The biodistribution and kinetics of the Samarium-153 labeled avidin,streptavidin and biotin

Objective:To labelavidin(Av)or streptavidin(SA)with 153 Sm by takingadvantageof thehighbindingaffin-ityof biotinto Av or SA.Methods:A biotinderivative(DTPA-biotin)wasradiolabelledwith 153 Sm andthenboundto Av or SA.Thein vivo kineticsandbiodistributionof 153 Sm-labeledAv,SA andDTPA-biotinwerestudiedinratsandmice.Results:153 Sm-Avwascharacterizedby rapidclearancefromthebloodwithhighliverandrenaluptake;153 Sm-SAwas clearedfromthebloodslowlywithhighretentionintheliver,spleenandkidney,whereas 153 Sm-DTPA-biotinmetabolismwas accelerated,anditsexcretionwasmainlythroughthekidney.Conclu sion:Thebiodistributiondifferenceof SAandAvmay providean experimentalbasisfor theselectionof differentcomponentsof avidin-biotinsystemin pretagetingradioim-munoimagingandradioimmunotherapy.

Adjunction of Avidin to a Cysteamine Self-Assembled Monolayer for Impedimetric Immunosensor

In this work an impedimetric immunosensor based on affinity immobilization method of a biotin labelled anti-human IgG antibody, used as a model system, was reported. The experimental procedure involves the growth of a self-assembled monolayer of a thiol (cysteamine) carrying terminal amine groups on gold electrodes. Glutarardehyde, a homobifunctional cross-linker, was used as a coupling reagent for the covalent linking of avidin to the amine groups of cysteamine. The attachment of the biotin labeled antibodies (anti-Human IgG) to the subsequent modified gold electrode was achieved by affinity interactions tacking advantage of the strong avidin-biotin bridge. The stepwise assembly process of the electrode was interrogated by means of cyclic voltammetry, electrochemical impedance spectroscopy and contact angle measurements. The response of the antibody modified electrode to their target antigens was investigated in the presence of BSA (bovin serum albumin) in order to alleviate non-specific adsorption problems. A proposed electrical model was used to analyse the experimental data. The resulting immunosensor has a linear dynamic range of 100 - 900 ng?ml–1 of antigen and a detection limit of 100 ng?ml–1.

鸡蛋清粉制品残存抗生物素蛋白(Avidin)的分离和分析

对市售冲饮用的鸡蛋清粉中残存的抗生物素蛋白进行了分离和鉴定,结果表明鸡蛋清粉中仍具有一定比活性10U/mg)的抗生物素蛋白,含量达0．040~0．055 mg/g。

Avidin-biotin system pretargeting radioimmunoimaging and radioimmunotherapy and its application in mouse model of human colon carcinoma

AIM: To evaluate the multi-step pretargeting radioimmunoimaging (RII) and radioimmunotherapy (RIT) in nude mice bearing human colon carcinoma with avidin-biotin system labeled with 153Sm.METHODS: Two- and three-step strategies for avidinbiotin system pretargeting techniques were established.In a three-step procedure, human colon carcinoma bearing nude mice were first injected with biotinylated monoclonal antibody (McAb-Bt) followed by cold avidin (Av) 48 h later and then 153Sm-DB2 24 h thereafter;whereas the twostep procedure consisted of injection of 153Sm-SA 48 h after pretargeting with biotinylated anti-CEA monoclonal antibody (CEA McAb-Bt). SPECT imaging and biodistribution were performed at 4, 24, 48, or 72 h after injection of 153Sm-labeled compounds. Five groups of nude mice subcutaneously grafted with human colon carcinoma were treated 3 d after grafting. One group received the injection with 100 μg CEA McAb-Bt followed by cold avidin (80 μg)after 2 d and 11.1 MBq 153Sm-DB2 after 1d. Four control groups were treated respectively with 11.1 MBq 153SmCEA McAb, 11.1 MBq 153Sm-nmIgG, 11.1 MBq 153Sm-DB2,100 Μl normal saline. Toxicity was evaluated by changes of leukocyte count, and the efficacy by variation in tumor volume. Histological analyses of tumors were performed.RESULTS: The three-step procedure allowed faster blood clearance and yielded higher tumor blood ratios (5.76 at4 h and 12.94 at 24 h) of the 153Sm-DB2. The tumor was clearly visualized at 4 h in y-imaging after the injection of 153Sm-DB2, while a significant accumulation of 153Sm-SA in the tumor was observed only 24 h after the injection and tumor blood ratios at 4 and 24 h were 1.00 and 2.03,respectively, in the two-step procedure. Pretargeting RIT and 153Sm-CEA McAb had a strong tumor-inhibiting effect.The tumor inhibitory rate was 80.67% and 78.44%,respectively, five weeks after therapy. Histopathological evidence also indicated radioactive damage in tumor tissues as necrosis of tumor cells, while in the other organs such as liver and kidney no radioactive damage was observed. Leukocyte counts showed significant decrease after treatment in groups of 153Sm-CEA Mc Ab and 153SmnmIgG.CONCLUSION: The two kinds of pretargeting strategies can elevate the target-to-nontarget ratio, decrease the blood background and shorten the imaging time compared to 153Sm-CEA McAb. Three-step pretargeting RIT is as efficient as 153Sm-CEA Mc Ab, but markedly less toxic. This study provides experimental evidence for the clinical application of pretargeting RII and RIT.

