制备双特异性单克隆抗体的方法

双特异性抗体由于其独特的结构－2个不同抗原，引起研究机构的广泛重视。研究者将治疗药物和针对疾病部位的特异目标分别放在双特异性单克隆抗体的2个臂上。治疗药物可以是药品、毒素、酶、DNA和放射性核素等。双特异性单克隆抗体能使免疫效应细胞的细胞毒性作用于致病细胞或者诱导产生全身免疫应答。作者重点介绍了制备双特异性单克隆抗体的3种方法，分别是化学方法、生物方法和基因工程，为制备临床需要的抗体奠定坚实的理论基础。     

单个B细胞抗体制备技术研究进展

单克隆抗体在疾病预防、诊断和治疗方面具有重要意义，尤其在免疫机制研究方面具有突出贡献。随着单克隆抗体制备技术的发展，单个B细胞抗体制备技术成为新一代快速制备单克隆抗体的方法。该技术利用每个B细胞仅可产生一种特异性抗体的特性，直接筛选出分泌特异性抗体的B细胞，对其表达抗体重链和轻链的基因进行克隆和体外表达，从而获得特异性单克隆抗体。相较传统抗体制备技术，该技术具有快速、高效、产量高等优点，且表达的抗体具有天然构象，不仅可用于病原微生物相关抗原的抗体开发、病毒跨种传播机制等研究，还在抗肿瘤治疗、抗自身免疫病等方面发挥重要作用。本文主要对单个B细胞抗体制备过程和应用现状进行综述，以期为单克隆抗体制备技术的发展和完善提供参考，促进其更好地应用于相关领域。     

单克隆抗体在动物疾病治疗中的应用及前景

将抗原注入Balb/c小鼠体内诱导产生特异性B细胞,与骨髓瘤细胞融合形成杂交瘤细胞,经筛选分离出单个杂交瘤细胞,将其扩大培养,最终分离纯化抗体。筛选出的单个杂交瘤细胞具有骨髓瘤细胞和特异性B细胞的双重特性,既能持续繁殖,又能分泌针对单一抗原表位的特异性抗体,即单克隆抗体。单克隆抗体具有便于制备、理化性质均一、生物活性单一、特异性强、易于标准化等优点。使用单克隆抗体,能提高免疫学检测和临床治疗的特异性和精确性,对生物、化学、医学、免疫学等领域有着重要的意义。1单克隆抗体在动物疾病治疗中的应用单克隆抗体的特性使其能…     

免疫技术在水产动物养殖中的应用

由于化学药物施用后会存在鱼体残留、抗药株出现及破坏生态环境等诸多不足，就极大地推进了鱼类免疫学及免疫疫苗等免疫技术的研究和发展。但由于水产动物是较低等的动物，免疫系统和免疫反应较高等动物原始，其研究进展也比较缓慢。近几年的研究表明，免疫技术在水产动物的疾病诊断、疾病防治及营养等多方面均有广阔的应用前景。１免疫诊断技术免疫诊断技术具有高特异性、高灵敏性等特点，将抗原抗体的免疫反应相结合，缩短水产养殖过程中疾病的诊断时间，提高了诊断的准确性。１．１单克隆抗体技术单克隆抗体（MonoclonalAntibodyMcAb）…     

DNA疫苗的研究进展

大约十年前,人类进入第三次疫苗革命阶段,其起始标志是Wolff在一次实验中偶然发现,他将裸露的DNA注射到肌肉中结果得到了表达的蛋白产物.自1993年起,Robinson首次报道了禽流感病毒(AIV)DNA疫苗研究成功,紧接着Ulmer证实了表达流感病毒核蛋白的质粒DNA不仅能诱导小鼠的特异性细胞免疫反应,而且可对不同血清亚型的异源流感病毒产生交叉免疫保护.在大量实验工作的基础上,一种新的疫苗——DNA疫苗诞生了,其概念可表述为将编码某种抗原蛋白的外源基因(DNA)与质粒重组后,直接导人动物细胞内,并通过宿主细胞的转录系统合成抗原蛋白,诱导宿主细胞产生对该抗原蛋白的免疫应答,从而达到预防和治疗疾病的目的.     

