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.

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.

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.

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.

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.

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.

Characterization and potential diagnostic application of monoclonal antibodies specific to rabies virus

Objective：Rabies is invariably a fatal encephalomyelitis that is considered to be a serious public health problem.It is necessary to develop standard rabies virus diagnostic tools,especially for diagnosing the strains prevalent in China.Methods：Monoclonal antibodies（MAbs）specific to rabies virus were produced and characterized by enzyme linked immunosorbent assay（ELISA）,isotyping,affinity assay,immunofluorescence assay（IFA）,and immunocytochemistry.The MAb,whose affinity was higher for antigen,was used to establish an antigen captureELISA（AC-ELISA）detection system and test the efficiency by using clinical samples.Results：The heavy chain subclasses of two MAbs were all determined to be IgG2a.The 3C7 MAb showed stronger reactivity with rabies virus protein than the 2C5 MAb in an ELISA analysis,whereas the 3C7 MAb showed the highest affinity for antigen.IFA and immunocytochemistry results also indicated that the two MAbs could recognize rabies virus protein in its native form in cell samples.Data obtained using clinical samples showed that rabies virus could be detected by AC-ELISA detection system using the 3C7 MAb.Conclusion：It was potentially useful for the further development of highly sensitive,easily handled,and relatively rapid detection kits/tools for rabies surveillance in those areas where rabies is endemic,especially in China.

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。

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。

A型塞内卡病毒VP2蛋白多克隆抗体的制备及间接ELISA检测方法的建立

[目的]制备A型塞内卡病毒(Senecavirus A,SVA)VP2蛋白多克隆抗体,建立检测SVA抗体的间接ELISA方法,以期为SVA致病机制及诊断提供研究基础。[方法]利用同源重组技术将VP2基因克隆至原核表达载体pET-28a中,构建重组表达质粒pET-28a-VP2,并转化大肠杆菌BL21(DE3)感受态细胞进行IPTG诱导表达,表达产物经纯化后皮下多点注射新西兰大白兔制备多克隆抗体。利用间接免疫荧光试验(IFA)、Western blotting和中和试验鉴定多克隆抗体的特异性、反应性和中和活性。以VP2为抗原包被酶标板,通过矩阵优化、临界值确定、特异性鉴定及敏感性和重复性分析建立检测SVA抗体的间接ELISA方法。采集50份临床血清样品,分别用间接ELISA与IFA方法进行检测,分析间接ELISA方法的符合率。[结果]重组VP2蛋白以包涵体形式表达,大小为40 ku。制备的多克隆抗体能与重组VP2蛋白和SVA特异性结合,与其他病毒无交叉反应,且具有较高的中和活性。经对间接ELISA条件的优化,确定VP2包被浓度为4μg/mL,阳性血清稀释浓度为1∶250,封闭液为5%脱脂乳+5%BSA,血清样品和二抗孵育时间均为90 min, TMB底物反应时间为10 min,临界值为0.182。建立的间接ELISA方法与常见的猪病毒阳性血清不反应,与IFA符合率达94.0%。[结论]原核表达系统表达的VP2蛋白具有良好的免疫原性,制备的多克隆抗体能与SVA和VP2发生特异性反应。建立的间接ELISA方法特异性高,与IFA符合率高,适用于临床SVA抗体检测和疫苗效力评价。

牛病毒性腹泻病毒E^(rns)-ELISA抗体检测试剂盒的制备

为建立快速检测牛病毒性腹泻病毒(BVDV)的血清学方法,本试验以BVDV NADL毒株结构蛋白E^(rns)作为包被抗原,优化反应条件并组装BVDV酶联免疫吸附试验(ELISA)抗体检测试剂盒,通过重复性试验、灵敏性试验、符合率试验和保存期试验验证该试剂盒的准确性、灵敏性和稳定性。结果显示,抗原包被浓度为4μg/mL,1%酪蛋白溶液37℃封闭45 min,被检血清以1∶400稀释孵育30 min,兔抗牛HRP标记二抗以1∶20000稀释孵育30 min,TMB底物反应5 min时,ELISA的反应背景下降,区分度最好。优化后方法的批内重复性试验和批间重复性试验的变异系数均在10%以内,表明该试剂盒具有较好的稳定性;灵敏性试验结果显示,当阳性血清稀释度达到1∶6400时仍可以检测为阳性,说明该试剂盒灵敏度较高;该试剂盒与美国爱德士生物科技公司(IDEXX)BVDV总抗体检测试剂盒共同检测466份临床血清样品,两者总符合率为89.1%;保存期试验结果显示,第4个月时该试剂盒仍能检测到阳性血清,说明稳定性较好。综上表明,本试验利用E^(rns)蛋白建立的BVDV间接ELISA抗体检测试剂盒灵敏度高且重复性好,可为BVDV抗体检测和流行性调查提供新的方法。

