一种新型电流型免疫传感器的ABA最优测定条件研究

研究了一种基于纳米金/硫堇修饰金电极的ABA电流型免疫传感器的最优条件.该传感器利用硫堇是HRP与电极之间电子传递媒介体的原理,将硫堇和酶标抗体共价吸附于电聚合纳米金的金电极上,用BSA封闭可能存在的活性住点以避免非特异性吸附,最后加入ABA与一部分抗体结合降低电化学信号,用循环伏安法测得加入不同浓度ABA前后电化学信号差值,从而测得ABA的含量.主要对使用此传感器测定ABA时的最优化条件进行了研究.     

玉米素核苷的酶标免疫测定法

牛血清白蛋白-玉米素核苷(BSA-ZR)的兔抗血清对玉米素核苷具有很高的亲和性,而且专一性强,除了玉米素外,对其它一些细胞分裂素如激动素(KT)的交叉反应甚微。用辣根过氧化物酶(HRP)作为标记物的酶标免疫法,由于它的灵敏度高,相当于几十毫克量的样品就可以测出细胞分裂素的含量。测定范围在0.25—50pmol之间,测定范围较广。由于该方法专一性高,植物组织的粗提取物可以直接用于测定。避免了提取分离的繁琐程序,使得测定方法较简便、快速,可成批进行,适用于一般实验室。用该方法测得风信子(Hyacinthus orientalis L.)各部分的细胞分裂含量(以玉米素核苷计)在10—60×10~(-9)克/克鲜重,即10—60ng/g F.W.。     

毛细管内均相辣根过氧化物酶(HRP)与核酸杂交偶联HRP 催化化学 发光比较研究

目的:了解毛细管内核酸偶联和游离辣根过氧化物酶(HRP)催化化学发光差异。方法:不同浓度HRP和经核酸杂交固定于毛细管内壁HRP催化化学发光反应。结果:①游离HRP催化化学发光线性检测范围窄(2.7×10-5-1.3×10-6mg/ml,R2>0.96),下限为1.0×10-7mg/ml;②2.0×1014-2.0×106copies/ml的5μl单链DNA杂交后,1.0-10min时DNA浓度M对数(lgM)与化学发光I值线性相关(R2>0.99),且大于阴性I值平均数+3倍标准偏差(s.d);③5.0×1011-5.0×106copies/ml的5μl PCR产物杂交后,10min内PCR产物对数lgM与I值线性相关(R2>0.97),且大于阴性I值平均数+3倍标准偏差(s.d)。4.0-7.0min内lgM与I值的R2>0.99,3次平行检测标准偏差<5.0%。结论:毛细管内核酸杂交的HRP催化化学发光检测线性范围宽、灵敏度高、底物用量少,有望用于临床核酸分子杂交检测。     

霍乱疫苗中残余霍乱毒素检测方法的建立及验证

采用间接酶联免疫法,即用神经节苷脂包被,加入待检样品,再加入兔抗霍乱毒素B亚单位抗体,用标准样品的吸光值(A值)对标准样品的浓度绘制4-参数拟合曲线,根据标准曲线计算出待测样品中的CT浓度。结果显示,在浓度范围(0.6～16)ng/ml之间,CT标准浓度和检测浓度成线性关系,r2=0.9986。精确度在浓度范围(0.6～16)ng/ml,CT的平均回收率在96.24%～114.44%之间。精密度:批内变异CV%≤12.98%,批间变异CV%≤18.48%。特异性CT浓度在10ng/ml时,平均回收率为102.6%;CT浓度在5ng/ml时,平均回收率为111.17%;CT浓度在2.5ng/ml时,平均回收率为123.83%。实验表明该方法可检测霍乱疫苗原液中CT的含量。     

微量定磷法测定红细胞钙调素含量

 磷酸二酯酶在钙调素(CaM)激活下水解cAMP为AMP。后者经蛇毒水解为无机磷酸和腺苷。用孔雀绿显色测定无机磷酸量与标准CaM比较即可测出CaM活性。该法测定CaM灵敏度3.4ng,可测范围3.4—48ng,批间变异系数3.8％—6.4％。 纯化的红细胞(RBC)经超声破碎,沸水浴加热,离心上清与标准CaM所作的磷酸二脂酶平行激活曲线呈正相关(r=0.9900,p<0.0005)。RBC CaM含量测定,批间变异系数11.5％,回收率97.8％±2.0％。本法测得30例正常人RBC CaM含量为6.31±1.5fg/cell,性别之间无显著差异(p>0.5)     

