免疫标记技术的进展及纳米技术在其中的应用

酶免疫标记、胶体金免疫标记及荧光免疫标记技术是目前应用最广泛的免疫反应检测技术。随着科学技术，尤其是纳米技术的发展和生物医药等方面的需求，人们在提高检测灵敏度、简化操作步骤、有效缩短检测时间等方面，取得了很多可喜的进展。本文将分别针对酶免疫标记、胶体金免疫标记及荧光免疫标记技术三方面的进展情况，尤其是纳米标记技术在免疫反应检测中的应用进展进行综述。     

胶体金免疫层析技术在兽医学中的应用

胶体金免疫层析技术是一种固相免疫标记技术,具有操作简单、反应灵敏迅速、判定结果直观、无需特殊设备和专业人员等优点。胶体金检测技术一直以来在人类医疗领域中应用较为广泛,近年来该项技术也逐渐应用到兽医临床检测等方面中。目前在兽医领域内,该项技术已经在细菌性疾病、病毒性疾病及寄生虫性疾病等多方面都有应用。本综述对胶体金免疫层析技术进行简述,并对该项技术在兽医领域中的应用加以总结和展望,以期能为今后胶体金的相关研究提供参考。     

胶体金标记技术及应用

胶体金是继三大标记技术后对免疫标记技术的又一大贡献,近10年来在药物检测、生物医学等领域发展迅速。主要介绍了近年来胶体金标记技术在免疫光镜、免疫电镜及免疫层析分析上的研究进展。     

以膜为固相载体的免疫胶体金快速试验

免疫胶体金标记技术是现在免疫标记技术的一个重要范畴,20世纪80年代末期,人们把胶体金免疫技术与固相膜结合发展了胶体金免疫快速试验,由于其简便、快速、结果直观从而得到了广泛的发展和应用,且其商业前景广阔,目前人们对其需求越来越多。本文对其基本原理及其中应注意的问题做了一些介绍,并对其应用领域及发展方向进行了探讨。     

胶体金标记抗体技术

胶体金标记抗体技术是近十多年来免疫组织(或细胞)化学迅速发展起来的一项免疫标记技术。在国外许多研究领域中已经得到发展,并逐渐受到国内同行的重视。本文介绍了胶体金标记抗体技术的原理、方法及其发展概况。     

蓖麻毒素检测技术研究进展

蓖麻毒素是从蓖麻种子的胚乳中提取的一种核糖体失活蛋白。基于其潜在的威胁,建立快速、灵敏的蓖麻毒素检测技术受到人们的高度关注。根据蓖麻毒素的理化性质、免疫原性,已经建立了免疫荧光技术、夹心免疫PCR技术、免疫胶体金标记技术、蛋白芯片技术和生物传感器技术等用于检测蓖麻毒素。     

胶体金免疫层析试纸条技术在病毒检测领域的应用研究现状北大核心CSCD

胶体金免疫层析试纸条技术是一种快速、灵敏和精准的固相标记检测技术,胶体金免疫层析试纸条具有价格低廉、操作简便、检测快捷和特异性强的优点,具有在短时间内灵敏、准确地定性检测出相关病毒的潜在能力,有效解决传统检测方法在医学、兽医、动植物病毒检测和农药残留检测等领域存在检测时间长、设备不便和专业性强的弊端。目前在检测领域,该技术在检测细菌性疾病、病毒性疾病和预防传染性疾病大面积扩散等方面都有应用,因此,该技术在检验方面具有巨大的发展空间。文中主要对胶体金免疫层析技术进行综述,并对该技术在生物病毒检测方面进行总结和展望。     

胶体金免疫层析快速诊断技术

免疫胶体金技术是一种新的免疫学方法,该技术是一种将胶体金颗粒与包括抗原、抗体在内的许多蛋白质标记形成免疫金复合物的技术.目前在生物医学各领域得到了日益广泛的应用.在过去的十年里,基于膜上的快速诊断技术的发展为金标免疫试剂创造了巨大的市场.使用免疫金标技术制备的标记物作为检测系统中的指示试剂,应用在检测传染病、环境污染物、毒品、早孕以及兽医等领域.这两种技术的应用很大程度上提高了体外诊断技术的简便性和快速性.本文对其基本原理及其中应注意的问题做了一些介绍,并对其应用领域及发展方向进行了探讨.     

随机扩增多态性DNA原理及其在动物研究中的应用

RAPD(随机扩增多态性DNA)技术是一项应用日益广泛的遗传标记技术。与PCR和RFLP相比,该技术具有简便、快速、准确以及成本相对较低等特点,已成为众多分子标记技术中走向普及的先锋技术。阐述了RAPD的原理和方法,综述了RAPD目前在动物研究中的应用进展,并对RAPD技术应用前景作进一步阐述。     

胶体金免疫层析技术的研究进展

胶体金免疫层析技术是一种常用的免疫层析检测方法,由于其操作简单、省时、制造成本较低、结果易判读等特点,非常适合于现场检测,广泛用于生物、医药、食品等领域。但该技术也存在一些不足,如质量控制困难、灵敏度低、不易定量等,在一定程度上限制了其应用。本文,简述了该技术的原理、特点和国内外研究进展,重点综述了该方法的不足及改进的可能性,为拓宽其应用范围提供帮助。     

Autometallographic silver-enhancement of colloidal gold particles used to label phagocytic cells.

