Detection of bluetongue virus group-specific antigen using monoclonal antibody based sandwich ELISA

A monoclonal antibody (MAb) specific for the bluetongue virus (BTV) group specific antigen (VP7) was characterized for its reactivity with purified virus and recombinant BTV VP7 (rVP7) protein and its suitability for use in the sandwich ELISA. The MAb, designated as 5B5 was specific to VP7 and belongs to IgG2a subclass and was selected for the development of the sELISA in this study. The MAb had a titer of 1:25 with BTV and 1:2 with the rVP7 protein. The sELISA is based on capturing of BTV antigen with VP7 specific MAb followed by detection using BTV polyclonal antiserum raised in rabbits. The assay was evaluated with six cell culture adapted serotypes of BTV that have been isolated from India, 1, 2, 15, 17, 18 and 23. The assay could detect BTV antigen as early as day 8 in blood. It was also successfully applied for the detection of BTV group specific antigen in clinical samples of blood, washed RBCs, buffy coat and plasma. A total of 102 field samples from animals, suspected of being infected with BTV, were tested and 29.42% were positive. The blood samples were also amplified in cell culture which improved the sensitivity of the assay. Results confirmed that the sELISA is rapid and specific.     

双抗体夹心ELISA法检测狂犬病疫苗抗原活性组分

采用多克隆双抗体夹心ＥＬＩＳＡ法快速检测狂犬病毒抗原含量。结果表明,灵敏度达到１５μｇ／ｍｌ,同时特异性及变异系数均合乎要求。本方法用于精制狂苗制备过程中有效抗原活性组分的检测准确、快速、重复性好,且抗原的ＥＬＩＳＡ效价与ＮＩＨ动物法测定效价具有平行趋势。     

应用双抗原ELISA夹心法检测破伤风抗毒素效力的试验

作者建立了一套适用于破伤风抗毒素效力检测的双抗原夹心ELISA法,对破伤风类毒素免疫后的豚鼠和马匹分别进行了血清效价测定,并和传统的小鼠攻毒法的效力检测进行了比较,结果表明：该检测方法与小鼠攻毒法对不同水平的破伤风抗毒素效力检测,结果是一致的,有很好的相关性;此法省时、特异、灵敏,可成为另一种检测破伤风抗毒素的有效方法。     

Molecular cloning and expression of the hepatitis delta virus genotype IIb genome

Analysis of hepatitis delta virus (HDV) genome sequences has revealed multiple genotypes with different geographical distributions and associated disease patterns. To date, replication-competent cDNA clones of HDV genotypes I, II, and III have been reported. HDV genotypes I, II, and IIb have been found in Taiwan. Although full-length sequences of genotype IIb have been published, its replication competence in cultured cells has yet to be reported. In order to examine this, we obtained a full-length cDNA clone, Taiwan-IIb-1, from a Taiwanese HDV genotype IIb isolate. Comparison of the complete nucleic acid sequence of Taiwan-IIb-1 with previously published genotype IIb isolates indicated that Taiwan-IIb-1 shares 98% identity with another Taiwanese isolate and 92% identity with a Japanese isolate. Transfection of Taiwan-IIb-1 into COS7 cells resulted in accumulation of the HDV genome and appearance of delta antigens, showing that cloned HDV genotype IIb can replicate in cultured cells.     

利用异种动物抗血清双抗夹心法检测小麦黄花叶病毒

小麦黄花叶病毒(Wheat yellow mosaic virus,WYMV),属于马铃薯Y病毒科(Potyviridae),大麦黄花叶病毒属(Bymovirus),传播介体为禾谷多粘菌(Polymyxa graminis),与发生在欧美的小麦梭条花叶病毒(WSSMV)为同一属内的两种病毒。该病毒在我国分布广泛,在长江流域各省份以及济南、陕西等都有分布,对小麦生长.发育构成严重危害。一般可引起小麦减产10％～30％,严重时达70％,甚至绝产。以往对该病害的诊断主要是根据田间的症状表现,有时很难与由其他病原或环境因子引致症状相区分,目前,关于WYMV的问接酶     

Substitution rates in hepatitis delta virus

Substitution rates were estimated for the coding and noncoding regions of the hepatitis delta virus (HDV). The estimated rates of synonymous substitution in HDV were lower than the rates of substitution at nonsynonymous sites and in the noncoding region. HDV has lower synonymous substitution rates than the hepatitis C virus, though both are RNA viruses. The relatively low rate of synonymous substitution in HDV may be due to a strong preference of G and C nucleotides at third codon positions. Variation in substitution rate among HDV lineages may be correlated with the clinical development of the HDV-induced hepatitis. The phylogenetic tree inferred for 24 HDV strains reveals similarities between lineages isolated from the same geographic region. Correspondence to: W.-H. Li     

Enhanced chemiluminescence ELISA for the detection of antibody to hepatitis B virus surface antigen

A chemiluminescent enzyme linked immunosorbent assay (ELISA) for the detection of antibody to hepatitis B virus surface antigen (anti-HBs) in human serum has been developed. Polystyrene microtitre plates were coated with recombinant, yeast-derived hepatitis B surface antigen (rec-HBsAg). Patient serum samples and appropriate controls were added to the rec-HBsAg-coated wells and incubated to bind anti-HBs. The wells were then washed and a fluorescein isothiocyanate (FITC) conjugate of a human plasma-derived hepatitis B surface antigen (HBsAg) was added. Following incubation and further washing the bound FITC-labelled HBsAg was detected after addition of a horseradish peroxidase (HRP) conjugate of a monoclonal anti-FITC antibody and assaying for the enzyme. The activity of the HRP was measured using luminol and hydrogen peroxide as substrates and iodophenol as a chemiluminescence enhancer. The luminescence was recorded using a camera luminometer. Preliminary tests have shown the assay to be suitable for the detection of antibody in sera from both vaccinees and also from individuals with a past hepatitis B virus infection. The use of the FITC-anti-FITC system together with the measurement of a chemiluminescence signal makes possible the completion of this assay in a few hours. The assay has been shown to be both specific and sensitive and provides a permanent photographic record.     

抗原抗体复合物型治疗性疫苗(乙克)临床研究中患者血清乙型肝炎病毒表面抗原下降的分析与启示

近年来全球慢性乙型肝炎(chronic hepatitis B,CHB)防治指南提出了“功能性治愈”(functional cure)的概念,即患者经过治疗达到血清乙型肝炎病毒表面抗原(hepatitis B virus surface antigen,HBsAg)消失,但现有抗病毒治疗很难实现这一目标。本研究对既往临床试验中经抗原抗体复合物型治疗性疫苗(乙克)治疗后的CHB患者HBsAg下降情况进行了归纳分析,结果显示,经乙克治疗随访后达到乙型肝炎e抗原(hepatitis B e antigen,HBeAg)血清学转换者的HBsAg下降高达0.95log₁₀IU/mL,显著高于未达到HBeAg血清学转换者的0.32log₁₀IU/mL(P<0.01),而经氢氧化铝佐剂治疗随访后发生HBeAg血清学转换(0.49log₁₀IU/mL)者与未发生HBeAg血清学转换者(0.36log₁₀IU/mL)之间HBsAg下降无统计学差异。乙克组治疗过程中,丙氨酸氨基转移酶(alanine aminotransferase,ALT)骤升(ALT flare)在HBsAg下降>1.0log₁₀IU/mL者中较多见,氢氧化铝组未观察到此现象。回归分析显示,乙克治疗后HBsAg下降的影响因素有患者出现HBeAg血清学转换、感染的HBV为B基因型、治疗过程中ALT出现10倍增高,以及基线血清HBsAg为高水平。结果提示,乙克诱导的特异性免疫对降低CHB患者血清HBsAg水平有一定效果,采用“抗病毒药物治疗＋针对HBsAg的中和性抗体被动免疫＋乙克主动免疫”的“三明治”治疗策略可能会提高“功能性治愈”率。     

RNA editing in the replication cycle of human hepatitis delta virus

For some time it has been known that the RNA genome of human hepatitis delta virus (HDV) undergoes a specific RNA editing event. This review describes the editing phenomenon and its potential biological significance, and evaluates the data regarding the mechanism involved, including the possible relationship to other RNA editing phenomena.     

