质粒的研究进展做出综述。显示：淋病奈瑟氏菌的耐药除可由染色体介导外,更主要是由质粒介导;研究较多的质粒携带的耐药基因是TEM-1基因和TetM基因;常用核酸杂交法和聚合酶链反应进行耐药基因的检测。     

抗极低密度脂蛋白受体多克隆抗体的制备及鉴定

为制备抗极低密度脂蛋白受体的多克隆抗体，以极低密度脂蛋白受体配体结合域上特定片段的合成肽免疫新西兰家兔，收集免疫前后动物的血清，通过酶联免疫吸附法及免疫印迹法对血清中抗体进行分析和鉴定。结果发现，已获得高效价的抗极低密度脂蛋白受体血清，可利用免疫印迹法检测人、鼠组织和细胞中的天然极低密度脂蛋白受体及其亚型的蛋白。此结果提示，抗极低密度脂蛋白受体多克隆抗体的制备为从蛋白水平研究极低密度脂蛋白受体提供了有效的工具。     

Development of an Effective Double Antigen Sandwich ELISA Based on p30 Protein to Detect Antibodies against African Swine Fever Virus

African swine fever (ASF), the highly lethal swine infectious disease caused by the African swine fever virus (ASFV), is a great threat to the swine industry. There is no effective vaccine or diagnostic method to prevent and control this disease currently. The p30 protein of ASFV is an important target for serological diagnosis, expressed in the early stage of viral replication and has high immunogenicity and sequence conservatism. Here, the CP204L gene was cloned into the expression vector pET-30a (+), and the soluble p30 protein was successfully expressed in the E. coli prokaryotic expression system and then labeled with horseradish peroxidase (HRP) to be the enzyme-labeled antigen. Using the purified recombinant p30 protein, a double-antigen sandwich ELISA for ASFV antibody detection was developed. This method exhibits excellent specificity, sensitivity and reproducibility in clinical sample detection with lower cost and shorter production cycles. Taken together, this study provides technical support for antibody detection for ASFV.     

Bibliographical Study of the Concurrent Existence of Anticentromere and Antitopoisomerase I Antibodies

Some connective tissue diseases are characterised by specific autoantibodies. Although anticentromere or antikinetochore antibodies (ACA), and antitopoisomerase-I or anti-Scl-70 antibodies (ATA), have disease-specific meanings for systemic sclerosis and its CREST variant, respectively, the clinical significance of their concurrent existence has not been clarified. We investigated this condition in our case and with reference to the literature. For this purpose published reports between 1980 and 1998, where both ACA and ATA were measured simultaneously, were analysed by a MEDLINE search. In 10 papers 24 patients had both antibodies. In a further 25 reports, covering 3509 subjects who had either ACA or ATA, no concurrent existence was found. Prevalences of ACA (P(ACA)) and ATA (P(ATA)) in exclusive cases varied from 8.8% to 54.5%, and from 11.8% to 87.5%, respectively, whereas P(ACA) varied from 20.0% to 56.6%, and P(ATA) from 16.8% to 63.7% in the reports with patients positive for both. The actual prevalence of simultaneous presence was between 0.7% and 5.6%, significantly lower than the expected probabilities if both antibodies were to occur independently (p<0.005). In concurrently positive cases visceral involvement was characteristic, especially affecting the vascular system, with deterioration of oesophageal function and cutaneous lesions. We suggest that ATA and ACA do not coexist by chance, and that clinical characteristics with coexistence have a significance for the classification of scleroderma. Received: 20 September 1999 / Accepted: 5 July 2000     

重组人神经肽Y融合蛋白的原核表达、纯化及其多克隆抗体制备与鉴定

目的：构建人神经肽Y（NPY）基因的原核表达载体,诱导表达重组蛋白,制备兔抗人NPY抗血清。方法：取已构建好且经测序确认无误的再组质粒pET28a—NPY转化大肠杆菌BL21（DE3）,IPTG诱导表达融合蛋白,并经SDS—PAGE检测和Western印迹鉴定,表达产物包涵体经Ni^2＋-NTA亲和层析纯化,以纯化后的融合蛋白pET28a—NPY为抗原免疫家兔,获得抗血清,Western blotting、ELISA法鉴定获得的抗血清。结果经IPTG诱导含有pET28a—NPY重组质粒的DE3菌,表达出重组人NPY融合蛋白。重组蛋白经Ni^2＋-NTA亲和层析进行纯化后,得到了较高纯度的融合蛋白,用纯化的融合蛋白免疫家兔制备了多克隆抗体,Western blotting、ELISA法证实多克隆抗体制备成功。结论：成功表达了人NPY蛋白并获得了其多克隆抗体,为进一步研究人NPY蛋白的功能奠定了基础。     

