猪生长激素抗独特型抗体的制备及免疫学鉴定

应用猪生长激素（Ag）纯品，通过杂交瘤技术制备了猪生长激素单克隆抗体（Ab1）。Ab1经免疫亲和层析柱纯化后免疫新西兰白兔，其血清经饱和硫酸铵纯化后获得猪生长激素抗独特型抗体（Ab2）。再应用ELISA等方法进行鉴别。Ab2与Ab1呈阳性反应，与BALB／c小鼠r球蛋白呈阴性反应；Ab2与Ag竞争结合Ab1；Ab2抑制Ab1与Ag的结合。Ab1是针对猪生长激素纯品的单克隆抗体。Ab2是具有猪生长激素抗原内影像的抗独特型抗体。     

MD病毒抗独特型抗体的研制

制备鸡马立克氏病病毒(MDV)抗独特型抗体(Ab2),探讨Ab2模拟抗原诱导免疫应答的能力.用MDV免疫SPF鸡,分离提纯鸡抗MD-IgG(Ab1),Ab1免疫BALB/C鼠,用鼠脾细胞与SP2/0细胞制备Ab2杂交瘤细胞株,间接ELISA筛选阳性杂交瘤细胞株,经克隆、纯化2×株抗MDV独特型抗体杂交瘤细胞株(6E5,7A12),其中6E5培养上清、腹水ELISA效价分别为4000×、10000×,免疫20 d攻毒,具有抗MDV感染保护.     

旋毛虫不同发育时期排泄分泌物抗原在免疫学诊断中的应用研究

[目的]系统了解并比较旋毛虫不同发育时期排泄分泌物(ES)抗原的免疫学特性,探索可用于临床检测出栏猪旋毛虫感染的血清学诊断技术。[方法]分别以旋毛虫肠道期10 h肌幼虫(10 h ML)、肠道期30 h成虫(30 h Ad)、3 d成虫(Ad3)、6 d成虫与新生幼虫混合(Ad6+NBL)以及肌幼虫(ML)五个不同发育时期的ES作为包被抗原,应用ELISA方法,检测感染不同剂量、不同天数的猪血清中的抗旋毛虫抗体Ig M和Ig G水平,绘制抗体消长规律曲线并进行数据分析。[结果]10 h ML ES和ML ES作为包被抗原适合检测不同感染剂量、感染35 d之前的猪抗旋毛虫Ig M抗体,低剂量感染10 d左右可以检出,高剂量感染5 d也可以检出;Ad3 ES作为包被抗原对高剂量感染35 d之前的猪抗旋毛虫Ig M抗体检测敏感;Ad3和ML的ES作为包被抗原可检测不同剂量、感染35 d之后的猪抗旋毛虫Ig G抗体,其中Ad3 ES抗原检测低剂量感染的效果优于ML ES抗原。[结论]肠道期肌幼虫、成虫和肌幼虫的ES抗原可用于检测旋毛虫的早期感染,成虫和肌幼虫的ES抗原可用于检测出栏猪的旋毛虫感染。本研究为进一步合理有效利用旋毛虫不同发育时期的ES抗原,建立更有效的检测屠宰动物旋毛虫感染的方法提供了重要理论基础和参考。     

抗H9亚型禽流感病毒独特型抗体的制备与应用

用纯化的鸡抗H9亚型禽流感病毒（AIV）免疫球蛋白G（IgG）作为抗原免疫家兔，制备兔源多克隆抗独特型抗体（Pab2），同时免疫小鼠，应用细胞融合技术建立了分泌抗AIV独特型单克隆抗体（Mab2）的杂交瘤细胞系。经鉴定，2类抗独特型抗体均能竞争性地抑制鸡抗AIVIgG与AIV的结合。保护试验证实，兔源多克隆抗独特型抗体疫苗较单克隆抗独特型抗体疫苗具有更高的保护作用。     

