抗腹泻性贝毒软海绵酸多克隆抗体的制备与检测

制备了腹泻性贝毒软海绵酸(okadaic acid,0A)的多克隆抗体,鉴定了抗体特性.利用半抗原BSA偶联小分子毒素OA,制备完全抗原OA-BSA,免疫两只新西兰白兔,分离、纯化抗血清.用间接ELISA方法测定抗体效价,两个兔抗血清效价均达到128,000以上.IC50为2.852 ng/mL.DOT BLOT测定抗体特异性,结果表明,抗体与抗原有特异性反应.试验表明,抗OA多克隆抗体制备成功,其质量较好,可用于将来的免疫检测研究.     

三聚氰胺半抗原及完全抗原的合成与鉴定

本研究合成了三聚氰胺半抗原及完全抗原并鉴定合成是否成功,为制备三聚氰胺抗体奠定基础。将2-氯-4,6-二氨基-1,3,5-三嗪和3-巯基丙酸的反应合成半抗原3-(4,6-二氨基-1,3,5-三嗪-2-基硫代)丙酸,采用质谱鉴定和红外光谱鉴定;通过活性酯法将半抗原分别与牛血清白蛋白、卵清蛋白进行偶联制备完全抗原,采用紫外光谱法初步鉴定,后经动物免疫进一步验证。结果表明半抗原与完全抗原合成成功,为后续的抗体制备奠定了基础。     

新型喹乙醇残留标志物人工半抗原的设计及其在动物组织残留检测中的应用

为提高酶联免疫方法测定喹乙醇残留标志物(MQCA)的检测性能,设计了一种新型MQCA人工半抗原,该半抗原既完整保留MQCA分子特征性基团和结构,又具有便于和蛋白质载体偶联的基团-NH2。通过三步化学反应合成该半抗原,将其与蛋白质载体偶联成为免疫抗原后,进一步制备特异性抗体,研制了MQCA酶联免疫试剂盒,灵敏度高,特异性好,IC50为1.94μg/L。建立了测定动物性产品中MQCA残留量的直接竞争酶联免疫法,测定猪肝、猪肉、鸡肉、鸡蛋中MQCA的检测限分别为0.42、0.23、0.28、0.28μg/kg。猪肉中不同添加浓度MQCA(1,2,4μg/kg)的回收率为70%~97%;批内、批间变异系数均低于12%。本试剂盒可同时快速检测大批样品,有望在MQCA残留检测中发挥重要作用。研究有助于指导小分子药物半抗原的合理设计,为现有的半抗原设计方法提供新的理念和思路。     

三聚氰胺抗原合成及其多克隆抗体的制备

以2-氯-4,6-二氨基-1,3,5-三嗪为原料,强碱条件下与3-巯基丙酸反应,制备三聚氰胺半抗原并经质谱和红外鉴定;应用碳二亚胺法将半抗原与BSA和OVA偶联制备三聚氰胺免疫原和包被原,完全抗原经紫外扫描鉴定偶联成功,偶联比分别为16:1和10:1,通过免疫新西兰大白兔成功制备三聚氰胺多克隆抗体,抗体效价可达1:128000以上。本研究为三聚氰胺人工抗原的制备提供了有效途径,为三聚氰胺免疫检测方法的研发奠定了基础。     

兽药人工抗原合成的研究进展

在兽药的免疫化学残留分析中,能否合成稳定的具有良好免疫原性的人工抗原是制备抗体和建立免疫分析方法的最关键步骤.综述了国内外兽药人工抗原合成过程中载体的选择、半抗原的设计（强调偶联部位和间隔臂的引入）、半抗原与载体的偶联方法、人工抗原的纯化与鉴定等方面的研究进展.     

小分子化合物单克隆抗体的制备及其在动物医学领域的应用

农药、兽药、重金属、生物毒素等残留物质的痕量检测在医学诊断、食品安全、环境监测等领域均具有重要意义，其中基于抗原-抗体特异性识别的免疫分析技术具有特异性强、灵敏度高、稳定性好、操作简便快捷等优点，但如何获得小分子化合物的特异性抗体是研制免疫分析技术的关键。作者介绍了基于杂交瘤技术制备小分子化合物单克隆抗体的方法，分析了影响杂交瘤技术制备小分子化合物单克隆抗体的因素，然后综述了小分子化合物单克隆抗体在兽药残留、药物开发、环境保护等动物医学领域的应用，最后展望了小分子化合物单克隆抗体生产和应用的发展趋势，为小分子化合物单克隆抗体的制备与应用相关研究提供指导。     