IgG-Biotin-Avidin-HRP信号转导探针结合免疫比色法快速检测克罗诺阪崎肠杆菌

本文建立了一种快速检测克罗诺阪崎肠杆菌的新型免疫比色法。用抗体修饰的磁球(MNPs-IgG)作为捕获探针分离、富集克罗诺阪崎肠杆菌,再通过免疫夹心法分别结合生物素标记的克罗诺阪崎肠杆菌抗体(Biotin-IgG)和亲和素标记的辣根过氧化物酶(Avidin-HRP),形成的复合物与底物四甲基联苯胺(3,3′,5,5′-Tetramethylbenzidine,TMB)发生显色反应,经硫酸终止后,有色产物的吸光度值与克罗诺阪崎肠杆菌浓度的对数有良好的线性关系,在103~107CFU/mL范围内,线性方程为y=0.2353x-0.2307。本研究成功设计优化了基于比色法的克罗诺阪崎肠杆菌定量检测新方法,检测限为8.68×10^2CFU/mL(S/N=3),加标样品检出率96%,检测时间不超过2.5 h,为实际应用与商品化提供了理论支撑。

Avidin chase reduces side effects of radioimmunotherapy in nude mice bearing human colon carcinoma

AIM: To evaluate the influence of avidin chase on the side effects of radioimmunotherapy (RIT) in nude mice bearing human colon carcinoma and therapeutic outcome.METHODS: Purified anti-CEA monoclonal antibody (McAb)was biotinylated with NHS-biotin, and then radiolabeled with 188Re by the direct method. 188Re-labeledbiotinylated anti-CEA McAb (188Re-CEA McAb-Bt) was intravenously injected followed by intravenous injection of avidin after 24 h. SPECT imaging and biodistribution study were performed at 28-48 h after the injection of 188Re-CEA McAb-Bt. Three groups of nude mice subcutaneously grafted with human colon carcinoma were treated 7 d after the graft. Mice in the avidin chase group received intravenous injection of 188Re-CEA McAb-Bt (11.1 MBq/20 μg) followed by intravenous injection of cold avidin (80 μg) after 24 h. Mice in the control group (treated group without avidin chase) only received the injection of 188Re-CEA McAb-Bt (11.1 MBq/20 μg), another control group (non-treated group) only received 0.1 mL normal saline solution. Toxicity was evaluated on the basis of change of body weight and peripheral WBC counts, and therapy effects were determined by variation in tumor volume. Histological analysis of tumors was also performed.RESULTS: Avidin chase markedly accelerated the clearance of 188Re-CEA McAb-Bt from the blood and normal tissues. The tumor uptakes of 188Re-CEA Mc Ab-Bt at 28 h were 5.90 and 6.42% ID/g, respectively, in chase group and in non-chase group, while the tumor-to-background (T/NT) ratios were 3.19 and 0.56, respectively. The tumor uptake was slightly decreased by avidin chase, but the T/NT ratios were increased. In treated groups the growth rate of body weight and the number of WBC decreased after injection of 188Re-CEA McAb-Bt, and the WBC counts recovered earlier in the group with avidin chase than in the group without avidin chase. Compared to the nontreated group, treated groups with and without avidin chase showed significant anti-tumor effects.CONCLUSION: Avidin chase can effectively reduce the side effects of RIT, and improve therapeutic efficacy.