伊氏锥虫抗独特型抗体疫苗的研究Ⅱ．伊氏锥虫变异表面糖蛋白抗独特型抗体制备与检测

用纯化的伊氏锥虫变异表面糖蛋白（ＶＳＧ）免疫大白鼠制备出抗血清，经饱和硫酸铰盐析，ＤＥ－５２纤维素离子交换层析和Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析提取ＶＳＧ特异性多克隆ＩｇＧ抗体（Ａｂｌ）。用Ａｂｌ免疫大白鼠和家兔制备出抗独特型血清。ＥＬＩＳＡ抑制试验检测结果表明，８份大白鼠抗独特型血清的抑制率高于２５％。用正常大白鼠ＩｇＧ致敏的醛化绵羊红细胞吸收兔抗独特型血清除去异种抗体，采用Ａｂｌ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析，可从每毫升兔抗独特型血清中获得９７．４微克抗独特型抗体（Ａｂ２）。经ＥＬＩＳＡ抑制试验检测表明，提取的兔抗独特型抗体（Ａｂ２）对Ａｂｌ与ＶＳＧ的结合反应可产生明显的抑制效应，能识别Ａｂｌ的抗原结合部位，其配位的空间构型与ＶＳＧ表位具有相似性。     

旋毛虫感染兔血清抗体消长规律的研究

应用旋毛虫肌幼虫干燥冷浸可溶性抗原与聚醛化聚苯乙烯（ＰＡＰＳ）载体微球共价交联制成快诊试剂,对旋毛虫感染兔血清进行检测,初步探明其血清抗体的消长规律。实验显示,以１６０００条肌幼虫／只经口饲喂感染１０只兔,于第７天可检出抗体,并分别于感染后第４５￣４９天先后达到抗体最高水平,然后维持一段时间（２０￣３０天左右）,以后逐渐缓缓下降。此结果与旋毛虫肌幼虫感染宿主后的生活周期相吻合。肌幼虫在肠道内发育为     

抗PRRSVGP5和M蛋白独特型抗体替代抗原诱导机体产生中和抗体的研究

制备猪繁殖与呼吸综合征病毒(PRRSV)抗独特型抗体(Ab2),探讨Ab2替代抗原对机体免疫调节作用的研究。用纯化的PRRSV-GP5(GP5-Ab1)蛋白和抗PRRSV-M(M-Ab1)蛋白单抗免疫大白兔,当免疫血清琼扩效价达1:16以上,加强免疫后心脏采血分离血清,分别纯化兔抗PRRSV-GP5IgG(GP5-Ab2)和抗PRRSV-MIgG(M-Ab2)血清,分别用GP5-Ab2、M-Ab2以及GP5-Ab2与M-Ab2混合型三种分别免疫未免PRRS疫苗的小猪,同时设PRRS弱毒苗、灭活苗和空白组作对照,免疫后21d采集猪血清,间接ELISA检测免疫Ab2和PRRS疫苗的猪血清全为阳性,空白组仍然为阴性。经细胞中和试验证明免疫Ab2及PRRS疫苗的猪血清都具有中和抗体,说明Ab2在体内具有替代PRRSV的作用诱导机体产生具有中和效应的中和抗体,从而起到保护机体免受PRRSV的感染作用,为猪繁殖与呼吸综合征病新型疫苗的研究提供了新的思路。     

旋毛虫丝氨酸蛋白酶抑制剂定位及免疫保护效果评估

探讨旋毛虫丝氨酸蛋白酶抑制剂(Ts-serpin)的抗原性、定位及免疫保护性。将本实验室前期构建的旋毛虫丝氨酸蛋白酶抑制剂的原核表达载体进行大量表达纯化。利用不同感染时间的猪旋毛虫阳性血清,通过Western blot方法对纯化后的重组丝氨酸蛋白酶抑制剂(rTs-serpin)进行反应原性鉴定,并制备多克隆抗体,用间接免疫荧光法检测Ts-serpin在旋毛虫肌幼虫中的定位。随后将rTs-serpin免疫小鼠进行免疫保护效果评估。结果显示:rTs-serpin可被不同感染时间的猪旋毛虫阳性血清特异性识别,表明rTsserpin是高反应原性抗原;Western blot结果显示旋毛虫排泄分泌物和旋毛虫虫体粗提取物均存在Ts-serpin,间接免疫荧光显示Tsserpin定位在旋毛虫肌幼虫表皮中;免疫保护试验结果显示rTs-serpin免疫后的小鼠旋毛虫肌幼虫减虫率约为32.2%。综上所述,Tsserpin主要定位在旋毛虫肌幼虫表皮,rTs-serpin具有较强的抗原性,且对小鼠具有免疫保护作用。     

