对硫磷完全抗原的合成鉴定及其免疫效果

目的 合成免疫原性较强的人工抗原,为制备对硫磷单克隆抗体和建立免疫快速检测食品中农药残留方法奠定基础.方法 选用2种方法合成羧基化对硫磷:一种以对硫磷为原料,改造硝基一端;另一种以三氯硫磷为原料,改造磷硫键一端.EDC法偶联牛血清白蛋白(BSA)制备免疫原(PA-1-BSA和PA-2-BSA),并分别免疫Balb/c小鼠.间接ELISA测定抗血清效价,竞争ELISA测定检测灵敏度,比较免疫效果.结果 SDS-PAGE电泳表明,2种完全抗原均合成成功;质谱法鉴定免疫原的偶联比分别为:N(PA-1/BSA)=7.2,N(PA-2/BSA)=15.4;小鼠抗血清经间接ELISA检测表明,PA-2-BSA组效价明显高于PA-1-BSA组;间接竞争ELISA检测表明,PA-2-BSA组半数抑制浓度(IC50)为1μg/ml,明显优于PA-1-BSA组(IC50为＞100 μg/ml).结论 成功合成2种对硫磷完全抗原,免疫Balb/c小鼠PA-2-BSA组免疫应答效果好于PA-1-BSA组、     

甲基对硫磷人工抗原的合成及鉴定

为了合成甲基对硫磷人工抗原,用醋酸－锌粉－盐酸还原甲基对硫磷,制备氨基甲基对硫磷,重氮化法使氨基甲基对硫磷与牛血清白蛋白（ＢＳＡ）及中国鲎血蓝蛋白（ＴＴＨ）偶合,合成人工抗原Ｍ１６０５－ＢＳＡ,Ｍ１６０５－ＴＴＨ。Ｍ１６０５－ＢＳＡ免疫新西兰兔１０周后,双向琼脂扩散试验和间接ＥＬＩＳＡ检测,证明得到了高价且具有较好特异性的多抗血清,成功地合成了甲基对硫磷人工抗原,为其免疫分析方法的建立提供了条件。     

三聚氰胺人工抗原合成方法的研究

目的:研究三聚氰胺人工抗原的合成方法.方法:利用戊二醛法和EDC法分别合成了两种三聚氰胺-牛血清白蛋白(Mel-BSA1和Mel-BSA2).将Mel-BSA1和Mel-BSA2分别免疫两只新西兰大白兔,获得多克隆抗体.结果:红外光谱法鉴定结果表明,抗原合成成功.基质辅助激光解析电离飞行时间质谱鉴定表明,偶联物Mel-...     

磺胺胍和磺胺噻唑人工抗原的合成与鉴定

目的制备磺胺胍和磺胺噻唑的人工抗原以检测动物性食品中的磺胺胍和磺胺噻唑。方法通过重氮化法将磺胺胍和磺胺噻唑与牛血清白蛋白(BSA)偶联,制备磺胺胍和磺胺噻唑人工抗原,偶联比分别为10∶1和9∶1。用该抗原免疫小鼠,间接ELISA法检测抗血清效价。结果经过紫外光谱检测、红外光谱检测及免疫动物实验确证该人工抗原。结论磺胺胍和磺胺噻唑人工抗原合成成功,为获得抗磺胺胍和磺胺噻唑的特异性抗体奠定了基础。     

杀虫剂残杀威人工抗原的制备及免疫效价检测

[目的]制备杀虫剂残杀威(propoxur)人工抗原以及免疫效价的检测。[方法]将杀虫剂残杀威,通过戊二醛法与载体鸡卵清蛋白OVA相偶联,所得的半抗原偶联物为免疫原,用紫外光谱、红外光谱扫描分析所制备的人工抗原,然后免疫小鼠检测效价。[结果]将制备的抗原免疫BALB/c小鼠,免疫后采血测其效价,即通过酶联免疫法(ELISA)检测免疫小鼠所产生的抗体,检测结果为阳性。[结论]小鼠产生独特型抗体。     

