High resolution autoantibody detection by optimized protein G-horseradish peroxidase immunostaining in a western blotting assay.
We report a procedure for optimisation of Western blotting using protein G-horseradish peroxidase (protein G-HRP) which avoids the false positive reactions often caused by second antibodies and increases the detection of autoantibodies by protein G conjugate. A number of modifications were investigated. Higher concentrations of serum and protein G-HRP at 1:5 to 1:10 and 1:100, respectively, increased the detection to the same order as that obtained with second antibody systems and gold staining with silver enhancement. The role of various detergents in the procedure was established. 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) in incubation with protein G-HRP increased the binding between protein G and immunoglobulin G. Addition of Tween-20 for blocking produced little background so that protein blockers could be avoided. Prolonged incubation with serum increased markedly the sensitivity of the procedure when compared with the recommended 2 h incubation period. Polyvinylidene difluoride membrane provided better transfer effect, lower background and higher mechanical strength than nitrocellulose membrane. The utilization of only one antibody-specific ligand increased the simplicity, reliability, economy, efficiency and specificity of the method. These modifications make this method significantly better for detection and screening for autoantibodies.