Global analysis of antibody repertoires. 1. An immunoblot method for the quantitative screening of a large number of reactivities.
This paper describes a procedure for analysing multiple antibody reactivities that explores a commercially available immunoblot system, and is based on a double staining of nitrocellulose membranes, revealing both antibody reactivities and the migration position of the blotted proteins in the membrane. Quantification of both stainings by densitometry allowed the accurate superposition of the immunoreactivity and total protein profiles of each lane. Moreover, the protein stainings of the different lanes could be adjusted with a simple-scale transformation algorithm, correcting for possible distortions during electrophoretic migration, and allowing for the precise comparison of the immunoreactivity profiles in different lanes. The procedure is discriminatory enough to identify unique reactivity patterns in random pools of 10(4) activated B cells, and to define strain-specific natural antibody repertoires. The utility of this immunoblot method as an assay for simultaneously scoring multiple reactivities to hundreds of antigens in complex mixtures of antibodies, and thus defining antibody repertoires in a global manner, is discussed.
Haury
M
M
Unite d'Immunobiologie, CNRS URA 359, Institut Pasteur, Paris, France.
Grandien
A
A
Sundblad
A
A
Coutinho
A
A
Nobrega
A
A
D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology; D002478:Cells, Cultured; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D015151:Immunoblotting / Q000379:methods*; D007075:Immunoglobulin M / Q000032:analysis* / Q000145:classification; D008213:Lymphocyte Activation / Q000276:immunology; D051379:Mice; D008815:Mice, Inbred Strains; D009124:Muscle Proteins / Q000276:immunology; D013047:Specific Pathogen-Free Organisms
D007075:Immunoglobulin M; D009124:Muscle Proteins