杀虫脒衍生物的合成及其人工抗原的制备

本文以对硝基苯乙腈和对氯邻甲苯胺为主要原料，经五步反应合成了带苯胺基的杀虫脒衍生物（半抗原）。用ＩＲ、ＨＮＭＲ和ＭＳ证明了产物的结构。经重氮化－偶合反应，将半抗原与载体蛋白偶联制成抗原，为建立杀虫脒的免疫分析法打下了基础。     

有机磷农药共性结构人工抗原的合成与鉴定

目的建立有机磷农药多残留的免疫分析方法。方法以O,O-二甲基硫代磷酰氯和4-羟基苯甲酸甲酯为原料,经两步化学反应合成了与有机磷农药化学结构相类似的一类半抗原O,O-二甲基-O-(4-羧基苯基)硫代磷酸酯(HP),并分别通过活泼酯法和混合酸酐法将HP与牛血清白蛋白(BSA)和卵清蛋白(OVA)耦联制备了HP的免疫原(HP-BSA)和包被原(HP-OVA)。结果经质谱和紫外光谱表征,证明所合成的产物为目标半抗原HP,并且与载体蛋白耦联比可达到15.6∶1(HP-BSA)和16.2∶1(HP-OVA)。结论半抗原HP及人工抗原合成成功,可以用于制备该类半抗原的广谱特异性单克隆抗体。     

黄曲霉毒素B1人工抗原的高效率合成方法及抗体制备的研究

将ＡＦＴＢ１羧甲基肟制成活性酯，再与载体蛋白质结合制成ＡＦＴＢ１人工抗原。采用此法ＡＦＴＢ１羧甲基肟的利用率可达４７％，偶联物的产率（以偶联物与ＡＦＴＢ１肟的质量比表示）达到１０。用本法合成的ＡＦＴＢ１－ＢＳＡ偶联物免疫家兔，获得了高效价的特异性抗血清，经间接竞争抑制ＥＬＩＳＡ法测定，产生５０％抑制所需的ＡＦＴＢ１、Ｂ２、Ｇ１和Ｇ２浓度依次为１２．８、５７．６、２８．７和＞４００ｎｇ／ｍｌ。本文还讨论了偶联物分子中ＡＦＴＢ１残基数目对免疫效果的影响。     

毒死蜱人工抗原的合成与分析

目的 制备毒死蜱人工抗原,为建立其酶联免疫吸附检测方法提供技术储备.方法 以毒死蜱为原料,在弱碱性条件下与3-巯基丙酸反应,合成半抗原O,O-二乙基-O-[3,5-二氯-6-(2-羧乙基)硫代-2-吡啶基]硫代磷酸酯之后,分别与牛血清白蛋白(BSA)和卵清白蛋白(OVA)通过EDC方法合成人工抗原,前者为免疫原,后者为...     

壬基酚人工抗原及多克隆抗体的制备

目的制备壬基酚人工抗原和多克隆抗体,为采用免疫学方法检测壬基酚做准备。方法利用碳二亚胺(DCC)法制备人工抗原,并用紫外扫描法初步验证,用所制备的人工抗原对家兔免疫制备多克隆抗体,并用ELISA法检测抗体滴度。结果初步验证人工抗原中壬基酚与匙孔嘁血蓝蛋白的结合比为18∶1,ELISA法测定抗体效价为1∶32 000。结论用碳二亚胺法成功合成了壬基酚人工抗原,并获得了较高效价的多抗血清,制备壬基酚多克隆抗体所用的时间不足3 d。     

应用膜抗原荧光抗体试验检测人群水痘-带状疱疹病毒血清抗体阳性率

目的 应用膜抗原荧光抗体试验(FAMA)方法调查广州地区正常人群水痘-带状疱疹病毒(VZV)的流行现状.方法 采用以VZV感染细胞作为抗原、异硫氰酸荧光素(FITC)标记的羊抗人IgG作为二抗的FAMA试验,对随机抽取的592份正常人血清标本进行特异性VZV抗体检测.结果 FAMA试验检测VZV抗体与其他的疱疹类病毒的相应抗体不产生交叉反应.应用此法检测592份血清标本中,VZV抗体总体阳性率为76.52%;1～、4～、7～、14～、20～、30～、40～及≥50岁年龄组血清抗体阳性率分别是14.67%、51.56%、73.91%、91.26%、92.78%、95.65%、98.11%和100%.1～3岁年龄组血清抗体阳性率最低,血清抗体阳性率随年龄的增大而升高;不同性别间阳性率差异无统计学意义(P0.005),不同年龄组间阳性率差异有统计学意义(P<0.001).结论 采用FAMA法检测人群中VZV抗体与其他疱疹类病毒的相应抗体不产生交叉反应,是VZV抗体检测的可靠方法 .应以1～3岁儿童为VZV疫苗免疫的首选对象.     