The present paper demonstrates that colloidal gold silver-enhanced by autometallography (AMG) can be used to label phagocytic cells for light microscopic detection. Cultured macrophages were exposed to 0.5 microliters 6 nm colloidal gold particles for 24 or 48 h. Other cultures were exposed to 25 microliters of the same solution for 1 to 14 days. The staining was found to be stable also when new unmarked cells were applied. The colloidal gold had no adverse effect on the cells. The presented technique might also prove valuable for estimation of the total number of phagocytes in a culture or in an organism by applying labelled cells to culture or organism, and to ascertain the fate of a population of marked cells.     

Application of colloidal gold to ultrastructural studies

Progress in recognition of structure and function in a variety of cells has always been linked to development of new research techniques. Beyond doubt, immunocytochemical techniques belong to such new methods. However, the techniques had not been widely applied to electron microscopy until colloidal gold was introduced as a label. This paper presents the pre-embedding and the post-embedding techniques and the applied markers, which are used in immunocytochemical techniques at the ultrastructural level. The potential of colloidal gold techniques is discussed. Particular attention is paid to methods of reaction amplification. Application of gold is illustrated with our own results.     

Adsorption of horseradish peroxidase, ovomucoid and anti-immunoglobulin to colloidal gold for the indirect detection of concanavalin A, wheat germ agglutinin and goat anti-human immunoglobulin G on cell surfaces at the electron microscopic level: a new method, theory and application.

A method is described for the adsorption of selected macromolecules to colloidal gold which is then used as an electron dense marker for the indirect detection of specific cell surface molecules. Membrane bound concanavalin A, which binds specific sugars on horseradish peroxidase, and wheat germ agglutinin, which binds specific sugars on ovomucoid are detected indirectly with gold labeled horseradish peroxidase and ovomucoid, respectively. Goat anti-human IgM on blood lymphocytes is detected with gold labeled rabbit anti-goat IgG. In the preparation of colloidal gold labeled proteins, the problems of flocculation of colloidal gold by proteins and nonadsorption of proteins to colloidal gold, are solved through a combination of concentration of protein and pH variable adsorption isotherms, which allows one to determine the conditions for adsorption of proteins to colloidal gold. Adsorption is pH dependent, the pH conditions correlating with the isoelectric point(s) of the major protein fraction(s); adsorption is influenced by interfacial tension, solubility and by the electrical charge on the molecules. Colloidal gold is inexpensive and preparation of a useful label is rapid, reproducible and the results easily quantitated from electron micrographs.     

Autometallographic silver-enhancement of colloidal gold particles used to label phagocytic cells

Summary The present paper demonstrates that colloidal gold silver-enhanced by autometallography (AMG) can be used to label phagocytic cells for light microscopic detection. Cultured macrophages were exposed to 0.5 l 6 nm colloidal gold particles for 24 or 48 h. Other cultures were exposed to 25 l of the same solution for 1 to 14 days. The staining was found to be stable also when new unmarked cells were applied. The colloidal gold had no adverse effect on the cells. The presented technique might also prove valuable for estimation of the total number of phagocytes in a culture or in an organism by applying labelled cells to culture or organism, and to ascertain the fate of a population of marked cells.     

Cationic colloidal gold--a new probe for the detection of anionic cell surface sites by electron microscopy

Particles of colloidal gold were coated with poly-L-lysine to prepare cationic colloidal gold. Monodispersed colloidal gold with a particle diameter of 5, 8, or 15 nm and poly-L-lysine with a molecular weight of 350,000 or 1500-8000 were used. The resulting complexes were used to label red blood cell membranes. The labeling was sensitive to neuraminidase treatment or acid hydrolysis, demonstrating that cationic colloidal gold binds preferentially to anionic cell surface constituents. Cationic colloidal gold can be used at physiological pH values and ionic strength, as well as at low pH values, making it a flexible probe for detection of anionic cellular components.     

Colloidal gold as an electrochemical label of streptavidin-biotin interaction

A new electrochemical method to monitor biotin-streptavidin interaction, based on the use of colloidal gold as an electrochemical label, is investigated. Biotinylated albumin is adsorbed on the pretreated surface of a carbon paste electrode (CPE). This modified electrode is immersed in colloidal gold-streptavidin labelled solutions. Adsorptive voltammetry is used to monitor colloidal gold bound to streptavidin, obtaining a good reproducibility of the analytical signal (R.S.D. = 3.3%). A linear relationship between peak current and streptavidin concentration from 2.5 x 10(-9) to 2.5 x 10(-5) M is obtained when a sequential competitive assay between streptavidin and colloidal gold-labelled streptavidin is carried out. On the other hand, the adsorption of streptavidin on the electrode surface was performed, followed by the reaction with biotinylated albumin labelled with colloidal gold. In this way, a linear relationship between peak current and colloidal gold labelled biotinylated albumin concentration is achieved with a limit of detection of 7.3 x 10(9) gold particles per ml (5.29 x 10(-9) M in biotin).     

