Covalently bound antibody on polystyrene latex beads: Formation, stability, and use in analyses of white blood cell populations
Covalently bound antibody on polystyrene latex beads: Formation, stability, and use in analyses of white blood cell populations
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DOI：
10.1006/jcis.2000.7279
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发表时间：
2001-02-01
影响因子：
9.9
通讯作者：
Insausti, ME
中科院分区：
文献类型：
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作者：
Siiman, O;Burshteyn, A;Insausti, ME
通过活化的CD4或CD8单克隆抗体与直径为2微米、1,3-二氨基丙烷（DAP）偶联的聚苯乙烯醛/硫酸酯乳胶珠反应，将CD4或CD8抗体与乳胶珠共价结合。对未反应抗体的抗体-头偶联混合物滤液进行分光光度分析，构建抗体与聚苯乙烯头表面的结合曲线，并评估抗体与头的结合常数，CD4和CD8抗体的结合常数范围为1.5 × 10(7)至1.6 × 107 M-l。抗体巯基与活化的马来酰亚胺基在pH 7.2-7.3的条件下在10-15 min内完成反应。CD4或CD8单克隆抗体从共价偶联抗体-聚苯乙烯乳胶珠表面位移的动力学是作为温度（5度，22度和37度）和抗体包被珠的最终稀释液性质的函数，通过ELISA（酶联免疫吸附试验）测量偶联珠滤液中的抗体浓度。位移反应表现出伪零级速率依赖关系，常数k(1)在0.65 × 10（-17）到270 × 10(-17) M s（-1）之间。在全血生物细胞试验中，还监测了悬浮在各种介质中的抗体包被珠的功能。细胞测定依赖于在靶淋巴细胞周围形成一层珠，通过de或rf电导率或中角光散射的差异来区分它们与非靶淋巴细胞。在5度、22度和37度的培养液中储存16周的共价结合的CD4和CD8抗体珠在不同献血者的STKS试验中显示出优异的结果，与流式细胞术中荧光标记的参考结果具有良好的相关性（CD4数据的相关系数为0.99，CD8数据的相关系数为0.93）。在5-37℃的BSA缓冲液中储存2周的共价结合的CD4/CD8微球在提供CD4或CD8阳性细胞在总白细胞群中所占百分比的准确值方面表现得同样好，而在47-50℃的范围内储存的相同微球表现出一些性能上的失败。与吸附抗体头悬浮液滤液中的抗体浓度比较，在相当的时间、介质和温度条件下，游离抗体的量增加了2至10倍。游离抗体的阈值必须达到1-2马克杯/毫升，才会对生物细胞试验产生明显的不良影响。(C) 2001学术出版社。
CD4 or CD8 antibodies were covalently bound to latex beads by reaction of activated CD4 or CD8 monoclonal antibodies with 2-mum-diameter, 1,3-diaminopropane (DAP) coupled, polystyrene aldehyde/sulfate latex beads. Spectrophotometric analyses of the filtrates of the antibody-bead conjugation mixtures for unreacted antibody allowed construction of binding curves of antibody for the polystyrene bead surface and evaluation of binding constants for association of antibody with bead, ranging from 1.5 x 10(7) to 1.6 x 107 M-l for CD4 and CD8 antibodies. The reaction of the antibody thiol group with the activated maleimide group on the bead at pH 7.2-7.3 was complete within 10-15 min. The kinetics of CD4 or CD8 monoclonal antibody displacement from the surface of covalently conjugated antibody-polystyrene latex beads was followed as a function of temperature (5, 22, and 37 degreesC) and the nature of the final diluent for the antibody-coated beads by measuring the concentration of antibody in the filtrates of conjugated beads by an ELISA (enzyme-linked immunosorbent assay). The displacement reaction showed a pseudo-zero-order dependence of the rate, with constants, k(1), ranging from 0.65 x 10(-17) to 270 x 10(-17) M s(-1). The functionality of antibody-coated beads suspended in various media was also monitored in a biological cell assay with whole blood. The cell assay depends on forming a layer of beads around targeted lymphocytes to distinguish them from nontargeted Lymphocytes by differences in de or rf conductivity or median angle light scatter. Covalently bound CD4 and CD8 antibody beads stored in one set of media at 5, 22, and 37 degreesC over a period of 16 weeks showed excellent results in the STKS assay with various blood donors, which correlated well (correlation coefficients of 0.99 for CD4 data and 0.93 for CD8 data) with reference results obtained with fluorescent markers by flow cytometry. Covalently bound CD4/CD8 beads stored for 2 weeks in BSA buffer at 5-37 degreesC performed equally well in providing accurate values of the percentage of CD4- or CDS-positive cells in the total white blood cell population, whereas the same beads stored in the 47-50 degreesC range showed some failures in performance. Comparison with antibody concentrations in filtrates of adsorbed antibody-bead suspensions showed 2- to 10-fold greater amounts of free antibody at comparable elapsed time, media, and temperature conditions. A threshold of 1-2 mug/mL of free antibody was necessary before adverse effects on the biological cell assay were noticeable. (C) 2001 Academic Press.