双特异性单克隆抗体在治疗中的应用

双特异性抗体由于其独特的结构——两个不同抗原,引起研究机构广泛的重视。双特异性单克隆抗体有两个臂,研究者将治疗药物和针对疾病部位的特异目标分别放在两个臂上。治疗药物可以是药品、毒素、酶、DNA和放射性核素等。此外,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。双特异性单克隆抗体从治疗来看,有很大的发展前景,这是由于:(1)最近重组DNA技术的重大突破;(2)人类基因组图谱工程的完成,提高了疾病的鉴别水平;(3)对人类免疫系统机制有了更好的了解。     

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。     

Conjugation and Characterization of Latex Particles with Toxoplasma gondii–specific Immunoglobulin Y Antibodies for Diagnostic Aim and Evaluation Efficiency in In Vitro Culture

Toxoplasma gondii is a parasite that causes severe health problems in the world. Toxoplasmosis, an infection caused by T. gondii, leads to high risk of mortality in patients with immunodeficiency, transplantation, and cancer. Besides that, it causes miscarriages in pregnancy, various abnormalities such as hydrocephalus in infants and congenital diseases. Because the clinical indication of the disease is not specific, it is confused with many diseases, and this leads to the necessity of directly detecting the presence of the toxoplasmosis. Therefore, various diagnostic assays are needed for the diagnosis of the disease. Amongs them, latex agglutination assay is widely used for the detection of specific antibodies or antigens in samples. Latex particles are coated with immunogenic molecules (antigens) to detect antibodies in the blood or used to identify antigens when coated with specific antibodies. In both, aggregation of latex particles results in agglutination. Monoclonal antibodies are often used in latex agglutination assay as in other diagnostic methods. However, monoclonal antibodies can be produced in low quantities at a high cost. Besides, to produce monoclonal antibodies, an experienced staff, a well-equipped cell culture laboratory, a long period of time, and a burdened budget are needed. In recent years, as an alternative to monoclonal antibodies, immunoglobulin Y (IgY) antibodies, which are obtained from chicken eggs, and specifically produced against desired antigenic constructs, have become quite attractive in terms of both low cost and abundant production without requiring infrastructure. In contrast, the latex assay based on IgY antibodies for use in the diagnosis of T. gondii has not been developed. This study aimed to conjugate T. gondii-specific IgY antibodies to latex particles, characterize the particles by Fourier transform infrared spectroscopy, scanning electron microscopy, and spectroscopic methods, and finally demonstrate the interaction with T.gondii parasites in culture with scanning electron microscopy analysis.     

Passive immunization using purified IgYs against infectious bursal disease of chickens in Pakistan

Infectious bursal disease (IBD) is an acute and highly contagious disease of young chickens caused by Birnavirus. Mortality of infected birds can be best prevented if injected with antibodies. The present study was an attempt to raise specific hyper-immune polyclonal antibodies against IBD virus in Pakistan. Commercial layers divided into four groups were injected with IBD vaccine subcutaneously according to four different treatment regimens. Eggs were collected daily and antibodies were purified from yolk with dextran sulphate. Titers of antibodies in serum and yolk were evaluated with enzyme linked immunosorbant assay and agar gel precipitation test. Antibody titers were significantly higher in yolk than serum. Eggs collected at 28 days post-vaccination had maximum antibody titers. Of treatment regimens, T₃ was found to be most effective for hyperimmunization. Lyophilized antibodies stored at 4℃ did not lose their activity till the end of experiment. IBD virus infected birds were injected with purified antibodies which induced 92% recovery as compared to control birds. The study implicates that the purified antibodies may be useful as a therapeutic agent to cure IBD infected birds.     

Antinuclear antibodies: presence and specificity in autoimmune connective tissue disease in the dog.