基于衍生化3-氨基-2-恶唑烷酮的夹心酶联免疫分析

建立一种3-氨基-2-恶唑烷酮(3-amino-2-oxazolidinone,AOZ)夹心免疫检测方法。通过1,6-己二醇连接2-硝基-4-羧基苯甲醛和生物素合成针对AOZ的新型衍生试剂。在样品前处理时加入衍生试剂可对AOZ进行衍生,衍生产率为89%。在酶标板上包被抗AOZ单克隆抗体并以辣根过氧化物酶标记的亲和素或抗生物素抗体作为第二结合物可实现AOZ的夹心酶联免疫吸附检测(enzyme-linked immunosorbnent assay,ELISA)。从实际应用的角度,得到双抗夹心和抗体-亲和素夹心模式下两个表位的极限距离,分别为12Å和13Å,理想距离分别为16Å和17Å。在双抗夹心和抗体-亲和素夹心模式下的检出限分别达到1.8 pg/mL和0.8 pg/mL(以AOZ质量浓度计),相对于竞争ELISA,其灵敏度最高提高了25倍。平均回收率为73%~85%,平均相对标准偏差为9.0%。

Preparation of Surface-enhanced Raman Scattering（SERS）-active Optical Fiber Sensor by Laser-induced Ag Deposition and Its Application in Bioidentification of Biotin/Avidin

A surface-enhanced Raman scattering（SERS） optical fiber sensor was prepared by the laser-induced deposition ofAg nanoparticle membrane on a silica optical fiber tip, which was applied to the real time SERS spectral monitoring on the biorecognition of biotin/avidin. The bioidentification of biotin/avidin was carried out through a indirect method, in which the bioidentification is based on the SERS response signal of a labeled dye（Atto610） after its fluorescence has been quenched totally by the deposited Ag nanoparticle membrane. By SERS monitoring the bioidentification process of biotin/avidin, it has been found that this recognition process is finished in 40 min. The lowest detection concentration of biotin is 1.0 × 10^-7 mg/mL. This research is promising in the application of immunoassays on line and in vivo.

Study on Interaction of Avidin With Biotin Lipid Monolayer

1 Introduction X-ray diffraction is the major powerful method to resolve the problems on biological macromolecules in detail, but the large single crystals needed for analysis are usually.difficult to obtain. In contrast to forming three-dimensional(3D) crystals, many proteins can form two-dimensional (2D) crystals easily. There have been several approaches to making 2D crystals. Lipid layer crystallization is a novel 2D crystallization method developed by Kornberg group. Some water-soluble proteins have been crystallized successfully by this method, and it can be possibly used to crystallize some intrinsic membrane proteins. The formed 2D crystals are

Thermoresponsive dendronized copolymers for protein recognitions based on biotin-avidin interaction

Thermoresponsive biotinylated dendronized copolymers carrying dendritic oligoethylene glycol（OEG）pendants were prepared via free radical polymerization,and their protein recognitions based on biotin-avidin interaction investigated.Both first（PG1） and second generation（PG2） dendronized copolymers were designed to examine possible thickness effects on the interaction between biotin and avidin.Inherited from the outstanding thermoresponsive properties from OEG dendrons,these biotinylated cylindrical copolymers show characteristic thermoresponsive behavior which provides an envelope to capture avidin through switching temperatures above or below their phase transition temperatures（T_（cp）s）.Thus,the recognition of polymer-supported biotin with avidin was investigated with UV/vis spectroscopy and dynamic laser light scattering.In contrast to the case for PG1,the increased thickness for copolymer PG2 hinders partially and inhibits the recognition of biotin moieties with avidin either below or above its T_（cp）.This demonstrates the significant architecture effects from dendronized polymers on the biotin moieties to shift onto periphery of the collapsed aggregates,which should be a prerequisite for protein recognition.These kinds of novel thermoresponsive copolymers may pave a way for the interesting biological applications in areas such as reversible activity control of enzyme or proteins,and for controlled delivery of drugs or genes.