抗体夹心酶联免疫吸附法测定重组溶葡萄球菌酶研究

用重组溶葡萄球菌酶免疫家兔获得抗血清,经亲和层析纯化后用HRP标记,以双向免疫扩散法确定抗血清效价,以Westernblot鉴定抗体的特异性,建立双抗夹心法标准曲线,鉴定其最小检出限、精确度、回收率。实验显示多克隆抗体能与溶葡萄球菌酶特异性结合,双抗夹心ELISA法检测抗原的最小检出限为0·98ng/mL,标准曲线在0·98~500ng/mL范围内线性良好。3份同批样本分别重复6次测定,平均批内变异系数为6·4%;3份不同批样本分别重复6次测定,平均批间变异系数为6·5%。血清中加入已知量的标准抗原,测得平均回收率为98·6%。此法检测重组溶葡萄球菌酶的可测范围广,灵敏度和精密度高,变异系数较小。结果证实建立的检测血清中重组溶葡萄球菌酶含量的双抗夹心酶联免疫吸附测定法(Enzyme-linkedimmunosorbentassay,ELISA)灵敏、准确、可靠。     

牛血清蛋白酶联免疫试剂盒性能的验证

为了全面验证研制的牛血清蛋白(BSP)酶联免疫试剂盒的性能,由3个部门6人协作进行了验证。结果表明,,6人进行的18次试验全部满足BSP-ELISA试剂盒的质控标准,达标率100%;6名实验人员对3、4、5、10、20ng/ml浓度的BSP标准品进行测定,结果变异系数在1.96%~6.24%之间,精密度较好;回收率在94.8%~98.7%之间,准确度理想,测量限量为3ng/ml。该试剂盒与人血白蛋白、卵清蛋白等均无交叉反应,对BSA、B-IgG特异性蛋白的检测回收率为101.2%和94.7%;与牛血清白蛋白试剂盒对比测定11种疫苗总计108批,符合率为96.3%,针对不同疫苗,BSP残余量约为BSA的1.12~3.13倍,验证结果表明,该试剂盒可检测疫苗中BSP而不仅是BSA,能更全面客观地反映疫苗中牛血清的真实情况,有利于疫苗生产的严格质量监控。     

提高二苯胺反应灵敏度的简易方法

在生物学研究中,脱氧戊糖-二苯胺反应是常用于定量测定DNA含量的方法。但是如果溶液中DNA浓度低于每毫升5微克时,这一反应往往不够灵敏。据报道,如果用乙酸戊酯把反应中形成的蓝色脱氧戊糖-二苯胺络合物从溶液中抽提出来,灵敏度就可提高4倍,能精确地测定溶液中浓度低至每毫升1.5微克的DNA。由于乙酸戊酯能有效的把溶液中二苯胺反应特异     

金霉素单克隆抗体的制备及检测方法的建立

采用羰基二咪唑法,将半抗原金霉素(AM)分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联制备金霉素免疫抗原AM-BSA和检测抗原AM-OVA,通过紫外光谱扫描检测偶联产物。采用细胞杂交瘤技术,制备抗金霉素单克隆抗体杂交瘤细胞株,建立了金霉素竞争ELISA检测方法,其灵敏度达到50ng/ml,且呈现良好的线性关系(r=0.9812),并且与其他抗生素无交叉反应。     

甲醇酵母Pichia pastoris高水平表达有活性的辣根过氧化物酶

表达有活性的辣根过氧化物酶(HRP)不仅可以深入揭示HRP结构与功能及其生理作用规律,而且为HRP的广泛需要提供新的来源.为了在甲醇酵母P.pastoris中成功表达,将编码HRP-C成熟肽的cDNA构建到pPIC9上,再转化到P.pastoris中,筛选到了分泌表达非糖基化HRP和高糖基化HRP(分子质量超过100 ku)两种主要产物的重组细胞株.优化表达条件,目标产物在摇瓶发酵液中高效表达,可达4～6 g/L.并且直接从发酵液中可获得具有活性的高糖基化HRP,每毫升发酵液中酶活力约有2 U,经初步的纯化HRP具有最大吸收峰403 nm.     

