Development and Validation of an Indirect Whole-Virus ELISA Using a Predominant Genotype VI Velogenic NewCastle Disease Virus Isolated from Lebanese Poultry

Commercial ELISA kits are commonly used to assess the levels of chicken antibodies against NewCastle Disease Virus (NDV) and trace a field strain infection. Nevertheless, the specificity of these kits vis-à-vis endemic strains in Lebanon remains in question. This study developed an in-house indirect ELISA system to evaluate the level of chicken antibodies against a predominant velogenic NDV strain belonging to Genotype VI. A checkerboard analysis comprised a five-factorial multivariate experiment to optimize the protocol: coating antigen concentration, blocking buffer utilization, serum and conjugate dilution levels, and OD reading wavelength. The developed test was optimized and then validated through parallel testing of the sera of 20 broilers and 5 layers using standard serological assays. There was a strong correlation between the developed ELISA results and those obtained with the Hemagglutination Inhibition test (P < 0.01), and a commercial NDV ELISA kit (P < 0.05). The specificity, sensitivity, and reproducibility of the developed ELISA suggest that it can be used as the test of choice for the assessment of chicken antibody titers against locally circulating velogenic NDV strains, specifically those belonging to Genotype VI. It also offers better help in the serological detection of birds’ exposure to the said strains.

Establishment of an Indirect ELISA with the Major Epitope Domain of ApxⅡ of Actinobacillus pleuropneumoniae Expressed in Prokaryotic Cells

[Objective] This study aimed to develop an indirect ELISA to detect the antibodies against Actinobacil us pleuropneumoniae （APP） using the recombinant ApxⅡA1 protein expressed in prokaryotic cells as the antigen. [Method] The major epi-tope domain of ApxⅡ was cloned into the prokaryotic expression vector pET-28a （＋） to obtain the recombinant plasmid pET-ApxⅡA1, which was then transformed into E. coli BL21 （DE3） for expression. The immunogenicity of the recombinant pro-tein was analyzed by western-blotting. After that, the purified recombinant protein was used as the coating antigen in the indirect ELISA for detecting the antibodies against APP. Final y, the concentration of coated antigen and the dilution of serum were optimized. [Result] Proved by enzyme digestion and sequencing, the recombi-nant plasmid pET-ApxⅡA1 was constructed successful y. The recombinant protein was highly expressed in prokaryotic cells, and Western-blotting analysis showed that it was recognized specifical y by positive serum of APP. The indirect ELISA could detect the antibody against APP with the purified recombinant protein as the coating antigen. The optimal concentration of coated antigen was 1.23 μg/ml and the opti-mal dilution of serum was 1：100. Compared with a commercial ELISA kit detecting antibody against ApxⅣ, the coincidence rate of the indirect ELISA was 90.4%. [Conclusion] Our results indicated that the indirect ELISA is sensitive and specific, and suitable for evaluating the effect of APP vaccine and epidemiological surveys.

Construction of Recombinant Baculovirus Containing Peste des Petits Ruminants Virus N Gene and Establishment of Indirect ELISA for Detecting Serum Antibodies

This experiment was conducted to diagnose Peste des Petits Ruminants based on the eukaryotically-expressed PPRV nucleoprotein through an indirect ELISA. The full-length PPRV nucleoprotein gene was obtained from viral genome RNA by RT-PCR. The amplified fragments were cloned into baculovirus donor vectors pFastHTA of the Bac-to-Bac system. These recombinant plasmids, pFastHTA-PPRV-N, were transformed into DH10Bac host bacteria to obtain recombinant shuttle plasmids, pBacmid-PPRV-N. Recombinant baculovirus, Bacmid-PPRV-N, was generated for expression of the PPRV nucleoprotein by transfecting recombinant pBacmid-PPRV-N with Lipofectamine 2000 into Sf21 insect cells. The efficient expression of PPRV Nucleoprotein by baculovirus in Sf21 cells was verified by SDS-PAGE and Western blot. An indirect ELISA was developed using recombinant PPRV nucleoprotein as the coating antigen. 37 goat sera from an epidemic area in Tibet and 92 goat sera from a non-infected area in Qinghai Province were simultaneously detected by the indirect ELISA, developed here, and the international standard cELISA kit. The sensitivity and specificity of the indirect ELISA was 100% and 96.2% compared with the cELISA kit. The coincidence rate of the two methods was 96.9%. The results demonstrated that the indirect ELISA established in this study works well for diagnosis of PPR.