抗独特型抗体检测H9亚型禽流感血清抗体

为了探讨用抗独特型抗体替代病毒或病毒亚单位检测H9亚型禽流感血清抗体的可行性,本试验分别用全病毒抗原和抗独特型抗体(Ab2)作检测原,通过琼脂免疫扩散试验(AGP)和间接酶联免疫吸附试验(ELISA)检测了鸡的血清样品。在AGP试验中,分别用Ab2和禽流感病毒(AIV)对10份血清样品检测的符合率为70%。在间接ELISA试验中,Ab2和AIV抗原对10份血清样品检测的符合率为90%。用Ab2间接ELISA试验检测出的血清阳性率(8/10)高于血凝抑制试验(6/10)和AGP试验(5/10)。结果表明,在一定的情况下Ab2可以用来代替具有污染环境风险的病毒抗原来检测抗病毒抗体。     

抗独特型抗体对猪繁殖与呼吸综合征病毒感染的免疫作用

用PRRSV感染SPF猪，血清检测结果显示，机体不仅产生抗PRRSV抗原的各种抗体(Ab1)，而且产生针对这些抗体的抗独特型抗体(Ab2)。根据各种蛋白质的等电点不同，应用IEF技术分离纯化出PRRSV感染猪血清中的不同IgG。分别以纯化的抗PRRSV—GP5蛋白、抗PRRSV-M蛋白的Ab2免疫SPF猪各5头，7d后经鼻腔感染PRRSV，定期采集血样进行病毒分离或鉴定试验。抗PRRSV-GP5蛋白的Ab2免疫的猪，其血样自感染后3～7d均检出PRRSV；3头猪在感染后14～63d未检出PRRSV；2头猪在感染后14～35d检出PRRSV，从42～56d转为阴性，其中1头猪在63d时检出PRRSV。抗PRRSV—M蛋白的Ab2免疫的猪，其血样自感染后3～7d均检出PRRSV；2头猪在感染后14～63d未检出PRRSV；3头猪在感染后14～35d检出PRRSV，从42～56d转为阴性，其中1头猪在63d时检出PRRSV。抗PRRSV—GP5和抗PRRSV—M蛋白的Ab2免疫作用显著，可作为PRRSV-GP5和PRRSV—M蛋白的替代抗原产生具有中和效应的抗体，保护机体免受PRRSV的感染。     

鸡传染性法氏囊病模拟抗原苗免疫作用的探讨

用鸡传染性法氏囊病毒（IBDV）免疫雏鸡，纯化其IgG(Ab1)免疫兔制备Ab2,Ab2经正常鸡IgG(NC-IgG）和IBD抗原（Ag）吸收后，加佐剂免疫雏鸡，免疫保护指数：Ab2组分别为：100％、100％、90％；对照组：0％。重复实验：50只雏鸡接种3次Ab2免疫，能抵抗IBD强毒攻击，该试验证实：IBD模拟抗原苗在IBD免疫中的应用价值。     

Production and characterization of rabbit anti-idiotypic antibodies directed against a murine monoclonal anti-B. abortus antibody

The protective anti-B. abortus monoclonal antibody ISS/32 (Ab1) was used as an immunogen to induce anti-idiotypic antibodies (Ab2) in rabbits. The purpose was to produce and characterize anti-idiotypic antibodies that share conformational similarity with the corresponding bacterial epitope recognized by Ab1. The rabbit anti-IdAb so induced was isolated and affinity-purified. Its specificity for the paratope of Ab1 was determined by evaluating its ability to compete withB. abortus for binding to Ab1 in a competitive ELISA assay. The anti-idiotypic ISS/32 antibodies were able to compete withB. abortus for binding to Ab1 in a dose-dependent manner. Hence, the data indicated that the rabbit anti-Id ISS/32 antibodies reacted with or near the antigen-binding site of Ab1.Abbreviations Ab antibody - anti-Id anti-idiotypic - ELISA enzyme linked immunosorbent assay - IgG immunoglobulin - i.p. intraperitoneal - mAb monoclonal antibody - PBS phosphate-buffered saline - s.c. subcutaneously - TD PBS + 0.05% Tween 20% + 1% yeast extract     

Possible origin of CSF antibodies induced by intrathecal immunization and prophylactic effects against intracerebral rabies virus infection

