犬瘟热病毒单克隆抗体夹心ELISA检测方法的建立

为建立方便快捷的犬瘟热病毒(CDV)检测方法,本实验应用杂交瘤细胞融合技术,建立了4株分泌抗CDV单克隆抗体(MAb)的杂交瘤细胞株,分别命名为A2、F7、H4和G12.相加ELISA试验显示4株MAb作用于CDV不同的抗原表位.选取相加指数较高的两株MAb G12和A2分别作为捕获抗体和酶标检测抗体,建立检测CDV的夹心ELISA检测方法,并对实验条件进行了优化.结果显示,该方法与犬细小病毒、犬副流感病毒、犬腺病毒1型、犬冠状病毒等犬类病毒无交叉反应,敏感度为5 μg/mL,变异系数小于6％.利用该方法与RT-PCR方法同时检测57份临床样品,两种方法的符合率为100％.本实验建立的MAb夹心ELISA方法具有特异、敏感、方便快捷等优点,适用于大批量临床样品检测.     

重组牛γ-干扰素单克隆抗体的研制

为制备针对重组牛γ-干扰素(rBoIFN-γ)的单克隆抗体(mAb),以纯化的原核表达融合蛋白rHis-BOIFN-γ为免疫原,用纯化的rGST-BoIFN-γ为筛选抗原,应用淋巴细胞杂交瘤技术,研制抗rBoIFN-γ的mAb.结果获得13株稳定分泌抗BoIFN-γ mAb的细胞株,命名为1C12、1F7、1G5、3E6、4D5、5E11、5G4、6F8、6G6、7E9、8D3、8F8和9G11.腹水的效价除单抗9G11外,其余效价都在1:80000以上;除5G4 mAb亚类为IgG2b外,其余均为IgG1.Dot-ELISA结果表明,所得单抗只与融合蛋白rHis-BoIFN-γ和rGST-BoIFN-γ反应,而不与其它重组细胞因子和对照细菌反应,显示其良好的特异性.Western blot显示所获单抗能与相应的融合蛋白发生反应,出现特异性务带.同时所获单抗均能与牛IFN-γ标准品反应.13株单抗均为针对rBoIFN-γ的特异性mAb,为进一步研究rBoIFN-γmAb在牛的免疫调控、牛疫病的致病机制及其诊断中的应用奠定基础.     

犬副流感病毒单克隆抗体的研制及其特性鉴定

为研制犬副流感特异性诊断试剂,我们以犬副流感病毒(CPIV)免疫8周龄BALB/c小鼠,采用淋巴细胞杂交瘤技术获得4株稳定分泌针对CPIV的单克隆抗体(MAb)细胞株,分别命名为4F386、584C9、4G7F4和4C9D8.4株MAb腹水针对CPIV的间接ELISA抗体效价达1:10~5～1:10~6,与犬瘟热病毒(CDV)和犬细小病毒(CPV)均不发生交叉反应.MAb 4F386和4C9D8为IgG,5B4C9和4G7F4为IgM.Western blot检测表明,4F386与CPIV的F蛋白发生特异性反应,4G7F4与CPW的HN蛋白发生特异性反应,而584C9和4C9D8不与变性的CPIV蛋白发生反应.4株MAb均具有中和病毒活性,间接免疫荧光检测均呈为阳性.本研究为进一步研制CPIV特异性诊断和治疗制剂创造了条件.     

抗A型口蹄疫病毒单克隆抗体的制备及抗原捕获ELISA检测方法的建立

为建立A型口蹄疫病毒(FMDV)病原学诊断方法,本研究应用纯化的A型FMDV免疫BALB/c小鼠,取其脾细胞与SP2/0细胞融合,筛选获得一株稳定分泌单克隆抗体(MAb)的杂交瘤细胞株5F7.经IFA特异性鉴定,5F7为一株血清型特异性MAb.亚类为IgG1/k.间接ELISA检测杂交瘤细胞上清和腹水抗体效价分别为1:12 800和1:105.硫氰酸盐洗脱法测定其相对亲和力常数为1.5 mol/L.应用该A型特异性MAb及实验室已制备的MAb 3D9初步建立了检测A型FMDV的抗原捕获ELISA方法.该方法可以检出2.0 ng纯化FMDV和1.0×104 TCID50病毒,与O型、Asial型FMDV及牛肠道病毒、牛呼肠孤病毒、牛传染性鼻气管炎病毒等均无交叉反应,组内及组间重复性试验显示变异系数小于8％.本研究建立的抗原捕获ELISA方法为A型FMDV抗原检测提供了一种实用有效的技术手段.     

