immunoassay for antibody - أطلس مختبرات العرب

Immunoassay for antibody. (1) Antigen in saline is incubated on a plastic plate or tube, and small quantities become absorbed onto the plastic surface. (2) Free antigen is washed away. (The plate may then be blocked with excess of an irrelevant protein to prevent any subsequent non-specific binding of proteins.) (3) Test antibody is added, which binds to the antigen. (4) Unbound proteins are washed away. (5) The antibody is detected by a labelled ligand. The ligand may be a molecule such as staphylococcal protein A which binds to the Fc region of IgG – more often it is another antibody specific for the test antibody. By using a ligand which binds to particular classes or subclasses of test antibody it is possible to distinguish isotypes. (6) Unbound ligand is washed away. (7) The label bound to the plate is measured. A typical titration curve is shown in the graph above. With increasing amounts of test antibody the signal rises from a background level through a linear range to a plateau. Antibody titres can only be detected correctly within the linear range. Typically the plateau binding is 20–100 times the background. The sensitivity of the technique is usually about 1–50 ng/ml of specific antibody. Specificity of the assay may be checked by adding increasing concentrations of free test antigen to the test antibody at step 3; this binds to the antibody and blocks it from binding to the antigen on the plate. Addition of increasing amounts of free antigen reduces the signal.