双特异性抗体的研制方法及其在奶牛乳腺炎治疗中的应用

双特异性抗体具有两个不同的抗原结合位点,能分别与靶细胞和免疫细胞结合,从而引导免疫细胞定向杀灭靶细胞。双特异性抗体的研制经历了鼠源性单克隆抗体的化学偶联,双杂交瘤和基因工程3个发展阶段,基因工程双特异性抗体与以往鼠源性双特异性抗体相比具有较低的免疫原性和较好的组织穿透能力。knobs into holes技术是基因工程双特异性抗体研制过程中出现的一种新技术,该技术能引导异源二聚体的配对,提高双特异性抗体的产量和质量。目前已有一些Bs(Fab)2和diabody等类型双特异性抗体进入了临床试验阶段,其中以抗CD3/抗肿瘤双特异性抗体最为常见。抗中性粒细胞/金黄色葡萄球菌的试验结果表明,双特异性抗体对中性粒细胞体外抗金黄色葡萄球菌具有明显的导向作用,且对中性粒细胞的免疫功能具有诱导性。     

家蚕微孢子虫单克隆抗体的研制

以家蚕(Bombyx mori)微孢子虫(Nosema bombycis)免疫BALB/C小鼠的脾细胞与骨髓瘤细胞融合后制备杂交瘤细胞,分泌对N.bombycis孢子表面抗原特异性的单克隆抗体。用酶联吸附免疫分析(ELISA)以筛选杂交瘤细胞,经3次有限稀释法克隆后,用包括N.bombycis等6种微孢子虫及正常蚕体组织匀浆作抗原筛选出1株分泌仅对N.bombycis孢子有特异性单克隆抗体的杂交瘤细胞株(LC_2)。小鼠腹水滴度为1:640O,用ELISA最少检出100O个孢子的样本。     

马立克氏病毒单克隆抗体杂交瘤细胞株的建立与鉴定

以马立克氏病毒814株(MDV-814)作为免疫用抗原,建立了2个MDV特异性单克隆抗体(McAb)杂交瘤细胞株—4A6、3D7。鉴定结果表明;两株细胞所分泌的McAb均为IgM类免疫球蛋白,具有MDVⅠ型病毒特异性,无中和反应特性和沉淀反应特性。利用两种McAb对国内标准强毒MDV-京1株进行鉴定,肯定其为MDV-Ⅰ型毒株。     

分泌抗山羊γ干扰素单克隆抗体杂交瘤细胞株的制备与鉴定

为筛选分泌山羊γ干扰素(IFN-γ)单克隆抗体的杂交瘤细胞,以原核表达的山羊rIFN-γ蛋白免疫Balb/c小鼠,取其脾脏细胞与SP2/0瘤细胞进行细胞融合,以间接ELISA方法筛选分泌山羊IFN-γ单克隆抗体杂交瘤细胞,采用山羊外周血淋巴细胞产生的IFN-γ包被酶标板对IFN-γ的特异性进行鉴定,用试纸条对IFN-γ单克隆抗体类别和亚型进行鉴定。结果获得了1株能特异性识别山羊IFN-γ的杂交瘤细胞3C,3C分泌的单克隆抗体能够特异性识别天然结构的山羊IFN-γ,单克隆抗体类别为IgG,轻链为κ型。     

一种细胞因子检测新方法--ELISPOT技术

抗原特异性细胞毒T细胞在控制病毒感染和某些肿瘤中具有重要作用 ,如何准确地定量、定性抗原特异的T细胞反应及评价其作用 ,是细胞免疫学中的一项重要课题。ELISPOT技术是近年来正为众多研究者所认可的一项通过检测细胞因子 ,评价T淋巴细胞功能和特异性的新方法。与其他细胞免疫学方法相比较 ,该方法特异性强、灵敏度高、能够直接检测分泌细胞因子细胞 ,而无需体外扩增。ELISPOT技术对病毒性疾病和肿瘤的发病机制以及体内细胞免疫功能的了解具有重要的意义 ,也为这些疾病的细胞免疫治疗提供了理论依据。目前 ,有研究显示该方法不仅应用于肿瘤免疫及其治疗中 ,而且在细胞内感染微生物、自身免疫性疾病发病机制、治疗等方面均有巨大的发展潜力     

Comparison of intramuscular and footpad subcutaneous immunization with DNA vaccine encoding HSV-gD2 in mice

Herpes simplex virus type 2 is the most common infectious agent in humans that causes genital herpes disease and vaccination is a desirable method to prevent herpes infections. An effective therapeutic vaccine will need to elicit virus-specific immune responses. The route of immunization has important role in immune responses. In this study, DNA vaccine encoding glycoprotein D of herpes simplex virus type 2 (HSV-gD2) was prepared and injected via intramuscular and footpad routes to determine the optimal method of delivery for immune stimulation. The control manipulation of immune response by concerning route of administration is highly appreciated issue by researches. Although DNA vaccine containing HSV-gD2 is effective in both intramuscular and footpad injection routes, the latter could induce significantly higher cellular responses against HSV-2.     

