It has been tested some diluted HBsAg and anti-HBs positive sera by modified ELISA test. We have found a correlation between HBsAg titer and spectrophotometric reading value and we have shown it in a curve. On the other hand, by neutralization of different dilutions of a RIA anti-HBs positive serum with different dilutions of a HBsAg positive serum we have carried out a checkboard and we have found that the best titer for neutralizing HBsAg is 30 ng/ml. This HBsAg has been called "standard". Using HBsAg "standard" we have tested 50 sera of hemophiliacs and we have found 1 HBsAg positive (2%) and 38 anti-HBs positive patients (76%).
We reported previously that TG19318, a synthetic ligand deduced from the screening of combinatorial libraries, displays specific and selective recognition properties for immunoglobulins of the G class and can be used conveniently for affinity chromatography purification of monoclonal and polyclonal antibodies. In this study we have extended the ligand characterization, examining its ability to bind IgA from cell culture supernatants and from IgG-deprived serum. Affinity columns prepared by immobilizing TG19318 on Sepharose allowed convenient one-step purification of monoclonal IgA directly from crude feedstocks, in high yield and with full recovery of immunoreactivity. Optimal column adsorption occurred with phosphate buffer at neutral pH, while elution of adsorbed IgA could be accomplished by a buffer pH change to acidic or basic conditions. Column capacity was close to 7 mg IgA/ml support.
While antibodies of the G class can be conveniently purified by affinity chromatography using immobilized protein A or G even at large scale, scaling up purification of IgM, IgA, and IgE and IgY still presents several problems, as specific and cost-effective ligands for these classes of immunoglobulins are not available. Protein A (1), which is widely used for the affinity purification of antibodies from sera or cell culture supernatants, does not recognize immunoglobulins of the M, A, E, and Y classes well and is not used to capture and purify these immunoglobulins from crude sources. Moreover, these two proteins are obtained from microorganisms or genetically modified bacteria, which carries the risk of affecting the safety of the purified antibodies through the presence of contaminants such as viruses, pirogens, or DNA fragments. As a result, the availability of alternative ligands for the affinity purification of antibodies is highly important from an industrial aspect. After immobilization on solid supports, the mannan binding protein (MBP), an affinity ligand for IgM, provides affinity media useful for IgM isolation based on a temperature-dependent interaction of the ligand with the immunoglobulins (2). The use of immobilized MBP for the purification of IgM is based on the adsorption in the presence of calcium at a temperature of 4°C, and the elution at room temperature of adsorbed immunoglobulins in the presence of ethylene- diamineotetraacetic acid (EDTA). This ligand shows low binding affinity for IgG, but binds to bovine and human IgM with lower affinity than murine IgM. However, in addition to the complexity of MBP isolation, functional binding capacities of MBP columns are limited to 1 or 2 mg of IgM per milliliter of support. IgA, which is involved in the first specific defense against natural infection (3) and represents the second most abundant immunoglobulin in serum (4), can be purified through the combination of different fractionation techniques such as ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration (5, 6). All these procedures are time consuming, labor intensive, and are not compatible with industrial scaling up. Lectin jacalin, isolated from jackfruit seeds (7), binds to IgA and can be conveniently used for the affinity purification of IgA from colostrum or serum (8). However, several aspects limit the use of this lectin for large-scale purification of monoclonal IgA from cell culture supernatants. First, jacalin is a biologically active lectin, being a potent T cell mitogen and a strong B cell polyclonal activator (9), thus requiring a careful control for ligand leakage into the purified preparation. Second, jacalin binds to the carbohydrate moiety of IgA, and d-galactose is required to elute IgA from affinity columns, which is costly and impractical for large-scale operations.
Abstract: An eptapeptide phage library was used to isolate peptides interacting specifically with a-1 antitrypsin through a subtractive biopanning procedure. The peptide ligand identified after three selection cycles [SITPLVH] was chemically synthesized in a tetrameric form and analyzed for its capacity to recognize a-1 antitrypsin by ELISA assays. Multimeric ligand immobilization on solid supports for the preparation of a'°ffinity columns proved useful for the purification of a-1 antitrypsin in the presence of large amounts of albumin.
In the pretargeted antibody-guided radioimmunotherapy (PAGRIT) system, the combined use of two different antibodies directed against the same tumor antigen could represent a valid approach for improving tumor targeting and therapeutic efficacy. We developed a novel monoclonal antitenascin antibody, ST2485, and studied its biochemical and functional properties by in vitro and in vivo assays. We then investigated the first of the three-step therapy combining ST2485 with another antitenascin antibody, ST2146, previously described, to increase accumulation of biotinylated antibodies at the tumor site.Studies of immunoreactivity, affinity, immunohistochemistry, and biodistribution in xenograft model were carried out on ST2485. Analysis of the ST2485 and ST2146 combination was preliminary carried out by ELISA and BiaCore tests and then by in vivo distribution studies after administration of the radiolabeled biotinylated antibodies, followed by a chase with avidin as clearing agent.ST2485 was found to be a suitable antibody for therapeutic applications. Indeed, for its behavior in all tests, it was comparable with ST2146 and better than BC2, an antibody already used for clinical trials. The additivity of ST2146 and ST2485 in tenascin C binding, shown by in vitro tests, was confirmed by biodistribution studies in a xenograft model where tumor localization of the antibodies was near the sum of each antibody alone, with a tumor-to-blood ratio higher than 24.The results reported in this study suggest that a monoclonal antitenascin antibody mixture can improve tumor targeting. This strategy could represent progress for therapeutic approaches such as PAGRIT.