Avidin-dendrimer对Hela细胞的靶向研究

树状大分子对肿瘤有显著的EPR效应,本文在5代聚酰胺-胺类树状大分子表面连接对肿瘤有主动靶向作用的抗生物素蛋白(亲和素),然后用放射性同位素99mTc标记这种兼具被动靶向与主动靶向作用的纳米粒子。Hela细胞体外摄取实验表明连接靶向分子亲和素(Avidin)的Biotin-PAMAM G5-1B4M-99mTc纳米复合物对Hela细胞的摄取远高于未连接靶向分子Avidin的同类纳米粒子。如进一步在小鼠SPECT成像试验中表现出良好的显像效果,这种同位素标记的纳米粒子将有望用于肿瘤的早期诊断或治疗。

基因传感器表面两种探针固定法的比较

目的 比较基因传感器表面两种探针固定法的优劣。方法 分别用巯基固定法及生物素 亲和素固定法将人类乳头瘤病毒 (HumanPapillomaVirus,HPV)的探针固定在基因传感器的金膜表面 ,比较不同探针浓度、不同靶序列浓度下反应所引起的频率的变化以及所用的时间。结果 两种固定方法检测靶序列的灵敏度相当。生物素 亲和素法反应所需的时间较短 ,且具有放大效应 ,能结合上更多的探针并检测到更多的靶序列 ,但操作较为繁琐。巯基法操作较为简单 ,但所需的反应时间较长。结论 两种探针固定法各有利弊 ,实际操作中应根据不同的需要选择合适的固定方法。

夹心免疫PCR方法检测蓖麻毒素

目的 建立了双抗夹心免疫PCR的检测技术,检测极微量的抗原。方法 以亲和素作为连接分子连接生物素化抗体和生物素化DNA,应用于免疫PCR中,检测极微量的抗原。结果 建立了双抗夹心兔疫PCR的检测技术,检测蓖麻毒素的灵敏度达0.1pg/mL,与夹心ELISA方法比较灵敏度高103。结论 建立的免疫PCR系统灵敏度高,可用于各种微量抗原的检测。

生物素-亲和素放大酶联免疫吸附法测定双酚A

建立了可用于快速、灵敏地检测双酚A的生物素-亲和素放大酶联免疫吸附测定法,测得最佳实验条件为包被抗原浓度为13.8 mg/L、抗体稀释为2.4×105倍,生物素化二抗和酶标亲和素的最佳稀释倍数分别为2000倍和500倍.在优化条件下,方法的线性范围0.2～1000 μg/L;最低检出限0.05 μg/L.所建立的方法用于食品和唾液中双酚A含量的测定,样品处理简单,结果满意.

生物素化壳聚糖微球的体外抗癌活性

目的 研究一套有预定位及肿瘤导向作用的药物载体系统。方法 以生物素化无唾液酸胎球蛋白为分子识别系统 ,与肝癌细胞特异性结合 ,将包封有 5 Fu的壳聚糖微球生物素化 ,作为药物载体 ,通过生物素 亲和素系统将二者桥连 ,用“三步法”研究微球体外抗肿瘤作用。结果 亲和素 生物素化受体介导的生物素化壳聚糖微球能与体外培养的肝癌细胞特异结合并有显著的抗癌作用。结论 生物素化微球 亲和素 生物素化受体 (或单克隆抗体 )系统可望在体内有预定位及肿瘤靶向作用 ,有广泛的应用前景。

