用间接ELISA检测抗H9亚型AIV独特型单克隆抗体的研究

目的为制备抗H9亚型A型流感病毒(AIV)独特型杂交瘤细胞系，运用异种动物间共有独特型理论建立的间接ELISA进行检测。方法本试验采用兔抗H9亚型低致病力AIV IgG作为检测抗原，通过预试验确定了包被抗原的工作浓度为800μg/mL，以间接ELISA方法检测融合细胞培养上清液，筛选到产生抗AIV鸡和兔种间共有独特型抗体的杂交瘤细胞。结果间接ELISA试验中P／N值达12，效价达到2^-4，证明杂交瘤细胞株分泌目的单克隆抗体的能力强。结论该检测方法排除了分泌抗鸡同种型表位以及超变区其他表位抗体的杂交瘤细胞，从而成为筛选分泌抗H9亚型AIV独特型单克隆抗体杂交瘤细胞株的可靠方法。     

抗AIV(H9)独特型单克隆抗体的制备及其免疫原性的研究

用H9N2亚型AIV作为抗原免疫接种SPF鸡制备高免血清,用饱和硫酸铵法从该血清中提取免疫球蛋白G(IgG)。以该IgG制备弗氏佐剂疫苗免疫SPFBALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞NS-1在聚乙二醇(PEG)作用下融合,采用间接ELISA检测法,筛选获得3株(2H1D1、2H1G4、3H2D7)分泌特异性单克隆抗体的杂交瘤细胞。3株融合细胞培养上清液中的单克隆抗体效价均为2-4,小鼠腹水中的单抗效价是细胞培养上清液中的300～500倍。经检测表明,3株细胞系产生的单克隆抗体仅与相应的鸡抗H9N2亚型AIV血清发生特异性反应。用2H1D1分泌的抗独特型单克隆抗体制备疫苗与抗AIV灭活疫苗同时免疫SPF鸡收获的高免血清与AIV在MDCK细胞上进行中和实验,中和效价分别为10-1.8/10μl、10-1.6/10μl。由此表明,该抗独特型抗体疫苗具有良好的免疫原性。     

抗禽流感病毒M2蛋白单克隆抗体的制备及鉴定

目的:制备抗禽流感病毒M2蛋白的单克隆抗体(mAb)并进行特性鉴定.方法:利用纯化的融合蛋白GST-M2免疫BALB/c小鼠,然后以GST-M2 和 GST分别作为ELISA抗原进行筛选,选择GST-M2抗原检测强阳性、GST抗原检测阴性的杂交瘤细胞进行克隆,建立能稳定分泌抗AIV M2 mAb的杂交瘤细胞株.mAb的效价采用间接ELISA和琼脂扩散试验(AGP)测定,用夹心ELISA测定Ig亚类,Western blot、抗原捕获ELISA、间接免疫荧光及免疫组化染色法检测 mAb 的特性.结果:得到4株分泌抗禽流感病毒M2蛋白的mAb的杂交瘤细胞株1E1、2F8、4E3 和 5D6,小鼠腹水中抗体的ELISA效价大于210×100、AGP效价大于1:4.抗体亚类鉴定1E1和4E3为IgG2a,2178和5G6为IgG2b.抗原捕获ELISA表明,2F8 wAb能与H5、H9亚型AIV发生特异性反应,而不能与鸡新城疫病毒(NDV)和鸡传染性法氏囊病毒(IBDV)反应.IFA和免疫组化试验表明,2F8 mAb能够与感染了禽流感病毒的MDCK细胞以及鸡体组织细胞发生特异性结合.结论:本研究获得4株抗禽流感病毒M2蛋白的mAb,其中2F8mAb的效价高、特异性强,可作为检测AIV方法的核心试剂.     