Autoimmune connective tissue disease in both animals and humans present as multi-systemic disorders. Clinical signs referable to joints, musculoskeletal system and Dermatopathies are present. Autoantibody production is a regular feature of systemic autoimmune disease and these antibodies have diagnostic value in both human and veterinary medicine. In this study, we demonstrate that many of the commonly used diagnostic techniques developed for use with human patients may also be adapted for use in dogs. However, in some instances, 'canine specific' autoantibodies, distinct from analogous human antibodies may be detected in sera from dogs with a diagnosis of autoimmune disease. This finding indicates that dogs may develop 'dog-specific' autoimmune connective tissue disease.     

Defence mechanisms against viral infection in poultry: a review

Defence against viral infections in poultry consists of innate and adaptive mechanisms. The innate defence is mainly formed by natural killer cells, granulocytes, and macrophages and their secreted products, such as nitric oxide and various cytokines. The innate defence is of crucial importance early in viral infections. Natural killer cell activity can be routinely determined in chickens of 4 weeks and older using the RP9 tumour cell line. In vitro assays to determine the phagocytosis and killing activity of granulocytes and macrophages towards bacteria have been developed for chickens, but they have not been used with respect to virally infected animals. Cytokines, such as interleukin (IL)-1, IL-6 and tumour necrosis factor (TNF)-alpha, are indicators of macrophage activity during viral infections, and assays to measure IL-1 and IL-6 have been applied to chicken-derived materials. The adaptive defence can be divided into humoral and cellular immunity and both take time to develop and thus are more important later on during viral infections. Various enzyme-linked immunosorbent assays (ELISAs) to measure humoral immunity specific for the viruses that most commonly infect poultry in the field are now commercially available. These ELISAs are based on a coating of a certain virus on the plate. After incubation with chicken sera, the bound virus-specific antibodies are recognized by conjugates specific for chicken IgM and IgG. Cytotoxic T lymphocyte activity can be measured using a recently developed in vitro assay based on reticuloendotheliosis virus-transformed target cells that are loaded with viral antigens, e.g. Newcastle disease virus. This assay is still in an experimental stage, but will offer great opportunities in the near future for research into the cellular defence mechanisms during viral infections.     

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。     

Bovine herpesvirus glycoprotein D: a review of its structural characteristics and applications in vaccinology

The viral envelope glycoprotein D from bovine herpesviruses 1 and 5 (BoHV-1 and -5), two important pathogens of cattle, is a major component of the virion and plays a critical role in the pathogenesis of herpesviruses. Glycoprotein D is essential for virus penetration into permissive cells and thus is a major target for virus neutralizing antibodies during infection. In view of its role in the induction of protective immunity, gD has been tested in new vaccine development strategies against both viruses. Subunit, DNA and vectored vaccine candidates have been developed using this glycoprotein as the primary antigen, demonstrating that gD has the capacity to induce robust virus neutralizing antibodies and strong cell-mediated immune responses, as well as protection from clinical symptoms, in target species. This review highlights the structural and functional characteristics of BoHV-1, BoHV-5 and where appropriate, Human herpesvirus gD, as well as its role in viral entry and interactions with host cell receptors. Furthermore, the interactions of gD with the host immune system are discussed. Finally, the application of this glycoprotein in new vaccine design is reviewed, taking its structural and functional characteristics into consideration.     

Outbreak of malignant catarrhal fever in Welsh Black cattle in Carmarthenshire

An outbreak of malignant catarrhal fever (MCF) resulted in the deaths of 12 cattle in a herd of 77 animals during seven weeks in 1999; in addition, one cow developed a milder disease which was confirmed as MCF by PCR for ovine herpesvirus 2 DNA and an immunofluorescent antibody test for antibodies to the virus, but recovered. Further PCR and serological testing revealed the infection in three other animals, none of which developed clinical disease. Hypocuprosis and the possibility of a genetic predisposition were identified as factors which may have contributed to the outbreak.     

