苯乙醇胺A人工抗原的合成及鉴定

采用重氮化法将苯乙醇胺A分别与牛血清白蛋白(BSA)和卵清白蛋白(OVA)偶联,制备免疫抗原苯乙醇胺A(PEAA)-BSA和检测抗原PEAA-OVA。经紫外扫描法和SDS-PAGE鉴定表明偶联成功,偶联比分别为15∶1、17.4∶1。用PEAA-BSA免疫BALB/c小鼠,采用间接ELISA法检测抗体效价,采用竞争ELISA法检测IC50值,获得了效价4.1×105以上、IC50值为0.2μg/L的多抗血清,证明合成了具有免疫原性的人工免疫原。     

盐酸克伦特罗人工抗原及抗体的制备

为了制备盐酸克伦特罗(CL)人工抗原及抗体,试验采用重氮化法将盐酸克伦特罗与经过活化的牛血清白蛋白(BSA)和卵清蛋白(OVA)分别偶联,合成免疫抗原(CL-BSA)和检测抗原(CLOVA),经紫外光谱扫描对偶联物进行鉴定;用免疫抗原免疫BALB/c小鼠制备盐酸克伦特罗多克隆抗体,间接酶联免疫吸附试验(ELISA)法测定抗体效价。结果表明:盐酸克伦特罗人工抗原合成成功,制备的鼠源多克隆抗体效价达1∶64 000,该抗体可用于下一步试验。     

盐酸四环素人工抗原的合成及鉴定

本试验旨在合成盐酸四环素人工抗原,为其单克隆抗体的制备奠定试验基础。将盐酸四环素(TCH)发生霍夫曼降解和重氮化反应生成重氮盐,再与蛋白质载体(BSA,OVA)偶联生成免疫原和包被原,对偶联产物采用三氯化铁及紫外扫描法鉴定偶联成功,且免疫原和包被原的摩尔结合比分别约为3.4∶1和4.2∶1。用免疫原免疫BALB/c小鼠,用间接ELISA检测抗体效价,采用竞争ELISA法测IC50,获得了效价为1∶51200I、C50为16.91 ng/mL的多克隆抗体,最终证明人工抗原制备成功。     

多西环素人工抗原的合成与鉴定

将多西环素（doxycycline,DOX）进行化学修饰引入羧基或氨基等活性基团,然后与牛血清白蛋白（BSA）和卵清蛋白（OVA）偶联合成人工免疫原DOX—PABA—BSA、DOX—BSA和包被原DOX—PABA—OVA、DOX～OVA,并用紫外吸收（UV）、凝胶电泳（SDS—PAGE）和EUSA方法对人工抗原进行鉴定;将合成的人工抗原DOX—PABA—BSA、DOX—BSA分别免疫BALB／C小鼠,用间接ELISA方法测定多抗（pAb）效价,用竞争ELISA方法鉴定其敏感性,用交叉反应试验鉴定其特异性。结果表明,二个免疫组6只小鼠血清抗体效价均在1：6400以上,DOXpAb对DOX的50％抑制浓度（IC50）在39．79～53．13μg/L,抗血清与四环素类药物交叉反应很低。本实验为建立多西环素ELISA残留免疫学检测方法和并制备多西环素试剂盒奠定了基础。     

氯霉素人工抗原的合成及其抗体的制备

为了合成氯霉素(CAP)人工抗原并制备抗体,试验建立CAP残留的酶联免疫吸附试验(ELISA)法,采用重氮化法将半抗原CAP与牛血清白蛋白(BSA)进行偶联,制得免疫抗原,然后与卵清白蛋白(OVA)进行偶联,制得包被抗原,通过紫外光谱扫描鉴定是否偶联成功;用免疫抗原免疫KM小鼠,制备CAP多克隆抗体,并通过间接ELISA法测定抗体效价。结果表明:通过紫外光谱扫描,CAP-BSA在275 nm处为最大吸收峰,CAP-OVA在276 nm处为最大吸收峰;并且免疫KM小鼠得到的抗体效价高达1∶128 000。说明试验成功制备了CAP人工抗原,并且获得了高效价的多克隆抗体,也进一步证明了药物偶联成功。     