一次融合同时获得抗独特型和抗抗独特型单克隆抗体的杂交瘤

本文在研制带有HBsAg表位内影象的单克隆抗独特型抗体的过程中,用抗-HBs单克隆抗体(Ab_1)免疫同系鼠,除筛选分泌Ab_2β的杂交瘤细胞外,还同时设计了筛选分泌Ab_3的杂交瘤。结果在一次免疫,一只免疫鼠的脾脏细胞,一次融合实验中,同时获得了分泌Ab_2β及Ab_3抗体的杂交瘤细胞株。本文报告筛选Ab_3的方法及对Ab_3所作的免疫化学鉴定。结果获得一株分泌Ab_3单克隆抗体的杂交瘤细胞株,命名为M4。证明Ab_3具有Ab_1的特异性。这一结果不仅提示独特型网络的确在同一个体内客观存在,而且也为研制单克隆抗独特型抗体杂交瘤提供了一条简单、经济、事半功倍的经验。     

H4亚型禽流感血凝素单克隆抗体的研制及夹心ELISA的建立

目的制备H4亚型禽流感病毒(AIV)血凝素单克隆抗体(mAb),基于mAb建立H4亚型AIV夹心ELISA检测方法。方法用灭活H4亚型AIV免疫BALB/c小鼠,通过细胞融合、血凝抑制(HI)试验筛选和亚克隆,得到了一株抗H4亚型AIV mAb(6G4)。免疫荧光细胞化学染色和Western blot法验证6G4与H4亚型AIV的反应性, HI试验鉴定6G4特异性、广谱性和稳定性,试剂盒鉴定6G4亚类。以鸡抗H4亚型AIV多克隆抗体作为包被抗体, 6G4作为捕获抗体, HRP标记的山羊抗小鼠IgG作为酶标抗体,通过条件优化和一系列验证,建立H4亚型AIV夹心ELISA检测方法。结果 6G4亚类为IgG1,轻链为κ链,能稳定分泌抗体,具有良好的反应性、特异性、广谱性和稳定性;基于6G4建立的ELISA特异性强、敏感性高、重复性好,适合大批量检测。结论成功制备了抗H4亚型AIV的mAb,建立了检测H4亚型AIV的夹心ELISA。     

抗乙肝病毒核心抗体的单克隆抗独特型抗体杂交瘤细胞株的建立及鉴定

用单克隆抗体HBc免疫BALB/c小鼠,取脾细胞与Sp2/0骨髓瘤细胞融合,筛选到1C12、2G12两株分泌抗独特型抗体的杂交瘤细胞株,经鉴定属于IgG2a亚类。经ELISA独特型结合试验,抗原—抗体结合抑制试验进行鉴定,证实为抗乙肝病毒核心     

人绒毛膜促性腺激素单克隆抗体的研制

目的获得针对人绒毛膜促性腺激素单克隆抗体。方法hCG蛋白免疫Balb／c小鼠,取其脾细胞与同系小鼠骨髓瘤细胞NS-1按8：1比例融合,间接ELISA法筛选阳性克隆,有限稀释法进行克隆化培养;制备腹水抗体;采用间接ELISA法鉴定抗体亚型和测定抗体效价。结果得到6株能稳定分泌单克隆抗体的杂交瘤细胞株;抗体经鉴定均为IgG1、κ型,效价均达10^-5以上。结论所获得的6株杂交瘤细胞株均有较强稳定分泌抗-hCG单克隆抗体的能力。这为有关hCG的检测、hCG本身相关研究以及避孕疫苗的研制打下了基础。     

C-myc单克隆抗体的制备及鉴定

目的制备与鉴定鼠抗C-myc蛋白单克隆抗体。方法利用C-myc蛋白做免疫原免疫Balb/c小鼠,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行细胞融合,经间接ELISA法筛选、有限稀释法克隆、单克隆抗体Ig亚型鉴定,获得2株能够稳定分泌抗C-myc特异性抗体的杂交瘤细胞株,再将杂交瘤细胞注射入小鼠腹腔诱使小鼠产生腹水。结果获得2株可稳定分泌抗C-myc抗体的杂交瘤细胞株,命名为SHH-1H11,SHH-2A9,Ig亚型分别为IgG1亚类和IgM。细胞培养上清效价分别为1:10240、1:5 120;小鼠腹水效价分别为1:64 000、1:32 000。结论制备的C-myc单克隆抗体仅与C-myc蛋白反应,与其他蛋白没有交叉反应,表明获得的单克隆抗体的特异性很高。     

