微板酶联夹心杂交法定量检测人IL-18 mRNA

目的：建立一种高灵敏度、高特异性、精确、简便的微孔板酶联夹心杂交技术，定量检测人IL-18mRNN。方法：针对人IL-18mRNA RT-PCR产物—条链的不同区域序列设计—对特异探针，其中一条为捕获探针，5’端为氨基修饰，可以与微量DNA结合板表面的NOS基团共价结合，“竖直”地包被在微孔板内；另一条为检测探针，3’端标记生物素。提取人外周血单个核细胞中总RNA，进行RT-PCR扩增IL-18 mRNA，产物热变性后加入已包被捕获探针的微孔板内进行杂交，再加入检测探针与已杂交的产物结合，最后经亲和素-辣根过氧化物酶(SAV-HRP)系统检测杂交信号。结果：该法检测IL-18mRNA RT-PCR产物的灵敏度明显高于琼脂糖凝胶电泳，重复性良好，且结果数据化。结论：该方法具有操作简单、灵敏度高、特异性强等优点，适合于PCR扩增产物的定量检测。     

微量酶联夹心杂交法定量检测hIL-8 mRNA PCR扩增产物

目的:建立一种灵敏度高、特异性强、定量、精确、操作简便的微孔板酶联夹心杂交技术,定量检测人IL-8 mR-NA。方法:针对人IL-8 mRNA逆转录聚合酶链反应产物一条链的不同区域序列设计一对特异探针,其中一条为捕获探针,5′端用活性氨基修饰,与微量DNA结合板表面的NOS基团共价结合,“竖直”地包被在微孔板内;另一条检测探针的3′端标记生物素,和辣根过氧化物酶结合。提取人外周血单个核细胞总RNA,进行RT-PCR扩增IL-8 mRNA,双链DNA产物经热变性后加入已包被捕获探针的微孔板内进行杂交,加入检测探针与已杂交的产物结合,经亲和素-辣根过氧化物酶系统检测杂交信号。结果:该法灵敏度为检出16个循环的PCR产物、5×103个PBMCs中的IL-8 mRNA、检测PCR终产物的最高稀释倍数为1∶256阳性;特异性试验非目的扩增片段酶联杂交检测未检出阳性杂交信号;精密度试验CV为5.2%。结论:该方法具有操作简便、灵敏度高、特异性强等优点,适合IL-8 mRNA PCR扩增产物的定量检测。     

微量酶联夹心杂交法定量检测TNF-α mRNA

目的:建立一种高灵敏度、高特异性、精确、简便的微板酶联夹心杂交技术, 对人单核细胞分泌的TNF-α mRNA进行定量检测。方法:设计两条特异探针, 其中一条为捕获探针, 5'端带有氨基修饰, 可以与微量DNA结合板表面的NOS基团共价结合, 而且是"竖直"地包被在微孔内, 另一个为检测探针, 3'端带有生物素标记。提取人单个核细胞总RNA, RT-PCR后的扩增产物经变性后加入已包被好捕获探针的微孔板中, 加入检测探针与已杂交的产物结合, 最后加入亲和素-辣根过氧化物酶(avidin－HRP)显色系统催化底物显色, 450 nm波长下检测吸光度进行定量。结果:该方法检测TNF-αmRNA的灵敏度, 明显高于琼脂糖凝胶电泳, 而且具有良好的重复性, 等量PCR产物作10个复孔, 吸光度的CV值为7.2%, 批间的CV值为9.1%。在本实验中, 我们首次尝试着用客观的数据来表示正常人中1×10⁶个PBMCs中TNF－αmRNA的平均表达量, 发现该表示方法直观明了, 结果稳定, 而且简便可行, 不需要特殊的仪器及昂贵的试剂。结论:本方法具有高灵敏度、高特异性、精确、简单易操作, 结果数据化等优点, 适合于微量细胞因子mRNA的定量检测。     