乙型肝炎病毒e抗原的生物学功能及血清学转换的免疫学基础

目前在临床乙型肝炎的治疗中,乙型肝炎病毒e抗原(HBeAg)消失及其抗体的出现已成为重要的疗效指标.本文回顾了HBeAg的发现及其生物学和医学意义,对HBeAg与乙型肝炎病毒核心抗原(HBcAg)的免疫原性进行了比较,阐述了HBeAg血清学转换的免疫学基础,并对出现HBeAg血清学转换的意义作了分析.     

双抗体夹心ELISA法检测肾综合征出血热汉滩病毒糖膜蛋白

用双抗体夹心ELISA法检测肾综合征出血热（HFRS）汉滩病毒LRI株糖膜蛋白（GP）,并用SDS－PAGE电泳对ELISA检测结果进行验证,实验结果显示,双抗体夹心ELISA法特异性强,重复性好,简便易行,是HTN病毒糖膜蛋白较为理想的检测方法,对HFRS疫苗的生产和质量控制有重要意义。     

用ELISA一步法检测白色念珠菌

用白色念珠菌临床分离株免疫家兔并经纯化获得特异性抗体,建立了检测临床标本中白色念珠菌抗原的ＥＬＩＳＡ一步法,此法的敏感性为９９％,特异性为９８３％,与其它念珠菌和临床常见细菌均无交叉反应,整个反应只需１小时即可完成,可望成为一种取代培养法的快速诊断方法     

hGH单克隆抗体的筛选及双抗体夹心ELISA检测方法的建立

采用杂交瘤法制备单克隆抗体,并用辛酸-硫酸铵法纯化单抗,通过ELISA方法和Western blotting测定抗体的效价与特异性,并进行抗体类型、相对亲和力测定;应用纯化的单抗建立hGH双抗体夹心ELISA检测方法。筛选出两株可以稳定分泌抗hGH单抗的杂交瘤细胞株,分别命名为3E11、2G9,抗体类型均为IgG1,抗体滴度均可达10-10,特异性好,相对亲和力高,以筛选到的两株单抗建立的双抗夹心ELISA法线性范围为0.09～1.5625ng/mL,R2>0.9,灵敏度为0.09ng/mL。筛选出高效抗hGH的单抗,并建立了hGH双抗体夹心ELISA检测方法。     

Development of a multi-epitope antigen of S protein-based ELISA for antibodies detection against infectious bronchitis virus

An indirect enzyme-linked immunosorbent assay (ELISA) method based on a novel multi-epitope antigen of S protein (SE) was developed for antibodies detection against infectious bronchitis virus (IBV). The multi-epitope antigen SE protein was designed by arranging three S gene fragments (166–247 aa, S1 gene; 501–515 aa, S1 gene; 8–30 aa, S2 gene) in tandem. It was identified to be approximately 32 kDa as a His-tagged fusion protein and can bind IBV positive serum by western blot analysis. The conditions of the SE-ELISA method were optimized. The optimal concentration of the coating antigen SE was 3.689 μg/mL and the dilution of the primary antibodies was identified as 1:1000 using a checkerboard titration. The cut-off OD₄₅₀ value was established at 0.332. The relative sensitivity and specificity between the SE-ELISA and IDEXX ELISA kit were 92.38 and 89.83%, respectively, with an accuracy of 91.46%. This assay is sensitive and specific for detection of antibodies against IBV.     

Detection of Legionella pneumophila serogroup 1 urinary antigen using an enhanced chemiluminescence ELISA

An enhanced chemiluminescence enzyme-linked immunosorbent assay has been developed for the detection of soluble antigen in the urine of patients with Legionnaires' disease (LD). In the assay antigen(s) in the urine samples are captured by a rabbit anti-L. pneumophila antibody coated onto microtitre strips. A fluorescein-isothiocyanate (FITC) conjugate of the same antibody is then added which binds to the captured antigen. Any immobilized FITC-labelled antibody is then detected with a horseradish peroxidase (HRP) conjugate of a monoclonal anti-FITC antibody. HRP activity is monitored after oxidation of luminol in the presence of H₂O₂ and iodophenol. The resulting luminescence is recorded using a camera luminometer. Urine specimens were available for testing from 31 patients with evidence of ongoing L. pneumophila serogroup 1 infection. A positive result was obtained in the cases of 12/12 specimens from culture-proven LD patients, and 16/19 specimens from patients with serological evidence of LD. Thus the sensitivity is estimated to be 28/31 (90%) The specificity was estimated using urine specimens from eight patients with non-L. pneumophila pneumonias of known aetiology. All eight specimens gave a negative result.     

