Brandt D W, Wittliff J L
Department of Biochemistry, University of Louisville, KY 40292.
J Chromatogr. 1987 Jun 26;397:287-97. doi: 10.1016/s0021-9673(01)85011-1.
High performance liquid chromatography (HPLC) was employed as a means of analyzing estrogen receptor (ER)-antibody recognition. This technique takes advantage of the fact that the majority of gamma-globulin-antigen complexes do not interact with the anion-exchange resins selected. A variety of monoclonal (MAb) and polyclonal antibodies (PAb) raised against ER and ER-associated proteins were assessed for their chromatographic behaviour after association with charged ER, based upon properties of size, shape, and surface charge. ER exhibits polymorphism, several isoforms being present in target cells. The monoclonal antibody H222Sp gamma demonstrated discrete specificity for the 150 mM ER isoform (normally eluting at 150 mM phosphate) from the high-performance ion-exchange chromatography column which was eluted unretained when complexed with antibody. However, the monoclonal reagent D547Sp gamma interacted directly with anion-exchange columns (SynChropak AX-1000 and Altex DEAE-5PW), complicating a clear evaluation of ER-MAb association. Only 50-60% of the 150 mM ER isoform was eluted at a lower salt concentration. Few conclusions could be drawn with respect to MAb interaction with the 50-60 mM ER isoform (normally eluting at 50-60 mM phosphate) since the antibody-receptor complex was also eluted at the same phosphate concentration. In addition, polyclonal and monoclonal antibodies to the ER and ER-associated proteins were assessed by HPLC. At present, heat shock proteins and protein kinase activity have been shown by other techniques to be associated with the ER. Size-exclusion resins, such as TSK 3000 SW, were employed in a fast method of determining ER isoform-antibody recognition. Thus, HPLC may be used to analyze soluble antibody-antigen interactions rapidly, with high recovery of biological activity.
高效液相色谱法（HPLC）被用作分析雌激素受体（ER）-抗体识别的一种手段。该技术利用了大多数γ-球蛋白-抗原复合物不与所选阴离子交换树脂相互作用这一事实。基于大小、形状和表面电荷特性，对多种针对ER和ER相关蛋白产生的单克隆抗体（MAb）和多克隆抗体（PAb）与带电ER结合后的色谱行为进行了评估。ER表现出多态性，在靶细胞中存在几种异构体。单克隆抗体H222Spγ对来自高效离子交换色谱柱的150 mM ER异构体（通常在150 mM磷酸盐中洗脱）表现出离散特异性，当与抗体复合时该异构体未保留地被洗脱。然而，单克隆试剂D547Spγ直接与阴离子交换柱（SynChropak AX - 1000和Altex DEAE - 5PW）相互作用，使得对ER - MAb结合的清晰评估变得复杂。只有50 - 60%的150 mM ER异构体在较低盐浓度下被洗脱。关于MAb与50 - 60 mM ER异构体（通常在50 - 60 mM磷酸盐中洗脱）的相互作用几乎无法得出结论，因为抗体 - 受体复合物也在相同的磷酸盐浓度下被洗脱。此外，通过HPLC对ER和ER相关蛋白的多克隆和单克隆抗体进行了评估。目前，其他技术已表明热休克蛋白和蛋白激酶活性与ER相关。尺寸排阻树脂，如TSK 3000 SW，被用于一种快速测定ER异构体 - 抗体识别的方法中。因此，HPLC可用于快速分析可溶性抗体 - 抗原相互作用，生物活性回收率高。