Nucleoprotein-based indirect enzyme-linked immunosorbent assay(indirect ELISA) for detecting antibodies specific to Ebola virus and Marbug virus

Full-length nucleoproteins from Ebola and Marburg viruses were expressed as His-tagged recombinant proteins in Escherichia coli and nucleoprotein-based enzyme-linked immunosorbent assays(ELISAs) were established for the detection of antibodies specific to Ebola and Marburg viruses. The ELISAs were evaluated by testing antisera collected from rabbit immunized with Ebola and Marburg virus nucleoproteins. Although little cross-reactivity of antibodies was observed in antiEbola virus nucleoprotein rabbit antisera, the highest reactions to immunoglobulin G(Ig G) were uniformly detected against the nucleoprotein antigens of homologous viruses. We further evaluated the ELISA's ability to detect antibodies to Ebola and Marburg viruses using human sera samples collected from individuals passing through the Guangdong port of entry. With a threshold set at the mean plus three standard deviations of average optical densities of sera tested, the ELISA systems using these two recombinant nucleoproteins have good sensitivity and specificity. These results demonstrate the usefulness of ELISA for diagnostics as well as ecological and serosurvey studies of Ebola and Marburg virus infection.

An Indirect ELISA of Classical Swine Fever Virus Based on Quadruple Antigenic Epitope Peptide Expressed in E.coli

In this study,a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV)E2 glycoprotein was expressed in E.coli to a obtain target protein.This target protein was used as a coating antigen to establish an indirect ELISA for specifically detecting anti-CSFV antibodies in serum samples from pigs.The P/N cut-off value of this assay was 1.92 by receiver operating characteristic curve(ROC)analysis based on 30 negative sera and 80 positive samples.The test gave 97.5%sensitivity and 96.7%specificity compared with the indirect hemagglutination(IHA)test.The inter-assay and intra-assay coefficients of variation (CVs)for 16 sera were both≤6.8%.No cross-reactivity between the coating antigen and anti-bovine viral diarrhoea virus(BVDV)antibodies was observed.

Secretory Expression of E2 Main Antigen Domain of CSFV C Strain and the Establishment of Indirect ELISA Assay

The sequence encoding an E2 main antigen glycoprotein of the C strain of classical swine fever virus (CSFV) was highly expressed in the host cell E. coli BL21–CodonPlus (DE3)–RIL using the pGEX-4T-1 expression vector and the soluble recombinant product was purified with Glutathione Sepharose TM4B by centrifugation. The soluble recombinant protein showed good immune reactions and was confirmed by Western blot using anti-CSFV-specific antibodies. Then an indirect ELISA with the purified E2 protein as the coating antigen was established to detect antibody against CSFV. The result revealed that the optimal concentration of coated antigen was 0.6 μg/well and the optimal dilution of serum was 1:80. The positive cut-off value of this ELISA assay was OD tested serum / OD negative serum≥2.1. The E2-ELISA method was evaluated by comparison with the indirect hemagglutination test (IHAT). When a total of 100 field serum samples were tested the sensitivity and specificity were 90.3% and 94.7% respectively. Specificity analysis showed that there were no cross-reactions between BVD serum and the purified E2 protein in the E2-ELISA.