可提取核抗原抗体检测的性能验证

目的 可提取核抗原抗体(ENA)的性能验证.方法 对免疫印迹法检测的可溶性核蛋白抗体重复性和准确度进行评价.批内重复性验证:用6份特征性患者血清样本(5份阳性,1份阴性)对同一批号的产品进行检测,每份血清检测5次,比较阳性血清检测的结果,要求特异性抗体检出结果基本一致,阴性血清检测的结果为阴性;批间重复性验证:在3d内,用6份特征性患者血清样本(5份阳性、1份阴性)对同一批号的产品进行检测,每份血清每次复管检测1次,统计其阴阳性符合率;批批重复性验证:用6份特征性患者血清样本(5份阳性、1份阴性)对近3个月内所有不同批号的试剂进行检测,统计1年内所检测不同批号试剂的阴阳性符合率;阴性、阳性符合率:将100份已检测过的患者标本,其中阴性50份,阳性50份,重新检测1次,分别统计阴性、阳性重复检测结果的一致率.准确度验证:统计分析近3年参加卫生部临床检验中心及其他单位组织的室间质评结果与本室检测结果的阴性和阳性符合率.结果 批内、批间、批批及100份患者标本的阴性、阳性符合率均为100％.结论 本室免疫印迹法检测可溶性核蛋白抗体的重复性、准确度能满足实验性能验证的要求.     

白假丝酵母菌天冬氨酸蛋白酶Sap2多克隆抗体的制备、纯化及鉴定

目的制备白假丝酵母菌天冬氨酸蛋白酶Sap2多克隆抗体,并对其进行鉴定。方法提取白假丝酵母菌基因组DNA为模板,经聚合酶链反应（PCR）获取SAP2目的基因;双酶切SAP2基因与原核表达载体pMAL c2x（+）,构建pMAL c2x/SAP2重组质粒;在大肠埃希菌BL21(DE3)感受态细胞中经IPTG诱导表达出可溶性的融合蛋白;用可溶性Sap2免疫小鼠制备抗血清,间接酶联免疫吸附试验(ELISA)检测抗血清效价,亲和层析法纯化抗血清后,利用Western免疫印迹(Western Blot)检测抗体特异性。结果该研究成功制备了抗Sap2多克隆抗体,抗体效价＞1∶51 200;Western Blot检测结果表明,该抗体可特异性识别Sap2蛋白。结论纯化后的Sap2作为抗原免疫小鼠,有较好的抗原性和免疫原性,可成功制备多克隆抗体,并具有高特异性,为快速检测侵袭性白假丝酵母菌感染奠定了基础。     

Preparation and testing of a Haemophilus influenzae Type b/Hepatitis B surface antigen conjugate vaccine

The majority of conjugate vaccines focus on inducing an antibody response to the polysaccharide antigen and the carrier protein is present primarily to induce a T-cell dependent response. In this study conjugates consisting of poly(ribosylribitolphosphate) (PRP) purified from Haemophilus influenzae Type b bound to Hepatitis B virus surface antigen (HBsAg) virus like particles were prepared with the aim of inducing an antibody response to not only the PRP but also the HBsAg. A conjugate consisting of PRP bound to HBsAg via an adipic acid dihydrazide (ADH) spacer induced strong IgG antibodies to both the PRP and HBsAg. When conjugation was performed without the ADH spacer the induction of an anti-PRP response was equivalent to that seen by conjugate with the ADH spacer, however, a negligible anti-HBsAg response was induced. For comparison, PRP was conjugated to diphtheria toxoid (DT) and Vi polysaccharide purified from Salmonella Typhi conjugated to HBsAg both using an ADH spacer. The PRP_AH–DT conjugate induced strong anti-PRP and anti-DT responses, the Vi–_AHHBsAg conjugate induced a good anti-HBsAg response but not as strong as that induced by the PRP_AH–HBsAg conjugate. This study demonstrated that in mice it was possible to induce robust antibody responses to both polysaccharide and carrier protein provided the conjugate has certain physico-chemical properties. A PRP_AH–HBsAg conjugate with the capacity to induce anti-PRP and anti-HBsAg responses could be incorporated into a multivalent pediatric vaccine and simplify formulation of such a vaccine.     

