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 Line. Moreover, the protein stainings ofthe different lanes could be adjusted with a simple‐scale transformation algorithm, correcting for possible distortions during electrophoretic migration, and allowing for the precise comparison ofthe immunoreactivity profiles in different lanes. The procedure is discriminatory enough to identify unique reactivity patterns in random pools of 104 activated B cells, and to define strain‐specific natural antibody repertoires. The utilily 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.

[1]  A. Coutinho,et al.  Global analysis of antibody repertoires. II. Evidence for specificity, self‐selection and the immunological “homunculus” of antibodies in normal serum , 1993, European journal of immunology.

[2]  D. Klinman.  Similarities in B cell repertoire development between autoimmune and aging normal mice. , 1992, Journal of immunology.

[3]  T. Ternynck,et al.  Natural mouse IgG reacts with self antigens including molecules involved in the immune response , 1992, European journal of immunology.

[4]  J. Dwyer.  Manipulating the immune system with immune globulin. , 1992, The New England journal of medicine.

[5]  A. Coutinho,et al.  Normal serum immunoglobulins participate in the selection of peripheral B-cell repertoires. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[6]  K. Rajewsky,et al.  Most peripheral B cells in mice are ligand selected , 1991, The Journal of experimental medicine.

[7]  G. Winter,et al.  Phage antibodies: filamentous phage displaying antibody variable domains , 1990, Nature.

[8]  J. Devlin,et al.  Random peptide libraries: a source of specific protein binding molecules. , 1990, Science.

[9]  J. Scott,et al.  Searching for peptide ligands with an epitope library. , 1990, Science.

[10]  A. Coutinho,et al.  Selective peripheral expansion and activation of B cells expressing endogenous immunoglobulin in μ‐transgenic mice , 1990, European journal of immunology.

[11]  A. Coutinho,et al.  Expression of antibody V-regions is genetically and developmentally controlled and modulated by the B lymphocyte environment. , 1989, International immunology.

[12]  M. Kazatchkine,et al.  Anti‐idiotypes against Autoantibodies in Normal Immunoglobulins: Evidence for Network Regulation of Human Autoimmune Responses , 1989, Immunological reviews.

[13]  D. Stott,et al.  Immunoblotting and dot blotting , 1989, Journal of Immunological Methods.

[14]  A. Francoeur.  Antibody Fingerprinting: A Novel Method for Identifying Individual People and Animals , 1988, Bio/Technology.

[15]  R. Schwartz,et al.  Preferential autoantibody reactivity of the preimmune B cell repertoire in normal mice. , 1988, Journal of immunology.

[16]  J. Kearney,et al.  Idiotype‐Directed Interactions during Ontogeny Play a Major Role in the Establishment of the Adult B Cell Repertoire , 1986, Immunological reviews.

[17]  S. Avrameas,et al.  Murine Natural Monoclonal Autoantibodies: A Study of their Polyspecificities and their Affinities , 1986, Immunological reviews.

[18]  A. Coutinho,et al.  Antibody Repertoires of Normal BALB/c Mice: B Lymphocyte Populations Defined by State of Activation , 1986, Immunological reviews.

[19]  A. Coutinho,et al.  Immunocompetent autoreactive B lymphocytes are activated cycling cells in normal mice , 1986, The Journal of experimental medicine.

[20]  A. Coutinho,et al.  Lymphocyte Population Kinetics in the Mouse , 1986, Immunological reviews.

[21]  A. Coutinho,et al.  High frequency of natural autoantibodies in normal newborn mice. , 1985, Journal of immunology.

[22]  J. Kyhse-Andersen.  Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. , 1984, Journal of biochemical and biophysical methods.

[23]  H. Towbin,et al.  Immunoblotting and dot immunobinding--current status and outlook. , 1984, Journal of immunological methods.

[24]  K. Rajewsky,et al.  Mechanism of Neonatally Induced Idiotype Suppression and its Relevance for the Acquisition of Self‐tolerance , 1984, Immunological reviews.

[25]  K. Hancock,et al.  India ink staining of proteins on nitrocellulose paper. , 1983, Analytical biochemistry.

[26]  A. Coutinho,et al.  Clonal growth and maturation to immunoglobulin secretion in vitro of every growth-inducible B lymphocyte , 1977, Cell.

[27]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[28]  D. Cornblath.  Manipulating the immune system with immune globulin. , 1992, The New England journal of medicine.

[29]  S. Rusconi,et al.  Transmission and expression of a specific pair of rearranged immunoglobulin μ and κ genes in a transgenic mouse line , 1985, Nature.

[30]  G. L. Peterson.  [12] Determination of total protein , 1983 .

[31]  J. Gordon,et al.  A multidot immunobinding assay for autoimmunity and the demonstration of novel antibodies against retroviral antigens in the sera of MRL mice. , 1983, Journal of immunological methods.

[32]  G. L. Peterson.  Determination of total protein. , 1983, Methods in enzymology.