糖化酶单抗的研制及其抗原识别位点的分析

为建立掺糖造假蜂蜜中残留糖化酶的检测方法,用从黑曲霉中提取的糖化酶作为免疫原,免疫BALB/c小鼠,利用杂交瘤技术获得了12株稳定分泌针对糖化酶抗体的杂交瘤细胞株。单克隆抗体亚型鉴定结果显示,10株为IgG1,2株为IgG2b,轻链均为κ轻链。Western blotting分析结果表明,12株抗体均可特异性结合糖化酶。其中6株单抗(McAb-2H4F9、6H9D8、8F2F11、8F2E9、1A8G6、1C4D5)细胞株采用体内诱生法制备的腹水效价均1∶1×10⁴以上。采用抗体叠加试验对这6株抗糖化酶单抗的抗原识别位点进行检测,反应增殖结果表明,6株单抗分别针对4类不同抗原位点,McAb-6H9D8和McAb-8F2F11针对第Ⅰ种抗原决定簇;McAb-1A8G6和McAb-1C4D5针对第Ⅱ种抗原决定簇;McAb-8F2E9针对第Ⅲ种抗原决定簇;McAb-2H4F9针对第Ⅳ种抗原决定簇。制备的抗体针对不同的抗原表位,为双抗夹心ELISA方法的建立提供前提。     

猫杯状病毒VP1蛋白单克隆抗体的制备及其抗原表位的鉴定

为制备抗猫杯状病毒(FCV)衣壳蛋白VP1的单克隆抗体(MAb)及鉴定其表位,本研究以原核表达的重组VP1-E蛋白(aa427~aa524)免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞(SP2/0)进行融合制备杂交瘤细胞;以纯化的FCV-2280病毒粒子作为检测抗原,采用间接ELISA检测方法筛选杂交瘤细胞株,分析杂交瘤细胞所分泌的抗体亚型、中和活性及所识别的抗原表位等。结果表明,获得两株稳定分泌抗FCV VP1-E MAbs的杂交瘤细胞E2F1和E2F6,抗体均为IgG1亚型,轻链为κ链。杂交瘤细胞分泌的抗体无中和活性。Western blot结果显示,两种抗体可以特异性识别FCV-2280,表明其针对的抗原表位为线性表位。两株MAb识别VP1-E序列均为~(467)YICGSLQRAWG~(477)。生物信息学分析显示该抗原表位在FCV VP1蛋白中相对保守。该MAb为建立特异性强、灵敏度高的FCV的检测方法奠定基础。     

马传染性贫血病毒基质蛋白p15单克隆抗体的制备

为制备抗马传染性贫病毒(EIAV)的单克隆抗体(MAb),本研究用纯化的重组EIAV基质蛋白作为免疫原免疫BALB/c小鼠,采用淋巴细胞杂交瘤技术,建立了抗EIAV基质蛋白p15抗体的杂交瘤细胞.应用重组p15和EIAV总蛋白为抗原建立的ELISA以及EIAV感染细胞的间接免疫荧光法,对杂交瘤细胞进行了有限稀释法筛选,获得了7株稳定分泌抗p15抗体的杂交瘤细胞株,分别命名为:1F12、1E2、1E3、4B10、4H7、5E5、4G5.这些抗p15 MAb均能与感染驴胎皮肤细胞的EIAV和经SDS-PAGE分离的EIAV总蛋白中的相应蛋白结合.抗体效价叠加实验表明,这7株MAb中含有至少3种识别不同抗原决定簇的抗体.这些p15 MAb的获得有助于对EIAV和慢病毒的深入研究.     