DNA Fragment Encoding Human IL-1β 163–171 Peptide Enhances the Immune Responses Elicited in Mice by DNA Vaccine against Foot-and-Mouth Disease

DNA vaccine has been tested for protection against foot-and-mouth disease. However, the relatively low efficacy of DNA vaccine in inducing immune responses in large animals has restricted its practical use. Interleukin-1 plays an essential role in amplifying both the cellular and humoral immune responses to foreign antigens, and may therefore represent a good candidate as an adjuvant of DNA vaccines. Since the inflammatory activity of IL-I may restrict its application in DNA vaccine treatment, we explored the possibilities of augmenting immune responses without unwanted inflammatory effects using the IL-1beta fragment (amino acids (aa) 163-171), which is essential for IL-1 receptor-1 binding. The DNA fragment encoding the human IL-1beta fragment (aa 163-171) was fused to foot-and-mouth disease virus (FMDV) DNA vaccine, and injected into mice to analyse its immune response. Compared with control mice receiving FMDV DNA vaccine alone, significant increases in the FMDV-specific antibody response and also in T cell proliferation were observed in mice receiving IL-1beta (163-171)-FMDV. These results suggested that DNA fragment encoding IL-1beta 163-171 peptide might represent a good candidate for an adjuvant of FMDV DNA vaccine.     

New gene-based approaches for an AIDS vaccine

Vaccine approaches against AIDS have focused on inducing cellular immune responses, since many studies revealed the role of T cell responses in the control of human immunodeficiency virus or simian immunodeficiency virus (SIV) infections. The experimental infection of rhesus macaques with SIV or chimeric SHIV is routinely used as a model for AIDS. In such models, DNA immunization is a tool to elicit specific T cell responses and to study their protective efficacy. DNA immunogenicity in primates depends on parameters such as level of antigen expression, choice of the antigen among SIV proteins, use of fusion proteins, route of immunization, and addition of adjuvants. Recent results suggest that priming with DNA and boosting with attenuated recombinant viral vectors, each expressing corresponding SIV antigens, leads to improved specific immunity and, in some cases, affords protection against pathogenic challenge. After preclinical evaluations, DNA has entered clinical trials for a therapeutic or prophylactic gene-based AIDS vaccine.     

Immunisation strategies against prion diseases: prime-boost immunisation with a PrP DNA vaccine containing foreign helper T-cell epitopes does not prevent mouse scrapie

Vaccination against prion diseases constitutes a promising approach for the treatment and prevention of the disease. Passive immunisation with antibodies binding to the cellular prion protein (PrP(C)) can protect against prion disease. However, immunotherapeutic strategies with active immunisation are limited due to the immune tolerance against the self-antigen. In order to develop an anti-prion vaccine, we designed a novel DNA fusion vaccine composed of mouse PrP and immune stimulatory helper T-cell epitopes of the tetanus toxin that have previously been reported to break tolerance to other self-antigens. This approach provoked a strong PrP(C)-specific humoral and cellular immune response in PrP null mice, but only low antibody titres were found in vaccinated wild-type mice. Furthermore, prime-boost immunisation with the DNA vaccine and recombinant PrP protein increased antibody titres in PrP null mice, but failed to protect wild-type mice from mouse scrapie.     

Proteomic analysis of soluble protein extract of adult Toxocara cati

Toxocara cati is a cat roundworm and the causative agent of toxocariasis as a cosmopolitan zoonotic disease. As no information has been reported so far, identification of T. cati proteins can be useful for the development of new diagnostic strategies. This study was conducted to identify the major proteins in the adult T. cati tegument using bi-dimensional electrophoresis (2-DE) and shotgun proteomics. A total proteins were identified, among them the metabolic enzymes were the largest group, including: Enolase, triose phosphate isomerase, fructose-bisphosphate aldolase, aldehyde dehydrogenase. The other important protein groups recognized in T. cati, belong to the HSP-family, the structure and motor proteins, such as actin. The role of these proteins have been implicated in parasite–host interactions and modulating cellular immune response, immune regulation in evasion mechanisms of the host immune response. Characterizing T. cati adult proteins play a key role not only in host-parasite interactions, but also in the discovery of drug targets, subunit vaccines against toxocariasis, immunodiagnostic kits for toxocariasis and the identification of novel immuno-modulators that can form the next generation of therapeutic possibilities for inflammatory diseases.     

