Special remarks:

INTRODUCTION

The insulin-like growth factors (IGF)-I and –II play a pivotal role in the regulation of proliferation and differentiation of several tissue types (1-3). IGF-I also called Somatomedin C (4) has a molecular weight of 7.469 kDa (5). Its expression is mainly regulated by Growth Hormone and nutrition (6). But several hormones and peptide factors are known to influence IGF-II synthesis in different tissues. Bioavailability of the IGFs is regulated by specific binding proteins (IGFBP). Beside the high affinity Insulin-like Growth Factor Binding Proteins 1-6, IGFs are also bound be IGFBP-related Proteins (7, 8, 22). These binding proteins bind IGF-I and IGF-II with the same affinity or prefer IGF-II (9, 10). Direct measurement of IGFs in serum samples without pretreatment results in false values because of the extremely slow dissociation of the IGF/IGFBP complexes during the assay incubation only a part of the IGF-II in the specimen can bind to the antibodies and be detected.

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.

This assay is easy, fast and results do not depend on the binding protein concentration of the sample. Its based on the high specificity of the employed antibodies for IGF-II. There is virtually no cross-reactivity with IGF-I. This allows the separation of IGF-II from the binding proteins by acidification and blocking of the free binding proteins with IGF-I. Thus, the endogenous IGF-II is free in solution.

INTENDED USE

Scientific investigations in the field of neonatal hypertrophy (IGF-II is a foetal growth factor) and malignancies (IGF-II is an monogenic growth factor). Age dependent reference values are shown in Table 4.

IGF-II seems to be of use in differential diagnostics of malignancies. Thus, it is possible to differentiate by IGF-II between adrenocortical carcinomas and adenomas (24). Further tumor staging and differentiation between hyperplasia and carcinoma can be improved by IGF-II measurements in prostate tumors (25). The IGF-System seem to be of relevance in neurodegeneration as well, e.g. Alzheimer´s and Parkinson’s diseases (26).

PRINCIPLE

The Demeditec ELISA for IGF-II DEE030 is a so-called Sandwich-Assay. It utilizes two specific and high affinity antibodies for this protein. The first antibody, immobilized on the microtiter plate, and the added second biotinylated antibody are binding the IGF-II in the sample. The Streptavidin-Peroxidase Enzyme Conjugate subsequently binds to the complex. In the closing substrate reaction the turn of the colour will be high specific catalysed, quantitatively depending on the IGF-II-level of the samples.

IGF-II-IGFBP complex is dissociated by dilution in an acidic buffer. IGFBPs are blocked by IGF-I excess, thus allowing the measurement of free IGF-II. With this method, the IGFBPs are not removed, but their function and therefore their interference in the assay is neutralized.

Due to the low cross-reactivity of the IGF-II antibody with IGF-I, excess IGF-I does not disturb the interaction of the first antibody with IGF-II