Optimizing the solid-phase immunofiltration assay. A rapid alternative to immunoassays.
The technical variables of the solid-phase immunofiltration assay (SPIA) for the detection of antibodies bound to antigens on a solid-phase filter have been investigated. The binding to solid-phase filters of 125I-labelled axial filament proteins derived from Treponema phagedenis and the optimal conditions for blocking non-specific protein binding were analysed. Axial filament was applied to nitrocellulose, Hybond Nylon and Zeta Probe. After extensive rinsing, the highest amount (68%) of axial filament was observed bound to Zeta Probe. However, blocking non-specific protein binding by pre-wetting the filter with rinsing buffer containing 0.5% Tween 20, prevented the binding of protein to the filter only when nitrocellulose was used as solid phase. Tween 20 (0.5%) in the rinsing and incubation solutions was found to be necessary for the reduction of non-specific binding of contaminants in turbid sera. However, the use of such solutions resulted in a substantial leakage of antigen (47%) during rinsing procedures. Binding of antigen-specific antibody was analysed using 125I-labelled protein A. The maximal possible binding of the antibody occurred within 5 min when the antibody solution was filtered. For optimal binding of 125I-labelled protein A an incubation time of 1 h was needed. It is suggested that solid-phase immunofiltration may provide a rapid alternative for radioimmunoassays or enzyme immunoassays for the detection of specific antibodies.
IJsselmuiden
O E
OE
Department of Dermato-venereology, University Hospital Dijkzigt, Rotterdam, The Netherlands.
Herbrink
P
P
Meddens
M J
MJ
Tank
B
B
Stolz
E
E
Van Eijk
R V
RV
D000818:Animals; D000907:Antibodies, Bacterial / Q000032:analysis; D000942:Antigens, Bacterial / Q000032:analysis*; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D003101:Collodion; D005374:Filtration / Q000295:instrumentation / Q000379:methods; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods*; D007136:Immunoglobulins; D011817:Rabbits; D013205:Staphylococcal Protein A; D014208:Treponema / Q000032:analysis* / Q000276:immunology
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D007136:Immunoglobulins; D013205:Staphylococcal Protein A; D003101:Collodion