甲基对硫磷与灭多威杀虫剂混配中毒的实验治疗

目的 探讨肟类药物氯解膦定（ＰＡＭ＿Ｃ１）和（吡啶－２－醛肟－１－１－甲基）－（４－氨基甲酰基－吡啶－１－甲基）醚氯盐（ＨＩ－６）对甲基对硫磷与灭多威杀虫剂混配中毒动物的保护作用，并比较肟类、阿托品（Ａt）及Ａt与肟类联合应用的疗效。方法 小鼠和大鼠灌胃染毒后，经腹泻注射生理盐水、肟类、Ａt及Ａt与肟类药物，记录各组动物的中毒表现和死亡结局，并测定血和脑中胆碱酯酶（ＣhＥ）活力。结果 各治疗组和对照组相     

Exposure of neonatal rats to parathion elicits sex-selective impairment of acetylcholine systems in brain regions during adolescence and adulthood

Background

Organophosphates elicit developmental neurotoxicity through multiple mechanisms other than their shared property as cholinesterase inhibitors. Accordingly, these agents may differ in their effects on specific brain circuits.

Objectives

We gave parathion to neonatal rats [postnatal days (PNDs) 1–4], at daily doses of 0.1 or 0.2 mg/kg, spanning the threshold for barely detectable cholinesterase inhibition and systemic effects.

Methods

We assessed neurochemical indices related to the function of acetylcholine (ACh) synapses (choline acetyltransferase, presynaptic high-affinity choline transporter, nicotinic cholinergic receptors) in brain regions comprising all the major ACh projections, with determinations carried out from adolescence to adulthood (PNDs 30, 60, and 100).

Results

Parathion exposure elicited lasting alterations in ACh markers in the frontal/parietal cortex, temporal/occipital cortex, midbrain, hippocampus, and striatum. In cerebrocortical areas, midbrain, and hippocampus, effects in males were generally greater than in females, whereas in the striatum, females were targeted preferentially. Superimposed on this general pattern, the cerebrocortical effects showed a nonmonotonic dose–response relationship, with regression of the defects at the higher parathion dose; this relationship has been seen also after comparable treatments with chlorpyrifos and diazinon and likely represents the involvement of cholinesterase-related actions that mask or offset the effects of lower doses.

Conclusions

Neonatal exposure to parathion, at doses straddling the threshold for cholinesterase inhibition, compromises indices of ACh synaptic function in adolescence and adulthood. Differences between the effects of parathion compared with chlorpyrifos or diazinon and the non-monotonic dose–effect relationships reinforce the conclusion that various organophosphates diverge in their effects on neurodevelopment, unrelated to their anticholinesterase actions.     

癌胚抗原、腺苷脱氨酶、结核抗体联合检测在诊断结核性和肿瘤性胸水中的意义

目的探讨胸腔积液患者胸水癌胚抗原（CEA）、腺苷脱氨酶（ADA）、结核抗体水平在诊断结核性胸水和恶性胸水中的临床意义。方法分别测定70例恶性胸水与62例结核性胸水患者胸水中的癌胚抗原、腺苷脱氨酶、结核抗体。结果恶性组患者胸水CEA水平明显高于结核性组,两组差异有统计学意义（P〈0．01）,而结核性胸水ADA和结核抗体阳性率高于恶性胸水。结论检测患者胸水癌胚抗原（CEA）、腺苷脱氨酶（ADA）、结核抗体阳性率水平有助于临床对胸水性质的诊断。     

