再生丝素固定的酶标免疫传感器的研究

所研究的酶标免疫传感器是采用再生丝素将待测抗原 (兔IgG)固定在石墨电极表面 ,选用抗体 (山羊抗兔IgG HRP)与其识别结合。利用H₂O₂ 将抗原抗体结合的电位响应信号放大采用直接电位法检测IgG的浓度。该传感器测定IgG的最低浓度可达 1.2×10^-10 mol/L ,标准曲线的线形范围在4.1×10^-7～1.2×10^-10 mol/L ,回归方程为: E=-1049+721g[IgG],响应时间为 15s。通过电泳的方法加速抗原抗体的识别结合 ,反应时间由原来的 90min缩短到 3 0min。这种以固定化抗原结合酶标抗体量的多少作为检测抗原标准的新型酶标免疫传感器 ,在临床检测、环境监测、HLA个人身份鉴定等领域都有着广阔的应用前景。     

黄曲霉毒素B1的免疫检测Ⅱ．抗体的产生及应用

将黄曲霉毒素B₁肟(AFB₁O)与牛血清白蛋白(BSA)的连接物,通过多点、多次免疫法注射免疫兔子。分析了抗体的产生进程、效价以及特异性。注射抗原后的第60天开始有较明显抗体产生,第120天达到高峰,维持15天左右后开始下降;抗体的ELISA效价高达1：30000;和黄曲霉毒素B₁(AFB₁)的结构类似物的竞争ELISA表明,抗体有很好的特异性。运用该抗体,以ELISA分析检测了几种农产品及饲料中污染AFB₁,的含量,并和薄层层析法的分析结构进行了比较,结果表明当AFB₁．的含量大于等于5ng／ml时。两者间有很好的相关性。     

三碘甲状腺原氨酸(T3)和甲状腺素(T4)固相放射免疫联合测定法

三碘甲状腺原氨酸(T₃)、甲状腺素(T₄)固相放射免疫联合测定法,是将₃抗体涂布在塑料小球上,将T₄抗体涂布在塑料管上。这种联合测定主要解决了T₃抗体对T₄的交叉反应。本法能在同一试管内进行,只需50μl血清样品,90分钟反应,可同时精确地测出两种激素水平,它比以往单独测定T₃或T₄的PEG双抗法更精确、快速和简便。     

鸡肝6-磷酸果糖-2-激酶/果糖-2-,6-二磷酸酯酶单克隆抗体制备及对酶活力影响

利用鸡肝-6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酯酶(6PF-2-K/Fru-2,6-P₂ase)的单克隆抗体对其结构和功能进行了初步研究.用鸡肝6PF-2-K/Fru-2,6-P₂ase为抗原免疫Balb/C小鼠,最后获得7株单克隆抗体.其中6株抗体的抗原决定簇位于6PF-2-K/Fru-2,6-P₂ase的酯酶结构域部分,而另一株H₂的抗原决定簇则位于其激酶结构域部分.7株单克隆抗体都能引起鸡肝6PF-2-K/Fru-2,6-P₂ase的激酶活力提高2倍左右,而对酯酶活力的影响大致相同.它们激活该酶的酯酶活力至4倍左右,但却不影响分离的鸡肝果糖-2,6-二磷酸酯酶结构域的酯酶活力.以上结果再次提示6PF-2-K/Fru-2,6-P₂ase双功能酶和分离的Fru-2,6-P₂ase结构域的酯酶处于2种不同的构象和活性状态.     

ELISA法分析抗血清中链霉素抗体抗原结合部位的特异性

用链霉素抗血清对链霉素的亲和力(K_SM)和链霉素抗血清对二链霉胺的亲和力(K_B)之比(K_SM/K_B)为指标,表征链霉素抗血清抗原结合部位的特异性;建立了快速分析该指标的ELISA(酶联免疫吸附法)法;并对链霉素抗体抗原结合部位的特异性进行了分类。     

高温和H2O2诱导酵母细胞产生活性衍生物的研究*

对高温和H₂O₂ 应激条件下产生活性酵母细胞衍生物 (Live Yeast Cell Derivative ,简称LYCD)进行了研究。结果表明 :低剂量的预处理 ( 37℃和 0.2mmol LH₂O₂ )能够增加细胞内谷胱甘肽 (GSH)含量 ,提高超氧化物歧化酶 (SOD)、过氧化氢酶 (CAT)活性。两种预处理均可以诱导对致死浓度H₂O₂ 的抗性。通过 37℃和 0.     