The treatment of multicentric canine lymphoma

Canine LSA is a fatal disease if untreated, but fortunately it is also a disease that is very responsive to therapeutic intervention. It is likely that most cases seen and treated by practitioners will be managed with the currently effective drugs and with new protocols as they are developed. Other approaches, including immunotherapy and BMT, are likely to remain more in the arena of the academic institution but should be available in the referral setting for appropriate cases. Great strides have been made in the less than 30 years that canine LSA has been widely treated; it is reasonable that similar progress is to be expected in the years to come.     

口蹄疫自然感染动物与免疫动物鉴别诊断研究进展

口蹄疫是偶蹄动物高度接触性传染病 ,能引起巨大经济损失。国际兽医局将其列为 A类动物传染病之首。除发达国家外 ,大多数发展中国家都采用注射疫苗的办法来控制该病的流行。因此如何区分感染动物和免疫动物是口蹄疫防制中迫切需要解决的问题。目前口蹄疫灭活疫苗的生产工艺可以将绝大部分的非结构蛋白除去 ,因而灭活疫苗免疫动物只能产生结构蛋白抗体 ,而感染动物能产生结构蛋白抗体 ,也能产生非结构蛋白抗体 ,因此 ,检测非结构蛋白抗体为鉴别口蹄疫感染动物与免疫动物提供了美好前景。文章从鉴别诊断的原理 ,非结构蛋白的免疫特性 ,鉴别诊断所面临的问题及解决方案 ,应用非结构蛋白作为鉴别诊断抗原的研究现状等方面进行了综述。     

Feline systemic hypertension: Classification and pathogenesis

PRACTICAL RELEVANCE: the increased availability of indirect blood pressure monitoring devices in clinical practice over the past decade has highlighted the significance of systemic hypertension in the feline population. Without routine monitoring and appropriate intervention, cats with undiagnosed systemic hypertension may first be presented with sudden-onset blindness as a consequence of either hyphaema or retinal detachment. CLINICAL CHALLENGES: the primary aim in the early diagnosis and treatment of systemic hypertension is prevention of hypertensive target organ damage (with respect to the eye, kidney, cardiovascular and central nervous systems, in particular). A prerequisite is a knowledge of the pathophysiological mechanisms and disease conditions that may contribute to the development of hypertension. This allows the clinician to determine those cases in which blood pressure assessment and longitudinal monitoring is essential and can assist in determining appropriate therapeutic strategies for control of blood pressure. Recent studies have also begun to explore the relationship that systemic hypertension may have with proteinuria and the progression of kidney disease. PATIENT GROUP: the geriatric cat appears most susceptible to the development of systemic hypertension, and monitoring of systolic blood pressure is often advocated as part of a routine health screen in cats over 9-12 years old. Consideration must also be given to cats suspected of having an underlying disease such as chronic kidney disease or hyperthyroidism, or which are receiving therapeutic agents, irrespective of their age. EVIDENCE BASE: much of our understanding of the pathogenesis of feline hypertension is extrapolated from studies performed in experimental animal models or in human patients, and interspecies differences are often poorly understood.     

Development and functional characterization of monoclonal antibodies recognizing chicken lymphocytes with natural killer cell activity.

We have developed two monoclonal antibodies which detect cell surface antigens present on chicken lymphocytes mediating natural killer (NK) cell activity against the avian tumor cell target. The monoclonal antibodies, K-14 and K-108, stained 17 and 6% of splenic lymphocytes, and 11 and 14% of peripheral blood lymphocytes (PBL), respectively, and fewer than 5% of thymic and bursal lymphocytes. Neither of these monoclonal antibodies stained adherent macrophages or the MC29-virus transformed monocytic cell line. Both monoclonal antibodies significantly inhibited NK cell activity in a standard 4 h 51Cr-release cytotoxicity assay using the LSCC-RP9 tumor cell line as target cells at an effector to target ratio of 50:1. Pretreatment of splenocytes with either monoclonal antibody in the presence of rabbit complement (C) resulted in a significant reduction in NK cell activity. However, the monoclonal antibody K-1 which detects normal chicken macrophages did not interfere with NK cell activity. The monoclonal antibody K-108 significantly blocked Fc receptor-mediated rosette formation of sheep red blood cells coated with IgG antibodies (EA) by 56% while the monoclonal antibody K-14 did not show a significant blocking. These results indicate that the monoclonal antibodies K-108 and K-14 identify different epitopes present on the surface of chicken splenic lymphocytes which mediate spontaneous NK cytotoxicity.     

