Insulin-like Growth Factor-II (IGF-II) (IGFBP Blocked) RIA

Radioimmunoassay for the determination of human IGF-II in serum or plasma.<br><br>

Insulin-like growth factors (IGF) I and II play a pivotal role in regulating the proliferation, differentiation and specific functions of many cell types (1-3). IGF-II has a molecular weight of 7469 daltons (5). Its major regulators are growth hormone (GH) and nutrition (6), although its production in specific tissues is affected by a multitude of tropic hormones and other peptide growth factors. In contrast to many other peptide hormones, IGFs are avidly bound to specific binding proteins (IGFBP). The seven classes of IGFBPs which are known at present (7,8,22) either bind IGF-I and IGF-II with similar affinities or show a preference for IGF-II (9,10).<br><br>

A major problem of IGF-II measurement results from the interference of IGFBPs in the assay. Direct determinations in untreated serum samples (11) give false values because of the extremely slow dissociation of the IGF/IGFBP complexes during the assay incubation. Depending on the ratio IGF to IGFBP the following errors may occur:<br><br>

- in samples with low IGF concentration, IGFBP-complexation will take place predominantly with the IGF tracer, thus leading to false-high results in a competitive RIA. Effect: Overestimation of low IGF levels.<br><br>

- in samples with high IGF concentration, unmarked IGF from the sample will be predominantly complexed by IGFBPs and therefore withdrawn from measurement. Effect: Underestimation of high IGF levels.<br><br>

Therefore, various techniques were applied to physically separate IGF-II from its binding proteins before measurement, including (a) size exclusion chromatography under acidic conditions, (b) solid-phase extraction and (c) acid-ethanol extraction (2,12,13). These techniques, however, are either inconvenient or time-consuming or give incomplete and not-reproducible recoveries. The most widely used method is the acid-ethanol extraction (13,14) with a recovery of only 70-80 % of IGFBP-bound IGF as a result of co-precipitation. The absolute results of such an extraction are therefore false low (15). The extraction removes the IGFBPs only insufficiently and leads to reduction in sensitivity of the assay due to pre-dilution of the samples by the extraction procedure.<br><br>

Furthermore, the remaining IGFBP may still interfere in the assay. In addition, the acid-ethanol extraction is ineffective in specimens other than serum or plasma (e.g. cell culture media), in which determination of IGF is already difficult enough due to the fact that IGFBPs are frequently present at large excess.<br><br>

To avoid these difficulties, an uncomplicated assay was developed, in which special sample preparation is not required before measurement. (except dilution and/or acidification in a specially-composed buffer system). For research use only, not for use in diagnostic procedures.