马波沙星人工抗原的合成及其免疫原性的鉴定

合成、鉴定了马波沙星(MBF)人工抗原,为马波沙星单克隆抗体的制备及其免疫学检测方法的建立奠定基础。以牛血清白蛋白(BSA)、鸡卵清蛋白(OVA)为载体蛋白,采用碳二亚胺法分别合成马波沙星免疫抗原(MBF-BSA)和包被抗原(MBF-OVA),并使用紫外分光光度法、SDS-PAGE、ELISA方法鉴定人工抗原合成效果。初步鉴定结果显示,MBF-BSA与MBF-OVA均偶联成功;用免疫抗原MBF-BSA免疫小鼠,经ELISA方法检测,五免后血清中抗体效价可达1∶5.12×105,半数抑制率为90.20 ng/m L。研究表明,马波沙星人工抗原合成成功,其免疫抗原具有良好的免疫原性,免疫小鼠可获得高效价、高特异性多克隆抗体。     

磺胺甲恶唑人工抗原的合成与鉴定

为检测磺胺甲恶唑(SMZ)的残留,用重氮化方法将SMZ偶联于载体蛋白BSA和OVA上,合成了免疫抗原BSA-SMZ和包被抗原OVA-SMZ。经紫外扫描、SDS-PAGE电泳鉴定后,免疫BALB/c小鼠,制备多抗血清;利用间接ELISA法测定多抗血清效价,竞争ELISA测定多抗血清的敏感性和特异性。结果表明,所合成的免疫抗原效果较好,6只免疫小鼠多抗血清的效价均大于1∶1.28×104,5号小鼠的多抗血清敏感性最高,半数抑制浓度为47.1ng/mL,与其他磺胺类药物无交叉反应,具备良好的特异性。试验成功合成了SMZ人工抗原,并制得了敏感性高、特异性好的鼠源多抗血清,为SMZ单克隆抗体制备及其免疫学快速检测方法的建立奠定了基础。     

呋喃唑酮人工抗原的制备

[目的]制备呋喃唑酮人工抗原。[方法]通过重氮化法和戊二醛法将半抗原FZ与载体蛋白质牛血清白蛋白(BSA)和卵清蛋白(OVA)耦联,制备人工免疫抗原和检测抗原,经SDS-PAGE电泳鉴定人工抗原是否耦联成功,用紫外可见分光光度计法测定耦联蛋白浓度。[结果]SDS-PAGE结果显示,用2种方法制备的人工抗原FZ-BSA和BSA、FZ-OVA和OVA的2组蛋白条带迁移距离均发生了变化,耦联物的迁移距离均小于其相对应的载体蛋白。[结论]通过SDS-PAGE鉴定呋喃唑酮人工抗原制备成功。     

氨基脲人工抗原的合成及其多克隆抗体的制备

目的合成氨基脲人工抗原并制备其多克隆抗体。方法以氨基脲(SEM)和对醛基苯甲酸(CP)为原料合成半抗原(CP-SEM),并采用碳化二亚胺法和混合酸酐法将其分别与牛血清白蛋白(BSA)和鸡卵清蛋白(OVA)偶联,合成氨基脲人工抗原。以氨基脲人工抗原(CPSEM-BSA)为免疫原免疫Balb/c小鼠,利用间接ELISA法测定其抗体效价。结果经薄层层析、红外光谱和元素分析等方法鉴定合成半抗原即为CP-SEM。紫外扫描、聚丙烯酰胺凝胶电泳结果表明人工抗原合成成功。免疫获得抗氨基脲的多克隆抗体,其抗体效价为1:64000。结论氨基脲人工抗原合成成功,并获得抗氨基脲的多克隆抗体。     

氨苄西林人工抗原的合成及鼠源多克隆抗体的制备

为了建立氨苄西林(AMP)残留检测的免疫分析方法,试验采用碳二亚胺(EDC)法将AMP与经活化的牛血清白蛋白(BSA)和卵清蛋白(OVA)分别进行偶联,合成人工抗原AMP-BSA和AMPOVA,通过紫外光谱扫描对人工抗原进行鉴定,将AMP-BSA免疫BALB/c小鼠获得多克隆抗体,利用间接酶联免疫吸附试验(ELISA)法测定抗体效价。结果表明:AMP与载体蛋白BSA和OVA偶联成功,并成功制备了鼠源AMP多克隆抗体,效价高达1∶64 000。     

Antigen requirements and specificity of enzyme-linked immunosorbent assay for detection of canine IgG against canine distemper viral antigens

An enzyme-linked immunosorbent assay (ELISA) was developed for detection of specific immunoglobulin G (IgG) against canine distemper virus (CDV) antigens. Sucrose gradient separation of viral and cellular proteins was required to produce coating antigens for the ELISA. The specificity of the ELISA was demonstrated by blocking CDV-positive canine sera with CDV-specific antisera produced in goats and rabbits and adsorption of positive sera with CDV antigens. A comparison of the ELISA with the serum-neutralization technique for the detection of CDV antibodies was conducted. Anti-CDV IgG was detected in conventional dogs as early as 6 days after inoculation with a commercial vaccine to CDV. Paired sera from the immunized dogs were evaluated by both techniques and a statistically (P less than 0.01) significant agreement between the ELISA and the serum-neutralization technique was shown (r = 0.6121, n = 75).     