Induction of immune responses to glycoprotein gD of Aujeszky's disease virus with DNA immunization.

In an attempt to produce a DNA vaccine to prevent Aujeszky's disease, the induction of immune responses against Aujeszky's disease virus (ADV) gD was investigated in mice. The plasmid was constructed by placing ADV gD gene downstream of murine cytomegalovirus immediate early promoter of expression vector pMYK, which was injected twice on the skin of mice by using a gene-gun. All mice showed neutralizing antibodies against ADV gD at 4 weeks after immunization. The induction of cytotoxic T lymphocytes and splenic natural killer cells was also observed at 6 weeks post immunization. These results indicate that ADV gD gene in the form of DNA vaccine may induce specific as well as non-specific immune responses in vivo.     

Mechanisms of immunosuppression.

Immunosuppresive disease is a major economic concern in domestic poultry production. Although many immunosuppressive agents have been described, mechanisms of how infectious and noninfectious agents compromise the immune system are poorly understood in avian species. Two categories, generalized and antigen-specific immunosuppression have been described in mammals. Generalized immunosuppression produces overall reduced responsiveness and increased susceptibility to a wide variety of infectious and neoplastic diseases. The best characterized immunosuppressive mechanisms are described in HIV-1 infections that lead to acquired immune deficiency syndrome (AIDS) in humans. In contrast, the antigen-specific suppression observed in human leprosy illustrates how an infecting agent selectively suppresses host responses against itself favoring bacterial spread. Both diseases have well-defined clinical staging classifications that correlate with specific immunological defects. An approach to studying immunosuppressive mechanisms in the avian suggests the need for relating pathogenesis with tests of immune responsiveness using a series of increasingly more specific immunological assays to pinpoint defects.     

Induction of protective immunity in chickens immunised with plasmid DNA encoding infectious bursal disease virus antigens

Direct DNA inoculations were used to determine the efficacy of gene immunisation of chickens to elicit protective immune responses against infectious bursal disease virus (IBDV). The vp2 gene of IBDV strains GP40 and D78, and the vp2-vp4-vp3 encoding segment of strain D78 were cloned in an expression vector which consisted of human cytomegalovirus (HCMV) immediate early enhancer and promoter, adenovirus tripartite leader sequences and SV40 polyadenylation signal. For purification of vaccine-quality plasmid DNA from E. coli, an effective method was developed. Chickens were vaccinated by inoculation of DNA by two routes (intramuscular and intraperitoneal). Two weeks later, chickens were boosted with DNA, and at 2 weeks post-boost, they were challenged with virulent IBDV strain. Low to undetectable levels of IBDV-specific antibodies and no protection were observed with DNA encoding VP2. However, plasmids encoding VP2-VP4-VP3 induced IBDV-specific antibodies and protection in the chickens. DNA immunisation opens a new approach to the development of gene vaccines for chickens against infectious diseases.     

猪肺炎支原体P46基因的原核表达与间接ELISA方法的建立

猪肺炎支原体是猪喘气病的病原体,本研究选择猪肺炎支原体P46膜蛋白基因亲水区序列进行克隆,并将其内3个编码Trp的TGA突变成TGG,然后再克隆到pET28a(+)载体中,在大肠杆菌BL21 (DE3)细胞内实现了高效表达,表达产物相对分子质量约为31 ku,约占菌体总蛋白35％,表达形式为包涵体,通过Western blotting 证明表达产物与猪肺炎支原体高免血清具有很好的反应原性和特异性.将大肠杆菌表达的猪肺炎支原体P46重组蛋白经过洗涤、过柱纯化后,作为间接ELISA包被抗原用于检测猪血清中猪肺炎支原体抗体,通过对各参数和试剂的优化建立了rP46-ELISA方法,获得了较好的效果,通过与现有ELISA检测方法的比较,结果表明二者间具有较高的符合率.