The silver anniversary of gold: 25 years of the colloidal gold marker system for immunocytochemistry and histochemistry

Since 1971, when W.P. Faulk and G.M. Taylor published “An immunocolloid method for the electron microscope”, colloidal gold has become a very widely used marker in microscopy. It has been used to detect a huge range of cellular and extracellular constituents by in situ hybridization, immunogold, lectin-gold, and enzyme-gold labeling. Besides its use in light microscopic immunogold and lectin-gold silver staining, colloidal gold remains the label of choice for transmission electron microscopy studying thin sections, freeze-etch, and surface replicas, as well as for scanning electron microscopy. The year 1996 is the 25th anniversary of the introduction of colloidal gold as a marker in immunoelectron microscopy and this overview outlines some of the major milestones in the development of the colloidal gold marker system.     

Endocytosis by African trypanosomes. II. Occurrence in different life-cycle stages and intracellular sorting

Horseradish peroxidase (HRP) and colloidal gold-labeled proteins enter many of the endocytic organelles of bloodstream forms of Trypanosoma brucei and T. congolense. However, the colloidal gold markers were excluded from substantial parts of the pathway that contained HRP. Morphometric studies revealed that HRP entered organelles that accounted for approximately 5% of the total cell volume while transferrin-gold entered organelles that comprised approximately 2% of the total cell volume. In addition, large colloidal gold particles were excluded from organelles that contained smaller gold particles. Antibodies, raised against the variable surface glycoprotein, when applied to thawed cryosections were found to label structures from which endocytosed colloidal gold coupled to bovine serum albumin (BSA) was excluded. Endocytosis was shown to occur in two in vitro propagated forms of trypanosomes, similar to those found in the insect vector (Glossina spp.). The mammal-infective metacyclic forms were similar to bloodstream forms in that they endocytosed HRP and colloidal gold markers but excluded colloidal gold from approximately 3% of the endocytic organelles. Estimation of the flagellar pocket volumes of bloodstream form T. brucei showed that this organelle occupied 0.5% to 1.4% of the total cell volume. The flagellar pocket volume of T. congolense varied between life-cycle stages, with a fractional volume of 4.4% for bloodstream forms, 2.3% for metacyclic forms and 1.4% for procyclic forms. Endocytosis of HRP, but not of protein-gold markers, occurred in procyclic (uncoated) forms. Endocytosis by procyclic forms has heretofore not been reported.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunogenic Properties of Colloidal Gold

We studied the capacity of colloidal gold for enhancing specific and nonspecific immune response in laboratory animals (rabbits, rats, and mice) immunized with antigens of various nature. The antibody titers obtained with colloidal gold as a carrier were higher as compared to the standard immunization techniques (free antigen or its combination with Freund's adjuvant). Application of colloidal gold also enhanced nonspecific immune responses, such as lysozyme concentration in the blood, activity of the complement system proteins, as well as phagocytic and bactericidal activities. The antibodies were tested by immunodot assay using gold markers. Immunization of the animals with colloidal gold conjugates with haptens or complete antigens (without other adjuvants) was shown to induce the production of highly active antibodies. In addition, the amount of antigen used for animal immunization with colloidal gold was an order of magnitude lower, compared to immunization with complete Freund's adjuvant. This fact can be evidence for adjuvant properties of colloidal gold proper.     

Silver-enhanced colloidal gold as a cell surface marker for photoelectron microscopy

Colloidal gold labeling in conjunction with silver enhancement was investigated as a labeling technique for photoelectron microscopy (PEM). PEM uses UV-stimulated electron emission to image uncoated cell surfaces, and markers for cell surfaces need to be sufficiently photoemissive to be clearly visible against this background. Label contrast provided by 6 nm or 20 nm colloidal gold markers alone was compared to that provided by 6 nm markers after silver enhancement, using both direct and indirect labeling methods for fibronectin on human fibroblast cell surfaces. In all cases, details of the fibrillar fibronectin labeling distribution which were barely discernible before silver enhancement became highly visible against the cellular surface features. Two factors evidently contribute to the pronounced increase in label contrast with silver enhancement: (1) Increased particle size, which was documented by transmission electron microscopy, and (2) increased photoemission resulting from a silver coating on the enhanced gold markers, compared with the protein coating on the unenhanced gold markers. These data demonstrate that silver enhancement of colloidal gold labeling patterns in PEM images is a highly effective method for localization of specific sites on cell surfaces.