Enzyme-linked immunosorbent assay for detection of serum antibody in children vaccinated with Japanese encephalitis vaccine

Serum antibodies in children who had been vaccinated with Japanese encephalitis (JE) vaccine were measured by enzyme-linked immunosorbent assay (ELISA) and neutralization (N) and hemagglutination-inhibition (HI) tests. Of 20 serum samples obtained after two shots of JE vaccine in the first year, all but one showed positive titers in the ELISA and N test, but five showed negative titers in the HI test. All 12 serum samples obtained after booster immunization with JE vaccine in the second year showed positive and considerably higher titers in all three tests. Moreover, a high correlation was found between the ELISA, N and HI titers. These results indicate that the ELISA is useful for detecting antibodies in subjects immunized with JE vaccine.     

轮状病毒灭活疫苗和减毒活疫苗序贯免疫的体液免疫应答效果

目的:评价采用轮状病毒灭活疫苗进行初始免疫,减毒活疫苗进行加强免疫的序贯免疫方案的体液免疫应答效果。方法:将实验小鼠随机分为4组(口服疫苗组、序贯疫苗组、口服对照组及序贯对照组),按相应方案免疫后,ELISA检测血清轮状病毒特异性IgG和IgA、肠道轮状病毒特异性IgA;微量中和实验检测血清病毒特异性中和抗体;同时采用ELISA分析口服活疫苗后病毒排出情况。结果:与对照组相比,序贯疫苗组小鼠产生的轮状病毒特异性血清IgG、IgA、中和抗体及肠道IgA水平显著升高。与口服疫苗组相比,序贯疫苗组的免疫方案诱发的轮状病毒特异性血清IgG、IgA、中和抗体水平显著升高,肠道IgA水平两组间没有显著差异。同时,与口服疫苗组相比,序贯疫苗组中轮状病毒灭活疫苗进行的初始免疫未影响第一次口服活疫苗后病毒的排出量和排出时间,但序贯疫苗组第二次口服活疫苗后病毒的排出量迅速减少,排毒时间快速缩短,与口服疫苗组第三次服苗后病毒的排出量和排出时间相似。结论: 轮状病毒灭活疫苗和减毒活疫苗序贯免疫可有效诱发小鼠全身和黏膜局部的体液免疫应答,该方案将有可能成为轮状病毒疫苗临床应用的候选方案。     

Genetically engineered Bifidobacterium animalis expressing the Salmonella flagellin gene for the mucosal immunization in a mouse model

BACKGROUND: A critical component of the host defense against enteric infections is the immunological response of the mucosal membrane, a major starting point of infectious disease, such as typhoid fever. The mucosal immune system consists of an integrated network of lymphoid tissues, mucous membrane-associated cells, and effector molecules. In the present study, we developed a recombinant Bifidobacterium animalis (B. animalis) genetically modified with the Salmonella flagellin gene for mucosal immunization as an oral typhoid vaccine. METHODS: We constructed an oral vaccine against Salmonella typhimurium, consisting of recombinant B. animalis containing the flagellin gene of Salmonella. The recombinant B. animalis was administered orally to mice every other day for 6 weeks. Anti-flagellin antibodies in the serum and stools were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: We detected significantly higher levels of flagellin-specific IgA in the serum and stools of the mice treated with the recombinant B. animalis containing the flagellin gene than was seen in those treated with parental B. animalis. CONCLUSIONS: Our findings suggest that an oral vaccination using recombinant B. animalis genetically modified with the flagellin gene of Salmonella may be effective against Salmonella infections.     

Evaluation of an anti-rPA IgG ELISA for measuring the antibody response in mice.

A recombinant protective antigen (rPA)-based enzyme-linked immunosorbent assay (ELISA) was developed to measure the serological response of female A/J mice after inoculation with the new rPA-based anthrax vaccine. Several fundamental parameters of the ELISA were evaluated: specificity, precision, accuracy, linearity, and stability. Experimental results suggested that the quantitative anti-rPA IgG ELISA could be used to measure antibody levels in female A/J mice and may be useful as a potency assay to monitor consistency of manufacture of a rPA-based vaccine for planned clinical trials.     