Serodiagnosis of Lyme disease in UK dogs

Lyme disease is a chronic, multisystemic, inflammatory disorder of man and animals associated with infection by the tick-borne spirochaete, Borrelia burgdorferi. Lyme disease was recently reported for the first time in a dog in the UK (May and others 1990). Using an enzyme-linked immunosorbent assay (ELISA), we have performed a serological survey to investigate the prevalence of antibodies to B burgdorferi in UK dogs. The survey has shown that dogs from many areas in the UK have serum antibodies to B burgdorferi, that the presence of serum antibodies is associated with known exposure to ticks and that some dogs seropositive for B burgdorferi have clinical signs consistent with Lyme disease. High levels of serum anti-Borrelia antibodies are not diagnostic for canine Lyme disease, but, in association with appropriate clinical signs, they help to confirm the diagnosis in suspected cases.     

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.     

Development of a monoclonal antibody based competitive-ELISA for detection and titration of antibodies to peste des petits ruminants (PPR) virus

Peste des petits ruminants (PPR) is an acute febrile, viral, disease of small ruminants with great economic importance. A competitive-ELISA (c-ELISA) test was developed for detection of antibodies to PPR virus in the sera samples of goats and sheep. The test uses monoclonal antibody to a neutralizing epitope of haemagglutinin protein of the virus. Based on the distribution of known negative sera samples (n=933) in respect of PPR virus antibodies in the test, a cut-off value was set as 38%. This value was the result of mean of negative population added with two times the standard deviations. A total of 1668 sera samples from goat and sheep and 32 sera from cattle were screened by c-ELISA and virus neutralization test (VNT). Efficacy of c-ELISA compared very well with VNT having high relative specificity (98.4%) and sensitivity (92.4%). The sensitivity of c-ELISA for PPR sero-surveillance could further be increased (95.4%), if the target population is non-vaccinated. c-ELISA test correlated well with VNT (r=0.845) for end-point titration of PPR virus antibody in 64 goat sera samples. It could clearly separate infected population from uninfected in field sera. Using c-ELISA test paired sera samples from 13 goats provided a clear diagnosis of PPR virus infection. Furthermore, antibodies to PPR virus could be successfully detected during 1 year after vaccination in four goats inoculated with an experimental PPR vaccine. Findings suggest that the c-ELISA test developed can easily replace VNT for sero-surveillance, sero-monitoring, diagnosis from paired sera samples and end-point titration of PPR virus antibodies.     

Differentiating infection from vaccination in foot-and-mouth-disease: evaluation of an ELISA based on recombinant 3ABC

Recent devastating outbreaks of foot-and-mouth disease (FMD) in Europe have reopened the discussion about the adequacy of the non-vaccination strategy implemented by the EU in 1991. Here we describe the evaluation of a new commercially available test kit for the discrimination between vaccination and infection. The test is based on the detection of antibodies against the recombinant non-structural (NS) protein 3ABC. In contrast to immunization with vaccines free of 3ABC, these antibodies are elicited as a consequence of infection. Testing more than 3600 negative sera from several countries revealed a specificity of > 99% for bovine, ovine, and porcine samples. Antibodies specific for 3ABC can be detected as soon as 10 days post-infection. As compared with the occurrence of antibodies against structural proteins of FMDV, anti-3ABC antibodies can be detected 5-10 days later, depending on the species. No anti-3ABC antibodies were detected in sera from vaccination experiments or in field sera from vaccinated animals. However, anti-3ABC antibodies can be detected in vaccinated animals upon challenge. These results provide evidence that this test can facilitate the use of vaccines in new strategies against FMD.