Tandem conjugation of enzyme and antibody on silica nanoparticle for enzyme immunoassay

We present a new type of enzyme-antibody conjugate that simplifies the labeling procedure and increases the sensitivity of enzyme-linked immunosorbent assay (ELISA). The conjugates were prepared through layer-by-layer immobilization of enzyme and antibody on a silica nanoparticle scaffold. A maximal amount of enzyme was immobilized on the nanoparticle, followed by antibody linkage through Dextran 500. The conjugate could be easily purified from unreacted reagents by simple centrifugations. In comparison with the conventional antibody-enzyme conjugate used in ELISA, which often has one or two enzyme molecules per antibody, the new type of conjugate contained more enzyme molecules per antibody and provided a much higher signal and increased sensitivity. When used in an ELISA detection of the hepatitis B surface antigen (HBsAg), the detection limit was three times lower than that of the commercially available ELISA kit.     

Enzyme immunoassay of testosterone in plasma, with use of polyethylene glycol to separate antibody-bound and free hormone.

We describe an enzyme immunoassay for testosterone in which we use a testosterone-3-(carboxymethyl)oxime horseradish peroxidase conjugate as the label and an antiserum, raised in rabbits, to testosterone-3-(carboxymethyl)oxime-bovine serum albumin conjugate. Polyethylene glycol (Carbowax 6000) is used to separate antibody-bound and free steroid. The assay has a sensitivity of 12 pg/assay tube and satisfies the usual criteria of specificity, precision and accuracy. The results agree well with those obtained with a comparable radioimmunoassay.     

Quantitation of aflatoxin B1 and aflatoxin B1 antibody by an enzyme-linked immunosorbent microassay.

A specific microtest plate enzyme immunoassay has been developed for the rapid quantitation of aflatoxin B1 at levels as low as 25 pg per assay. Multiple-site injection of rabbits with an aflatoxin B1 carboxymethyloxime-bovine serum albumin conjugate was used for the production of hyperimmune sera. Dilutions of the purified antibody were air dried onto microplates previously treated with bovine serum albumin and glutaraldehyde and then incubated with an aflatoxin B1 carboxymethyloxime-horseradish peroxidase conjugate. The amount of enzyme bound to antibody was determined by monitoring the change in absorbance at 414 nm after the addition of a substrate solution consisting of hydrogen peroxide and 2,2'-azino-di-3-ethyl-benzthiazoline-6-sulfonate. Antibody titers determined in this manner closely correlated with those determined by radioimmunoassay. Competition assays as performed by incubation of different aflatoxin analogs with the peroxidase conjugate showed that aflatoxins B1 and B2 and aflatoxicol caused the most inhibition of conjugate binding to antibody. Aflatoxins G1 and G2 inhibited the conjugate binding to a lesser degree, whereas aflatoxins M1 and B2a had no effect of the assay.     

Bioluminescent enzyme immunoassay for progesterone using monoclonal antibodies and glucose-6-phosphate dehydrogenase labels

Bacterial luciferase, NAD(P): FMN oxidoreductase and anti-mouse immunoglobulin were co-immobilized on Sepharose 4B. This reagent together with a progesterone glucose-6-phosphate dehydrogenase conjugate and various anti-progesterone monoclonal antibodies was used to develop a non-separation bioluminescent immunoassay for progesterone. This monoclonal antibody based assay was sensitive and reliable and using the tracer progesterone-11-acetate-glucose-6-phosphate dehydrogenase, the majority of the monoclonal antibodies give a better sensitivity with this enzymatic tracer than that obtained with an iodinated tracer. In a second assay design progesterone-glutathione was co-immobilized with bacterial luciferase and NAD(P): FMN oxidoreductase on Sepharose 4B and three monoclonal antibodies were labelled with glucose-6-phosphate dehydrogenase. With aqueous progester-one standards, this assay gave comparable sensitivity to the bioluminescent enzyme immunoassay using the second antibody immunoadsorbant and to an RIA but was unsuitable for plasma samples.     

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.     

Action of natural abscisic acid precursors and catabolites on abscisic acid receptor complexes