大观霉素人工抗原的合成及其多克隆抗体制备

Using spectinomycin (SPE), carboxyl methyl hydroxylamine hydrochloride （CMO）， glycol diglycidyl ether （EDE）, bovine serum albumin (BSA) and ovalbumin （OVA） as the main stuff, four artificial-immunogens had been obtained by the N-hydroxysuccinimide-activated ester method and the EDE double functional group method. Ten female rabbits were immunized with this two artificial-immunogens. Animal immune test demonstrated that there were antibodies against SPE in rabbits sera. The polyclonal antibody was measured with indirect ELISA test. The titer was 1∶160000 and the IC₅₀ was 0.484 ng/mL. This study laid the basis for further research on the immune test kit for the SPE remnant.     

Identification by transfer blot of antigens reactive in the enzyme-linked immunosorbent assay (ELISA) in rabbits immunized and a calf infected with Cryptosporidium sp

Soluble and particulate fractions of Cryptosporidium sp. oocysts from cattle were obtained by homogenization and sonication. Electrophoresis of the soluble fraction in polyacrylamide gels with sodium dodecyl sulfate and silver staining revealed the presence of 41 bands. Enzyme-linked immunosorbent assay (ELISA) of sera from rabbits immunized with either fraction and from a calf 40 days after infection showed that the animals produced specific antibodies. Enzyme-linked immunoelectrotransfer blot tests revealed the presence of five antigens with the rabbit sera and nine with the calf serum. ELISA proved to be an appropriate test for diagnosis of cryptosporidiosis. Selection of reactive antigens may improve the quality of diagnosis and/or reveal the presence of protective materials in the parasite.     

Preparation of heavy chain specific antisera to bovine IgA, IgM, IgG1 and IgG2

Goats, guinea pigs and rabbits were immunized with bovine IgM or with intact molecules, heavy chains, Fc portions or light chains of bovine IgG1 and IgG2. Rabbits and guinea pigs were immunized with bovine secretory IgA. Goats and guinea pigs produced heavy chain specific antisera to intact IgM whereas rabbits produced anti-light chain antibody and in one instance anti-alpha 2-macroglobulin antibody in addition to the anti-mu response. Goats and guinea pigs produced antisera to bovine IgG1 and IgG2 and their Fc portions that needed little absorption to render them monospecific for the heavy chain. In addition to antibody to the heavy chains, rabbits produced anti-light chain antibody when immunized with intact IgG1 or IgG2 molecules. These latter sera, and those produced by rabbits immunized with Fc portions of IgG1 or IgG2 required extensive absorption before they were monospecific for their respective heavy chains. Heavy chains were poor immunogens in all three species. Rabbits immunized with IgA produced both anti-alpha and anti-light chain antibodies while guinea pigs produced sera with antibody activity to the alpha chain only.     

结核分枝杆菌CFP10-ESAT-6融合基因的表达及其多克隆抗体的制备

以结核分枝杆菌标准株H37Rv的基因组DNA为模板，经重叠PCR扩增获得CFP10-ESAT-6融合基因片段，克隆于pET21a表达载体中，获得了重组质粒pET21a—CFP10—ESAT—6，将其转化大肠杆菌BL21细胞，经IPTG诱导表达，得到了25ku的目的蛋白。表达产物经纯化和定量分析后免疫新西兰大白兔，收集兔血清进行抗体ELISA检测。结果显示，成功构建了CFP10-ESAT-6融合基因的原核表达质粒，获得了CFP10-ESAT-6融合蛋白，以此蛋白免疫动物可产生高效价的抗体。     

猪抗克伦特罗(CL)抗血清的制备及其IgG纯化与鉴定

为了获得高特异性、高纯度的猪抗克伦特罗 (CL)IgG ,将克伦特罗跟牛血清白蛋白 (BSA)偶联后 ,选取 1 0头大×大×约三元杂交猪 (5头为免疫组 ,5头为对照组 ) ,用偶联物BSA CL对其进行免疫 ,来制备猪抗CL抗血清。以卵清蛋白 (OVA)跟CL的偶联物OVA CL为包被抗原 ,采用ELISA法测定抗血清效价和血清阻断率。结果表明 ,有 4头猪体内产生了抗CL抗体 ,且在第 2次加强免疫后达到最大 ,效价为 1∶2 0 0 0 0 ;而当血清稀释率为 1∶40 0 ,CL的PBS液浓度在 1 8× 1 0 - 4 ～ 7 0× 1 0 - 7之间时 ,其阻断率为 80 %～ 1 8%。随后用正辛酸 硫酸铵法对血清抗体进行纯化 ,经紫外吸收法测定和SDS PAGE电泳试验结果表明IgG成份得到了纯化 ,可用于进一步的免疫试验     