Establishment of Indirect ELISA Diagnosis Technique based on the VP1 Protein of Foot and Mouth Disease Virus Serotype A

The VP1 protein of foot-and-mouth disease virus serotype A was prokaryotically expressed and purified to replace the traditional virus antigen for estab- lishing a fast, safe, effective indirect ELISA method, so as to detecting antibody of foot-and-mouth disease virus serotype A. Western-Blot test showed that the VP1 recombinant protein could be used as detective antigen as it can be specifically recognized by bovine positive serum of FMDV serotype A. By employing matrix titra- tion method, the optimal parameters were obtained as follows： 1 mg/L VP1 protein as coating antigen, Vserum：Vblocking solution = 1：50 dilution for serum and Vsecondary enzyme-linked antibedies：Vblocking solution ---1：2 000 for enzyme combined antibodies. The results showod that the sensitivity and specificity of this method were 94.32% and 99.09% respectively, the coefficients of variations in intra-assay and inter-assay reproducibility tests was lower than 8%. Compared with liquid phase blocking ELISA kits, the agreement of 201 serum samples reached 92.54%. The VP1-ELISA method established here is specific, sensitive, stable and simple, which can be used to monitor the antibody level of FMD serotype A.

Development of a recombinant pB602L-based indirect ELISA assay for detecting antibodies against African swine fever virus in pigs

African swine fever(ASF),caused by the African swine fever virus(ASFV),is a devastating disease of domestic and wild pigs.There is no effective vaccine,and the control of the disease relies mainly on surveillance and early detection of infected pigs.Previously,serological assays,such as ELISA,have been developed mainly based on recombinant structural viral proteins of ASFV,including p72,p54,and p30.However,the antibodies against these proteins do not provide efficient protection against ASFV infection in pigs.Therefore,new serological assays that can be applied for clinical diagnosis and evaluating serological immune response in vaccinated pigs are still required.In this study,we expressed and purified a recombinant p B602 L protein.The purified p B602 L protein was then used as an antigen to develop an indirect ELISA assay.This assay has no cross-reaction with the anti-sera against the 15 most common pig pathogens in China,such as classical swine fever virus,pseudorabies virus,and porcine parvovirus.This assay and a commercial ELISA kit were then used to detect 60 field pig serum samples,including an unknown number of antiASFV sera.The coincidence of the two assays was 95%.Furthermore,the p B602 L-based ELISA was employed to test the antibody responses to the seven-gene-deleted ASFV strain HLJ/18-7 GD in pigs.The results showed that the antibody levels in all vaccinated pigs,starting from the 10 th day post-inoculation,have increased continuously during the observation period of 45 days.Our results indicate that this p B602 L-based indirect ELISA assay can be employed potentially in the field of ASFV diagnosis.

Expression of Pseudorabies Virus gE Core Epitopes in Escherichia coli Strain BL21 and Utilization of Indirect PRV gE-ELISA

Pseudorabies virus glycoprotein E （PRV gE） has been recognized as a suitable diagnostic antigen for pseudorabies. In order to produce gE antigen in large quantities and at low cost, a gene fragment encoding PRV gE core epitopes was expressed in E. coli BL21 expression system. SDS-PAGE and Western Blotting revealed that the expression product in culture supematant of E. coli BL21 was a recombinant protein, approximately 51.8 Kd. At 5 h post-induction, protein concentration assay showed that the expression product amounted to 1.65 mg/ml, accounting for 24. 17% of total proteins in the culture supematant. An indirect PRV gE-ELISA was established by using the recombinant expression product as a coating antigen. Cross-reactivity assay showed that this antigen was PRV specific. In addition, the assay was consistently reproducible. Comparison of detection results of 240 serum samples between PRV gE-ELISA and a commercially available PRV diagnostic kit showed that there was no significant difference between these two methods （P 〉 0.05 ）.

