Diluent Buffer Ions and pH: Their Influence on the Performance of Monoclonal Antibodies in Immunohistochemistry

In immunohistochemistry, buffers near physiologic pH are traditionally used for the dilution of primary antibodies. Because this pH lies within the range of isoelectric points of immunoglobulins, the possibility of creating unfavorable conditions for the optimal immunoreactivity of some antibodies may exist. Twelve monoclonal and two polyclonal antibodies were therefore examined as primary antibodies in immunohistochemistry within the pH range of 6.0–8.6. For this, the antibodies were studied in several dilutions by using either Tris or phosphate buffers with some containing various molarities of NaCl. It was found that at dilutions higher than those recommended by the vendor, the staining properties of almost all investigated monoclonal antibodies responded selectively to changes in pH by retaining their maximal immunoreactivities at one pH, while losing the same at another. In addition to pH, the presence of Na+ ions in both diluent buffers also affected the performance of most monoclonal antibodies. The higher the concentration of this cation or the higher the pH in its presence, the more the immunoreactivity of the affected antibodies decreased. Dilutions made with phosphate-buffered 0.15 M NaCl resulted in the lowest sensitivity. For optimal staining intensities, phosphate buffers and use of NaCl are therefore not recommended. The two polyclonal antibodies, when subjected to the same experimental conditions, showed none of the responses to pH or solutes. It is therefore proposed that new monoclonal antibodies be first examined by immunohistochemistry in several dilutions of twofold increments beyond those generally recommended by vendors using 0.05 M Tris buffers of pH 6.0 and 8.6. The highest dilution and the pH at which maximal staining is retained may subsequently be used routinely. By following these guidelines, the majority of monoclonal antibodies could be used in dilutions up to eightfold higher than those recommended by the vendor, thereby affording greater economy.