Chapter 9

To produce polyclonal antisera for HLC assays, sheep are immunised using individual monoclonal immunoglobulins of a single class and light chain type (e.g. IgGκ) [127]. Sheep are simultaneously tolerised with: 1) monoclonal immunoglobulins of the same class but opposite light chain type (i.e. IgGλ); 2) monoclonal immunoglobulins of different classes but the same light chain type (i.e. IgAκ); and, 3) monoclonal free light chains (FLCs) of the same light chain type (i.e. κ FLC). Antigens for immunisation and tolerisation are purified from the serum or urine of patients with monoclonal gammopathies. Antigen purity is assessed using silver-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis.