阪崎肠杆菌单克隆抗体的制备与特性鉴定

目的:制备抗阪崎肠杆菌的单克隆抗体并对其生物学特性进行鉴定。方法:采用灭活的阪崎肠杆菌菌体为抗原,免疫BALB/c小鼠,取血清效价高的小鼠脾细胞与SP2/0骨髓瘤细胞进行融合,间接ELISA法筛选阳性杂交瘤细胞,mAb亚类检测试剂盒鉴定单克隆抗体的亚型,通过Western blot和间接ELISA法鉴定该单克隆抗体的特性、效价及mAb相对亲和力。结果:获得2株能稳定分泌抗阪崎肠杆菌mAb的杂交瘤细胞株,分别命名为1H7、2B12,抗体Ig亚类分别为IgG1和IgG2b;交叉反应显示单抗具有良好的特异性;ELISA分析表明制备的单抗效价在1×107～2×107,相对亲和常数达109L/mol,染色体鉴定分别为104和106条,符合杂交瘤细胞的特性。结论:抗阪崎肠杆菌单克隆抗体的成功制备为其快速检测方法的建立奠定了基础。     

抗狂犬病病毒CVS-11株单克隆抗体的制备及鉴定

目的 制备抗狂犬病毒单克隆抗体,为建立快速准确的狂犬病毒抗原检测方法奠定基础.方法 将狂犬病病毒CVS-11株纯化浓缩后免疫6～8周龄雌性BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞进行融合,经细胞克隆和间接ELISA筛选,获得稳定分泌抗狂犬病毒单克隆抗体的杂交瘤细胞株.小鼠腹腔注射法制备大量单克隆抗体并测定腹水效价,用G蛋白亲和层析柱进行纯化,间接ELISA法和间接荧光法鉴定单克隆抗体的类型、特异性及敏感性.结果 细胞融合率达100%,经克隆筛选获4株稳定分泌抗狂犬病病毒抗体的杂交瘤细胞株,其腹水效价分别为1×104,1×105,1×104和1×105;4株单抗均为IgG类型且特异性好.结论 制备的单抗具有良好特异性和敏感性.     

抗3-硝基酪氨酸单克隆抗体的制备及免疫学特性分析

目的制备抗3-硝基酪氨酸(3-NT)单克隆抗体并鉴定其免疫学特性。方法用化学合成方法将3-NT与OVA用戊二醛偶联合成的人工抗原3硝基酪氨酸-戊二醛-鸡卵白蛋白(3NT-G-OVA)作为免疫原,皮下注射免疫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,用间接ELISA法筛选阳性杂交瘤细胞株,扩大培养并对抗体进行免疫学鉴定与分析。结果获得1株稳定分泌抗3-硝基酪氨酸单克隆抗体的杂交瘤细胞株,所分泌的单抗亚型为Ig G2a;纯化后的抗体效价为1∶(6.14×106);单抗亲和力常数为7.81×107(L·mol-1);Western blot检测证明该单抗与含有3-硝基酪氨酸的蛋白能特异地结合。结论制备了1株具有较高免疫学活性和特异性的抗3-NT单克隆抗体,并可用于Western blot检测病人样本中的蛋白硝基化水平。     

一对高亲和力抗人肌钙蛋白Ⅰ单克隆抗体的制备及鉴定

目的 获取抗人肌钙蛋白Ⅰ（cTnI）的单克隆抗体（McAb）.方法 以人cTnI作为抗原,免疫Balb/c小鼠,通过杂交瘤技术制备了抗人cTnI高亲和力、高特异性单克隆抗体.随后采用间接ELISA法测定抗血清效价,用protein G亲和纯化法纯化抗体,抗原竞争ELISA法鉴定抗体亲和力,SDS-PAGE法鉴定纯度,Western blotting鉴定抗cTnI单克隆抗体的特异性,竞争ELISA法分析抗原结合位点.结果 筛选出9株稳定分泌抗cTnI的单抗杂交瘤细胞株,其中A3、A9两株免疫球蛋白亚类均为IgG2a,分泌的抗体纯度高,与CK-MB、cTnT无交叉反应,效价均为：1：1024000,亲和力分别为4.21×10^8mol/L、1.07×10^8mol/L,抗原结合位点不同.结论 成功制备出了一对高亲和力、高特异性抗人cTnI单克隆抗体.     

