Methodological aspects of Universal immuno-PCR on standard tubes.

One of the most used formats in inmuno-polymerase chain reaction (IPCR) is known as "Universal" IPCR (signal-generating complexes is based on conjugates of biotinylated DNA, biotinylated IgG and avidin). In the present study, we evaluated the utility of using mono- and bi-biotinylated DNA probes, pre-self-assembled DNA-neutravidin complex, blocking step and glutaraldehyde pretreatment of standard PCR tubes to improve the analytical performance of the hTSH-IPCR assay. The use of pre-self-assembled mono-biotinylated DNA-neutravidin complex enhances both the sensitivity and the reproducibility of the hTSH-IPCR assay, even without blocking step: hTSH-IPCR assay showed an improved limit of detection (LOD: 0.01 µIU/ml), calibration sensitivity (SEN: 2.4) and analytic sensitivity (γ: 9 µIU/ml-1) in comparison with both a self-made ELISA and a commercial one.

A scFv antibody fragment as a therapeutic candidate to neutralize a broad diversity of human IFN-alpha subtypes.

Despite their significant role in maintaining the normal physiology, cytokines may cause pathological conditions when they are overproduced. In this way, the increased expression of human interferon alpha (hIFN-alpha) is associated with acute viral infections, inflammatory disorders and several autoimmune illnesses, where the cytokine may be a factor in either initiating or maintaining the disease. Currently, there are several mAbs marketed for a variety of indications and many more in clinical trials, in which IFN-alpha represents a potential target for antibody-based therapy. A panel of 11 murine mAbs was prepared using recombinant hIFN-alpha2b as immunogen, all of which bound to the native form of the cytokine with affinity constants ranging from 1.7x10(7) M(-1) to 1.4x10(10) M(-1). An epitope mapping protocol demonstrated four spatially distinct areas of the protein recognized by the mAbs. Taking into account the characterization of the antibodies and their ability to inhibit the IFN-alpha biological activity, four mAbs were selected to produce scFv fragments. One of these fragments (CA5E6) was able to neutralize a wide spectrum of subtypes of the IFN-alpha family, including the recombinant cytokines hIFN-alpha2a and hIFN-alpha2b and a heterogeneous collection of IFN-alpha produced by activated leukocytes and Namalwa cells. With the aim of improving the affinity of the selected fragment, a standard error-prone PCR method was carried out. By using this strategy, it was possible to generate a new fragment (EP18) with increased affinity and ability to neutralize a broad diversity of IFN-alpha subtypes. Consequently, the scFv EP18 represents a potential therapeutic agent for those immune and inflammatory diseases which are associated with an increased IFN-alpha expression.

Novel long-lasting interferon alpha derivatives designed by glycoengineering.

Human alpha interferons (hIFN-alpha) comprise a family of closely related proteins that block viral infection, inhibit cell proliferation and modulate cell differentiation. Recombinant hIFN-alpha2 has proved useful for the treatment of a variety of human viral diseases and cancers. However, the clinical use of this cytokine has been restricted due to its short circulating half-life, which makes frequent dosing over an extended period necessary. To circumvent this problem, a glycoengineering strategy was carried out using site-directed mutagenesis. Fourteen mutants were constructed by the insertion of one N-glycosylation consensus sequence into different positions of the cytokine. Mutations were focused on amino acid positions that were believed not to be critical for the protein's structure or function. Taking into account the retained specific in vitro bioactivity and the higher carbohydrate content, five N-glycosylation positions were selected to be introduced into the molecule. Successive increases in molecular weight were observed after each addition of a functional consensus sequence, resulting in analogs with 4 and 5 N-linked carbohydrates (4N- and 5N-IFN) with increased size and charge, factors that reduce renal clearance of proteins. Pharmacokinetic experiments showed a similar behavior of 4N- and 5N-IFN variants, with a 25-fold increase in the elimination half-life and a 20-fold decrease in the systemic clearance rate compared with the non-glycosylated rhIFN-alpha2 following subcutaneous administration to rats. Besides, both distribution and elimination half-lives of the 4N analog were longer in comparison with the non-glycosylated cytokine, determining a 10-fold increase in the area under the curve after intravenous inoculation. Thus, herein we describe for the first time heavily glycosylated IFN analogs with a remarkable improvement in pharmacokinetic properties, which allow us to project drugs that combine less frequency of administration with enhanced therapeutic efficacy.