牛奶中林可霉素磁免疫化学发光检测方法的建立

林可霉素药物中的醇羟基经PDC氧化后得到酮基林可霉素,然后与氨基丁酸进行缩合反应,得到四碳链羧基半抗原产物,即林可霉素半抗原;将辣根过氧化物酶标记林可霉素半抗原,制备得到酶标抗原;将林可霉素半抗原分别与BSA和OVA偶联,制备得到免疫原和包被原,进而制备得到林可霉素单克隆抗体,测得抗体效价为1:180000;将林可霉素单克隆抗体与磁珠偶联得到磁标抗体;配合全自动磁免疫化学发光仪建立了化学发光免疫检测方法,建立了林可霉素磁免疫化学发光检测试剂盒标准曲线,测得该方法对牛奶中林可霉素的检测限为5.88μg/L,该方法对林可霉素具有较高的特异性,对与林可霉素结构或者功能相似竞争的9种药物均无交叉反应。进行牛奶空白样品添加试验时回收率为85.8%~113.3%,批内变异系数为5.4%~8.6%,批间变异系数为8.4%~9.1%。该方法试剂盒配合全自动磁免疫化学发光仪检测,具有灵敏度高、特异性好、操作简单、检测时间短等优点,适合对乳制品安全监管和企业内控工作中推广应用,为我国乳制品监管工作提供了技术支持。     

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。     

呋喃唑酮代谢物人工抗原的合成及抗体的制备

为改进和完善免疫检测呋喃唑酮代谢物方法,制备了抗呋喃唑酮代谢物(AOZ)特异性抗体.采用对醛基苯甲酸对AOZ进行衍生化得到CPAOZ,还原CPAOZ结构中的C=N双键得到半抗原,半抗原用N-羟基琥珀酰亚胺活化酯法与BSA偶联制备免疫原,免疫新西兰大白兔制得特异性抗体,采用间接(竞争)ELISA法评价抗血清效价及特异性.结果显示,试验获得了较高效价(1∶1280000)的抗AOZ血清,AOZ半抑制浓度为5.9 ng/mL;抗血清与结构类似物呋喃它酮代谢物的交叉反应率仅为0.76％,与呋喃妥因代谢物和呋喃西林代谢物无交叉反应;利用该抗体建立的间接竞争ELISA检测法,AOZ在1～27 ng/mL与抑制率呈线性关系.结果表明,该特抗体虽然灵敏度较低,却对AOZ而非AOZ衍生物有特异性,可为畜产品中AOZ残留检测提供新的思路和方法.     

甲硝唑人工抗原的制备及其ELISA检测

采用碳二亚胺法将甲硝唑半抗原与牛血清白蛋白(BSA)连接制备人工免疫原,同样方法将其与卵清蛋白(OVA)连接制备人工包被原.经紫外扫描分析,两种方法合成的免疫原和包被原的结合比分别为8∶1、2∶1和6∶1、2∶1;动物免疫试验分析偶联物,小鼠抗体效价达1∶5 000,与3种硝基咪唑类药物的交叉反应率均小于10%.表明制备的抗体可以用于甲硝唑残留的检测,同时为检测试剂盒的研制奠定了基础.     

单链抗体在小分子化学物质残留检测中的研究进展

兽药、农药及生物毒素等小分子化学物质在食品、饲料或水体中的过量残留对人、动物的健康及生态环境的平衡造成一定程度的危害,因此建立一种针对上述小分子化学物质残留的快速检测方法十分必要。以抗原抗体特异性结合为基础的免疫分析技术是残留检测的常用手段,该技术的关键是制备可用来检测特异性抗原的抗体。随着对抗体结构的解析及重组DNA技术的发展,单链抗体为小分子化学物质的残留检测提供了新的技术手段。单链抗体具有分子质量小、免疫原性低、可塑性强、可批量生产等优点,具有广阔的发展空间。近年来,以单链抗体为基础建立的酶联免疫吸附法检测小分子化学物质的残留检测成为最常用的分析工具,且单链抗体的筛选策略、表达策略、突变策略及与碱性磷酸酶形成的融合蛋白等进一步提高了抗体产量、灵敏度、广谱性,并使免疫分析方法简便化,从而推进单链抗体免疫分析方法在小分子化学物质残留检测的应用。作者综述了以单链抗体为基础的免疫分析方法在兽药、农药及生物毒素等小分子化学物质方面的研究进展,以期为小分子化学物质残留检测提供参考。     

