Isoelectric focusing‐affinity immunoblot analysis of mouse monoclonal antibodies to the four human IgG subclasses

Isoelectric point (pI) microheterogeneity, immunoreactivity and specificity of murine monoclonal anti‐human IgG subclass antisera (MoAb) were evaluated in parallel by isoelectric focusing (IEF)‐affinity immunoblotting and enzyme‐linked immunosorbent assay (ELISA). Ascites protein bands in the pH 3 to 9 region of Coomassie Blue‐stained IEF‐polyacrylamide gels corresponded to mouse host proteins such as albumin, transferrin and immunoglobulins. Bands in the pH 5.5 to 8.0 region were shown to be murine IgG by direct blotting onto nitrocellulose followed by detection with conjugated anti‐mouse IgG. Use of IgG myeloma (antigen)‐coated nitrocellulose in the IEF‐affinity immunoblot allowed detection of the charge micro‐heterogeneity of the MoAbs. As a group, the MoAbs had pIs ranging from 6.1 to 7.8 with a 0.1 to 0.6 pH unit spread. There were 1 to 5 major dense bands flanked by up to 4 minor fainter bands. The pI values and banding patterns as determined by IEF‐affinity immunoblot analysis were consistent both within and between blots. Semi‐quantitative estimates of binding specificity in the IEF‐affinity blot compared well with cross‐reactivity data obtained from a quantitative ELISA. IEF‐affinity immunoblotting is a useful analytical tool for defining the microheterogeneity of monoclonal antibody pI's and for monitoring antibody specificity. Both parameters are indicators of consistency of antibody produced in culture or in ascites by hybridomas which have been repeatedly frozen and thawed over several years.