H5N1流感病毒M1蛋白免疫制备的单克隆抗体的研究

目的 制备抗人流感病毒H5N1株M1蛋白的单克隆抗体,为流感的快速诊断和研究提供新的工具.方法 应用在大肠埃希菌中表达的人H5N1亚型禽流感病毒(A/Anhui/1/2005)株M1蛋白,以纯化的表达产物免疫BALB/c小鼠,取脾细胞与sp2/0细胞系作细胞融合后,间接ELISA法筛选阳性的杂交瘤细胞,并应用间接免疫荧光法对抗体的特异性进行鉴定.结果 获得3株能稳定分泌抗禽流感病毒M1抗原的McAb杂交瘤细胞株,交叉反应试验及间接免疫荧光检测表明,三株McAb具有型特异性.结论 用H5N1禽流感病毒M1蛋白免疫制备的单克隆抗体,具有一定的交叉反应性,可用于多种亚型甲型流感病毒的检测.     

一种狂犬病实验室诊断用单克隆抗体制备新路线的探索

目的 探索采用合成肽作为免疫原制备狂犬病实验室诊断用单克隆抗体的可行性.方法 以狂犬病病毒CVS-11核蛋白355-369位B细胞线性抗原表位合成肽与钥孔戚血蓝蛋白（Keyhole Limpe hemocyanin,KLH）大分子耦联后免疫BALB/c小鼠,利用经典杂交瘤细胞技术制备单克隆抗体.采用间接酶联免疫吸附试验（enzyme-linked immunosorbent assay,ELISA）和间接荧光试验（indirect fluorescent assay,IFA）筛选和鉴定杂交瘤细胞株.结果 经过对杂交瘤细胞株上清的间接ELISA和IFA筛选获得阳性杂交瘤细胞株2B1D11,该杂交瘤细胞株产生的抗体经纯化后在IFA中可以有效检出感染犬脑组织和BHK-21细胞的狂犬病病毒.结论 采用合成肽作为免疫原制备狂犬病实验室诊断用抗体在技术上是可行的.     

Renal dysplasia and asplenia in two sibs

A family is reported in which two sibs, one male and the other female, both died within 24 hours of birth with enlarged polycystic kidneys. Postmortem histology in the second child showed gross renal dysplasia. In both children the pancreas was enlarged, nodular and cystic but the liver appeared macroscopically normal. In the second child, histological examination confirmed pancreatic fibrosis with cystic dilation of ducts, but showed portal fibrosis with bile duct proliferation in the liver.
This combination of findings is very reminiscent of those in a girl and her brother reported by Ivemark et al. (1959). The children reported here also showed absence or hypoplasia of the spleen, cardiac anomalies and other features of the Ivemark syndrome (Ivemark 1955), a quite different, usually sporadic, congenital disorder. It is suggested that the children described here have a distinct lethal congenital disorder, probably inherited in an autosomal recessive manner.     

Schizophrenia in a North Swedish geographical isolate, 1900–1977. Epidemiology, genetics and biochemistry

