Non-covalent adsorption of antigens to plastic

The attachment of proteins to plastic by non-covalent bonds is often little affected by the buffer. Many different protocols can be indiscriminately used without any significant modification in the procedures. Some recent methods were designed to expose more of the hydro-phobic regions of the proteins and seemed to have a beneficial effect on the end-results. The three most important variables for the adsorption of proteins on a solid phase are temperature, time and concentration. 

The most widely used coating buffer is 50 mM carbonate, pH 9.6. Other buffers are 10 mM Tris-HCl, pH 8.5, containing 100 mM NaCl or PBS (10 mM sodium phosphate buffer, pH 7.2, containing 100 mM NaCl). 

Non-ionic detergents (Triton X-100, Tween 20) should be avoided during coating since they compete strongly with the protein for the solid phase and prevent the formation of hydrophobic interactions. 

Coating of polystyrene plates with densonucleosis virus (DNV) at different concentrations (0.2 ml/well) and incubation at 4 C or 37°C. The dilution of antiserum giving an optical density of 0,15 (cut-off value) after 30 min was plotted against the incubation period. For DNV, incubation at 37 C has the advantages that short incubation periods are needed (e.g., 20 min) and that relatively more virus is coated if present in low concentrations (from Tijssen et al., 1982 courtesy Archives of 

Partial denaturation of IgG is carried out with samples containing 10 pg/ml in 50 mM glycine-HCl buffer, pH 2.5, containing 100 mM NaCl, incubated for 10 min at room temperature and neutralized with 500 mM Tris. The sample is then dialyzed against the coating buffer. Alternatively, IgG may be denatured in neutral buffer by the addition of an equal volume of 6 M urea and incubation overnight at room temperature, followed by extensive dialysis. Thermal denaturation is carried out for 10 min at 70°C for sheep antibodies or at 82°C for rabbit antibodies (Conradie et al., 1983). This treatment may be different for IgG preparations from other species. 

---