猪胞内劳森菌抗体间接ELISA检测方法的建立及应用

猪增生性肠病(PPE),通常被称为猪回肠炎(PI),由胞内劳森氏菌(LI)感染引起。为建立检测LI抗体的间接ELISA方法,本研究构建LI外膜蛋白基因(LI0841)的重组表达质粒p ET28a-LI0841,经测序无误后转化BL21(DE3)宿主菌,经IPTG诱导表达,采用SDS-PAGE检测重组蛋白LI0841蛋白(rLI0841)的表达形式,经Ni柱纯化后采用western blot鉴定其反应原性。SDS-PAGE结果显示,在32 ku处出现目的条带,且其主要以可溶性形式表达;western blot结果显示,r LI0841能够与兔LI多克隆抗体特异性反应,表明纯化的r LI0841具有较强的反应原性,可作为包被抗原用于建立间接ELISA检测方法,经各反应条件优化初步建立LI抗体的间接ELISA检测方法。各反应条件的优化结果显示,4.38 ng/孔的r LI0841以4℃过夜包被最佳;血清最佳稀释度为1∶100,37℃反应0.5 h;羊抗猪Ig G-HRP最佳稀释度为1∶10000,37℃作用0.5 h;TMB底物37℃显色15 min。利用建立的间接ELISA方法检测猪繁殖与呼吸障碍综合征病毒、猪伪狂犬病病毒、副猪嗜血杆菌、猪链球菌、传染性胸膜肺炎放线杆菌及经美国Biostone PPE抗体检测试剂盒检测为阳性的猪血清,评估该方法的特异性;将LI阳性血清2倍倍比稀释(1∶100~1∶51200)后,采用本研究建立的间接ELISA方法检测,评估该方法的敏感性;以同一批次和不同批次包被的酶标板分别检测5份不同LI抗体水平的猪血清,评估该方法的重复性。结果显示,该方法除能检测到LI阳性血清外,其余相关病原的阳性血清均为阴性,特异性较强;LI阳性血清1∶800稀释时检测结果仍为阳性,敏感性较高;对5份不同抗体水平的LI阳性血清的批内、批间重复性试验的变异系数均小于10%,重复性较好。利用该ELISA方法与美国Biostone公司PPE抗体检测试剂盒同时检测104份临床猪血清样品,比较二者的检测结果,并计算二者的符合率;采用建立的间接ELISA方法检测黑龙江、吉林等地区413份临床猪血清样品,分析LI在上述地区的流行状况。结果显示,两种方法的阳性符合率为90.91%,阴性符合率为91.84%,总符合率为91.35%;413份临床猪血清样品中LI的阳性检出率为59.81%(247/413),表明LI在黑龙江、吉林等地区的猪群中普遍存在。本研究建立了检测LI抗体的间接ELISA方法,该方法特异性强、敏感性高、重复性与准确性均较好,为临床PI血清流行病学调查提供技术支持。

PCV4 Cap抗体ELISA检测方法的建立及血清流行病学调查

旨在建立一种猪圆环病毒4(porcine circovirus type 4, PCV4)Cap蛋白间接ELISA抗体检测方法,对猪群中PCV4抗体进行检测,并与以往的关于PCV4血清流行病学的调查结果进行对比研究,从而更全面地了解PCV4在猪群中的感染和流行情况。本研究利用原核表达系统成功表达PCV4 Cap蛋白,对反应条件进行优化,建立了基于PCV4 Cap蛋白的间接ELISA方法。采用建立的ELISA方法对中国18个省份的不同阶段的2 298份猪血清样本进行检测,以调查中国猪群中PCV4的血清学流行情况。同时将Cap-ELISA检测方法与之前建立的Rep-ELISA进行对比,平行检测845份血清样本。结果显示,在18个省份中,17个省的血清样本中检测到PCV4抗体。PCV4总血清阳性率为34.94%,母猪和育肥猪阳性率分别为59.95%和31.64%,仔猪阳性率为21.14%,保育猪中检出的阳性率最低,为4.20%。两种方法共同检测845份血清样本,符合率为82.84%,其中Cap-ELISA检测总阳性率为31.83%,Rep-ELISA检测总阳性率为35.03%。研究表明,PCV4在中国广泛传播,不同年龄阶段的猪只均能感染。Cap-ELISA和Rep-ELISA两种方法一致性高,从抗体消长规律来分析,两种方法都说明猪群感染PCV4主要在育肥猪和母猪阶段。本研究提供了中国PCV4的最新血清流行病学特征和PCV4在不同阶段猪群中的感染情况,进一步证实了PCV4在我国猪群中有一定的感染率,其危害值得进一步研究和关注。