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。     

自制NSE单抗的鉴定及其双抗夹心ELISA方法的建立

目的:制备并鉴定NSE(Neuron-specific enolase)单克隆抗体,建立可检测NSE蛋白的双抗夹心ELISA方法。方法:用本实验室已表达纯化的NSE融合蛋白免疫BALB/c小鼠,采用杂交瘤技术制备单克隆抗体。采用WB、IP、IF、IHC等方法对获得的NSE单抗进行鉴定及亚型检测。利用辣根过氧化物酶标记纯化后的NSE单抗,建立一个可检测NSE蛋白的双抗夹心ELISA法。结果:通过分析和鉴定,选定2株可稳定分泌抗NSE抗体的杂交瘤细胞株,效价达4.2×107～6.5×107,亚型为IgG2b。免疫印迹结果显示,该抗体不仅能识别细胞内源NSE蛋白,还能识别分泌到细胞培养上清液中的NSE蛋白,此外还可用于免疫荧光及免疫组化检测。文中所建立的双抗夹心ELISA法,最低检测极限为8.85 ng/ml。结论:成功获得了效价高、灵敏度好及特异性强的NSE单抗,建立了一个双抗体夹心ELISA检测系统,具有良好的临床应用前景。     

抗人BPI23单克隆抗体的制备和鉴定

目的 采用杂交瘤技术制备抗人BPI23单克隆抗体,并对其应用进行初步分析。方法 免疫小鼠脾细胞与小鼠骨髓瘤细胞按常规方法融合;用间接ELISA法和Western - blot筛选分泌抗体的杂交瘤细胞株;阳性克隆用有限稀释法亚克隆3次获得稳定分泌抗人BPI23单克隆抗体的杂交瘤细胞株;扩增杂交瘤细胞注入小鼠腹腔后制备腹水;纯化腹水中的单抗并对抗体类型进行鉴定;用Western-blot分析抗体的特异性;用间接ELISA法测抗体效价;将分离纯化的正常人外周血中性粒细胞和单个核细胞制成涂片,用抗人BPI23单克隆抗体进行免疫染色。结果 获得3个（1B4、9C12和2H11）稳定分泌抗人BPI23单克隆抗体的杂交瘤细胞株,所分泌的单抗类型分别为κ型IgM、κ型IgG1和κ型IgG1;抗体效价分别为1.28×105、1.28×105和4.1×106,纯化后抗体含量分别为0.208g/L、2.03g/L和3.88g/L;3种纯化抗体均能与本实验制备的人BPI23和市售人BPI55标准品特异性结合,而不能与小鼠BPI25和人LBP结合;在免疫组化实验中,1B4、9C12和2H11单抗均能与人中性粒细胞中的BPI特异性结合。结论 成功制备了人BPI23特异性单克隆抗体,为BPI检测试剂盒的研制奠定了基础。     

抗ssDNA单克隆抗体的研制和在检测凋亡细胞中的应用

为了制备抗单链DNA单克隆抗体和建立检测细胞凋亡的新方法 ,采用杂交瘤细胞融合技术用于筛选分泌抗单链DNA抗体的单克隆杂交瘤细胞株 ,并用斑点印迹和竞争ELISA法鉴定单抗的特异性 ,进而结合免疫组织化学和免疫荧光方法用于检测凋亡细胞。通过筛选得到单克隆杂交瘤细胞株B17,其分泌的抗体与单链DNA结合而与双链DNA无交叉反应 ,用此单抗建立的凋亡细胞检测方法能够检测出凋亡细胞 ,区分非凋亡细胞和坏死细胞。实验结果表明 ,成功地获得一株分泌特异性抗单链DNA抗体的单克隆杂交瘤细胞株 ,以此建立的凋亡细胞检测方法特异性高、敏感性强     

呼吸道合胞病毒单克隆抗体的制备及鉴定

目的：建立能稳定分泌抗呼吸道合胞病毒（RSV-long株,国际标准株）单克隆抗体的杂交瘤细胞株。方法：杂交瘤技术制备单抗,鉴定各项特性。结果：培育出稳定分泌抗RSV蛋白的单克隆抗体的杂交瘤细胞5株。杂交瘤细胞株染色体数目在89～104条之间,其分泌的抗体分别属于鼠IgG1、IgG2a、IgG2b亚类,各种腹水单抗的荧光效价在1∶4000～1∶16000之间。一种单抗识别RSV基质蛋白（M）,两种单抗识别RSV融合蛋白（F）,另两种单抗识别RSV核衣壳蛋白（N）。单抗中和效价最高达1∶64。相加指数证实5种单抗识别不相关的抗原表位。用硫氰酸铵洗脱法比较了五种单抗相对亲和力的大小,依次为：1#〉2#〉4#〉3#〉5#。所有杂交瘤细胞株,经连续3个月培养及冻存半年后复苏,细胞生长良好,检测上清,其分泌抗体效价稳定。结论：已获得抗呼吸道合胞病毒的单克隆抗体,为RSV感染的早期诊断及进一步研究奠定了基础。     

抗人肝再生增强因子单克隆抗体的研制

目的　利用纯化的重组人肝再生增强因子(hALR)制备抗hALR的单克隆抗体,建立hALR的检测方法。方法 采用杂交瘤技术制备单克隆抗体,经ELISA和免疫印迹证明其特异性。结果获得了4株分泌抗hALR特异性抗体的杂交瘤 细胞系;无血清培养液效价为1×10-2,腹水效价为1×10-5;亚类鉴定表明2株单克隆抗体为IgGl,另2株为IgG2b;免疫印迹 显示抗体结合抗原的分子量与hALR相符,为特异性抗hALR抗体。结论通过杂交瘤技术,获得了抗hALR的单克隆抗体 为以后的工作奠定了基础。     

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.