Screening for hybridomas producing antibodies to steroid hormone receptors: interference from phenol red in the hybridoma culture supernates.

The culture supernates from a variety of mouse hybridomas which were known to secrete antibodies to antigens unrelated to steroid hormone receptors were examined for routine use as experimental controls for monoclonal anti-oestrogen receptor (ER) antibodies. During this process, we made an observation which is important for all investigations on monoclonal anti-receptor antibodies. Our results indicated that the phenol red dye present in these hybridoma culture fluids by binding to the immunoglobulins (Ig) secreted by the hybridoma cells, may impart to the Ig-dye complex, a capacity to interact with ER/progesterone receptors(PR) which are present in the cytosols or in the sections of ER+ tumors. The Ig-dye may thus mimic anti-receptor antibody activity. Growth media containing phenol red and Ig-free bovine calf serum supplement or culture fluids from a non-Ig secreting hybridoma failed to show similar binding to ER+ tissues. This observation documents the need for Ig-dye complex formation to obtain such ER-Ig interaction. Complete dialysis of phenol red from the Ig positive supernates removed the observed interference of the dye. In this report we describe the nature of this interference and the results obtained before and after the removal of the dye from Ig+ culture fluids. In addition, we suggest some remedial measures that should be considered, while screening and testing the specificity of any hybridoma culture product for anti-receptor antibody activity (particularly, anti-ER or anti-PR antibodies).