The Development and Application of an Indirect ELISA Test for the Detection of Chicken Anaemia Virus (CAV) by VP1 in Chicken Flock Serum

Chicken anaemia virus (CAV) causes a viral disease in chickens worldwide and thus has economic importance. The main aim of this study was to develop a rapid, sensitive and specific VP1-CAVI indirect ELISA for the detection of CAV infection. The CAV-VP1, was separately cloned and expressed in recombinant E. coli. The purified recombinant CAV-VP1 protein was then coated as an antigen on an ELISA plates to evaluate its reactivity against chicken sera. The resulting indirect ELISA was then compared with a commercial ELISA. The specificity and sensitivity of the indirect ELISA were measured as 93.3% and 100%, respectively. A t-test produced a t-value of 15.805 for the indirect ELISA and revealed a significant difference between CAV-positive serum and CAV-negative serum (p-value of 0.001). For the second variable (i.e., a commercial ELISA), the t-test yielded a t-value of 5.063, which revealed a significant difference between CAV-positive serum and CAV-negative serum (p-value of 0.015). This intervention produces statistically significant improvements in both variables (p-values < 0.05). The correlation coefficient for the indirect ELISA was r = 0.93. Therefore, this work can be considered as a new achievement in diagnosis for Chicken anaemia virus in chicken flocks.

Efficient and Soluble Expression of N Protein of Peste Des Petits Ruminants Virus and Development of Indirect ELISA

[ Objective] The paper was to get effective soluble N protein to establish indirect ELISA method for Peste des petits ruminants （PPR）. [ Method] Soluble N protein with high expression was obtained from Escherichia coli expression system through codon optimization and optimization of expression conditions, and indirect ELISA detection method based on N protein was further established. [ Result] The assay had no cross reaction with other sheep pathogens. The intra- and inter-batch variation coefficients were less than 9%, indicating the method had good repeatability. Furthermore, totally 480 clinical serum samples were detec- ted by the assay, and the agreement rate with commercial ELISA kit （IDVET） was 98.33%. [ Conclusion] The study laid a foundation for further development of mature PPRV antibody detection kits.

Prokaryotic Expression of IBV N Protein and Development of Indirect IBV N Protein-mediated ELISA

Avian infectious bronchitis(IB)is an acute and highly contagious disease caused by infectious bronchitis virus(IBV).In the study,according to IBV gene sequences published in Gen Bank,specific primers were designed to clone N gene by RT-PCR,and this gene was inserted into p ET-30a(+)vector resulting in a prokaryotic expression plasma p ET-30a-N.The results of SDS-PAGE and Western Blot analysis showed that the recombinant protein was expressed successfully and had good reactivity with IBV positive serum.Using purified recombinant N protein as a coating antigen,the indirect ELISA protocol was established and optimized,in which N protein was 2.5μg·m L^-1 of concentration,sample serum of 1:40 dilution.For clinical specimen,the IBV antibodies could be detected by this method efficiently and got nearly the same results as those of IBV-mediated ELISA.It would provide a good tool for rapid diagnosis and epidemiological study of avian infectious bronchitis.

Development of an Indirect ELISA for Detection of T.solium Based on Recombinant 18 kDa Protein

The gene encoding the 18 kDa protein of Taenia solium metacestodes was amplified by RT-PCR and cloned into the pGEM-T vector for sequencing.The recombinant plasmid named pGEX-CE18 was constructed and transformed into E.coli BL21 for in vitro expression.SDS-PAGE and Western blot were employed for analyzing the recombinant protein,which was then used for development of an indirect ELISA for detection of anti-cysticercosis antibodies.The results showed that the recombinant protein of interest was 35 kDa in size,accounting for 28%of total bacteria proteins,and reacted with positive sera against cysticercosis.Using the newly-constructed indirect ELISA and a commercially available ELISA kit,paired analyses of 178 serum samples indicated that the concordant rate was 98.83%and the ELISA exhibited good specificity and sensitivity,supporting its utility and application for diagnosis of cysticercosis.