Development and Primary Application of an Indirect ELISA Based on Rep Protein to Analyze Antibodies against Porcine Cocirvirus-like Virus (PCLV)

Porcine circovirus-like virus (PCLV) is a member of circovirus that contains a single-strand DNA genome, which may be one of the pathogens that causes diarrheal symptoms in pigs. The Rep protein encoded by the genome of PCLV may be responsible for viral genome replication. The development of serological detection methods for PCLV is of great necessity for clinical diagnosis, as well as epidemiological investigations. Therefore, this study attempted to build an indirect enzyme-linked immunosorbent assay (ELISA) to examine antibodies against PCLV based on the His-tagged recombinant Rep protein. Full-length PCLV Rep protein was induced and expressed in E. coli and was purified as an antigen to establish an ELISA detection kit. The purified Rep protein was used to inject into mice to produce specific antibodies. There was no cross-reaction of Rep-based ELISA with antisera against other porcine viruses. The intra-assay and inter-assay coefficient variations (CVs) were 0.644–8.211% and 0.859–7.246%, respectively, indicating good repeatability. The non-cross-reaction with TGEV, PRRSV and PCV2 testing showed high sensitivity and high specificity for this ELISA assay. A total of 1593 serum samples collected from different pig farms in Jiangxi Province were tested for anti-PCLV Rep antibodies, and 284 (17.83%) of the 1593 samples were Rep antibody positive. Altogether, the indirect ELISA detection tool developed in this study could be applied to examine serum of PCLV antibodies with good repeatability, high sensitivity and high specificity. In addition, field sample detection results suggested that the PCLV antibody has a low prevalence in pig populations in Jiangxi Province of China.     

Preparation of Monoclonal Antibodies against the Viral p54 Protein and a Blocking ELISA for Detection of the Antibody against African Swine Fever Virus

African swine fever virus (ASFV) causes a highly contagious viral disease in domestic and wild pigs, leading to serious economic losses. As there are no vaccines or drugs available, early accurate diagnosis and eradiation of infected animals are the most important measures for ASFV prevention and control. Therefore, improvement of available diagnostic assays and development of novel effective techniques are required. This study is devoted to generating a new detection platform of blocking monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) against ASFV p54 protein. Seven monoclonal antibodies against recombinant p54 protein were produced and four epitopes were identified. Three blocking ELISAs were developed with 6A5 and 6F9 mAbs labeled with HRP, respectively, of which the 6A5/6F9-based blocking ELISA displayed the best detection performance, with an AUC of 0.986, sensitivity of 98.36% and specificity of 92.36% in ROC analysis. Moreover, it has an excellent agreement at 96.59% (198/205) when compared to the commercial blocking ELISA (kappa value = 0.920). The method also has high repeatability, with CV <10%, and no cross reaction with the serum antibodies against PRV, PRRSV, CSFV, PCV2 or SVA. This indicates that the 6A5/6F9-based blocking ELISA has high accuracy with good sensitivity and specificity, suitable for viral detection, field surveillance and epidemiological studies.     

抗环子孢子蛋白保守II~ 区单克隆抗体的制备及鉴定

目的　获得针对恶性疟原虫环子孢子蛋白 (CSP)保守II 区 (RegionII )的单克隆抗体。　方法　采用恶性疟原虫保守II 区十二肽 (EWSPCSVTCGNG)免疫BALB/c小鼠 ,经融合 ,ELISA 3次筛选 ,获得 3株分泌针对保守II 区单克隆抗体的杂交瘤细胞株。　结果　ELISA检测结果显示单抗能与重组表达的恶性疟原虫CSP片段及天然CSP特异性反应。间接荧光抗体检测显示 ,单抗不仅能识别恶性疟原虫子孢子 ,也能识别约氏疟原虫子孢子。　结论　成功地获得了针对恶性疟原虫CSP保守II 区的单克隆抗体     

抗环子孢子蛋白保守II+区单克隆抗体的制备及鉴定

目的　获得针对恶性疟原虫环子孢子蛋白 (CSP)保守II⁺ 区 (RegionII⁺ )的单克隆抗体。　方法　采用恶性疟原虫保守II⁺ 区十二肽 (EWSPCSVTCGNG)免疫BALB/c小鼠 ,经融合 ,ELISA 3次筛选 ,获得 3株分泌针对保守II⁺ 区单克隆抗体的杂交瘤细胞株。　结果　ELISA检测结果显示单抗能与重组表达的恶性疟原虫CSP片段及天然CSP特异性反应。间接荧光抗体检测显示 ,单抗不仅能识别恶性疟原虫子孢子 ,也能识别约氏疟原虫子孢子。　结论　成功地获得了针对恶性疟原虫CSP保守II⁺ 区的单克隆抗体。