抗人VEGF受体Ⅱ胞外Ⅲ区单克隆抗体的制备

目的 制备人血管内皮细胞生长因子受体Ⅱ(Kinase insert domain tontaining receptor,KDR)胞外Ⅲ区的单克隆抗体(McAb)，探讨其抑制VEGF诱导的体外活性。方法 在大肠杆菌中表达重组KDR胞外Ⅲ区融合蛋白。融合蛋白经抗E—tag柱分离纯化后免疫Balb/c小鼠，采用传统杂交瘤技术制备抗KDR胞外Ⅲ区McAb，采用ELISA和Western免疫印迹鉴定其亚类和抗原结合特异性，并测定单克隆抗体对VEGF165刺激脐静脉内皮细胞株ECV304增殖的中和活性。结果 成功筛选出一株能稳定分泌KDR胞外Ⅲ区单克隆抗体的杂交瘤细胞株(1D3)，其分泌抗体亚类为IgGl。该McAb可明显阻断VEGF165对脐静脉内皮细胞株ECV304的刺激增生作用。结论 抗人血管内皮细胞生长因子受体KDR胞外Ⅲ区McAb可通过封闭KDR而抑制VEGF活性，McAb 1D3具有潜在治疗肿瘤的活性。     

利用基因重组抗原研制人CD20单克隆抗体及其功能的研究

目的　利用基因重组抗原免疫小鼠 ,制备人CD2 0单克隆抗体 ,研究其特异性和功能。方法　用表达重组人CD2 0基因的细胞NIH 3T3免疫BALB c小鼠 ,取其脾细胞与Sp2 0细胞融合 ,以间接免疫荧光法筛选杂交瘤上清。免疫沉淀法及流式细胞术鉴定其识别抗原的相对分子质量 (Mr)和特异性 ;以MTT法、流式细胞术及形态学方法检测诱导细胞凋亡和抑制细胞增殖的功能。结果　利用杂交瘤技术获得了 1株抗人CD2 0的单克隆抗体 1 2 8,它具有CD2 0抗体特异的细胞反应谱 ,其识别的抗原Mr 为 33× 10 3 。MTT实验证实 1 2 8能显著抑制Daudi和Raji细胞生长。结论　利用基因重组抗原制备了 1株能够稳定分泌抗人CD2 0的单克隆抗体的杂交瘤细胞株 1 2 8,此单抗具有抑制CD2 0阳性细胞增殖和诱导其凋亡的功能。     

抗人Toll样受体4单克隆抗体的制备及其生物活性研究

目的运用单克隆抗体技术制备抗人Toll样受体4(TLR4)单克隆抗体(mhTLR4抗体),并鉴定其生物学活性。方法以重组人TLR4(rhTLR4)作为抗原,腹腔注射免疫Balb/c小鼠。分离小鼠脾脏B淋巴细胞与骨髓瘤细胞融合并培养,挑选阳性杂交瘤细胞,扩大培养,制备和鉴定mhTLR4其生物学效应。结果经3次免疫后的小鼠血清中抗rhTLR4抗体的效价具有较高水平的表达。1∶500的小鼠免疫血清可明显抑制人PBMC的TNF-α表达。分离小鼠脾细胞与骨髓瘤细胞进行融合,获得3株阳性单克隆杂交瘤细胞。其中2株经过增殖培养、纯化浓缩制备出较高产量的鼠抗人TLR4单克隆抗体。生物学活性测定表明,产生的抗rhTLR4单克隆抗体,能够明显地阻断脂多糖(LPS)诱导的人PBMC细胞对TNF-α的表达。结论本文制备的鼠抗人TLR4单克隆抗体,经鉴定具有较高的阻断内毒素信号传导的效应。为进一步研制人源化抗TLR4抗体、研制新一代抗内毒素靶向药物奠定了基础。     

基因疫苗脾内免疫制备抗人c-kit单克隆抗体及其初步鉴定

目的：用基因脾内免疫方法制备抗人c-kil单克隆抗体，并通过对抗体生物学特性的初步研究，确定该方法制备抗体的可行性。方法：利用分子克隆方法构建重组质粒pcDNA3．1／c-kit胞外区，脾内免疫BALB／c小鼠一次，通过杂交瘤技术制备抗人c-kit单克隆抗体。采用流式细胞术、Western blot方法初步鉴定制备的抗体。结果：目的基因片段c-kit外区正确插入质粒pcDNA3．1，重组质粒免疫小鼠后通过杂交瘤技术获得了三株分泌抗人c-kit胞外区的杂交瘤细胞株6CA、2C5和5D5。其亚类均为IgM类，识别不同的抗原表位，其特异性与抗人c-kit抗体一致。结论：可用基因脾内一次免疫法制备抗人c-kit单克隆抗体。     

人SUMO1蛋白表达纯化及单克隆抗体的制备、鉴定

目的 表达重组人SUMO1(small ubiquitin-related modifier 1)蛋白并制备单克隆抗体.方法 构建含人SUMO1基因的重组表达质粒pET32a-HIS-SUMO1,在大肠杆菌中表达重组蛋白HIS-SUMO1;以纯化后的HIS-SUMO1蛋白为抗原免疫BALB/c小鼠,利用杂交瘤技术,通过ELISA和Western blot方法筛选稳定分泌抗体的杂交瘤细胞株,用免疫双向扩散法鉴定抗体Ig的类型及亚类;取一株筛选细胞按照常规方法制备腹水,利用Millipore抗体纯化试剂盒进行抗体纯化,Western blot方法检测抗体效价.结果 表达纯化了重组人HIS-SUMO1蛋白;3株稳定分泌特异性抗人SUMO1的单克隆抗体杂交瘤细胞株被筛选出,其免疫球蛋白类型均为IgG1类;通过腹水制备和纯化获得效价较高的鼠抗人SUMO1单克隆抗体.结论 通过表达纯化人SUMO1重组蛋白,制备出高效价鼠抗人SUMO1的单克隆抗体,该抗体可用于蛋白质SUMO化的研究.     

