Assessment of estrogen receptor-monoclonal antibody interaction by high-performance liquid chromatography

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.