鸡堆型艾美耳球虫特异性单抗轻链可变区基因的克隆与序列测定

将已扩增出的鸡堆型艾美耳球虫特异性单抗轻链可变区基因进行纯化,并用纯化的基因片段与pMD-18T载体连接,将重组载体转化于感受态细胞JM109,筛选出阳性重组子,提取阳性重组子质粒并进行测序,得到特异性单抗轻链可变区的基因序列。为鸡堆型艾美耳球虫特异性单链抗体基因的构建奠定了基础。     

纳米抗体的制备与临床应用研究进展

抗体是一类能特异性识别并结合抗原的免疫球蛋白，也是机体体液免疫的主要成分。随着科学技术的发展及对抗体研究的深入，抗体的应用领域不断扩展，可作为亲和配体或探针用于蛋白互相作用、蛋白与核酸相互作用、生物大分子定位与分布等研究，也可作为免疫抑制剂或治疗药物用于免疫性疾病、肿瘤、感染等疾病的治疗，还可结合各种标记技术用于疾病诊断、食品安全检测和环境监测。但传统抗体生产成本高、组织穿透力差、免疫排斥反应等缺点限制了其在疾病治疗等领域的广泛应用。近年来，科学家们致力于新型小分子抗体的寻找和研究，先后研制出嵌合抗体、小分子抗体、双特异性抗体等新型抗体。其中，纳米抗体（nanobody，Nb）又称重链可变区（variable heavy chain domain，VHH）抗体，是一种仅由一个重链可变区组成的单域抗体。纳米抗体保留了重链抗体完整的抗原结合能力，且具有分子质量小、组织穿透性强、抗原亲和力高、能识别隐藏表位、免疫原性低、结构稳定、水溶性好、生产成本低、易于产业化等优点，在疾病诊断、癌症和感染性疾病治疗、小分子药物及毒素残留检测等领域展现出巨大的应用前景。作者首先介绍了纳米抗体的特点，然后简述了纳米抗体的制备流程，重点综述了纳米抗体在疾病诊断、疾病治疗、食品安全和环境监测等领域的应用，最后对纳米抗体在兽医临床的应用前景进行了分析和展望。     

兽药残留免疫检测技术应用进展

免疫检测技术（Immunoassays,IAs)是利用抗原与抗体在体外的特异性反应建立起来的检测技术。兽药属于小分子物质（MW＜1000D），没有免疫原性，必须与大分子物质交联成人工抗原才能获得免疫原性。本文从兽药人工抗原的制备、抗体的制备、免疫检测方法的建立等三个方面对免疫技术在兽药残留检测的应用进行了综述，并展望了兽药免疫检测技术的发展趋势和前景。     

Characterization and epitope mapping of monoclonal antibodies to the nucleocapsid protein of severe acute respiratory syndrome coronavirus

The sudden emergence of severe acute respiratory syndrome (SARS) at the end of 2002 resulted in 774 reported deaths from more than 8000 cases worldwide. As no effective vaccines or antiviral agents are available, the most effective measure to prevent the expansion of a SARS epidemic is the rapid diagnosis and isolation of SARS patients. To establish specific diagnostic methods, we generated nine clones of monoclonal antibodies to nucleocapsid protein (NP) of SARS-coronavirus (SARS-CoV). On immunofluorescent antibody assay and Western blotting analysis, none of the monoclonal antibodies showed cross-reactivity to authentic and recombinant NPs of human coronavirus (HCoV) 229E strain. To determine the region on the NP molecule where the monoclonal antibodies bind, we generated four truncated recombinant NPs and analyzed the reactivity between monoclonal antibodies and truncated NPs. Two monoclonal antibodies reacted with a truncated NP covering from amino acid residues 111 to 230, and seven reacted with another truncated NP covering from amino acid residues 221 to 340. Epitope mapping analysis indicated that monoclonal antibody SN5-25 recognized the amino acid sequence Q(245)TVTKK(250) On SARS-NP. Within the epitope, Q245, T246, V247, K249, and K250 appeared to form an essential motif for monoclonal antibody SN5-25 to bind. The information about binding sites and epitopes of monoclonal antibodies may be useful for the development of new diagnostic methods for SARS and for analyzing the function of N protein of SARS-CoV.     