牛冠状病毒S1蛋白的真核表达及间接ELISA方法的建立与应用

为建立一种检测牛冠状病毒(BCoV)的抗体间接ELISA检测方法,本研究利用昆虫杆状病毒表达系统表达BCoV的S1蛋白,并作为包被抗原,建立BCoV的S1蛋白抗体间接ELISA检测方法,应用于临床样品检测。结果显示,S1蛋白大小约100 ku,可表达于昆虫High5细胞培养基上清,经Western blot鉴定S1蛋白抗原性良好;经反应条件优化,确定抗原包被浓度为0.125μg·mL^(-1),血清稀释度为1∶200;采用1%明胶,于37℃封闭3 h;血清样品于37℃孵育30 min;酶标二抗的稀释度1∶25000,37℃孵育45 min;37℃避光显色13 min;临界值为0.297;经检验,该方法特异性良好;批间及批内变异系数均小于10%。当临界值为OD_(450 nm)为0.297时,若以IFA为标准,ELISA的敏感性为96.88%(31/32),特异性为87.50%(7/8),与IFA具有很强的一致性(κ=0.844,P<0.01);若以中和试验为标准,ELISA的敏感性为100.00%(31/31),特异性为88.89%(8/9),与血清中和试验具有很强的一致性(κ=0.925,P<0.01)。采用该方法对303份牛血清进行检测,BCoV阳性率为74.91%。由此说明,本研究建立的ELISA方法特异性强,敏感性高,重复性好,可用于BCoV的血清学调查和临床诊断,并可以应用于替代中和试验检测BCoV免疫后抗体水平。

猪丁型冠状病毒NSP14蛋白截短表达及其间接ELISA抗体检测方法的建立

【目的】进行猪丁型冠状病毒(PDCoV)NSP14蛋白截短原核表达,制备PDCoV NSP14蛋白的多克隆抗体,并建立检测PDCoV抗体的间接酶联免疫吸附试验(ELISA),为探究PDCoV NSP14蛋白的生物学功能及进行PDCoV监测和诊断提供参考依据。【方法】以PDCoV 1468B毒株为模板,构建NSP14截短基因原核重组质粒pET32a-NSP14,经双酶切和测序鉴定后,通过原核表达系统获得NSP14融合蛋白,将其纯化后免疫新西兰大白兔获得NSP14蛋白的多克隆抗体,进行Western blotting验证,并用间接ELISA测定其效价。以NSP14融合蛋白为抗原包被酶标板,采用方阵滴定法优化一系列反应条件,构建检测PDCoV抗体的间接ELISA。【结果】在构建的pET32a-NSP14重组质粒中,NSP14蛋白在大肠杆菌BL21(DE3)中以包涵体形式表达,其大小约40.2 kD;制备的NSP14蛋白多克隆抗体效价在1∶512000以上,经Western blotting验证,能与纯化的NSP14融合蛋白发生特异性反应。经过筛选,确定所构建检测PDCoV抗体间接ELISA的最佳反应条件:NSP14融合蛋白最佳包被浓度为4.0μg/mL,包被条件为37℃、2.0 h;封闭条件为5%脱脂奶粉封闭1.0 h;待检血清按1∶400稀释,孵育2.0 h;酶标二抗1∶2500稀释,孵育60.0 min;TMB底物显色时间为30.0 min。特异性试验结果表明,猪病阳性血清PRRSV、JEV、PCV2、PPV、PEDV和CSFV的OD450均小于0.235,说明优化的间接ELISA检测的病原与其他猪病毒阳性血清无反应,特异性良好。重复性试验结果表明,优化的间接ELISA检测猪病阳性血清的批内和批间变异系数均小于10.000%,说明优化的ELISA具有较好的重复性。【结论】成功制备具有高效价和良好免疫反应性的PDCoV NSP14蛋白多克隆抗体,并建立检测该抗体的特异性、敏感性和重复性良好的间接ELISA,可供进一步研究PDCoV NSP14蛋白的生物学功能及进行PDCoV监测和诊断参考应用。