Establishment of Indirect ELISA Method for Detection of Penton Protein of Fowl Adenovirus GroupⅠ

[ Objective] To establish an indirect ELISA method which can detect fowl adenovirus group I （FAVI） antibody easily and rapidly. [ Method] The expressed and purified FAVI penton recombinant protein was used to be an antigen, optimized the reaction conditions, and then estab- lished the FAVI indirect penton-ELISA antibody detection method. [ Result] The optimal coating concentration of antigen was 1.5 μg/hole, the opti- mal coating condition was 37℃ 2 h and 4 ℃ overnight; the optimal dilution of serum was 1：100; the optimal working concentration of anti-chicken IgG-HRP was 1：2 000; the positive and negative critical value of ELISA was 0.335. Detected the 100 chicken serum samples by the established penton-ELISA method, the positive rate was 41%. [ Conclusion] Through the study, ~e established penton-ELISA method has a good specificity, sensitivity and reproducibility. And it offers an effective tool for the diagnosis of FAVI, the survey of antibody and epidemiology survey.

Development of an Indirect Sandwich ELISA for Detection of Duck Swollen Head Septicemia Virus

[Objective] To develop an indirect sandwich ELISA for rapid detection of duck swollen head septicemia virus （DSHSV）. [Method] DSHSV hyperimmune sara were prepared in ducks and rabbits by inoculation with DSHSV XD strain and then purified using saturated ammonium sulfate and Sephadex G-150 column chromatography to obtain anti-DSHSV IgG. An indirect sandwich ELISA was developed using the purified duck anti-DSHSV IgG and rabbit anti-DSHSV IgG after reaction conditions were optimized. Its specificity, sensitivity and repeatability were evaluated, and its accuracy was confirmed by observation with immunoelectron microscopy. Then, distribution of DSHSV in tissues of challenged ducks was also detected. [Result] Through optimizing conditions, the ELISA was developed. Only DSHSV could be detected by the developed method, but other pathogens could not be detected. Compared with agar gel diffusion test, the developed method was more sensitive. The coefficient of variation was less than 10%, and the developed method had good repeatability. In addition, the ELISA-positive samples contained DSHSV, as confirmed by im- munoelectron microscopy. All heart, liver, lung and kidney collected from the DSHSV-challenged ducks were positive when they were detected by the developed ELISA. [ Conclusion] The developed ELISA method is rapid, simple, specific and sensitive, and it is suitable for large-scale quaran- tine of DSHS. Heart, liver, lunq and kidney should be selected preferentially as specimens for diaclnosis of DSHS.

抗PCV4 Cap蛋白抗体间接ELISA检测方法的建立

为建立可应用于猪圆环病毒4型(PCV4)候选疫苗特异性抗体检测与评价方法,本研究应用PCV4 Cap蛋白作为抗原,以PCV4多克隆兔源抗体作为一抗,优化各反应的最佳条件并建立了针对PCV4 Cap蛋白抗体的间接ELISA方法。最佳条件为2μg/m L PCV4 Cap纯化蛋白,4℃包被过夜,5%脱脂乳封闭60 min,待检血清稀释比例为1∶800,反应条件为37℃、45 min,酶标抗体稀释比例为1∶5000,反应条件为37℃、60 min,底物显色时间为10 min,Cut of f值为0.157,灵敏度可达102400倍。成功建立的抗PCV4Cap蛋白抗体间接ELISA检测方法具有良好的敏感性、重复性和特异性。可为检测PCV4候选疫苗的特异性抗体水平提供一种精准、高效的方法。