调节性T细胞与肿瘤研究进展

调节性T细胞是机体内存在的一群具有免疫抑制性作用的细胞,对于维护机体免疫平衡具有重要作用,同时其与肿瘤的发生发展关系密切。关于调节性T细胞的研究很多,但其在体内如何调控肿瘤的发生发展仍不清楚。随着对调节性T细胞的表型以及其作用机制的深入研究,发现调节性T细胞可能在不同类型的肿瘤内对疾病起到完全相反的作用。同时通过其作用机制的研究,也筛选出了一批肿瘤免疫治疗的潜在靶点,为肿瘤免疫治疗领域,如单克隆抗体和小分子抑制剂开发等提供了新思路。     

Monoclonal antibodies: cell surface markers for canine keratinocytes

Plasma membranes were isolated from cultured canine keratinocytes by paraformaldehyde-induced membrane vesiculation. The isolated plasma membrane vesicles retained cell surface antigens (eg, a pemphigus vulgaris antigen). These membrane vesicles were used as an antigen source for the production of monoclonal antibodies. Eight antibodies that had specific reactivity to the cytoplasmic membrane of keratinocytes on frozen sections of canine esophagus were identified by use of an indirect immunoperoxidase method. The stratified squamous epithelium of the esophageal mucosa had 4 staining patterns. When applied to frozen sections of canine skin, lip, and tongue, the antibodies had different tissue specificities for differing stratified squamous epithelia. Using sodium dodecyl sulfate polyacrylamide-gel electrophoresis and the western blot technique, one of the antibodies was specific for a 60-kD cell surface molecule. Therefore, such monoclonal antibodies may be useful in defining heterogeneity between different stratified squamous epithelia, in identifying biologically important surface antigens, or in the diagnosis of tumors.     

Developments in diagnostic techniques for differentiating infection from vaccination in foot-and-mouth disease

Foot-and-mouth disease (FMD) is a highly contagious and economically significant disease of cattle, pigs, sheep, goats and wild ruminant species. The FMD virus genome encodes a unique polyprotein from which the different viral polypeptides are cleaved by viral proteases, including eight different non-structural proteins (NSPs). Both structural and non-structural antigens induce the production of antibodies in infected animals. In contrast, vaccinated animals which have not been exposed to replicating virus will develop antibodies only to the viral antigens in the inactivated material. Vaccination against FMD is a key element in the control of the disease in addition to slaughter and movement restrictions. However, countries that vaccinate in the event of an outbreak will have to re-establish their FMD free status to the satisfaction of their trading partners.Because currently available vaccines stimulate the production of antibodies indistinguishable from those produced by infected animals in response to live virus and because vaccinated animals can be infected and become carriers of FMD virus, efforts have been made to develop diagnostic test that can differentiate vaccinated animals from those that are convalescent and from those that have been vaccinated and become carriers following subsequent contact with live virus. Currently the detection of antibodies to non-structural protein's (NSPs) is the preferred diagnostic method to distinguish virus infected, carrier, animals from vaccinated animals. However this is currently only possible at the herd level because of the great variability in the initiation, specificity and duration of the immune response in individual animals to the NSPs shown in many studies. Considerable effort and attention is now being directed toward the development of new methods and techniques for the rapid and accurate detection of anti-NSP antibodies, harmonization and standardization of current diagnostic techniques, as well as the production of defined reagents.     

Immunization of sheep against modified peptides of gonadotropin releasing hormone conjugated to carriers

The efficacy of antigens based on modified GnRH peptides in stimulating the production of antibodies against GnRH in sheep was tested. In the first study cysteine-containing GnRH peptides were conjugated to keyhole limpet haemocyanin (KLH) in 3 different orientations. The 3 conjugates were prepared in an emulsion of Freund's complete adjuvant (FCA) and were injected into 3 groups of 6 castrated male lambs. The 3 vaccines efficiently induced anti-GnRH titers in all the animals treated. The specificity of the GnRH antisera raised varied depending on the orientation of the GnRH molecule in the antigen and on the individual animal.

In a second trial designed to evaluate carrier molecules, a cysteine-containing GnRH peptide was conjugated to either KLH, equine serum albumin, ovalbumin or tetanus toxoid. The conjugates were prepared with FCA and injected into intact male lambs. All 4 vaccines stimulated the production of antibodies against GnRH in all the animals treated. The conjugates prepared with equine serum albumin or ovalbumin were the most effective in raising high anti-GnRH titers. In 18 of 20 lambs treated, anti-GnRH titers resulted in a marked atrophy of the testes.

We conclude that: 1) the different epitopes of the GnRH molecule are equally immunogenic in sheep; 2) the GnRH antibody response is affected by the carrier used; and, 3) anti-GnRH vaccines based on cysteine-substituted GnRH analogues show potential for use in immunocastration of livestock.