The use of glutaraldehyde-treated erythrocytes for assaying the agglutinating activity of lectins.
The use of glutaraldehyde-treated erythrocytes for assaying the agglutinating activity of lectins.
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DOI：
10.1016/0003-2697(75)90663-6
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发表时间：
1975-10
影响因子：
2.9
通讯作者：
Richard H. Turner;Irvin E. Liener
中科院分区：
文献类型：
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作者：
Richard H. Turner;Irvin E. Liener
考虑到目前对使用植物凝集素或凝集素作为探测细胞表面结构的工具的兴趣(1)，对这类蛋白质活性的定量测量的关注如此之少确实令人惊讶。大多数研究人员似乎满足于通过常规的连续两倍稀释和目测凝集程度的方法来评估其制剂对各种类型细胞的凝集活性(2)。为了克服这种技术固有的不准确性，人们尝试了光度测定法(3)、光散射测量法(4)、细胞计数法(1)和自动分析仪系统的使用(6)。无论采用哪一种方法，最麻烦的特点是必须使用新鲜血细胞，因为在储存过程中细胞的劣化会显著影响检测的可重复性（3,7）。本通讯的目的是描述一种稳定红细胞的简单方法，该方法不仅可以提高其灵敏度，而且允许其长时间储存，而不会明显影响测定的可重复性。按照Lis和Sharon(7)的描述，将全血胰蛋白酶化并用生理盐水（0.9% NaCl）洗涤。然后将细胞悬浮在足够的生理盐水中，使其在l-cm比皿中在620 mm处的吸光度为1。在此悬浮液中加入一定量的实用级戊二醛（伊士曼，25%的水），轻轻搅拌，使终浓度为0.1%（v/v）。15分钟后固定完成，细胞现在应该完全抵抗低渗休克和机械损伤。必须注意避免过量的戊二醛，因为过量的戊二醛可能由于细胞间交联而导致细胞结块。固定后，过量的戊二醛可通过广泛的透析或用生理盐水反复洗涤离心后的细胞来去除。然后将细胞悬浮液储存在冰箱中，并在需要时直接用于所选择的分析系统。我们选择了本实验室开发的光度测定法(3)来说明戊二醛处理的兔红细胞用于测量大豆凝集素活性的有效性（8,9）。该技术测量在620nm处红细胞悬浮液吸光度的下降，这是由凝集素-的沉降引起的
Considering the current interest in the use of phytoagglutinins or lectins as a tool for probing the structure of cell surfaces (l), it is indeed surprising that so little attention is paid to the quantitative measurement of the activity of this class of proteins. Most investigators seem to be content to evaluate the agglutinating activity of their preparations toward various types of cells by the conventional procedure of serial twofold dilution with visual estimation of the degree of agglutination (2). Attempts to overcome the inherent inaccuracy of this technique have included a photometric assay (3), light scattering measurements (4), cell counting (I?), and the use of the autoanalyzer system (6). Regardless of which of these methods is employed, the most troublesome feature is the necessity for using fresh blood cells since deterioration of the cells during storage markedly affect the reproducibility of the assay (3, 7). The purpose of this communication is to describe a simple method for stabilizing red blood cells which not only enhances their sensitivity but also permits their storage for long periods of time without appreciably affecting the reproducibility of the assay. Whole blood is trypsinated and washed with saline (0.9% NaCl) as described by Lis and Sharon (7). The cells are then suspended in sufficient saline to give an absorbance of 1 at 620 mm in a l-cm cuvette. To this suspension is added with gentle stirring a volume of practical grade glutaraldehyde (Eastman, 25% in water) to give a final concentration of 0.1%(v/v). After lo-15 min fixation is complete, and the cells should now be fully resistant to hypotonic shock and mechanical injury. Care must be taken to avoid an excess of glutaraldehyde which can cause a clumping of the cells presumably due to intercellular cross-linking. Following fixation excess glutaraldehyde may be removed by extensive dialysis or by repeated washing of the centrifuged cells with saline. The suspension of cells may then be stored in the refrigerator and used directly when needed in the assay system of choice. We have chosen the photometric assay developed in this laboratory (3) to illustrate the usefulness of glutaraldehyde-treated rabbit erythrocytes for measuring the activity of the soybean agglutinin (8, 9). This technique measures the decrease in absorbancy at 620 nm of a suspension of erythrocytes which results from the sedimentation of lectin-in-