Intrathecal (IT) immunization involves injecting antigens directly into the intraventricular or subarachnoid spaces, or brain, to induce antigen-specific antibodies (Ab) in the cerebrospinal fluid (CSF). In the present study, rabbits were immunized IT with inactivated rabies virus to investigate the origins of CSF Ab. The time course of Ab induction and tumor necrosis factor-alpha expression suggested the possibility that the CSF Ab originated in the serum. In addition, Ab-producing cells infiltrated around the blood vessels of the brain, suggesting local production of Ab within the central nervous system (CNS). Furthermore, subcutaneous (SC) immunization prior to IT immunization induced a rapid and magnified Ab response in the CSF compared with IT immunization alone. These results were confirmed by the fact that mice immunized SC prior to IT were more resistant to intracerebral challenge with rabies virus than mice immunized via the IT route alone. Taken together, these results suggest that combined SC and IT immunization is a more effective vaccination protocol for prophylaxis and treatment of rabies.     

抗绿脓杆菌外毒素A酶标抗体的制备及其应用

从病死羊体内分离到绿脓杆菌并提取出外毒素A(PEA),再以此毒素作为抗原加油佐剂制成乳化抗原免疫家兔,获得高免血清并提取免疫球蛋白G(IgG);用过碘酸钠法将过氧化物酶标记抗PEA抗体(IgG),制成酶标抗体,经检验,酶标抗体结合物中的HRP浓度和Ab(IgG)浓度分别是0.0608 mg/mL和0.336 mg/mL;HRP/Ab(IgG)克分子比值为1.724%;酶(HRP)结合率是11.15%.利用该酶标抗体以ELISA夹心法对羊体内抗PEA抗体含量进行了检测,结果证明,用酶标抗体ELISA法比用平板凝集实验法检测的抗体效价平均高出二个滴度,表明制备的PEA酶标抗体具有灵敏度高、特异性强的优点.     

用抗原免疫构建分泌兔出血症病毒抗独特型抗体的杂交瘤细胞

用纯化的兔出血症病毒（ｒａｂｂｉｔｈａｅｍｏｒｈａｇｉｃｄｉｓｅａｓｅｖｉｒｕｓ,简称,ＲＨＤＶ）免疫Ｂａｌｂ／ｃ小鼠。正如网络学说所指出的,从免疫鼠的杂交瘤细胞克隆中,不仅筛选出能分泌抗ＲＨＤＶ抗体（Ａｂ１）的细胞克隆,同时也筛选出能分泌抗ＲＨＤＶ独特型抗体（ａｎｔｉ－ｉｄｉｏｔｙｐｉｃａｎｔｉｂｏｄｉｅｓ,Ａｂ２）的杂交瘤细胞,用抗原而不用单克隆抗体（Ａｂ１）免疫,诱导产生抗独特型抗体（Ａｂ２）是一种具有广阔的应用前景的新技术     

Induction and characterization of monoclonal anti-idiotypic antibodies reactive with idiotopes of canine parvovirus neutralizing monoclonal antibodies.

Monoclonal anti-idiotypic (anti-Id) antibodies (Ab2) were generated against idiotypes (Id) of canine parvovirus (CPV) specific monoclonal antibodies (MoAbs). The binding of most of these anti-Id antibodies to their corresponding Id could be inhibited by antigen, thus classifying these anti-Id antibodies as Ab2 gamma or Ab2 beta. By inhibiting experiments it was shown that these anti-Id antibodies did not recognize interspecies cross-reactive idiotopes, but recognized private idiotopes, uniquely associated with the Id of the anti-CPV MoAb used for immunization. This classifies these anti-Id antibodies as non-internal image Ab2 gamma. The potential use of these non-internal image anti-Id antibodies for the induction of antiviral antibodies in the CPV system is discussed.     