Over 200 schizophrenic patients belonging to three major and interrelated pedigree complexes have been investigated over the past 30 years in a North Swedish geographically isolated population, presently numbering about 6,000. An intensive investigation of a number of biochemical correlates and genetic markers in a few selected families belonging to one of the major pedigrees has indicated new strategies for the current research program.
Schizophrenia, as defined operationally, is significantly associated with decreased activities of two enzymes (1) blood platelet monoamine oxidase, (2) plasma dopamine-β-hydroxylase, and (3) with the genetic marker Gc² (group specific antigen). Both enzymes are subject to genetic variation. A positive score for linkage between schizophrenia and low plasma DBH activity has been calculated, but, so far, available data are insufficient for discrimination between linkage and partial contribution of genetically controlled low plasma DBH to the pathogenesis of the disease. Alternatively, both mechanisms could be involved.
As a model for continued research, schizophrenia is explained as based on a double dominant-recessive genotype (Aabb), representing a vulnerability which in about 50 % of cases develops into clinical schizophrenia. It is suggested that the dominant mutation (A) operates on or affects MAO activity, and that the recessive genotype (bb) is instrumental in low variates of DBH activity and very likely such variates within the normal range of physiological variation. Moreover, it is suggested that the combined effects of MAO- and DBH-reduced efficiency on the metabolism of e.g. dopamine could be an essential pathogenic mechanism for the schizophrenic illness which is segregating in this population.     

The dominant diseases of modernized societies as omega-3 essential fatty acid deficiency syndrome: Substrate beriberi

About 1900, modern food selection and processing caused widespread $\text{epidemics}$ of the B vitamin deficiency diseases of beriberi and pellagra which, for genetic reasons, often expressed as different diseases ranging from bowel and heart disease to dermatoses and psychoses. But the B vitamins merely help convert essential fatty acids (EFA) into the prostaglandin (PG) tissue regulators and it now turns out that, through hydrogenation, milling and selection of w3-poor southern foods, we have also been systematically depleting, by as much as 90%, a newly discovered trace Nordic EFA (w3) of special importance to primates and sole precursor of the PG3(4) series, even as a concurrent fiber deficiency increases body demand for EFA. Since substrate EFA is processed by many B vitamin catalysts, an EFA deficiency will mimic a $\text{panh}$ypovitaminosis B, i.e., a mixture of $\text{substrate}$ beriberi and $\text{substrate}$ pellagra resembling vitamin beriberi and pellagra but exhibiting as even more diverse $\text{endemic}$ disease. This would consitute a second stage of the Modern Malnutrition and explain why some workers now hold the dominant diseases of modermized societies to be new, nutritionally based, pellagraform yet lipid-related and to range, once again, from heart disease to psychosis. It is an assumption that our dominant diseases are unrelated to each other or are merely revealed by our diagnostic acumen and therapeutic success; and that hydrogenating millions of tons of food oils annually, to destroy the rancidity producing w3-EFA, is safe for $\text{primates}$. Extensive beriberiform disease is reported here in 32 typical cases taken from medical practice which responds strikingly to linseed oil supplements (60% w3-EFA) in confirmation of identical results in Capuchins.     

What will studies of Fulani individuals naturally exposed to malaria teach us about protective immunity to malaria?

There are an estimated over 200 million yearly cases of malaria worldwide. Despite concerted international effort to combat the disease, it still causes approximately half a million deaths every year, the majority of which are young children with Plasmodium falciparum infection in sub-Saharan Africa. Successes are largely attributed to malaria prevention strategies, such as insecticide-treated mosquito nets and indoor spraying, as well as improved access to existing treatments. One important hurdle to new approaches for the treatment and prevention of malaria is our limited understanding of the biology of Plasmodium infection and its complex interaction with the immune system of its human host. Therefore, the elimination of malaria in Africa not only relies on existing tools to reduce malaria burden, but also requires fundamental research to develop innovative approaches. Here, we summarize our discoveries from investigations of ethnic groups of West Africa who have different susceptibility to malaria.     

'Very sore nights and days': the child's experience of illness in early modern England, c.1580-1720

Sick children were ubiquitous in early modern England, and yet they have received very little attention from historians. Taking the elusive perspective of the child, this article explores the physical, emotional, and spiritual experience of illness in England between approximately 1580 and 1720. What was it like being ill and suffering pain? How did the young respond emotionally to the anticipation of death? It is argued that children’s experiences were characterised by profound ambivalence: illness could be terrifying and distressing, but also a source of emotional and spiritual fulfilment and joy. This interpretation challenges the common assumption amongst medical historians that the experiences of early modern patients were utterly miserable. It also sheds light on children’s emotional feelings for their parents, a subject often overlooked in the historiography of childhood. The primary sources used in this article include diaries, autobiographies, letters, the biographies of pious children, printed possession cases, doctors’ casebooks, and theological treatises concerning the afterlife.     