ELISA法和NIH法检测狂犬病疫苗抗原量的比较研究

建立优化了用于狂犬病疫苗抗原量检测的ＥＬＩＳＡ方法,并与目前所用的ＮＩＨ法进行了比较,两种方法测定的相对效力符合性良好。与ＮＩＨ法相比,ＥＬＩＳＡ方法具有操作简便、省时省工、降低成本、缩短周期、重复性好、结果可靠等优点,可取代疫苗原液、半成品检测所用的ＮＩＨ法。     

两种甲型肝炎病毒抗原快速检测方法的建立

目的建立两种甲型肝炎病毒抗原(HAV-Ag)检测试剂盒,并对其检测效果进行评价。方法生物素标记甲型肝炎病毒抗体(HAV-Ab)与辣根过氧化物酶标记亲和素联合应用建立甲型肝炎病毒抗原BA-ELISA检测法;同时使用辣根过氧化物酶标记HAV-Ab作放大系统建立双抗体夹心甲型肝炎病毒抗原ELISA检测试剂,对比两种检测方法的特异性、灵敏度及实际应用效果。结果用生物素标记甲型肝炎病毒抗体-辣根过氧化物酶标记亲和素作放大系统建立的甲型肝炎病毒抗原BA-ELISA检测法,较双抗体夹心ELISA检测方法灵敏度高1～2个稀释度;两种检测法均对10余种病毒无交叉,P/N值BA-ELISA检测法较高。结论甲型肝炎病毒抗原BA-ELISA检测法是一种灵敏度高,特异性好,方便快捷的检测方法,可广泛应用于甲型肝炎病毒研究及临床检测中。而甲型肝炎病毒抗原双抗体夹心ELISA检测法,检测灵敏度适中,操作简单,更适用于甲肝疫苗生产检定。     

Response and function of cutaneous mucosal and serum antibodies in barramundi (Lates calcarifer) acclimated in seawater and freshwater

Mucosal and serum antibody responses were studied in sibling barramundi (Lates calcarifer) acclimated in either seawater or freshwater following vaccination by intraperitoneal injection or direct immersion in an inactivated Streptococcus iniae vaccine. As expected, route of vaccination had a marked effect on immune response, with direct immersion resulting in low serum antibody levels against S. iniae by ELISA detected 21 days post vaccination at 26 degrees C, whilst a significant response was detected in mucus. A strong specific antibody response was detected in both mucus and serum 21 days following intraperitoneal injection. Fish acclimated in seawater prior to vaccination showed a markedly higher specific mucosal antibody response than sibling fish acclimated in freshwater, regardless of the route of vaccination, whilst the serum antibody response was not affected by salinity. Both mucosal and serum antibodies from fish in seawater and freshwater were capable of binding antigen at salinities similar to full strength seawater in a modified ELISA assay. These results indicate that this euryhaline fish species is not only able to mount significant specific antibody response in cutaneous mucus, but that these antibodies will function in the marine environment.     

伤寒Vi多糖结合疫苗免疫小鼠后的抗体类型及动态分析

伤寒Vi多糖结合疫苗和Vi多糖疫苗分别免疫小鼠,分离血清,采用间接ELISA法测定不同时点血清中特异性IgA、IgM、IgG及其亚类(IgG1、IgG2a、IgG3)的抗体滴度。结果显示,免疫一针后,Vi多糖结合疫苗组的IgG抗体GMT值明显升高,第二针有加强效应(P<0.01);所测3种IgG亚型中IgG2a抗体滴度升高明显;Vi多糖和结合疫苗免疫小鼠后,血清中IgA和IgM抗体滴度均有显著升高,但无加强应答。显示Vi多糖结合疫苗在诱导小鼠血清IgG应答方面有加强效应。     

Detection of postvaccination mumps virus antibody by neutralization test, enzyme-linked immunosorbent assay and sensitive hemagglutination inhibition test

A group of 251 children aged 2-3 years given live attenuated mumps virus vaccine PAVIVAC of Czechoslovak production were tested for antiparotitis antibody levels in pre- and postvaccination sera by neutralization test (NT), enzyme-linked immunosorbent assay (ELISA) and sensitive hemagglutination inhibition test, enhanced by heterologous antibody to human immunoglobulin G (E-HIT). The prevaccination findings were as follows: positive ELISA IgG titres, neutralization antibodies and hemagglutination inhibition antibodies were present in, respectively, 35%, 25.9% and 27.9% of the sera. Postvaccination seroconversions were evaluated in 159 susceptible vaccinees whose prevaccination sera had been negative by all three tests. The lowest seroconversion was detected by NT (74.2%), seroconversions by ELISA and E-HIT were appreciably higher (82.4% and 86.8%, respectively). The seven children showing a seroconversion by E-HIT but not by ELISA had a 4 fold increase of anti-mumps ELISA IgG antibodies as well, but the rise of antibody titres was at a level falling in the range below the positivity criterion for ELISA. The statistically evaluated detection rate for antibodies was significantly higher (significance test "t") by ELISA as compared with neutralization test. However, antibody levels (geometric mean titres) were 8-10 times lower in postvaccination sera than in convalescent sera of 30 children with mumps in all three tests.     

