Production of STC-1 protein and monoclonal antibodies for therapeutic or clinical response monitoring in acute lymphoblastic leukemia

Abstract

The use of immunodiagnostic kits is one of the fast and safe tools for early treatment of human diseases. To develop these kits, at first, it's necessary the production of recombinant proteins or peptides specific to pathogens (targets) with high purity and that replicate the structural and conformal characteristics of the native protein, and in a second moment, monoclonal antibodies with high affinity and sensitivity, ensuring early, efficient, quick and accurate diagnoses. Acute Lymphoblastic Leukemia (ALL) is the most widespread cancer in childhood. The Stanniocalcin 1 protein (STC-1) was identified as a potential serum marker of ALL, useful for purposes of monitoring clinical response to therapy. This project focuses to continue the aims of PIPE I and complete the detection system based on ELISA Sandwich, through the production of a panel of monoclonal antibodies from STC-1 recombinant protein and thus, optimize, standardize and validate an early diagnosis kit with high sensitivity and reliability capable of detecting and differentiating the current STC-1 levels in biological samples from leukemia patients and non-patients. Thus, from the expression and purification of STC-1 protein gene in baculovirus system / insect cells, two strategies will be adopted for the production of monoclonal antibodies: immunization with (1) Balb/c mice and (2) GANP transgenic (germinal center-associated protein nuclear). These transgenic mice have increased activity with somatic hypermutation of V region immunoglobulin genes, resulting in production of high-affinity and high-specific-antibodies. Full-lenght STC-1 protein will be produced by infecting cells with the baculovirus culture and purified by chromatography in three steps: 1. Ion exchange; 2. Nickel affinity chromatography and 3. Molecular Exclusion. Like all diagnostic kit has a positive control and a standard curve, the expression of STC-1 C-terminal portion in prokaryote system is another objective to be achieved. Then, E. coli strains as Roseta and C43 will be used in association with different solubilizing and extraction buffers, in the presence or not of urea, and also refolding protocols for the production of soluble form of C-terminal STC-1. The diagnostic kit will be evaluated with samples from normal patients (including control groups, such as pregnant women, men and young) and patients (including many types of cancer) for STC1 validation as LLA marker and, furthermore, will be submitted to stability, storage and limit detection assays. Thus, this project features a recombinant protein synthesis platform as well as monoclonal antibodies for the detection of STC-1 in ALL patients. (AU)