Four monoclonal antibodies belonging to different subclasses and with differing isoelectric points were coupled to horseradish peroxidase (HRP) and alkaline phosphatase (AP) using various conjugation procedures. The conjugates were tested by enzyme immunoassay and their efficiency was characterized by the antibody and enzyme concentrations needed to obtain an arbitrary OD value. The suitability of antibody for conjugation through NH 2 groups was tested by fluorodinitrobenzene (FDNB). HRP conjugates were produced by two variants of the sodium periodate procedure and two variants of the glutaraldehyde method, as well as by the heterobifunctional linker N-succinimidyl 3-(2-pyridyldithio)pro-pionate (SPDP). Two of the four antibodies were coupled by a third variant of the periodate method, through their carbohydrate moieties. The periodatemediated conjugations, using sugar moieties on the enzyme, provided the most efficient HRP conjugates, regardless of the antibody subclass or isoelectric point. The glutaraldehyde procedures consistently gave the worst results. AP conjugates were prepared using the same methods. The most efficient and reproducible AP conjugates with all four monoclonal antibodies were obtained using the SPDP procedure. The efficiency of the other methods differed from one antibody to another.

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The neuregulin-1 (Nrg-1) gene is translated into several protein isoforms, which are either secreted or membrane-anchored. In vitro, neural stem cells (NSC) express mainly the cystein-rich-domain NRG (CRD-NRG) isoform, a membrane-anchored type III form. This isoform exhibits a cystein-rich-domain, which constitutes a second transmembrane domain and can be cleaved to release both a signaling EGF-containing domain (ECD) at the cell surface and an intracellular domain (ICD). The main goal of this paper was to determine the exact role of ECD and ICD in NSC survival and differentiation. Using an siRNA approach, we demonstrated that CRD-NRG inhibition was followed by a decrease in NSC proliferation and of neuronal or oligodendroglial differentiation. Overexpression of ICD but not ECD was followed by a decrease in NSC proliferation and an increase in neuronal and oligodendroglial differentiation. Moreover, we showed that ICD physically interacted in cultured NSC with BRM and BAF57, two members of the Swi/Snf remodeling complex, and that ICD stimulation of neuronal cell differentiation is dependent on the presence of BAF57.