The immobilisation of antibodies (Ab) or antigens (Ag) to solid phases is always a critical step when developing a heterogeneous immunoassay. There are two main procedures: passive adsorption and covalent coupling. Adsorption is a simple method, but it has its disadvantages including denaturation of antibodies and their leakage from the surface. The covalent coupling requires reactive group's both on the solid phase and on the protein (in this study an Ab). The most commonly used group on the protein is the ε-amino group (-NH2) on the amino acid lysine and there are many different covalent coupling techniques available for coupling to reactive groups on the solid phases. It is often advantageous to use particles as solid phase or detector reagents in heterogeneous immunoassays since this simplifies separation of bound immuno complex from free immuno reactants. Particles with a vide variety of reactive groups and diameters are suitable and easily-accessible for use as solid phase, while dyed polystyrene particles or gold particles are often used as detector reagent.
In this work antibodies were immobilised to different solid phases, mostly particles, and the aim was to determine how the antibodies were affected by immobilisation and how this later influenced the results of an immunoassay. In paper I, Abs against Immunoglobuline E (IgE) were coupled to polystyrene particles by different covalent coupling methods and used as solid phases in an RIA determining total IgE. In the second paper (II) fluorescent polystyrene particles were used as detector reagents and antibodies against eosinophil protein X (EPX) and human neutrophil lipocaline (HNL) were coupled using two different covalent coupling techniques: The-antibodies used as capture sites in paper II were conjugated to polyethylene glycol (PEG) prior to immobilisation on a nitrocellulose membrane. The reagents were evaluated in a lateral immunochromatography (ICR) assay determining EPX and HNL. The choice of immobilisation method influences the result in the final assay and it is an advantage to have simple methods for evaluating the immobilised reagents before using them in the final assay. We thus developed different methods for evaluation of the immobilised antibodies and attempted to correlate the results from these methods with the results from the immunoassay finally used. The method used to measure the antigen binding capacity should ideally be the method of choice for evaluating differences between antibody particle derivatives. In actual fact, the results from this method did not show any subsequent correlation to the results from the immunoassay. We have therefore preferred to functionally evaluate the immobilised antibodies in the final immunoassay.