麻疹病毒抗原性变异及免疫保护效果研究

目的 了解浙江省麻疹野毒株的抗原性变异状况以及麻疹疫苗的免疫保护效果。方法 采用麻疹国际标准株Edmonston株、疫苗株沪19l与近几年浙江省分离的麻疹野毒株(浙98-5和浙00-4)分别制备免疫血清，与各毒株进行交叉中和试验，并分别对不同来源的人群血清测定中和抗体滴度。结果 麻疹野毒株浙98-5与疫苗株沪川和Edm株的抗原比分别为3．0和2．6，浙00-4与上述两毒株的抗原比分别为7．30和5．65，其抗原性存在着明显的差异。麻疹疫苗初免儿童和麻疹急性期病人血清对麻疹野毒株的中和抗体几何平均滴度(GMT)分别为15．03和5．04，明显低于疫苗株沪19l，GMT分别为68．12和11．76。但健康人群血清对两毒株的中和抗体滴度无显性差异。结论 浙江省麻疹野毒株已出现较明显的抗原性变异，现行麻疹疫苗的免疫保护效果也受到一定影响。     

Evaluation of single-round infectious,chimeric dengue type 1 virus as an antigen for dengue functional antibody assays

Dengue fever and dengue hemorrhagic fever are endemic throughout tropical and subtropical countries. Four serotypes of dengue viruses (DENV-1 to DENV-4), each with several genotypes including various subclades, are co-distributed in most endemic areas. Infection–neutralizing and -enhancing antibodies are believed to play protective and pathogenic roles, respectively. Measurement of these functional antibodies against a variety of viral strains is thus important for evaluating coverage and safety of dengue vaccine candidates. Although transportation of live virus materials beyond national borders is increasingly limited, this difficulty may be overcome using biotechnology that enables generation of an antibody-assay antigen equivalent to authentic virus based on viral sequence information. A rapid system to produce flavivirus single-round infectious particles (SRIPs) was recently developed using a Japanese encephalitis virus (JEV) subgenomic replicon plasmid. This system allows production of chimeric SRIPs that have surface proteins of other flaviviruses. In the present study, SRIPs of DENV-1 (D1-SRIPs) were evaluated as an antigen for functional antibody assays. Inclusion of the whole mature capsid gene of JEV into the replicon plasmid provided higher D1-SRIP yields than did its exclusion in cases where a DENV-1 surface-protein-expressing plasmid was used for co-transfection of 293T cells with the replicon plasmid. In an assay to measure the balance between neutralizing and enhancing activities, dose (antibody dilution)-dependent activity curves in dengue-immune human sera or mouse monoclonal antibodies obtained using D1-SRIP antigen were equivalent to those obtained using DENV-1 antigen. Similar results were obtained using additional DENV-2 and DENV-3 systems. In a conventional Vero-cell neutralization test, a significant correlation was shown between antibody titers obtained using D1-SRIP and DENV-1 antigens. These results demonstrate the utility of D1-SRIPs as an alternative antigen to authentic DENV-1 in functional antibody assays. SRIP antigens may contribute to dengue vaccine candidate evaluation, understanding of dengue pathogenesis, and development of serodiagnostic systems.     

应用流式细胞术研究甲基对硫磷对大鼠睾丸生精细胞的毒性作用

目的探讨有机磷农药甲基对硫磷对雄性大鼠的睾丸毒性作用。方法20只SD雄性大鼠（220～240g）随机分为对照组和低（1．2mg／kg）、中（6mg／kg）、高（30mg／kg）剂量甲基对硫磷组,灌胃染毒,容量为5ml／kg,每日1次,连续6周。染毒结束次日处死小鼠,用流式细胞仪对大鼠睾丸生精细胞进行分析。结果①高、中剂量甲基对硫磷组细胞凋亡率均显著高于对照组（P〈0．05或P〈0．01）;②与对照组相比,各剂量甲基对硫磷组1C细胞显著减少,2C细胞显著增加（P〈0．05或P〈0．01）;③与对照组相比,高剂量染毒组G0／G1期细胞比例显著降低,G2／M期细胞比例显著增加（P〈0．05或P〈0．01）。结论甲基对硫磷能够阻滞大鼠生精细胞的细胞周期进程,诱导生精细胞凋亡。