Novel chitosan derivative nanoparticles enhance the immunogenicity of a DNA vaccine encoding hepatitis B virus core antigen in mice

BACKGROUND: Chitosan has been shown to possess useful properties such as non-toxicity, high biocompatibility and non-antigenicity that offer advantages for vaccine delivery systems. In this study, we prepared novel chitosan derivative nanoparticles as DNA vaccine carriers and the potential and mechanism of the DNA-nanoparticle complexes in inducing augmented immune responses were explored. METHODS: The pVAX(HBc)DNA-nanoparticle complexes as vaccine delivery systems were studied in several aspects: the protection against DNase I degradation was measured by an in vitro inhibition assay; the sustained expression of the plasmid in vivo was determined by RT-PCR; the elevated uptake efficiency by phagocytes was observed with confocal microscopy; the biocompatibility was evaluated by cytotoxicity and histology assay; the complexes were administrated to C57BL/6 mice and the humoral and cellular immune responses were evaluated by ELISA, IFN-gamma production and cytolytic T lymphocyte (CTL)-specific lysis assay. RESULTS: The remaining relative activity of DNase I after inhibition varied from 32.3% to 77.6%. The complexes were observed with higher uptake efficiency by phagocytes than naked DNA. Three types of nanoparticles did not induce significant cytotoxicity at concentrations相似文献   

A study of autologous anti-idiotypic antibody-forming cells in mice of different ages and genetic backgrounds.

Antibody response to phosphorylcholine, an immunodominant epitope of Streptococcus pneumoniae R36a (Pn), is characterized by a public idiotype, T15, that is expressed on a large proportion of antibody molecules produced by all mouse inbred strains. The ability of the immune system to produce an autologous antibody to T15 upon immunization with Pn vaccine was investigated using a modified ELISA plaque assay for detection of single antibody-forming cells (AFC). The limit of ELISA assay for detection of specific anti-T15 AFC is approximately 300 cells/spleen. However, our studies failed to detect any autologous anti-T15 AFC in the course of the primary antibody response to Pn vaccine in young/adult (2-4 months) BALB/c and C57BL/6 mice. Aged mice (20-22 months) also failed to develop any specific auto-anti-T15 AFC upon the primary Pn immunization, despite the fact that the anti-Pn response in these animals changes both quantitatively and qualitatively. In order to generate specific anti-T15 AFC, BALB/c mice had to be immunized repeatedly with Pn vaccine (four weekly injections) or immunized directly with T15 protein in CFA. Different results were obtained with D1.LP mice that are low responders to Pn and express lower levels of T15 Id as compared to BALB/c. Young D1.LP mice produced high numbers of auto-anti-T15 AFC of both IgM and IgG isotypes following a single immunization with Pn vaccine. The kinetics of auto-anti-T15 response in D1.LP mice was similar to that of the antigen-specific response. These results demonstrate that the ability of the immune network to produce autologous antibody to a shared Id depends on the genetic makeup of the host, and that this response may be regulated by the level of Id expression.     

Significant Spike-Specific IgG and Neutralizing Antibodies in Mice Induced by a Novel Chimeric Virus-Like Particle Vaccine Candidate for Middle East Respiratory Syndrome Coronavirus

正Dear Editor,Middle East respiratory syndrome coronavirus (MERS-CoV), first isolated in 2012, has emerged zoonotically among humans (van Boheemen et al. 2012). Since then,MERS-CoV continues to be a public health concern, with a fatality rate of 35%. On-going MERS-CoV outbreaks highlight the urgent need for the development of inter-     

Ability of ELISA and a toxin neutralization assay to detect changes in immunogenicity of a recombinant Bacillus anthracis protective antigen vaccine upon storage

We examined the capability of a mouse immunogenicity assay to detect improper storage of a recombinant protective antigen (rPA)-based anthrax vaccine formulated with an aluminum adjuvant, using ELISA and a toxin neutralization assay (TNA) to measure the antibody response to rPA. The vaccine was stored at 4 °C, room temperature (RT) or 37 °C for one, four and eight weeks and used for immunization, along with freshly prepared vaccine. Results showed that, contrary to ELISA, TNA is suitable to detect a loss of immunogenicity of the rPA vaccine following its exposure to RT for a period of eight weeks and to 37 °C for a period as short as 1 week.     