禽白血病病毒p27抗原ELISA检测试剂盒的比较与评估

为了更好地进行禽白血病监测与净化,研究使用由蛋清、细胞培养物、胎粪等多种样品组成的样品盘对7种禽白血病病毒(Avian leukosis virus,ALV)p27抗原ELISA检测试剂盒(国产试剂盒DA、DB、DC,进口试剂盒IA、IB、IC、IDEXX)进行比较。结果显示:从分析特性上看,试剂盒分析特异性均较好,未观察到与其他常见禽源病毒的交叉反应;与IDEXX相比,DA、IA的分析敏感性较高,DB、IC其次,DC、IB较低,对于ALV不同亚群,各种试剂盒分析敏感性差异可达2~3个稀释度;从诊断特性上看,与IDEXX相比,DB、DC和IC的诊断敏感性和诊断特异性均高于90%;DA、IA和IB与IDEXX的诊断敏感性和诊断特异性均高于80%;各试剂盒对于不同类型样品(DF-1细胞培养物、蛋清、胎粪)的诊断敏感性和诊断特异性存在差异;从重复性上看,DA和IA的批内变异系数均在15%以内。综上所述,与IDEXX相比,当前国产p27抗原ELISA检测试剂盒DA和DB、进口p27抗原ELISA检测试剂盒IA和IC的各项性能可满足禽白血病净化各阶段对不同类型样品的检测需求。

基于Penton蛋白禽腺病毒血清4型间接ELISA抗体检测方法的建立

Penton蛋白是构成禽腺病毒血清4型(Fowl adenovirus serotype 4,FAdV-4)衣壳的主要结构蛋白,具备高度保守性与良好的免疫原性。以纯化的Penton蛋白为包被抗原,建立一种基于禽腺病毒血清4型Penton蛋白的间接ELISA抗体检测方法。结果显示,Penton蛋白最佳包被质量浓度为2μg/mL,血清稀释倍数为200倍,酶标抗体稀释倍数为1∶5000,阳性临界值为0.222,敏感性可达到1∶6400,与鸡传染性支气管炎病毒、鸡传染性法氏囊病病毒、鸡传染性喉气管炎病毒均无交叉反应,特异性良好,批间批内重复性变异系数均<10%,在100份临床血清中阳性检出率为77%,与商品化试剂盒检出符合率可达98%。综上,建立的ELISA抗体检测方法可以用于禽腺病毒4型临床抗体检测。

牛病毒性腹泻病毒E2蛋白的真核表达及间接ELISA抗体检测方法的建立

为建立检测牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)抗体的间接ELISA方法,利用昆虫细胞真核表达系统成功表达E2蛋白,将纯化后的E2蛋白作为包被抗原,用方阵滴定方法对影响ELISA的各个因素进行优化,并进行特异性、敏感性和重复性试验。结果表明,在昆虫细胞中表达了BVDV E2蛋白,Western blot证实目的蛋白可与BVDV阳性血清发生特异性反应。ELISA优化结果显示,E2蛋白最佳包被浓度为0.5μg/mL,最佳封闭液为1%明胶,最佳血清稀释度为1∶400,最佳血清作用方式为37℃作用30 min,酶标抗体的最佳作用方式为1∶2000稀释、37℃作用30 min,最佳底物作用时间为室温20 min,阳性临界值为OD 450≥0.423。与血清中和试验法进行比较,总符合率为97.8%,板内和板间重复性试验的变异系数均小于10%。该方法与牛常见病毒阳性血清均无交叉反应。说明建立的间接ELISA抗体检测方法特异性、敏感性和重复性良好,可用于大批量样本的临床检测和流行病学研究。

可变色ELISA样品稀释液制备及其性能检测

为解决酶联免疫吸附法(ELISA)检测过程中样品错加、漏检问题,研制开发了一种可变色ELISA试剂盒样品稀释指示剂,明确了其制备方法,并开展了性能检测评价及其在检测过程的应用特性。可变色稀释指示剂制备方法为:在0.01 mol/L PBS(pH=7.2)基础缓冲液中,加入血清蛋白组分、0.01%可变色指示剂和特效稳定剂,充分溶解后再添加Krovin750防腐剂,定容后即可。结果:制备的可变色ELISA样品稀释显色反应明显,可用于ELISA试剂盒检测中的血清或血浆样本稀释,有效避免检测过程样品跳孔和错加现象;同时对样品具有良好保护,显著提高了检测结果的准确性和稳定性,实现了对样品的有效保护,降低了样品基质背景干扰,提高了检测结果的准确性和稳定性。