鸟氨酸脱羧酶活性微量测定法

本文采用一种简单,微量反应系统,根据 ¹⁴C-鸟氨酸释放的 ¹⁴CO₂量测定鸟氨酸脱羧酶(ODC)的活性,酶反应在置于液闪计数瓶内的玻璃小管中进行,释放的 ¹⁴CO₂被瓶内滤纸片上的海胺吸收。实验结果表明,加酸释放 ¹⁴CO₂后30分钟 ¹⁴CO₂吸收已达最大值,且吸收量与释放量成正比,酶反应测定证明 ¹⁴CO₂释放速度在40分钟内保持恒定。ODC活性与酶浓度呈线性关系,此方法不仅用于ODC活性测定,而且亦可用于其他脱羧酶活性的测定。     

黄芪总黄酮对DNA损伤防护作用的研究

用DNA解旋荧光检测法(FADU)研究了黄芪总黄酮(TFA)对γ射线和H₂O₂所致V79细胞DNA链断裂的防护作用. 结果表明TFA对这两种损伤因子所致的DNA损伤均有不同程度的防护作用, 当TFA浓度达到0.4g/L和0.6g/L时, 分别对H₂O₂和γ射线所致的损伤有保护作用(P<0.05), 而浓度增至0.8g/L和1.2g/L时, 分别对两种因素所致的DNA链断裂损伤有非常显著的防护效果(P<0.01), 对H₂O₂的防护效果优于对γ射线.     

大气CO2浓度升高对B型烟粉虱大小、酶活及其寄主的选择性影响

以CO₂浓度升高为作用因子,观测了连续3代饲养在转Bt基因棉(GK-12)和亲本棉泗棉3号(S3)上B型烟粉虱的个体大小、解毒酶活性及第3代烟粉虱的选择性和产卵量。结果表明,CO₂浓度和棉花品种处理对第2、3代烟粉虱卵长、伪蛹大小、雌雄成虫大小的影响均不显著,但CO₂浓度升高使第1代烟粉虱的伪蛹长、雌虫翅展和雌、雄虫长显著低于对照CO₂浓度下生长的烟粉虱;取食S3的伪蛹长、雌虫长和雌虫翅展分别比相应的对照浓度下的低2.81%、2.95%、0.94%,取食GK-12的雌虫翅展和雄虫长均比相应的对照浓度下的低2.08%和2.58%。3种解毒酶的活性测定显示,取食高CO₂浓度处理的转基因棉GK-12上的F3代烟粉虱的谷胱甘肽S转移酶(Glutathione Stransferase, 简称GSTs)活性和取食亲本棉S3的F1代的乙酰胆碱酯酶(Acetylcholinesterase;简称AchE)的活性分别比对照处理的增加45.73%和27.68%;而羧酸酯酶(Carboxylic Ester hydrolase;简称 CarE)在取食4个处理的棉花上烟粉虱的活性均无显著的差异;而取食对照CO₂条件下GK-12棉花的F1代烟粉虱GSTs活性比取食S3棉花低35.12%。对3个因子的交互分析结果显示,C0₂浓度对3种酶活影响均不显著,品种对GSTs的影响显著,世代、CO₂×品种及CO₂×品种×世代间的交互作用对AchE酶活影响显著,各因子对CarE的活性影响均不显著。烟粉虱的选择实验结果表明,4种处理的F3代B型烟粉虱均喜好选择在高CO₂处理的棉花,而且选择高CO₂处理的GK-12的数量多于其他3个处理,其中对照浓度下生长的烟粉虱选择高CO₂的GK-12比高CO₂处理的S3、对照浓度处理的GK-12 和S3分别增加9.175%, 19.89%、27.93%; 而高浓度下生长的烟粉虱选择高CO₂的GK-12比高CO₂处理的S3、对照浓度处理的GK-12 和S3分别增加12.56%,21.05%,28.73%。72h的产卵量检测发现,烟粉虱在高CO₂处理的2种棉花上的产卵量也是多于对照条件下的棉花,对照条件下的棉花相比,高CO₂和对照浓度下生长的烟粉虱的在高CO₂处理的2种棉花上的产卵量分别增加24.55%和19.03%;在高CO₂浓度下生长的F3代烟粉虱更喜好在高CO₂浓度处理的GK-12上产卵,与对照浓度下的GK-12相比增加26.93%,差异达到显著水平。     