鼠疫耶尔森菌rV抗原单抗系统及其ELISA检测方法的建立

目的建立ELISA双抗体夹心法,测定重组毒力因子rV抗原含量。方法采用杂交瘤技术,制备鼠疫菌rV抗原的鼠单克隆抗体,对抗原表位和单抗特异性进行分析及鉴定,建立ELISA双抗体夹心法,并验证方法的专属性、准确性、精密度和线性范围。结果成功组建了鼠疫菌rV抗原诊断试剂,灵敏度最低检测值为10 ng/mL。结论该方法可用于免疫学检测鼠疫组分疫苗原液rV抗原含量及制备过程中抗原活性,是鼠疫组分疫苗制备中一种重要的质量控制手段,也为进一步开发鼠疫诊断试剂盒及其他相关研究奠定了基础。     

改良双抗体夹心酶联免疫法测定重组人活性蛋白C含量的研究

研究用ELISA法直接定量检测细胞培养液中的重组人活性蛋白C(rhAPC)。用改良双抗体夹心酶联免疫检测法进行定量分析。用棋盘滴定法对包被抗体、反应一抗和辣根过氧化物酶(HRP)标记二抗的适宜工作浓度进行了选择,并对实验条件进行优化,参考品标准曲线线性相关性较好。改良双抗体夹心酶联免疫检测法可用于直接定量分析细胞培养液中的rhAPC水平。     

Detection of circulating antigen in bancroftian filariasis by sandwich ELISA using filarial serum IgG

The utility of the IgG fraction of human filarial serum immunoglobulin in detecting circulating antigen by sandwich enzyme linked immunosorbent assay (ELISA) was studied. 27 of 33 sera from persons with microfilaraemia, 19 of 30 sera from clinical cases of filariasis, 4 of 30 sera from normal persons from a region endemic for filariasis showed the presence of circulating filarial antigen. All the 20 normal sera from the area where filariasis was not endemic gave negative reaction for filarial antigen. Those sera from persons with microfilaraemia that showed the presence of circulating antigen also showed an apparent positive correlation between the microfilarial density and the antigen titre.     

待孕夫妇乙肝表面抗原及乙肝表面抗体阳性情况分析

目的 分析与探讨待孕夫妇乙肝表面抗原及乙肝表面抗体检测结果,并研究其对临床孕前检查的影响及评价。方法 随机选取2015‒2017年度在我院进行孕前检查的夫妇2440对（4 880例）为研究对象,按照年度将待孕夫妇分为两组,每组2 440例,两组均加强孕前检查中的乙肝表面抗原（HBsAg）及乙肝表面抗体（HBsAb）的检测。A组为2015年3月‒2016年2月在我院进行乙肝表面抗原及乙肝表面抗体检查的待孕夫妇;B组为2016年3月‒2017年2月在我院进行乙肝表面抗原及乙肝表面抗体检查的待孕夫妇。比较两组待孕夫妇乙肝表面抗原及乙肝表面抗体检测的阳性结果。结果 B组HBsAg阳性率、HBsAb阳性率明显高于A组（6.43% vs 4.63%;62.99% vs 58.44%）,差异有统计学意义（P＜0.05）。B组、A组男性HBsAg阳性率明显高于同组女性（59.87% vs 40.13%;60.18% vs 39.82%）,HBsAb阳性率低于同组女性（46.52% vs 53.48%;47.41% vs 52.59%）,差异均有统计学意义（P＜0.05）。B组、A组高中及以上学历HBsAg阳性率明显低于同组高中以下学历（38.85% vs 61.95%;38.05% vs 61.15%）,高中及以上学历HBsAb阳性率高于同组高中以下学历（53.15% vs 46.84%;51.75% vs 48.25%）,差异均有统计学意义（P＜0.05）结论 目前夫妇乙肝感染仍处于增高趋势,对于进行孕前检查的待孕夫妇加强乙肝表面抗原及乙肝表面抗体的检测,有助于疾病的早期诊断、干预及治疗,能够减少乙肝传播,可有效降低新生儿乙肝发病率,促进优生优育,提高出生人口整体素质。