It is essential that FLC immunoassays utilise antibodies that have high specificity and affinity. Early FLC immunoassays, from other groups, used polyclonal antisera but good specificity was difficult to obtain (Chapter 2). Monoclonal antibodies seemed to be the obvious solution to the problem. However, considerable effort failed to produce antibodies that reliably recognised a full range of monoclonal FLCs (Chapter 3). Similar findings have been reported for other monoclonal antibody-based FLC immunoassays and are discussed further in Chapter 8.
In contrast, an assay based on polyclonal antisera can recognise a wide variety of FLC epitopes, including the diverse range of pathological monoclonal FLC produced by patients with monoclonal gammopathies. Therefore, research focussed on optimising polyclonal FLC antisera to ensure the reliable detection of the huge variety of monoclonal FLCs. The following description is an outline of the successful procedures involved in the development of Freelite sFLC assays. In summary, sheep were immunised with κ or λ molecules that had been purified from urine samples containing Bence Jones proteins. The resultant antisera were adsorbed against purified IgG, IgA and monoclonal proteins and then affinity purified against mixtures of the respective FLCs that had been immobilised onto Sepharose. Antisera requiring further adsorption, as judged by the tests described below, were recycled through the adsorption and testing procedures, resulting in antisera highly specific for FLCs and deemed satisfactory for assay use.