The use of gold reagents to quantitate antibodies eluted from nitrocellulose blots: application to electron microscopic immunocytochemistry.
An assay is described in which gold reagents were used to quantitate nanogram amounts of antibody that had been eluted from antigens immobilized on nitrocellulose paper. Standard curves were generated by the application of rabbit immunoglobulin G (IgG) to nitrocellulose sheets assembled in a dot blot matrix apparatus. Blots were stained using either colloidal gold or immunogold, enabling quantitation of IgG concentration by scanning densitometry. Linear and reproducible standard curves were obtained. As little as 1 ng IgG/dot could be quantified using either gold reagent. In contrast to colloidal gold, immunogold could be used specifically to quantitate rabbit IgG regardless of the presence of bovine serum albumin or antigen coeluted from the nitrocellulose blot. The applicability of the immunogold assay was demonstrated by fractionating a complex rabbit antiserum raised against the RIM protein of frog retinal rod outer segments. Anti-RIM antibody was affinity-purified, quantitated by the immunogold assay, and subsequently employed in immunocytochemical studies using thin sections of retina embedded in a hydrophilic plastic, LR-Gold.
Burton
M D
MD
Department of Ophthalmology, Good Samaritan Hospital and Medical Center, Portland, Oregon 97209.
Onstott
L T
LT
Polans
A S
AS
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D003102:Colloids; D006046:Gold; D015151:Immunoblotting / Q000379:methods; D007074:Immunoglobulin G / Q000032:analysis; D008565:Membrane Proteins / Q000276:immunology; D008832:Microchemistry / Q000379:methods; D008854:Microscopy, Electron; D011817:Rabbits; D011898:Ranidae; D012374:Rod Cell Outer Segment / Q000032:analysis; D012834:Silver; D013194:Staining and Labeling
D000906:Antibodies; D003102:Colloids; D007074:Immunoglobulin G; D008565:Membrane Proteins; D012834:Silver; D006046:Gold