纳米二氧化钛对生菜的生长效应分析

为了阐明纳米二氧化钛颗粒(TiO₂NPs)对生菜(Lactuca sativa)生长的影响，采用自行设计的水培装置探究不同浓度TiO₂NPs (300~1 200 mg/L)下，生菜生长和生理生化指标的变化。结果表明，300 mg/L TiO₂NPs能促进生菜幼苗的根长、茎长、叶表面积、鲜重和干重；随着TiO₂ NPs浓度增大，生菜的生长指标呈现下降趋势，但仍优于对照组。生菜体内的抗氧化酶(SOD、POD)在低TiO₂ NPs浓度(300 mg/L)时，活性明显下降；随着TiO₂ NPs浓度增大，这两种抗氧化酶活性逐渐增强。因此，生菜对TiO₂NPs胁迫具有浓度依赖性，表现为“低促高抑”，且能够通过抗氧化酶系统来减轻TiO₂NPs伤害。     

Enzyme-Linked Immunosorbent Assay of Substance P: A Study in the Eye

A solid phase enzyme-linked immunosorbent assay for quantitation of substance P is presented. The assay measures the capacity of soluble substance P to compete with the solid phase antigen for a limited quantity of specific substance P antibody. The solid-phase antigen consists of a synthetic substance P.poly-D-glutamic acid conjugate coated to polystyrene micro-ELISA plate wells. Soluble substance P and antibodies to substance P are first preincubated together and then added to the wells containing solid-phase antigen. Subsequently the wells are incubated with anti-antibodies conjugated to alkaline phosphatase. The wells are finally incubated with p-nitrophenyl phosphate an the absorbance is read in a spectrophotometer 16--24 hr after the start of the assay. The threshold for detection of substance P was 5--10 pg per well (0.25 ml). Substance P was extracted from rabbit eyes and the values obtained with the present method are compared with previously reported values based on radioimmunoassay.     

A radiometric immunosorbent assay for the detection of anti-hormone-binding protein antibodies

A radiometric immunosorbent assay (RISA) for the detection of monoclonal antibodies to hormone-binding proteins has been developed. The assay involves incubating hybridoma supernatants in microtiter wells that have been coated with goat anti-mouse IgG antibodies. Any mouse IgG in the test supernatant is thus specifically retained in the wells. Radioactive ligand-binding protein complexes are then incubated in the wells. The presence of anti-binding protein antibodies in the supernatant is indicated by specific retention of radioactive ligand-binding protein complexes in the wells. Crude antigen preparations, such as tissue homogenates, can be used to detect antibodies. The assay is capable of detecting antibody at concentrations 20 ng/ml (approximately 100 pM IgG). The RISA has been used successfully to screen for monoclonal antibodies to the intracellular receptor for 1,25-dihydroxyvitamin D3 and should be useful for the detection of antibodies to ligand-binding proteins in general.     

Enhanced chemiluminescence ELISA for the detection of antibody to hepatitis B virus surface antigen

A chemiluminescent enzyme linked immunosorbent assay (ELISA) for the detection of antibody to hepatitis B virus surface antigen (anti-HBs) in human serum has been developed. Polystyrene microtitre plates were coated with recombinant, yeast-derived hepatitis B surface antigen (rec-HBsAg). Patient serum samples and appropriate controls were added to the rec-HBsAg-coated wells and incubated to bind anti-HBs. The wells were then washed and a fluorescein isothiocyanate (FITC) conjugate of a human plasma-derived hepatitis B surface antigen (HBsAg) was added. Following incubation and further washing the bound FITC-labelled HBsAg was detected after addition of a horseradish peroxidase (HRP) conjugate of a monoclonal anti-FITC antibody and assaying for the enzyme. The activity of the HRP was measured using luminol and hydrogen peroxide as substrates and iodophenol as a chemiluminescence enhancer. The luminescence was recorded using a camera luminometer. Preliminary tests have shown the assay to be suitable for the detection of antibody in sera from both vaccinees and also from individuals with a past hepatitis B virus infection. The use of the FITC-anti-FITC system together with the measurement of a chemiluminescence signal makes possible the completion of this assay in a few hours. The assay has been shown to be both specific and sensitive and provides a permanent photographic record.     