鲤春病毒血症病毒单克隆抗体的制备及其特性鉴定

为获得针对鲤春病毒血症病毒(SVCV)特异性的单克隆抗体(MAb),以纯化的SVCV为抗原,免疫BALB/c小鼠.将免疫鼠的脾细胞与SP2/0骨髓瘤细胞融合,采用间接ELISA法筛选获得4个能稳定分泌抗SVCV MAb的杂交瘤细胞株;4个杂交瘤细胞制备腹水的MAb效价为1:160 000～1:640 000.亚型鉴定结果表明,这些MAb分属2个亚型(1F1、3E1,IgG2a;3F5、4F9,IgGl),轻链均为K链.Western blot分析显示,MAb1F1、3F5、4F9能特异性地识别SVCV的N蛋白(47 ku),3E1能特异性地识别SVCV的G蛋白(69 ku).采用相加ELISA法对抗原表位分析结果显示,1F1、3F5、4F9可能识别相同的表位,3E1则识别不同的表位.间接免疫荧光试验结果显示4株MAb均能对染毒病灶产生特异性的荧光染色.这些MAb的制备为SVCV免疫学检测方法的建立奠定了基础.     

光滑型布鲁氏菌LPS单克隆抗体的制备及鉴定

为制备抗布鲁氏菌单克隆抗体(MAb),本研究用灭活的布鲁氏菌疫苗M5-90株免疫BALB/c小鼠,利用细胞融合技术获得杂交瘤细胞.以布氏杆菌疫苗株M5-90、S-19及小肠结肠炎耶尔森菌0:9的脂多糖(LPS)分别作为抗原包被酶标板,对杂交瘤细胞进行筛选,获得两株稳定分泌抗光滑型布氏杆菌LPS(S-LPS)MAb的细胞4G6和16C5.特异性分析表明,MAb 4G6除与耶尔森0:9全菌体有轻微的交叉反应外与其它革兰氏阴性菌无交叉,而16C5完全排除了与其他各菌的交叉反应.Ig亚型分析表明,所制备MAb的轻链亚类均为κ,4G6重链为IgM.16C5重链为IgG3.用Protein G亲和层析纯化MAb 16C5腹水并初步用于竞争ELISA方法,检测布氏杆菌抗体水平,结果表明了该实验方法的有效性.本研究制备的MAb 16C5具有高度的特异性和敏感性,排除了与其他革兰氏阴性菌的交叉反应,为建立一种诊断布鲁氏病的快速、有效、敏感的方法奠定了有力的基础.     

抗阪崎肠杆菌LPS单克隆抗体的制备及初步应用

为制备抗阪崎肠杆菌(ES)脂多糖(LPS)抗原单克隆抗体(MAb)及建立双抗夹心ELISA检测方法,本研究以热灭活的Es(ATCC51329)菌体抗原作为免疫原,ES菌体结合LPS作为筛选抗原,采用杂交瘤细胞技术筛选获得9株稳定分泌抗ES LPS特异性MAb的杂交瘤细胞株.采用改良的过碘酸钠法制备辣根过氧化物酶(HRP)酶标抗体,并建立检测ES的双抗体夹心ELISA方法,该方法对ES ATCC51329具有良好的特异性,最低检测限可达103 cfu/mL,为食品中ES的检测提供特异性MAb及ELISA检测方法.     

牛γ-干扰素ELISA检测方法的建立与初步应用

以纯化的rHis-BoIFN-γ制备兔多抗血清,辛酸—硫酸铵两步法对体内诱生的抗rBoIFN-γ单抗腹水进行纯化,用美洲商陆素（Pokeweed mitogen,PWM）刺激奶牛全血产生的分泌性天然BoIFN-γ筛选出与之反应最佳的单抗5E11。将单抗5E11以40μg/mL浓度包被,与1∶3 000倍稀释的兔抗rHis-BoIFN-γ多抗血清（13.8μg/mL）配对,以1∶6 000稀释的商品化酶标羊抗兔IgG为指示抗体,建立了BoIFN-γ抗原捕获ELISA检测方法,该方法可以检出2.56 U/100μL（30.5 pg/100μL）的rHis-BoIFN-γ、8U/100μL的rBac-BoIFN-γ和1U/100μL的分泌性天然BoIFN-γ。以商品化试剂盒作为平行对照,将获得的奶牛临床检测血浆样品使用BoIFN-γ抗原捕获ELISA法进行检测,结果显示两种方法检测符合率达到83.9%。本研究建立的BoIFN-γ抗原捕获ELISA检测方法可有效检测分泌性IFN-γ,为进一步开发BoIFN-γELISA检测试剂盒奠定了基础。     