Guidelines for the Validation and Use of Immunoassays for Determination of Introduced Proteins in Biotechnology Enhanced Crops and Derived Food Ingredients

Recent advancements in agricultural biotechnology have created a need for analytical techniques to determine introduced proteins in crops enhanced through modern biotechnology techniques. These proteins are expressed in plant tissues and may be present in food ingredients. Immunoassays are ideally suited for protein detection and may be used as both quantitative and threshold methods. Microplate ELISA and lateral flow devices are two of the most commonly used immunoassay formats for agricultural biotechnology applications. This paper provides general background information and a discussion of criteria for the validation and application of immunochemical methods to the analysis of proteins introduced into plants and food ingredients using biotechnology methods. It is the result of a collaborative effort of members of the Analytical Environmental Immunochemical Consortium. This collaborative effort represents the combined expertise of several organizations to reach consensus on establishing guidelines for the validation and use of immunoassays. Further, the paper offers developers and users a consistent approach to adopting the technology as well as aid in producing accurate and meaningful results.     

Ultracryotomy: development and application (with examples from the yeast cytology) (author's transl)

The preparation steps usually necessary for obtaining ultrathin frozen sections of biological material (chemical prefixation, enclosing, cryoprotective treatment, freezing, sectioning, and post-staining the sections for transmission electron microscopy) are submitted to a critical analysis. The application of cryo-ultramicrotomy, in particularly for cytochemical purposes, is reviewed. Fundamental considerations of chemical prefixation and poststaining are supported by examples from yeast cytology. Furthermore, the efficiency of the cryo-ultramicrotomy (electron optical resolution of ultrastructural details) is demonstrated on yeast cells and protoplasts.     

HLA in North Colombia Chimila Amerindians

HLA-A,-B,-C,-DRB1 and -DQB1 alleles have been studied in Chimila Amerindians from Sabana de San Angel (North Colombian Coast) by using high resolution molecular typing. A frequent extended haplotype was found:HLA-A*24:02-B*51:10-C*15:02-BRB1*04:07-DQB1*03:02 (28.7%) which has also been described in Amerinndian Mayos Mexican population (Mexico, California Gulf, Pacific Ocean). Other haplotypes had already been found in Amerindians from Mexico (Pacific and Atlantic Coast), Peru (highlands and Amazon Basin), Bolivia and North USA. A geographic pattern according to HLA allele or haplotype frequencies is lacking in Amerindians, as already known. Also, five new extended haplotypes were found in Chimila Amerindians. Their HLA-A*24:02 high frequencies characteristic is shared with aboriginal populations of Taiwan; also, HLA-C*01:02 high frequencies are found in New Zealand Maoris, New Caledonians and Kimberly Aborigines from Australia. Finally, this study may show a model of evolutionary factors acting and rising one HLA allele frequency (-A*24:02), but not in others that belong to the same or different HLA loci.     

The case for allele‐specific recognition by the TCR

There is a sharp difference in how one views TCR structure–function–behaviour dependent on whether its recognition of major histocompatibility complex‐encoded restriction elements (R) is germline selected or somatically generated. The generally accepted or Standard model is built on the assumption that recognition of R is by the V regions of the αβ TCR, which is not driven by allele specificity, whereas the competing model posits that recognition of R is allele‐specific. The establishing of allele‐specific recognition of R by the TCR would rule out the Standard model and clear the road to a consideration of a competing construct, the Tritope model. Here, the case for allele‐specific recognition (germline selected) is detailed making it obvious that the Standard model is untenable.