Multi-analyte capillary immunosensor for the determination of hormones in human serum samples

In this work we present the development of a multi-analyte immunosensor for the determination of follitropin, human chorionic gonadotropin and prolactin in human serum. The immunosensor is based on plastic capillaries. According to the methodology, discrete areas of the internal capillary surface are coated with different antibodies, which are highly specific for each one of the analytes to be determined. The sample that will be analyzed along with a mixture of analyte-specific biotinylated antibodies is introduced into the capillary. The coated and the detection antibodies react with different epitopes of the analytes in the sample to form a 'sandwich'. The detection is based on reaction of the immobilized biotinylated antibody with streptavidin labeled with R-phycoerythrin. The fluorescent areas formed were quantified by scanning the capillary with a light beam of appropriate wavelength. A light sensor placed at the end of the capillary detects the emitted photons, that are trapped and waveguided into the capillary walls. The multi-analyte immunosensor assays were characterized by high specificity and short analysis time. In addition, the results obtained by the multi-analyte optical capillary immunosensor were comparable to those obtained by immunofluorimetric assays performed in microtitration wells. Potential applications of the proposed immunosensor include determination of several analyte panels in a broad spectrum of disciplines such as endocrinology, hematology, and oncology.     

Solid-phase assay for the detection of low-abundance enzymes, and antibodies to enzymes in immune reactions, using acid sphingomyelinase as a model

The development of a solid-phase immunosorbent assay, suitable for use with enzyme antigens, is described. Acid sphingomyelinase and a mouse monoclonal anti-sphingomyelinase antibody have been used to determine optimal conditions for the assay. The assay involves immobilization of a second antibody (anti-mouse IgG) in the wells of a polyvinyl microtiter plate. Soluble immune complexes of first antibody (monoclonal anti-sphingomyelinase) and antigen (sphingomyelinase), incubated in separate vials, are then reacted in the anti-mouse IgG-coated assay wells, and the extent of the cross-reaction between antibody and antigen is measured by direct assay of enzyme retained in the well. A necessary condition of the assay is that antibody must not inhibit enzyme activity, which makes it especially suitable for monoclonal antibodies. The assay finds useful application in hybridoma fluid screening, equivalence point determination, and demonstration of cross-reacting enzyme from various tissue sources.     

A monoclonal antibody to the phosphorylated form of glial fibrillary acidic protein: application to a non-radioactive method for measuring protein kinase activities

Monoclonal antibody YC10 showed specificity for the phosphorylated form of human, bovine and porcine glial fibrillary acidic proteins (GFAPs) and negligible reactivity towards the dephosphorylated form of the GFAPs. Analysis of species specificity and of the epitope, determined using synthetic phosphopeptides, indicated that this antibody recognized the local phosphorylation-site sequence Thr-phosphoSer-Ala-Ala-Arg-Arg (residues 7-12 of GFAP). Making use of this antibody we developed a non-radioactive method to measure protein kinase activities. After incubation of a protein kinase with non-radioactive ATP in ninety-six wells coated with the synthetic peptide Arg-Arg-Arg-Val-Thr-Ser-Ala-Ala-Arg-Arg-Ser-Cys (residues 3-13 of GFAP), the phosphorylated product was detected by using this mouse antibody and peroxidase-labeled goat anti-mouse IgG. This method proved to be equally as sensitive as the radioactive method for the measurement of protein kinase activities and was less affected by concentrations of ATP present in the reaction mixture.     

Applications of monoclonal antibodies to human follicle-stimulating hormone in enzyme immunoassays

Monoclonal antibodies against human follicle-stimulating hormone (hFSH) were generated by using an improved hybridoma technique with a semisolid medium in methylcellulose for initial cloning. The generated monoclonal antibodies were characterized with respect to their subunit and epitope specificity as well as cross-reactivity to other glycoprotein hormones. Monoclonal antibodies of high affinity and high specificity to hFSH were finally selected for applications in sandwich enzyme immunoassay. The monoclonal antibody specific to the alpha-subunit of FSH was coated on microtiter wells and served as the first antibody. The other high-affinity monoclonal antibody specific to beta-subunit of FSH was labeled with horseradish peroxidase and served as the second antibody. This immunoassay can be performed within 70 min at room temperature and has a minimum sensitivity of 2 mIU/ml for serum sample.     