猪肺炎支原体P46-P65重组蛋白的表达及间接ELISA抗体检测方法的建立

为建立猪肺炎支原体(Mhp)血清学调查及免疫评估方法,本研究利用DNAStar生物学软件对Mhp的P46和P65进行抗原表位分析,利用重叠延伸PCR(SOE-PCR)获得P46-P65融合基因,构建重组表达质粒pETP46-P65,将其转化大肠杆菌BL21(DE3)感受态细胞,经诱导后获得了可溶性表达的重组P46(aa33~aa419)-P65(aa307~aa627)蛋白(rP46-P65)。以纯化的r P46-P65为包被抗原,经优化各反应条件后建立了检测Mhp抗体的间接ELISA方法。特异性试验结果显示所建立的方法与CSFV、FMDV、PEDV、PCV2、PRV、PRRSV和APP等阳性血清均无交叉反应。该方法可检测到最高稀释至6 400倍的Mhp阳性血清,批内和批间变异系数均小于5%。利用IDEXX试剂盒和本研究建立的方法同时检测298份临床血清样品,前者的检测阳性率为62.4%(186/298),后者的检测阳性率为73.8%(220/298),两者检测结果的总符合率为88.6%。IDEXX检测为阳性的血清,采用本研究建立的ELISA方法检测也均为阳性;而部分Mhp阳性猪经IDEXX试剂盒检测为阴性的血清,该ELISA方法检测结果却为阳性,其中79.4%(27/34)检测结果有差异的血清经Mhp颜色变化试验证明均为阳性,表明本研究建立的ELISA方法的敏感性明显高于IDEXX方法。本研究建立的检测猪Mhp抗体的间接ELISA方法与目前广泛使用的方法相比优势明显,为临床血清流行病学调查及血清抗体水平评估提供了可行方法。

基于VP6蛋白的牛轮状病毒抗体间接ELISA检测方法的建立与应用

为了建立牛轮状病毒(BRV)血清抗体的检测方法,从病料中克隆牛轮状病毒VP6基因,构建其表达载体,利用原核表达技术表达重组VP6蛋白,建立牛轮状病毒血清抗体间接ELISA检测方法。结果显示,重组VP6蛋白大小为40 ku,以包涵体形式表达,Western blot证实重组VP6蛋白有良好的反应原性;以4μg/mL浓度的抗原包被酶标板,一抗血清50倍稀释,酶标二抗稀释10000倍稀释,为最佳工作条件,阴阳临界值为0.233,表明该方法具有良好的特异性和敏感性。建立的检测BRV抗体的间接ELISA可用于临床BRV感染的检测。

竞争ELISA和间接ELISA方法应用于牛布鲁氏菌病净化的研究

旨在评价竞争ELISA方法和间接ELISA方法联合使用在布鲁氏菌病(以下简称“布病”)净化中的效果,为布病防控提供技术支撑。采用本实验室开发的动物布鲁氏菌病竞争ELSIA(cELISA)抗体检测试剂盒、牛布鲁氏菌病间接ELISA(iELISA)抗体检测试剂盒及微量法补体结合试验(mCFT),对西北某牛场3 271份牛血清进行检测。本研究采用cELISA初筛、iELISA确诊的净化策略,对检测阳性牛进行淘汰,可疑牛和阴性牛在完全消毒后隔离饲养,并在前一次检测后每隔1个月重新对群体采样,进行多次连续的“检—淘”策略,在群体的个体阳性率低于2%或全部转阴后使用微量补体结合试验(mCFT)进行验证。并在群体全部转阴后继续检测2个月后确定净化结果。结果显示,首次检测阳性率35.36%的感染群体实施本净化策略后,在第1个月阳性率下降至25.41%,第2个月下降至7.16%,第3个月下降为1.86%,到第4个月则实现了布病阳性群体的全面转阴,mCFT验证个体阴性率100%。此后持续检测2个月,个体阳性率均为0,至此实现了感染群体的布病净化,使得群体内近一半的牛免于扑杀,大大减少了经济损失。综上发现,将cELISA用于布病初筛,iELISA用于布病确诊的联合使用,经多次连续检测并结合常规的隔离消毒措施,可以在短时间内实现对于布病感染群体的全面净化。