The phytohormone abscisic acid (ABA) regulates stress responses and controls numerous aspects of plant growth and development. Biosynthetic precursors and catabolites of ABA have been shown to trigger ABA responses in physiological assays, but it is not clear whether these are intrinsically active or whether they are converted into ABA in planta. In this study, we analyzed the effect of ABA precursors, conjugates, and catabolites on hormone signaling in Arabidopsis (Arabidopsis thaliana). The compounds were also tested in vitro for their ability to regulate the phosphatase moiety of ABA receptor complexes consisting of the protein phosphatase 2C ABI2 and the coreceptors RCAR1/PYL9, RCAR3/PYL8, and RCAR11/PYR1. Using mutants defective in ABA biosynthesis, we show that the physiological activity associated with ABA precursors derives predominantly from their bioconversion to ABA. The ABA glucose ester conjugate, which is the most widespread storage form of ABA, showed weak ABA-like activity in germination assays and in triggering ABA signaling in protoplasts. The ABA conjugate and precursors showed negligible activity as a regulatory ligand of the ABI2/RCAR receptor complexes. The majority of ABA catabolites were inactive in our assays. To analyze the chemically unstable 8'- and 9'-hydroxylated ABA catabolites, we used stable tetralone derivatives of these compounds, which did trigger selective ABA responses. ABA synthetic analogs exhibited differential activity as regulatory ligands of different ABA receptor complexes in vitro. The data show that ABA precursors, catabolites, and conjugates have limited intrinsic bioactivity and that both natural and synthetic ABA-related compounds can be used to probe the structural requirements of ABA ligand-receptor interactions.     

A rapid and sensitive assay for abscisic acid using ethyl abscisate as an internal standard.

A gas-liquid chromatographic (glc) assay for abscisic acid (ABA) is described that is both rapid and highly sensitive. The selectivity and sensitivity of an electroncapture detector are used to reduce the assay procedure to just four steps: extraction, thin-layer chromatography (tle), esterification, and gle. Quantification of the ABA present is by reference to ethyl abscisate added during the assay as an internal standard. The procedure enables samples containing as little as 100 pg of ABA to be assayed.     

Chemiluminescence enzyme immunoassay of dehydroepiandrosterone and its sulfate using peroxidase as label

We report a highly sensitive enzyme immunoassay for dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) using horseradish peroxidase as the label enzyme. Separation of free and bound DHEA-peroxidase conjugate was by insolubilized antibody, prepared by coupling purified IgG of goat anti-rabbit IgG serum with Sepharose 4B or a polystyrene tube. The enzyme activity was measured by the chemiluminescence reaction using luminol and hydrogen peroxide as substrate. The faint chemiluminescence was measured by a photon counter. The sensitivity was 25 pg/assay tube for DHEA and 100 pg/assay tube for DHEA-S. Upon comparison, results obtained by radioimmunoassay and this method showed good agreement; r = 0.86 for free DHEA, r = 0.92 for acid-hydrolyzed DHEA-S and r = 0.91 for solvolyzed DHEA-S. The present method is applicable in the routine determination of DHEA and DHEA-S in biological fluid.     

Enzyme immunoassay of pancreatic glucagon at the picogram level using beta-D-galactosidase as a label.

An enzyme immunoassay of pancreatic glucagon was established by using E. coli beta-D-galactosidease [EC 3.2.1.23] as a marker. In order to increase the sensitivity of the immunoassay, different peptides obtained from glucagon fragments were used to produce the enzyme conjugate and the immunogen. Antiserum N6E raised against C-terminal fragment peptide (15-29) could be diluted to more than 1 : 100,000 in the assay and was highly specific for pancreatic glucagon. The antiserum reacted well with the C-terminal fragment peptide (21-29) as well as another fragment peptide (15-29) and pancreatic glucagon. The enzyme immunoassay using antiserum N6E and fragment peptide (21-29)-enzyme conjugate could detect as little as 1 to 2 pg of glucagon. The mean recovery of glucagon added to serum specimens was 104% and the coefficients of variation were 3.7-14.5% (within assay) and 9.0-18.5% (between assay).     

The development of an indirect enzyme linked immunoassay for abscisic Acid

An indirect method of enzyme-linked-immunosorbent-assay (ELISA) is reported for abscisic acid (ABA), utilising a thyroglobulin-ABA conjugate for coating wells. The assay can use commercially available monoclonal antibodies, is sensitive to as little as 20 picograms ABA per well, and is much more conservative of antibody than direct methods. The most dilute ABA standards did not retain their antigenicity during storage, so ABA standard sets were diluted immediately prior to use. The indirect ELISA was used successfully to estimate ABA concentrations in developing cotyledons of Pisum sativum L., after only little preliminary purification. It was validated for this tissue through the use of gas chromatography-electron capture detection (GC-EC), and capillary GC-selected ion monitoring (GC-MS-SIM) using [²H₆]ABA as an internal standard. Full spectrum GC-mass spectrometry was also used to verify that ABA was present in a sample assayed quantitatively by both ELISA and GC-MS-SIM.