Hutchens T W, Magnuson J S, Yip T T
Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030.
Pediatr Res. 1989 Dec;26(6):623-8. doi: 10.1203/00006450-198912000-00022.
Investigations into the mechanisms by which Ig in human colostrum influence the development and maturation of both the gastrointestinal and the immune systems of human milk-fed term and preterm infants have been restricted by the paucity of purified human milk Ig. We have developed a simple adsorption procedure for the selective removal and quantitative recovery (95-100%) of intact Ig (secretory IgA, IgG, and IgM) present in human colostral whey. The procedure exploits the rapid, ionic-strength dependent, thiophilic adsorption of Ig during a single pass of colostral whey through a column of beaded agarose with immobilized thioether-sulfone ligands (Anal Biochem 1986;159:217-226). The purity and composition of the adsorbed Ig were verified by SDS-PAGE and sensitive silver-staining protein detection procedures; proteins of approximately 78-80 kD (secretory component), 50-60 kD (heavy chain), and 25 kD (light chain) were observed. The identity, structural integrity, and relative concentrations of the recovered Ig were confirmed by high-performance size-exclusion chromatography, Ouchterlony immunodiffusion, rocket immunoelectrophoresis and ELISA. These results were analyzed and compared with reported values for the concentration of human milk Ig. Thus, the use of thiophilic adsorption appears to facilitate 1) selective removal of Ig from colostrum, enabling the evaluation of remaining components for growth- and immune-potentiating properties, and 2) selective immobilization and recovery of Ig from colostrum under conditions consistent with preserved biologic activity.
由于纯化的人乳免疫球蛋白（Ig）匮乏，关于人初乳中Ig影响母乳喂养的足月儿和早产儿胃肠道及免疫系统发育与成熟机制的研究受到了限制。我们开发了一种简单的吸附程序，用于选择性去除和定量回收（95 - 100%）人初乳乳清中存在的完整Ig（分泌型IgA、IgG和IgM）。该程序利用了初乳乳清单次通过装有固定化硫醚砜配体的珠状琼脂糖柱时，Ig快速的、离子强度依赖性的嗜硫吸附作用（《分析生物化学》1986年；159：217 - 226）。通过SDS - PAGE和灵敏的银染蛋白质检测程序验证了吸附的Ig的纯度和组成；观察到了分子量约为78 - 80 kD（分泌成分）、50 - 60 kD（重链）和25 kD（轻链）的蛋白质。通过高效尺寸排阻色谱、双向免疫扩散、火箭免疫电泳和ELISA确认了回收的Ig的同一性、结构完整性和相对浓度。对这些结果进行了分析，并与报道的人乳Ig浓度值进行了比较。因此，嗜硫吸附的应用似乎有助于：1）从初乳中选择性去除Ig，从而能够评估其余成分的生长和免疫增强特性；2）在与保留生物活性一致的条件下，从初乳中选择性固定和回收Ig。