Genetic fusion protein 3×STa‐ovalbumin is an effective coating antigen in ELISA to titrate anti‐STa antibodies

Heat‐stable toxin type I (STa)‐ovalbumin chemical conjugates are currently used as the only coating antigen in ELISA to titrate anti‐STa antibodies for ETEC vaccine candidates. STa‐ovalbumin chemical conjugation requires STa toxin purification, a process that can be carried out by only a couple of laboratories and often with a low yield. Alternative ELISA coating antigens are needed for anti‐STa antibody titration for ETEC vaccine development. In the present study, we genetically fused STa toxin gene (three copies) to a modified chicken ovalbumin gene for genetic fusion 3×STa‐ovalbumin, and examined application of this fusion protein as an alternative coating antigen of anti‐STa antibody titration ELISA. Data showed fusion protein 3×STa‐ovalbumin was effectively expressed and extracted, and anti‐STa antibody titration ELISA using this recombinant protein (25 ng per well) or STa‐ovalbumin chemical conjugates (10 ng/well) showed the same levels of sensitivity and specificity. Furthermore, mice immunized with this fusion protein developed anti‐STa antibodies; induced antibodies showed in vitro neutralization activity against STa toxin. These results indicate that recombinant fusion protein 3×STa‐ovalbumin is an effective ELISA coating antigen for anti‐STa antibody titration, enabling a reliable reagent supply to make standardization of STa antibody titration assay feasible and to accelerate ETEC vaccine development.

     

Evaluation of a botulinum fragment C-based ELISA for measuring the humoral immune response in primates.

An enzyme-linked immunosorbent assay (ELISA) using botulinum neurotoxin serotype B recombinant fragment C (rBoNTB(HC)) was developed to measure specific humoral immune responses of monkeys vaccinated with a vaccine consisting of rBoNTB(HC). Several fundamental parameters for a bioassay were evaluated. The evaluation results demonstrated that using BoNTB(HC) as the capture antigen led to a specific and sensitive ELISA for botulinum type B antibody with excellent precision, accuracy, and linearity. There was a good correlation (r=0.91) between ELISA titers and neutralization bioassay titers. Experimental results suggested that the ELISA could be useful for detecting botulinum type B antibody levels and may supplement mouse neutralization bioassays during planned clinical manufacturing and clinical trials of rBoNTB(HC) vaccine.     

Transgenic Eimeria tenella expressing enhanced yellow fluorescent protein targeted to different cellular compartments stimulated dichotomic immune responses in chickens

Eimeria tenella, one of the seven species of chicken coccidia, elicits protective immunity against challenge infection with both homologous and heterologous strains. We endeavor to use recombinant E. tenella as a vaccine vehicle for expressing and delivering pathogen Ags and investigate immune responses against these foreign Ags. In this study, two lines of transgenic E. tenella expressing a model Ag, enhanced yellow fluorescent protein (EYFP), targeted to the micronemes and to the cytoplasm of the recombinant parasites were constructed to study the impact of Ag compartmentalization on immunogenicity. The MTT assay, intracellular cytokine staining, and real-time PCR were performed to detect the EYFP-specific proliferation and effector functions of splenic lymphocytes of immunized chickens. ELISA was used to measure anti-EYFP IgG and IgA responses. The results showed that both lines of transgenic parasites stimulated EYFP-specific lymphocyte proliferation and IFN-γ expression in CD4 and CD8 T cells, whereas a higher level of Ag-specific lymphocyte proliferation was elicited by the transgenic line expressing microneme-targeted EYFP. Furthermore, this line stimulated stronger IgA response than the one expressing cytoplasm-targeted EYFP after the second immunization. Our findings are encouraging for further investigation of the effect of Ag compartmentalization in transgenic Eimeria on immunogenicity and for the development of a eukaryotic vaccine vector using genetically modified Apicomplexa parasites.     

A two-stage, multilevel quality control system for serological assays in anthrax vaccine clinical trials

A two-stage, multilevel assay quality control (QC) system was designed and implemented for two high stringency QC anthrax serological assays; a quantitative anti-PA IgG enzyme-linked immunosorbent assay (ELISA) and an anthrax lethal toxin neutralization activity (TNA) assay. The QC system and the assays were applied for the congressionally mandated Centers for Disease Control and Prevention (CDC) Phase 4 human clinical trial of anthrax vaccine adsorbed (AVA, BioThrax). A total of 57,284 human serum samples were evaluated by anti-PA enzyme-linked immunosorbent assay (ELISA) and 11,685 samples by anthrax lethal toxin neutralization activity (TNA) assay. The QC system demonstrated overall sample acceptance rates of 86% for ELISA and 90% for the TNA assays respectively. Monitoring of multiple assay and test sample variables showed no significant long term trends or degradation in any of the critical assay reagents or reportable values for both assays. Assay quality control data establish the functionality of the quality control system and demonstrates the reliability of the serological data generated using these assays.     