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.     

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。     

人血管内皮钙黏蛋白5单克隆抗体的制备及应用

目的制备并鉴定人血管内皮钙黏蛋白5(VECDH5)的单克隆抗体(m Ab)。方法用重组原核人VECDH5蛋白免疫BALB/c小鼠,利用杂交瘤技术进行细胞融合;间接ELISA筛选阳性克隆,得到稳定分泌抗VECDH5抗体的杂交瘤细胞株,ELISA测定其效价;Western blot法、流式细胞术、免疫荧光细胞化学染色、免疫组织化学染色鉴定其特异性及抗原表位。结果制备并获得1株可分泌特异性抗VECDH5的杂交瘤细胞株(2C11)。ELISA测定腹水效价高达1∶10 000。多种方法均证实该抗体能特异性识别人VECDH5蛋白,并可在上述不同方法中应用。经抗原表位鉴定,研究制备的2C11识别的氨基酸序列为LDREVVPWYNLTVEA。结论成功制备了亲和力高、特异型好的抗人VECDH5的m Ab。     

FcεRⅠ受体α亚基的重组表达、单克隆抗体的制备及特异性鉴定

目的通过基因重组的方法表达IgE高亲和力受体FcεRⅠα亚基，并用重组蛋白制备单克隆抗体。方法用RT-PCR的方法从过敏性疾病病人外周血嗜碱性粒细胞调取FcεRⅠα蛋白基因，经T-A克隆、亚克隆至pET28a（＋）原核表达载体，将重组质粒转化到大肠杆菌BL．21．STAR（DE3），IPTG诱导表达FeaRIOt亚基蛋白，通过Ni-NTA亲和层析纯化融合蛋白；并用其免疫BALB／c小鼠，运用杂交瘤技术制备抗FeaRIOt亚基单克隆抗体，用细胞免疫荧光法对单克隆抗体的特异性进行鉴定。结果成功调取了人IgE高亲和力受体FcεRⅠα亚基的基因，且测序正确；构建原核表达质粒FcεRⅠα-pET28a（＋）；成功建立4株稳定分泌抗FcεRⅠα亚基的单克隆抗体杂交瘤细胞株，分别命名为G101、C22、G113、G42。通过细胞免疫荧光实验证实，4株单克隆抗体均能特异性结合人嗜碱性粒细胞表面的FcεRⅠα亚基。结论成功利用基因重组技术制备了FcεRⅠα亚基蛋白，制备了4株效价高、特异性好的抗FcεRⅠα亚基单克隆抗体，为FcεRⅠα亚基及其抗体在过敏性疾病中的生物学功能研究奠定了基础。     

人肝再生增强因子单克隆抗体的制备:以非纯化的大肠杆菌表达抗原筛选杂交瘤

目的 :利用杂交瘤技术、以非纯化的大肠杆菌表达重组蛋白作为筛选抗原 ,制备小鼠抗人肝再生增强因子(hALR)的单克隆抗体。方法 :用实验室纯化的重组hALR 硫氧环蛋白融合蛋白免疫BALB c小鼠 ;小鼠脾细胞与SP2 0骨髓瘤细胞融合后经HAT选择培养基筛选杂交瘤 ;以重组质粒pQE30 hALR与空质粒pQE30在大肠杆菌中诱导表达后的细菌裂解产物作为筛选抗原和对照抗原 ,用ELISA方法筛选能分泌抗hALR单克隆抗体的阳性杂交瘤细胞克隆 ;进一步以ELISA方法和免疫印迹方法检测该杂交瘤细胞产生的抗体对真核细胞表达的重组hALR及人体血清中天然hALR的反应性。结果 :成功筛选出一株能稳定分泌抗hALR单克隆抗体的杂交瘤细胞 ;其产生的抗体能对真核细胞表达的重组hALR及人体血清中天然的hALR发生特异的抗原抗体反应。结论 :大肠杆菌表达的重组蛋白在以空质粒表达产物作为对照下 ,不经过任何纯化步骤也能够用于单克隆抗体制备中的杂交瘤筛选 ;hALR单克隆抗体为深入研究hALR提供了研究手段。     

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.     

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.     

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.     

'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.     

The universal muscular chamber. A basic unit of the genitourinary, cardiovascular, gastro-intestinal, skeletal, cutaneous, respiratory and other systems, endocameral hypertension; and spasm, the key to their idiopathic diseases and arthropathies

Starting with the integument, we see many organs are contractile sacs or multiples thereof, which tubes or bags constitute the major part of the entire body. Recognition of this basic unit and its characteristics sheds new light, individually and collectively, on many disorders previously considered unrelated. Muscular tears and perforations develop in the walls of these chambers, being no way peculiar to those organs, wherein, hydrochloric acid occurs. So, it is not necessary to explain the absence of excessive acid from patients who exhibit holes in the gastric, uterine, aortic, duodenal, rectal, pulmonary, retina, and other walls. Muscle, not acid is the great common factor relating idiopathic disorders in the gastrointestinal tract to each other and to similar diseases in other systems. When the units are linked together, the lesions tend to appear as arthropathies, i.e. at the joints. Rephrasing common-place observations, frees us from conventional, conceptual cul-de-sacs. An observation is only as good as its interpretation, so all possibilities must be considered, otherwise, we will remain blinded by our misconceptions.