抗独特型抗体和吡喹酮协同预防绵羊日本血吸虫病的研究——Ⅰ.抗独特型抗体疫苗和吡喹酮对绵羊肝脏虫卵肉芽肿形成的影响

本研究将日本血吸虫单克隆抗独特型抗体（抗－Ｉｄ）结合佐剂ＦＣＡ／ＦＩＡ免疫的绵羊设为抗－Ｉｄ免疫组，将在攻击感染日本血吸虫尾蚴前２小时肌注低剂量吡喹酮（ＰＺＱ）的绵羊设为ＰＺＱ组，将既用抗－Ｉｄ免疫又在攻击感染前肌注ＰＺＱ的绵羊设为协同组，同时设空白和ＦＣＡ对照组。在攻击感染血吸虫后的第１２周，剖杀各组绵羊，收集肝脏病理组织块，经切片和染色后，用显微镜测量各组肝脏单个虫卵肉芽肿直径。结果发现，抗－Ｉｄ组、ＰＺＱ组和协同组的肉芽肿直径分别为０．４１、０．４２和０．４０ｍｍ，均明显小于空白对照组（０．５１ｍｍ，Ｐ＜０．０５），也小于ＦＣＡ对照组。组织病理观察结果表明，试验组虫卵周围的多核巨细胞数量明显高于对照组。实验结果提示，抗－Ｉｄ免疫和预防性注射ＰＺＱ均能直接影响虫卵肉芽肿的形成，并且二者存在协同作用，对探讨血吸虫病免疫预防机理的研究具有一定的意义。     

Evaluation of serologic and cellular immune responses of cattle to a nonlipopolysaccharide antigen from Brucella abortus

Cows naturally infected with Brucella abortus developed antibody (Ab) responses to a nonlipopolysaccharide antigen (NLA) purified from B abortus strain 1119-3. Sera from strain 19-vaccinated cows did not have detectable amounts of Ab. Weak lymphoproliferative responses to NLA were observed in blood mononuclear cell suspensions obtained from infected cows. There was no evidence of NLA-specific lymphoproliferation in cell suspensions from healthy cows. Nonlipopolysaccharide antigen binding to bovine blood mononuclear cells was observed by antigen-consumption assays and direct binding of radiolabeled antigen. Cells from infected cows bound less NLA than did cells from healthy cows when assays were conducted with intact blood mononuclear cell preparations (monocytes plus lymphocytes). Monocytes obtained from any group did not bind NLA. Purified B lymphocytes from infected and healthy vaccinated cows bound about 3 times more NLA than did T lymphocytes, but there were no apparent differences between the 2 groups in extent of binding. Results of the study indicate that bovine lymphocytes have binding sites for a NLA purified from B abortus strain 1119-3.     

Evaluation of the ELISA and comparison to the complement fixation test and radial immunodiffusion enzyme assay for detection of antibodies against Mycoplasma hyopneumoniae in swine serum

An enzyme-linked immunosorbent assay (ELISA) was evaluated for detection of antibodies (Ab) against Mycoplasma hyopneumoniae and M. flocculare in sera from swine experimentally infected with these agents. In addition, the ELISA was compared with the complement fixation test (CFT), and radial immunodiffusion enzyme assay (RIDEA) for the demonstration of Ab against M. hyopneumoniae. Twenty two 6-week-old swine from a respiratory disease-free herd were divided into five groups. Two or three pigs from each of the four groups were inoculated, respectively, with M. hyopneumoniae or with M. flocculare while two pigs in each group were contact exposed to the inoculated penmates. A fifth group, consisting of three pigs, served as inoculated controls. Pigs inoculated with M. hyopneumoniae began coughing 13 days post inoculation (PI). Antibodies were first detected 2 weeks PI with the CFT, 3 weeks PI with the ELISA, and 5 weeks PI with the RIDEA. With the ELISA and RIDEA, Ab were still detectable one year PI at a very low level. With the CFT, Ab were not detectable in sera from any swine beyond 5 months PI. At necropsy 1 year PI, no lesions were detected in lungs of any of the animals nor were mycoplasmas detected. M. flocculare inoculated or contact-exposed pigs never evidenced clinical signs. Antibodies against M. flocculare were first detected 5 to 12 weeks PI with CFT, and 6 to 12 weeks PI with the ELISA. Peak optical density (OD) values obtained in the ELISA with M. flocculare Ab were as high as the values obtained with peak M. hyopneumoniae Ab titers. Levels of Ab against M. flocculare were at relatively higher OD at 1 year PI than Ab against M. hyopneumoniae. Sera with high levels of Ab against M. flocculare cross-reacted slightly with M. hyopneumoniae antigen in immunoblotting and ELISA.