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.
Hu
G R
GR
Department of Medicine, University of Sydney, Australia.
Harrop
P
P
Warlow
R S
RS
Gacis
M L
ML
Walls
R S
RS
D000544:Alzheimer Disease / Q000276:immunology; D001323:Autoantibodies / Q000032:analysis*; D001426:Bacterial Proteins; D015153:Blotting, Western / Q000379:methods*; D001921:Brain / Q000276:immunology*; D002021:Buffers; D003902:Detergents; D006735:Horseradish Peroxidase; D006801:Humans; D009419:Nerve Tissue Proteins / Q000276:immunology
D001323:Autoantibodies; D001426:Bacterial Proteins; D002021:Buffers; D003902:Detergents; C034284:IgG Fc-binding protein, Streptococcus; D009419:Nerve Tissue Proteins; D006735:Horseradish Peroxidase