Modified dot assay with increased sensitivity: detection of small amounts of immunoglobulin molecules and the importance of different detection systems.
The original method of the dot assay described by Hawkes et al. enabled the detection of 100 pg of immunoglobulin molecules. We compared different enzyme detection systems based on the avidin-biotin complex, in conjunction with different substrates. Three commercial kits (Vectastain-ABC, Streptavidin Bridge, and Streptavidin-Peroxidase Complex) based on the same technique were also included in the comparison. In addition, we tested the Aureo-Probe kit, which is based on gold- and silver-enhanced staining. Various incubation times, blocking solutions (horse serum, bovine serum albumin, and milk), and commercial antihuman Ig antisera or monoclonal antibodies were tested. The greatest sensitivity was achieved by using the peroxidase-avidin-biotin complex together with 4-chloro-1-naphtol as substrate, which detected 1 pg of immunoglobulin molecules. Equal sensitivity was also shown by the Aureo-Probe system.
Lavanchy
D
D
Division of Immunology and Allergy, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland.
Stroun
J
J
Frei
P C
PC
D001360:Avidin; D001667:Binding, Competitive; D001710:Biotin; D005069:Evaluation Studies as Topic; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007124:Immunoenzyme Techniques; D007136:Immunoglobulins / Q000032:analysis*
D007136:Immunoglobulins; D001360:Avidin; D001710:Biotin