Estimation of the relative affinity of B cell receptor by flow cytometry.

We have developed a simple method using flow cytometry to estimate the relative affinity of B cell receptor (BCR) possessing the hapten-binding activity. Bovine serum albumin (BSA) was conjugated with a hapten, (4-hydroxy-3-nitrophenyl)acetyl (NP) and biotin (NP-BSA-bio). The interaction between NP-BSA-bio and anti-NP monoclonal antibodies (mAb) was studied as a model of the BCR reaction by surface plasmon resonance (SPR) using a biosensor chip immobilized with mAbs through anti-Fc antibody (Ab). The relative affinity of these mAbs was estimated on the basis of resonance units for the binding of NP(0.5)-BSA-bio(21) relative to that of NP(7.4)-BSA-bio(21) expressed as a ratio (NP(0.5)-BSA-bio(21)/NP(7.4)-BSA-bio(21)). In combination with streptavidin (SA)-R-phycoerythrin (PE), we measured the binding of NP-BSA-bio to BCR by flow cytometry and found that a high number of biotin molecules was necessary to improve the sensitivity of detection of the bound NP-BSA-bio without steric hindrance in the NP-BCR interaction. We demonstrated that the ratio of the mean fluorescence intensity (MFI) of NP(0.5)-BSA-bio(21)/NP(7.4)-BSA-bio(21) at a concentration of 10(-8) M could be used as a practical measure of the affinity. This method is expected to be useful for the study of affinity maturation on the cellular level.

[1]  T. Tokuhisa,et al.  Memory B cells at successive stages of differentiation. Affinity maturation and the role of IgD receptors , 1980, The Journal of experimental medicine.

[2]  D. Tarlinton,et al.  Kinetics of establishing the memory B cell population as revealed by CD38 expression. , 1998, Journal of immunology.

[3]  M. Reth,et al.  Membrane IgM and IgD molecules fail to transduce Ca2+ mobilizing signals when expressed on differentiated B lineage cells. , 1990, Journal of immunology.

[4]  H. Nakamura,et al.  Junctional amino acids determine the maturation pathway of an antibody. , 1999, Immunity.

[5]  A Cumano,et al.  Evolutionary and somatic selection of the antibody repertoire in the mouse. , 1987, Science.

[6]  R. Holmdahl,et al.  Binding of autoreactive mouse anti-type II collagen antibodies derived from the primary and the secondary immune response investigated with the biosensor technique. , 1995, Journal of immunological methods.

[7]  K. Rajewsky,et al.  Human Immunoglobulin (Ig)M+IgD+ Peripheral Blood B Cells Expressing the CD27 Cell Surface Antigen Carry Somatically Mutated Variable Region Genes: CD27 as a General Marker for Somatically Mutated (Memory) B Cells , 1998, The Journal of experimental medicine.

[8]  T. Honjo,et al.  Defects of somatic hypermutation and class switching in alymphoplasia (aly) mutant mice. , 1996, International immunology.

[9]  I. Maclennan,et al.  Maturation and Dispersal of B‐Cell Clones during T Cell‐Dependent Antibody Responses , 1992, Immunological reviews.

[10]  D. Baltimore,et al.  Heavy chain variable region contribution to the NPb family of antibodies: somatic mutation evident in a γ2a variable region , 1981, Cell.

[11]  Paddy Tigme.  Immunoglobulin Genes. 2nd edn , 1997 .

[12]  Leroy Hood,et al.  IgG antibodies to phosphorylcholine exhibit more diversity than their IgM counterparts , 1981, Nature.

[13]  D. Allen,et al.  Antibody engineering for the analysis of affinity maturation of an anti‐hapten response. , 1988, The EMBO journal.

[14]  C. Milstein,et al.  Somatic mutation and the maturation of immune response to 2-phenyl oxazolone , 1984, Nature.

[15]  R. Zinkernagel,et al.  Neutralizing antiviral B cell responses. , 1997, Annual review of immunology.

[16]  T. Manser,et al.  Somatic evolution of variable region structures during an immune response. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[17]  K. Rajewsky,et al.  Idiotypic analysis of the response of C57BL/6 mice to the (4‐hydroxy‐3‐nitrophenyl)acetyl group , 1977, European journal of immunology.

[18]  D. Stetson,et al.  Development and Maintenance of a B220− Memory B Cell Compartment1 , 2001, The Journal of Immunology.

[19]  I. Maclennan,et al.  Germinal centres in T-cell-dependent antibody responses. , 1992, Immunology today.

[20]  A. Cumano,et al.  Clonal recruitment and somatic mutation in the generation of immunological memory to the hapten NP. , 1986, The EMBO journal.

[21]  K. Rajewsky,et al.  Bcl-2 increases memory B cell recruitment but does not perturb selection in germinal centers. , 1994, Immunity.

[22]  G. Kelsoe.  Life and death in germinal centers (redux). , 1996, Immunity.

[23]  C. Milstein,et al.  3 – The maturation of the antibody response , 1995 .

[24]  N. Motoyama,et al.  Role of conserved amino acid residues in the complementarity determining regions on hapten-antibody interaction of anti-(4-hydroxy-3-nitrophenyl) acetyl antibodies. , 1995, Molecular immunology.

[25]  M. Reth,et al.  Molecular components of the B-cell antigen receptor complex of the IgM class , 1990, Nature.

[26]  Y. Adachi,et al.  Accumulation of somatic hypermutation and antigen‐driven selection in rapidly cycling surface Ig+ germinal center (GC) B cells which occupy GC at a high frequency during the primary antihapten response in mice , 1997, European journal of immunology.

[27]  Klaus Rajewsky,et al.  Somatic hypermutation in normal and transformed human B cells , 1998, Immunological reviews.

[28]  C. Arpin,et al.  Germinal center development , 1997, Immunological reviews.

[29]  L. McHeyzer-Williams,et al.  Antigen-Specific B Cell Memory , 2000, The Journal of experimental medicine.

[30]  A. Cumano,et al.  Structure of primary anti‐(4‐hydroxy‐3‐nitro‐phenyl)acetyl (NP) antibodies in normal and idiotypically suppressed C57BL/6 mice , 1985, European journal of immunology.

[31]  R. Noelle,et al.  Identification of Murine Germinal Center B Cell Subsets Defined by the Expression of Surface Isotypes and Differentiation Antigens1 , 2000, The Journal of Immunology.

[32]  R. Geha,et al.  Affinity maturation without germinal centres in lymphotoxin-α-deficient mice , 1996, Nature.

[33]  A. Prescott,et al.  Antigen endocytosis and presentation mediated by human membrane IgG1 in the absence of the Igα/Igβ dimer , 1997 .

[34]  C. Berek.  Somatic mutation and memory. , 1993, Current opinion in immunology.

[35]  K. Rajewsky.  Clonal selection and learning in the antibody system , 1996, Nature.

[36]  M. Neuberger,et al.  The B-cell antigen receptor of the five immunoglobulin classes , 1991, Nature.

[37]  M. Oda,et al.  Reevaluation of stoichiometry and affinity/avidity in interactions between anti-hapten antibodies and mono- or multi-valent antigens. , 2000, Molecular immunology.