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Enhanced Affinity Capture MALDI-TOF MS: Orientation of an Immunoglobulin G Using Recombinant Protein G

2002; American Chemical Society; Volume: 74; Issue: 15 Linguagem: Inglês

10.1021/ac025558z

1520-6882

Hendrik Neubert, Eli S. Jacoby, Sukhvinder S. Bansal, Ray K. Iles, David Cowan, Andrew T. Kicman,

Biosensors and Analytical Detection

Although immobilization of antigen-specific immunoglobulins onto matrix-assisted laser desorption/ionization (MALDI) targets allows the specific detection and enrichment of an antigen from complex biological fluids, the process of antibody immobilization is not optimal. The principal reason is that the antibody can bind to the template in various orientations, many of which block antigen recognition. An affinity capture MALDI mass spectrometry methodology was developed by covalently immobilizing an Fc receptor (recombinant protein G) onto MALDI gold targets for the purpose of orientating an immunoglobulin G, with the Fab domains pointing away from the target surface. The pregnancy and cancer marker, human chorionic gonadotropin β core fragment (hCGβcf), was our chosen test substance. To optimize the methodology, different surface densities of protein G and immunoglobulin were achieved by employing varying concentrations for immobilization. Captured amounts of hCGβcf were compared using an external standard (cytochrome c). Orientation of immunoglobulin resulted in an ∼3-fold increase in MALDI signal compared to using randomly immobilized antibody. Higher antibody concentrations resulted in diminished MALDI signals, which were explained by steric hindrance. Purification and enrichment of hCGβcf was achieved from a test solution containing contaminant peptides and proteins using oriented immunoglobulins on-target.