An immunoaffinity column with a monoclonal antibody as ligand for human follicle stimulating hormone.

A detailed characterization regarding the chromatographic and biochemical properties of an immunoaffinity column for sample pretreatment using a follicle stimulating hormone (FSH) beta-unit specific mAb has been performed. A maximum binding capacity of approximately 300 microg/mL was determined. A K(D) value of 4.5x10(-8) M could be obtained in batch isotherm measurements. The dynamic binding capacity at 10% did not change when residence time was at least 6 min. High selectivity could be demonstrated using crude culture supernatant from FSH producing Chinese hamster ovary (CHO) cells. A great stability of the matrix was confirmed by performing 100 consecutive chromatographic runs without loss of capacity.

Yeast cell surface display system for determination of humoral response to active immunization with a monoclonal antibody against EpCAM.

Even though an immunogenic formulation of the murine monoclonal anti-EpCAM (epithelian cell adhesion molecule) antibody Mab 17-1A, has been shown to evoke a strong humoral immune response in both, monkey studies and early clinical trials, conventional anti-EpCAM ELISA could not identify anti-EpCAM immune response in relation to treatment with Mab17-1A. In contrast, usage of cellulose membranes prepared by SPOT technology presenting overlapping EpCAM peptides allowed the unequivocal determination of EpCAM related antibodies present in monkeys sera after immunization with IGN101. Based on such contradictory results, it was of high interest to compare obtained data to a different method for better assessment of their possible interpretation. Therefore, in the present studies, some EpCAM peptides, determined as reactive by binding of IgG isolated from sera of treated monkeys on membranes prepared by SPOT technology, were represented on yeast surface using the pYD1 yeast display vector system. Binding of biotinylated IgG from sera was detected with streptavidin-FITC and quantity of binding was determined by FACS measurement. Though using this completely different method, experiments with pre-immune and immune sera of four monkeys exemplarily are comparable to the results obtained by analysis with synthetic peptide arrays.

Engineering properties of a camelid antibody affinity sorbent for Immunoglobulin G purification.

An IgG-specific camelid antibody matrix (BAC, Naarden, The Netherlands), developed from an immune phage display library, was characterized regarding engineering properties including mass transfer characteristics. Uptake kinetics and equilibrium binding capacity were determined by a finite bath method. Adsorption kinetic parameters were also determined using a real time biosensor. Slightly different properties to conventional Staphylococcal protein A affinity media were shown; especially a 2-2.5 times lower maximal binding capacity with a value of 26 mg/ml polyclonal IgG was obtained. Mass transfer could be described by using a film and pore diffusion model (D(e)=5 x 10(-8) cm(2)/s). Determined engineering parameters were used to predict breakthrough behaviour in column mode considering film and pore resistances. The dynamic binding capacity at 10% breakthrough did not change when residence time was at least 6 min.