免疫磁珠技术分离唾液A/B血型抗原并用于吸附血清A/B抗体

目的:应用免疫磁珠分离技术获得具有良好抗原性的A/B血型抗原,并探究其作为ABO血型抗体吸附剂去除A/B抗体的可行性。方法:将含有血型物质的唾液进行预处理,再与包被了抗体的磁珠混合,分离出纯度较高的A/B抗原,运用酶联免疫及凝集抑制试验验证所得抗原的抗原性及是否存在交叉反应。用未纯化A/B抗原和纯化A/B抗原包被磁珠,对含有抗A/B IgM、IgG的血清进行抗体吸附,用纯化A/B抗原对100份来自O型血孕妇的临床血清样本进行抗体吸附,分别评价其吸附效果。结果:纯化抗原与对应抗体反应后,其吸光度显著高于对照组(A抗原与A抗体0.85±0.12 vs.0.27±0.03,P0.01;B抗原与B抗体0.86±0.09 vs.0.24±0.06,P0.01),与其它类型抗体反应后的吸光度值与对照组比较差异无统计学意义(P0.05)。进行红细胞凝集抑制试验时,纯化抗原可显著抑制相应抗体与红细胞的凝集反应,对其它类型抗体与红细胞的凝集没有抑制作用。血清抗体吸附实验表明纯化抗原的吸附效率比未纯化抗原的高(97.00%vs.88.00%,P0.001)。临床样本抗体吸附实验显示,纯化A抗原对抗A IgM/IgG的吸附效率分别为96.88%、98.44%;纯化B抗原对抗B IgM/IgG的吸附效率分别为96.88%、98.44%。结论:磁珠纯化抗原能特异性地与对应抗体结合,有效吸附血清中的血型抗体,有望作为合成A/B抗原的替代品。     

A solid-phase assay for the quantitative analysis of hyaluronic acid at the nanogram level

A sensitive and accurate solid-phase assay for the quantitative determination of hyaluronic acid (HA) is described. The wells of the polystyrene microplates used were coated with glutaraldehyde followed, via a Schiff's base bond, with spermine to introduce amino groups. HA was added to the activated microwells in the presence of carbodiimide and left to bind via a peptide bond to the amino groups. Then aggrecan solution was added to the wells of the microtiter plates to interact with its G1 domain with hyaluronic acid, and the amounts of aggrecan bound were measured immunochemically. The inhibition of the binding between aggrecan and immobilized HA due to soluble HA present in reference solutions showed linearity in the range of concentrations 0.1 to 0.7 microg/ml. The reaction is specific and rapid and can be widely used for the calculation of HA in body fluids directly and in tissue samples after a brief digestion with a proteolytic enzyme.     

Development and application of competitive ELISA assays for rat LH and FSH

Rat LH (rLH) and FSH (rFSH) were measured by sensitive and specific competition ELISAs. The rat LH ELISA used rLH-I-9 coated plates, an antiserum against rLH and an antibody against rabbit IgG labeled with peroxidase. Using rLH-RP-3 as a standard, rat LH was determined by binding of the anti-LH antibody to rLH-I-9 coated plates. The sensitivity of the assay was 0.8 ng/mL. Similarly, the rat FSH-ELISA used rFSH-I-8 coated plates, an antiserum against rFSH and an antibody against rabbit IgG labeled with peroxidase. Using rFSH-RP-3 as a standard, the FSH-ELISA was also determined by binding of the anti-FSH antibody to rFSH-I-8 coated plates. The sensitivity of this assay was 1.25 ng/mL. Both rat LH and FSH ELISA assays are highly specific and provide accurate determination of gonadotrophins in buffers, sera, cell culture media, and anterior pituitary extracts. These assays were used for monitoring the gonadotrophin surge-attenuating factor (GnSAF) and inhibin activities present in human follicular fluid (hFF). The 2 new ELISA procedures have practical advantages (safety, convenience, economy) over the RIA methods, and they perform as well as the RIA techniques at the same range of concentrations.