微生物发酵液中生物素含量的ELISA测定

生物素与亲和素具有很强的亲和力，本文利用这一特性建立了一种直接竞争抑制的酶联免疫法（ＥＬＩＳＡ），用于检测微生物发酵液中生物素含量．当标准品生物素含量在０．５～２０μｍｏｌ／Ｌ范围内，它与抑制率（Ｉ）负对数呈线性关系，因此发酵液样品中生物素含量可从标准曲线上查得。该法简便、准确，而且不需对样品作任何预处理。

亲和素-生物素间接偶联的压电DNA传感器研究

采用 33′ 二巯基硫代丙酸的金电极自组装技术 ,用乙基 3 (3 二甲氨基 )碳二亚胺盐酸盐 (EDC)和N 羟基磺基琥珀酰亚胺 (NHS)偶联剂将亲和素固定于金电极上 ,联于生物素标记的探针 ,制备成压电DNA传感器的检测电极 ,和杂交液中的待检葡萄球菌肠毒素B的ssDNA进行杂交 ,通过频率信号检测DNA杂交的量 ,达到检测的目的 .采用不同长度的基因片段进行了研究 ,制作的传感器一致性、特异性都较好 ;杂交后的电极 ,电极再生后 。

铂电极表面生物素-亲和素固载单链脱氧核糖核酸的电化学传感器

通过直接吸附将亲和素固定在Pt电极表面,联于生物素标记的脱氧核糖核酸(DNA)探针,制备了电化学基因传感器,建立了Pt电极表面修饰单链脱氧核糖核酸(ssDNA)的方法。修饰电极与待测溶液中人工合成的转基因食品中常有的花椰菜花叶病毒35S启动子(CaMV35S)或根癌农杆菌终止子(NOS)DNA片段进行杂交,以邻菲罗林钴络合物[Co(phen)3+3]为杂交指示剂,循环伏安法测量,通过杂交前后指示剂峰电流的变化检测DNA杂交的量。研究了电极修饰、杂交反应及测量的适宜条件,在优化实验条件下,峰电流的差值与DNA杂交量之间有良好的线性关系,相关系数r=0.9996。杂交后的电极经热变性再生,可重复使用多次。

人血清癌胚抗原的生物素-亲和素ELISA检测方法的建立

目的:建立检测血清癌胚抗原(CEA)的高灵敏度生物素-亲和素酶联免疫检测(BA-ELISA)方法。方法:亲和层析纯化得到CEA,免疫新西兰家兔制备多克隆抗体。将得到的抗体连接生物素和辣根过氧化物酶,在生物素化BSA包被的96微孔板上建立生物素-亲和素ELISA(BA-ELISA)。对这一体系的线性、灵敏度、特异度、稳定性、回收率等参数进行鉴定,并和常规ELISA、放射免疫检测以及化学发光法相比较检测了临床标本中的CEA浓度。结果:线性检测范围为(0.42～50)U/mL,检测结果表明,体系稳定性良好;灵敏度为0.42 U/mL,批内、批间差异均小于10.0%。该研究建立的方法与常规ELISA对比差异有统计学意义,与放射免疫检测对比差别无统计学意义。与化学发光法相比较,回归方程为:y=0.04825+0.99674x,r=0.994,差异无统计学意义。结论:建立的CEA的BA-ELISA检测方法易于操作、灵敏度高、价格低廉,适合应用于临床检测。

鼠抗人sIL-6R单克隆抗体的制备及sIL-6R检测方法的建立

以重组人ｓＩＬ－６Ｒ蛋白作为抗原，获得３株分泌特异性ＭｃＡｂ的杂交瘤株，分别命名为ＳＩ１０，ＳＩ１１和ＳＩ１２。３株ＭｃＡｂ均属小鼠ＩｇＧ１亚类。竞争抑制试验的结果表明，３株ＭｃＡｂ识别两个不同的抗原表位。将杂交瘤细胞注入同系小鼠腹腔诱生的ＭｃＡｂ腹水，经ｐｒｏｔｅｉｎＧ纯化后用生物素标记，建立了双ＭｃＡｂ夹心ＥＬＩＳＡ法，该法用于血清ｓＩＬ－６Ｒ的特异性检测，灵敏度为５０μｇ／Ｌ。