Partial Antigenic Characterization of Buffalopox Virus

The present study was undertaken to antigenically characterize the buffalopox virus (BPV). Six monoclonal antibodies (MAbs) against the BP4 strain of BPV have been produced and characterized. All six MAbs appeared to be specific to BPV, as none of them showed cross-reactivity with other poxviruses in antigen capture ELISA. Only two MAbs (20AB8 and 20CD11) bound significantly with different BPV isolates in antigen capture ELISA, whereas the remaining four MAbs bound weakly with the BPV. In Western blot analysis with purified BPV-BP4, the rabbit hyperimmune serum against purified BPV-BP4 reacted with 15 immunodominant polypeptides (100 kDa to 25 kDa), whereas two MAbs (21CB6, 21DB11) reacted with 42 kDa and 45 kDa polypeptides, respectively. However, three MAbs (20AB8, 20CD11, 21CB5) reacted with three degraded polypeptides (100 kDa, 40 kDa and 87 kDa) of BPV-BP4. In radioimmunoprecipitation assay (RIPA) with the rabbit hyperimmune serum to BPV-BP4, three virus specific polypeptides (69 kDa, 34 kDa, 32 kDa) were recognized in BPV-BP4, whereas two polypeptides (69 kDa, 34 kDa) were recognized in other BPV isolates (BPV-Bly, BPV-Vij96, BPV-Vij97). In virus neutralization test, none of the six MAbs tested showed any significant neutralizing ability to infection with different BPV isolates. However, the hyperimmune serum showed weak neutralizing ability to BPV infection.     

蓝舌病病毒重组VP7蛋白单克隆抗体制备及竞争ELISA检测方法的建立

为了建立蓝舌病(BT)的血清学诊断方法,本研究利用原核表达的蓝舌病病毒(BTV)血清型12型VP7纯化蛋白免疫BALB/c小鼠,制备2株单克隆抗体(MAb),分别命名为BTV-2D10和BTV-4H7。IFA试验表明,2株MAb均能与BTV 24个血清型发生特异性反应,而与茨城病病毒(IBAV)、中山病病毒(CV)、赤羽病病毒(AKAV)、牛病毒性腹泻病毒(BVDV)、牛传染性鼻气管炎病毒(IBRV)、牛轮状病毒(BRV)、牛肠道病毒(BEV)、牛呼肠孤病毒(RV)及口蹄疫病毒(FMDV)无交叉反应,表明2株MAb均为BTV群特异性抗体。采用重组表达的VP7蛋白作为包被抗原建立的竞争ELISA方法证明,BTV-4H7 MAb对不同血清型BTV阳性血清具有良好的阻断效果,而对AKAV、IBAV、BRV和FMDV阳性血清无阻断作用。本研究建立的竞争ELISA方法与IDEXX公司的试剂盒检测包括65份已知背景血清和322份采自广西省的山羊血清样品,检测结果符合率分别达100%和98%。该竞争ELISA方法的建立为BTV抗体的监测提供了安全、快速、准确的技术手段。     

牛干扰素-γ基因的克隆、序列分析及表达研究

为了使干扰素-γ(interferon γ,IFN-γ)在牛结核等疾病的诊断和防制方面取得更有效的应用,试验采用RT-PCR法从云南本地黄牛外周血淋巴细胞总RNA中扩增牛IFN-γ(BoIFN-γ)基因,测序并进行序列分析;然后亚克隆至pET-28a,转化大肠杆菌BL21(DE3),用IPTG诱导表达。序列分析结果表明,克隆的IFN-γ基因与GenBank中荷斯坦牛IFN-γ序列同源性达99.60%。表达产物经SDS-PAGE分析,在大小约23 ku处可见目的蛋白表达条带,与预期结果一致;用ELISA检测,表达的蛋白具有明显的生物学活性,在菌体中的含量为244 ng/mL。本试验结果为牛IFN-γ蛋白的进一步研究和应用奠定了基础。     

