CHARACTERIZATION OF NEW MONOCLONAL-ANTIBODIES TO HUMAN INSULIN-LIKE GROWTH FACTOR-II AND THEIR APPLICATION IN WESTERN IMMUNOBLOT ANALYSIS

Four immunoglobulin G, class monoclonal antibodies (mAbs; 1D5, 1D9, 2B11, and 2H11) were produced against recombinant human insulin-like growth factor-II (rhIGF-II). Enzyme-linked immunosorbent assay established that these four mAbs specifically recognized rhIGF-II and hIGF-II, but not rhIGF-I. mAbs 1D5, ID9, and 2H11 did not cross-react with mouse rIGF-II, although there are only six amino acid differences between mouse IGF-II and human IGF-II. The epitope for each mAb was partially defined by enzyme-linked immunosorbent assay using mouse-human chimera IGF-II mutants and other IGF-II mutants that were prepared by site-directed mutagenic procedures. These results indicated that the epitopes of mAbs 1D5, 1D9, and 2H11 are in the C-domain of hIGF-II around Ala32 to Ser36, and that of 2B11 is in the carboxyl-terminal end of the B- and A-domains of hIGF-II. A specific and sensitive RIA was developed using mAb 2H11. In this RIA, IGF-II variant ([RLPG/S29]IGF-II) and rhIGF-II competed equally with [I-125]IGF-II for binding to mAb 2H11. Similar results were produced when mAbs 1D5, 1D9, and 2B11 substituted for 2H11. The potential usefulness of mAb 2H11 in an immunoblot procedure to characterize the heterogeneity of IGF-II in the sera and tumor tissues of patients with nonislet cell tumor hypoglycemia was evaluated. A procedure that combined acid-ethanol extraction of serum or tumor tissues and immunoaffinity concentration of the extracted IGF-II with mAb 2H11-immobilized resin was found to be an effective way to prepare the samples. In Western immunoblots, a quantity of rhIGF-II as low as 3 ng could be identified, whereas 200 ng rhlGF-I or rat IGF-II were not recognized. The levels of IGF-II in the sera of 12 patients with nonislet cell tumor hypoglycemia varied from normal to about twice normal. The MOI Wt (Mr) of this IGF-II was between 10-17K. There was little of the processed 7.5K M, IGF-II in the sera of these patients. Finally, the source of the high M(r) forms of IGF-II was the tumor, because the ratios of high M(r) forms of IGF-II to 7.5K IGF-II changed dramatically from 99:1 and 91:9 to 4:96 and 32:68 in two patients after successful excision of their tumors. In conclusion, mAb 2H11, which is specific for human IGF-II, should be an important reagent in future studies designed to monitor the changes in the quantities and heterogeneity of IGF-IIs during development and as a consequence of metabolic disease.