微量酶联杂交法定量检测HBV基因 竞争PCR扩增产物

目的 建立一种简便、敏感、精确的微反应板酶联杂交技术,以鉴定HBV基因的竞争PCR扩增产物。方法 设计了两种捕获探针,能分别与竞争PCR扩增产物中的野生片段和突变片段杂交。捕获探针通过3′－端修饰的氨基与微量DNA结合板孔表面的NOS基团化学结合而被“竖直”地包被在反应板上;将热变性后的产物加入两种捕获探针反应孔内,产物中带有生物素的野生或突变片段的一条单链与相应的捕获探针杂交;最后用链亲和素－碱性磷酸酶及底物检测杂交信号。结果 该方法检测PCR产物DNA的灵敏度为80ng/ml,大于琼脂糖凝胶电泳染色鉴定法。获得野生片段和突变片段杂交信号值后,可根据公式计算扩增前野生模板的初始量。结论 本方法操作简单、灵敏度高、结果数据化、特异性强,适用于竞争PCR产物分析。     

甲型肝炎病毒TZ84株单克隆抗体在甲肝病毒抗原检测中的应用

目的 研究甲型肝炎病毒TZ84株单克隆抗体在甲肝病毒抗原检测中的应用，以期代替现有试剂中的多克隆抗体，得到灵敏度高、特异性好、重复性和准确性好的甲肝抗原检测试剂。方法 采用双抗体夹心法将甲肝多抗包被96孔酶标板后，加入甲肝抗原，分别用酶标单抗和酶标多抗两种方法进行检测甲肝抗原．对单抗试剂的真实性和可靠性进行评价。结果 单抗试剂的灵敏度为100％、特异度为100％，变异系数均小于15％。结论 单抗试剂可用于甲肝抗原的定性和定量检测，结果准确可靠。     

建立固相Capture-ELISA技术诊断糖尿病人HCMV活动性感染

目的建立检测HCMV抗原特异性IgM的抗体捕捉酶联免疫吸附试验（IgM antibody capture enzyme-linked immunosorbent assay,Capture-ELISA）,并分析糖尿病患者HCMV活动性感染状态。方法利用抗人IgM（μ链特异性）抗体包被固相载体,作为＂捕捉抗体＂吸附待检血清中的IgM,经过洗涤除去血清中IgG及其它成分,再加入特异性抗原与＂捕捉＂到的相应IgM抗体结合,然后加入酶标抗体和底物显色进行测定。同时,应用建立的酶联免疫捕获法诊断糖尿病患者HCMV近期感染状态,并与常用的间接酶联免疫吸附试验（In-ELISA）及RT-PCR进行比较。结果 Capture-ELISA的特异性及敏感性均高于间接法,且不受RF因子的影响。结论 Capture-ELISA操作简单、快速,且具有较高的特异性和灵敏度,是检测IgM抗体理想的方法之一。     

应用酶联免疫吸附试验检测肠出血性大肠杆菌O157:H7

目的制备和纯化O157：H7抗原，免疫家兔和豚鼠，获得高效价的抗O157：H7免疫血清并进行纯化及酶标记。建立对O157：H7感染患者快速诊断方法，做到早期发现及时治疗，有效控制疫情的蔓延。方法ELISA双抗体夹心法检测O157：H7抗原。步骤：包被特异性抗体，加处理后的粪便等标本，然后加入抗O157：H7酶结合物，最后加入底物显色。结果本课题所研制的抗O157：H7酶结合物只对O157：H7呈阳性反应，而与其他相关细菌无交叉反应。结论应用酶联免疫吸附试验(即双抗体夹心法)对肠出血性大肠杆菌O157：H7抗原的检测较常规法实验程序简捷、快速、敏感。临床和现场验证结果表明，其方法具有灵敏度高，特异性强操作简单等特点，为肠出血性大肠杆菌O157：H7的鉴定及快速诊断提供了一种新的检测手段。     

双抗夹心化学发光免疫分析定量检测Ⅳ型胶原方法的建立

目的 建立基于双抗夹心微孔板化学发光酶免疫分析的人血清Ⅳ型胶原定量检测方法.方法 应用Ⅳ型胶原多抗进行包被,辣根过氧化物酶标记Ⅳ型胶原单抗,以鲁米诺化学发光体系检测,调整优化各种反应液的工作浓度和各类反应条件后建立双抗体夹心的检测方法;评价所建立方法的线性范围、特异性、灵敏度、稳定性等性能指标;应用肝纤维化患者血清与进口试剂进行比对实验.结果 所建立方法的灵敏度为15.5 ng/ml,线性范围25 ～ 850 ng/ml,批内、批间变异均小于8％.检测Ⅳ型胶原的临床高、中、低值血清回收率分别为98.5％、94.3％和102.6％;在4℃和37℃条件下分别进行了3、5、7d的稳定性考察,线性相关系数均＞0.99,标准偏差＜7％;比对实验分析显示与进口试剂相关性具有统计学意义.结论 成功建立了定量Ⅳ型胶原化学发光酶免疫分析方法,该方法有较好的准确性、灵敏度、重复性,与进口试剂检测结果等效.     