Characterization of monoclonal antibodies against avian reovirus S1133 protein sigmaA synthesized in Escherichia coli

Monoclonal antibodies (MAbs) were prepared against avian reovirus S1133 protein sigmaA (esigmaA) synthesized in Escherichia coli. MAbs were characterized and used to develop a diagnostic test. Ten MAbs were selected for competitive binding assay following coupling with horseradish peroxidase. The results indicated that these MAbs delineated two epitopes I and II of esigmaA. An immuno-dot binding assay was used to detect the effect of denaturation on antibody recognition of the epitopes. All MAbs bound to esigmaA in its native form. After denaturation by boiling in SDS and 2-mercaptoethanol, the binding of MAbs recognizing epitope I was fully abolished. However, the reactivity of MAbs recognizing epitope II was not affected. MAbs 31 and 32, recognizing epitopes I and II, respectively, were selected for the cross-reactivity to heterologous reovirus strains. The results suggest that the two epitopes are highly conserved among these virus strains. A MAb capture enzyme-linked immunosorbent assay (ELISA) procedure was developed using MAbs 32 and 31 to detect reovirus protein sigmaA in samples from tendon tissues of infected bird and chicken embryo fibroblast (CEF) cell cultures. Avian reovirus sigmaA antigens in tendon specimens were detected from the inoculated birds as early as 2 days post-inoculation (PI), approximated a peak at 7 days PI, and maintained this until 16 days PI, then decreased gradually. A clear difference in absorbance values between the tendon samples of the avian reovirus- and mock-infected birds is obtained. Positive results were also obtained from avian reovirus-infected CEF and from the tendon tissues of naturally infected broilers. These results indicated that the MAb capture ELISA is a useful methods for the detection of avian reovirus from chickens suspected to have avian reovirus infections.     

牛支原体单克隆抗体的制备与鉴定

以牛支原体(Mycoplasma bovis)湖北分离株HB0801作为抗原免疫8周龄BALB/c小鼠,利用杂交瘤技术筛选出了6株能稳定分泌抗牛支原体的单克隆抗体细胞株,分别生产腹水并对单抗进行了纯化和特性鉴定。经亚型测定,这些单抗都属IgG类。腹水ELISA效价在1×10⁵~1.6×10⁶。ELISA特异性分析结果表明,6株单抗与临床分离的牛支原体菌株以及ATCC标准株PG45都显阳性反应,但与牛的其他常见病原菌如多杀性巴氏杆菌、化脓隐秘杆菌等都显阴性反应。所有制备的单抗都与无乳支原体有交叉反应,其中两株单抗1A5和1C11只与无乳支原体有交叉反应,与其他支原体无交叉反应。经Western blotting验证,6株单抗分别识别牛支原体全菌蛋白中的不同条带,说明分别针对不同的蛋白抗原。这些牛支原体单克隆抗体为后期建立牛支原体检测方法及致病机理研究奠定了良好基础。     

Production and characterization of monoclonal antibodies detecting chicken interleukin-2 and the development of an antigen capture enzyme-linked immunosorbent assay

Eleven monoclonal antibodies (mAbs) which are specific for chicken interleukin-2 (chIL-2) were produced and characterized by enzyme-linked immunosorbent assay (ELISA), Western blotting and neutralizing assays. These mAbs were used to develop a mAb-based antigen capture ELISA specific for chicken IL-2 detection. Anti-IL-2 mAbs bound specifically to E. coli-derived rchIL-2 in ELISA and identified a 16 kDa IL-2 polypeptide band in Western blot. Several mAbs were shown to neutralize the biological activities of both rchIL-2 and native chicken IL-2 as measured by concanavalin A (ConA)-induced lymphocyte proliferation assay, IL-2 bioassay, and natural killer cell assay. Among the neutralizing mAbs, the mAb chIL-2/11 was most potent in neutralizing IL-2 activity. To develop a sensitive ELISA for the detection of chicken IL-2, an antigen capture ELISA was developed using the mAb chIL-2/16 as the antigen capture antibody and rabbit anti-IL-2 peptide antibody as the detection antibody. Using the mAb-based antigen capture ELISA, significant correlation between the level of IL-2 detected in bioassays and in ELISA was observed. These results showed that the mAb-based antigen capture ELISA is less time-consuming and more reliable compared to a conventional IL-2 bioassay for chicken IL-2. These neutralizing mAbs will facilitate basic immunobiological studies of the role of IL-2 in normal and disease states in chickens.     

Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples

A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was described and evaluated for use as a presumptive screening test for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples. A total of 725 diagnostic samples collected in the field and submitted in Clark's transport enrichment medium (TEM) were analyzed. Cultural isolation of C. fetus was used as the standard for comparison. After incubation of the TEM vials for 4-5 days, fluid was removed for culture and ELISA testing. A sandwich ELISA format was used and the target antigen was C. fetus lipopolysaccharides (LPS). A rabbit anti-C. fetus polyclonal antiserum was used as the capture antibody. Murine monoclonal antibodies (MAbs) to C. fetus serotype A and B LPS core and O-polysaccharides and a goat anti-mouse horseradish peroxidase conjugate were used as detection antibodies. ELISA and culture results for the diagnostic samples were in complete agreement. Seven hundred and eight samples were negative by both tests. All 17 culture positive samples were positive by ELISA with a MAb to LPS core. The ELISA with MAbs to LPS O-polysaccharides detected all culture positive samples with the homologous C. fetus serotype. Sixty-six preputial wash samples from three known C. fetus culture positive bulls were also analyzed. Forty-nine of these samples were positive by both ELISA and culture, 16 were positive by ELISA only, and one was negative by both ELISA and culture. The results indicate that this ELISA is useful as a screening test for the detection of C. fetus in diagnostic samples.     

Rapid methodology for antigenic profiling of FMDV field strains and for the control of identity, purity and viral integrity in commercial virus vaccines using monoclonal antibodies

Monoclonal antibodies (MAbs) developed against different foot-and-mouth disease virus (FMDV) vaccine strains were extensively used to study any possible antigenic variations during vaccine production in Argentine facilities. Additionally, a typing ELISA using strain specific MAbs was developed to detect potential cross contaminations among FMDV strains in master and working seeds with high specificity and sensitivity and to confirm strains identity in formulated vaccines. This assay was carried out for the South American strains currently in use in production facilities in Argentina (A24/Cruzeiro, A/Argentina/01, O1/Campos and C3/Indaial) and for the strain O/Taiwan, produced only for export to Asia. These non-cross reactive MAbs were also used to analyze the integrity of viral particles belonging to each one of the individual strains, following isolation of 140S virions by means of sucrose density gradients from the aqueous phase of commercial polyvalent vaccines. Antigenic profiles were defined for FMDV reference strains using panels of MAbs, and a coefficient of correlation of reactivity with these panels was calculated to establish consistent identity upon serial passages of master and production seeds. A comparison of vaccine and field strain antigenic profiles performed using coefficients of correlation allowed the rapid identification of two main groups of serotype A viruses collected during the last FMD epidemic in Argentina, whose reactivity matched closely to A/Argentina/2000 and A/Argentina/2001 strains.     

抗猪(嗜血)支原体MSG1蛋白单克隆抗体的制备及鉴定

以原核表达纯化的猪(嗜血)支原体MSG1蛋白免疫BALB/c小鼠,运用细胞融合技术筛选分泌针对MSG1蛋白抗体的融合细胞。通过间接ELISA方法筛选获得2株能稳定分泌抗体的融合细胞株,分别命名为1A7和3G6,Western blot结果证明这2株细胞分泌的抗体能够与重组MSG1蛋白发生特异性反应。细胞上清和腹水中的ELISA抗体效价分别为1∶4 096、1∶1 024和1∶1 638 400、1∶51 200,其单抗亚类鉴定均属于IgG1,轻链为κ型。抗原识别位点分析结果表明,2株单抗所识别的抗原位点相同。猪(嗜血)支原体MSG1蛋白特异性单克隆抗体的制备成功,为制备免疫诊断试剂盒和致病机制的研究奠定了基础。