新型佐剂DC-Chol对不同来源HBsAg促细胞免疫比较研究

目的： 筛选治疗性乙肝疫苗的候选抗原。方法： 分别用国产CHO乙肝表面抗原和酵母乙肝表面抗原结合DC-Chol脂质体佐剂免疫BALB/c小鼠,1周后检测其脾细胞产生的IL-2和IFN-γ的水平。ELISPOT法评价铝佐剂和DC-Chol脂质体佐剂结合HBsAg促小鼠细胞免疫反应水平。结果： 酵母乙肝表面抗原结合DC-Chol脂质体所诱导的IL-2和IFN-γ的水平分别为CHO乙肝表面抗原结合DC-Chol脂质体的20倍和119倍。酵母乙肝表面抗原结合DC-Chol脂质体所诱导的IL-4和IFN-γ的斑点数分别为酵母乙肝表面抗原结合铝佐剂的2.8倍和46.3倍。结论： 就DC-Chol脂质体佐剂而言,酵母表达的乙肝表面抗原细胞免疫原性强,是适合用于治疗性乙肝疫苗的候选疫苗。     

Sabin-IPV抗原性及免疫原性分析

目的比较3个不同厂家的Sabin-IPV抗原性及免疫原性特点。方法采用ELISA方法,利用血清型和抗原位点特异性的单克隆抗体检测Sabin株脊髓灰质炎病毒疫苗D抗原含量,分析疫苗相对D抗原含量和单克隆抗体的相对反应性,评估疫苗抗原性;利用大鼠体内效力试验分析Sabin株脊髓灰质炎病毒疫苗的免疫原性,评估疫苗效力。结果与英国国家c IPV标准品Pu91相比,3个厂家的Sabin株脊髓灰质炎病毒疫苗相对D抗原含量存在差异,其中C厂家的相对D抗原含量最高;3个厂家的血清Ⅰ型Sabin株脊髓灰质炎病毒疫苗抗原性差异无统计学意义;血清Ⅱ型中,除B厂家的Sabin株脊髓灰质炎病毒疫苗的抗原位点1的抗原性较弱以外,A、C其2个厂家的Sabin株脊髓灰质炎病毒疫苗抗原性差异无统计学意义;血清Ⅲ型中,3个厂家的Sabin株脊髓灰质炎病毒疫苗与抗原性差异有统计学意义。接种Sabin株脊髓灰质炎病毒疫苗的大鼠血清对Sabin株及Salk株病毒具有良好中和效力。结论除血清Ⅲ型外,血清Ⅰ型和Ⅱ型Sabin株脊髓灰质炎病毒疫苗的抗原性与疫苗免疫原性一致。ELISA检测疫苗抗原性的方法有望替代疫苗动物体内效力评价试验。     

Binding activity, structure, and immunogenicity of synthetic peptides derived from Plasmodium falciparum CelTOS and TRSP proteins

Several sporozoite proteins have been associated with Plasmodium falciparum cell traversal and hepatocyte invasion, including the cell-traversal protein for ookinetes and sporozoites (CelTOS), and thrombospondin-related sporozoite protein (TRSP). CelTOS and TRSP amino acid sequences have been finely mapped to identify regions specifically binding to HeLa and HepG2 cells, respectively. Three high-activity binding peptides (HABPs) were found in CelTOS and one HABP was found in TRSP, all of them having high α-helical structure content. These HABPs' specific binding was sensitive to HeLa and HepG2 cells' pre-treatment with heparinase I and chondroitinase ABC. Despite their similarity at three-dimensional (3D) structural level, TRSP and TRAP HABPs located in the TSR domain did not compete for the same binding sites. CelTOS and TRSP HABPs were used as a template for designing modified sequences to then be assessed in the Aotus monkey experimental model. Antibodies directed against these modified HABPs were able to recognize both the native parasite protein by immunofluorescence assay and the recombinant protein (expressed in Escherichia coli) by Western blot and ELISA assays. The results suggested that these modified HABPs could be promising targets in designing a fully effective, antimalarial vaccine.