RT-PCR-ELISA方法检测甲肝减毒活疫苗病毒滴度的初步研究

目的 应用核酸扩增产物测定的固相杂交酶联显色法(RT-PCR-ELISA)检测甲肝减毒活疫苗病毒滴度。方法 应用RT-PCR-ELISA。将标记有生物素的寡核苷酸引物所扩增的疫苗病毒基因产物。与微孔反应板上的特异性探针进行快速杂交，通过辣根过氧化物酶标记的链亲和素进行酶联显色。读取吸光度(A值)。判断结果。应用此法检测了11批甲肝活疫苗滴度。并与常规细胞培养法(CCID50)比较。结果 本方法与细胞培养法的敏感性相仿，具有简便、快速、特异的优点。结论 RTPCR-ELISA法有望代替常规细胞培养法应用于甲肝减毒活疫苗病毒滴度的检测。     

C-肽化学发光免疫分析方法学研究

目的：建立人血清C-肽化学发光免疫分析法。方法：采用两株高特异的抗C-肽单克隆抗体，以一株包被微孔板制成固相抗体，另一株标记碱性磷酸酶，以金刚烷衍生物为底物，双抗体夹心法测定人血清C-肽浓度。结果：以5μg/ml单克隆抗体包被微孔板，酶结合物以1：5000稀释，在C-肽浓度0．1～15ng／ml范围内线性良好，线性方程为y=0．9575x-0．0869，相关系数0．99；灵敏度为0．01ng／ml，批内、批间变异系数分别为5．8％、8．4％；平均回收率98．4％，范围91．5％～105．0％。结论：文中建立的人血清C-肽化学发光免疫分析法，首次建立小分子人血清C-肽双抗体夹心化学发光免疫法测定。线性好，灵敏度较常规高出一个数量级，准确度和精密性高，能够满足一般临床诊断和科研的应用。     

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.     

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.     

'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.     

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.     

Der Einfluß von Nahrungsmitteln und Rauchen auf die klinisch-chemische Diagnostik von Phäochromozytom,Neuroblastom und Karzinoid-Syndrom

Zusammenfassung Der Einfluß von verschiedenen Nahrungsmitteln auf Methoden zur Bestimmung von Adrenalin (AD), Noradrenalin (NA), Vanillinmandelsäure (VMS), Metanephrinen (MN), Homovanillinsäure (HVS) und 5-Hydroxyindolessigsäure (5-HIE) im 24 h-Harn zur Diagnose des Phäochromozytoms bzw. Karzinoid-Syndroms wurde untersucht. Die in die Untersuchung einbezogenen Nahrungsmittel waren: Tee, Kaffee, Mandeln, Ananas, Käse, Walnüsse, Vanillepudding, Bananen, Tomaten und Milchschokolade. Außerdem wurde der Einfluß des Zigarettenrauchens auf die Bestimmung von AD, NA, VMS und MN untersucht.Walnüsse führten zu einer starken Erhöhung der 5-HIE-Ausscheidung. Bananen erhöhten die Ausscheidung von AD, NA, VMS, MN und 5-HIE. Kaffee und Ananas bewirkten eine geringe Zunahme der MN-Werte. Rauchen von 20–30 Zigaretten/Tag beeinflußte keine der vier Variablen.Wenn die beschriebenen Methoden benutzt werden, sollte lediglich auf den Verzehr von Bananen und Walnüssen vor und während der Harnsammelperioden verzichtet werden, da die oberen Normgrenzen im Harn überschritten werden könnten. Ein Verzicht auf Kaffee und Ananas in normalen Mengen ist nicht erforderlich. Es besteht kein Anlaß, weiterhin die bisherigen umfangreichen Restriktionen der übrigen Nahrungsmittel beizubehalten.