Further purification and characterisation of horse IgE

Horse IgE was isolated from a serum pool collected from foals naturally infected with endoparasites. The serum was precipitated with ammonium sulfate, delipidated with dextran sulfate and further purified by gel filtration, anionic exchange, immunosorption or preparative polyacrylamide gelelectrophoresis. By these methods IgE could be isolated at a purity of 81%. The sera from rabbits immunized with the purified horse serum fractions were tested using reversed passive cutaneous anaphylaxis and an enzyme linked immunosorbent assay (ELISA). By the ELISA method cross reaction of rabbit anti horse IgE sera to human, mouse and rat myeloma IgE was demonstrated. Rat myeloma IgE also served to monitor the production of antibodies to horse IgE in rabbits.     

Characterization of monoclonal and polyclonal antibodies to bovine enteric coronavirus: establishment of an efficient ELISA for antigen detection in feces

Monoclonal antibodies to bovine enteric coronavirus (BEC) were produced. Additionally, polyclonal antibodies were made in rabbits and guinea pigs and extracted from the yolk of immunized hens. The antibodies were characterized by neutralization test, hemagglutination inhibition test, enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Neutralizing antibody titers of polyclonal antisera ranged from 1:1280 to 1:40,000. Only one out of 908 hybridoma colonies tested secreted antibodies with neutralizing activity. By ELISA, polyclonal sera exhibited high background reactions that could be significantly reduced by treatment with kaolin in the case of rabbit sera. Attempts to establish an ELISA for BEC antigen detection based on polyclonal sera failed due to low sensitivity and specificity. Optimal results were achieved when a mixture of two monoclonal antibodies was coated onto microplates for antigen capture, while rabbit hyperimmune serum served as detecting antibodies in an indirect assay. The combination of the two monoclonal antibodies did not increase sensitivity synergistically, but in a compensatory fashion, probably because of epitope differences between BEC field strains.     

己烯雌酚多克隆抗体的制备和鉴定

用混合酸酐法合成的BSA-DES免疫兔子,制备抗DES的特异性抗体,分别用混合酸酐法和碳二亚胺法合成的OVA-DES作为包被原检测抗体;用混合酸酐法合成的OVA-DES和碳二亚胺法合成的OVA-DES免疫小白鼠,制备抗DES的特异性抗体,分别用混合酸酐法合成的BSA-DES和碳二亚胺法合成的BSA-DES作为包被原检测抗体。检测结果表明:三种免疫原免疫动物,都产生了DES的特异性抗体,并建立了间接阻断ELISA法。这为残留DES酶免疫检测试剂盒的制备奠定了基础。     

Evaluation of serological tests for detecting tick-borne encephalitis virus (TBEV) antibodies in animals

Tick-borne encephalitis (TBE) in animals is not well understood yet. TBE virus (TBEV) serology in several host species could be valuable for epidemiological analyses in the field as well as for the detection of clinical cases. However, performance and suitability of the available test systems are not well assessed. Therefore, we evaluated two commercial TBEV-ELISA kits in a pilot study and compared them for their suitability in veterinary applications. For this purpose, we tested 163 field collected goat sera and evaluated the results by serum neutralization test (SNT) as "gold standard". Twenty-eight SNT positive sera (17.2%) were detected. The best suited ELISA kit was used for determination of a species-specific cutoff for horses, cattle, sheep, goats, pigs, mice, dogs, rabbits and monkeys with defined sera from animals without known or with improbable contact to TBEV. The level of non-specific ELISA results does not only differ between animal species but may also be influenced by the age of the tested animals. The number of sera which tested false positive by ELISA was higher in older than in young sheep. In order to obtain defined polyclonal sera as references, two dogs, cattle, goats, sheep, rabbits and pigs each, as well as one horse and 90 mice were immunized four times with a commercially available TBEV vaccine. In conclusion, our results demonstrated that commercial TBEV-ELISA kits are suitable for application in veterinary medicine for both, verification of clinical TBE cases and epidemiological screening. However, positive ELISA results should be verified by SNT. Only a very low number of false negative ELISA-results were found.