%0 Journal Article %1 citeulike:570869 %A Nieto, A. %A Gaya, A. %A Jansa, M. %A Moreno, C. %A Vives, J. %D 1984 %J Molecular Immunology %K methodology antibody kinetic %N 6 %P 537--543 %R 10.1016/0161-5890(84)90070-1 %T Direct measurement of antibody affinity distribution by hapten-inhibition enzyme immunoassay. %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=6379416 %V 21 %X A rapid, simple and reliable technique for determining the affinities of antibody subpopulations in a complex mixture is described. The principle of this method is that antigen conc can be represented as the amount of antigen immobilized on the polystyrene surface of a microwell containing a fixed vol of diluted antibody. Thus, by measuring the proportion of antibody bound to different wells coated with varying amounts of antigen, it is a straightforward matter to calculate an affinity distribution. We have verified that: (1) the amount of antigen bound to a polystyrene plate is proportional to the concn of antigen used for sensitization and follows a typical saturation curve; (2) the antibodies bound to plates sensitized with low concns of antigen are of higher affinity than those bound to plates sensitized with high concns of antigen; (3) an apparent affinity constant (aK) is defined as the reciprocal concn of free hapten required for 50% inhibition of antibody binding to immobilized antigen; (4) the aK determined by this method is in close agreement with the intrinsic affinity constant (K) measured by fluorescence quenching or the Farr assay; and (5) that during the course of immunization in vivo there is a clear shift to higher-affinity antibody subpopulations.

@article{citeulike:570869, abstract = {A rapid, simple and reliable technique for determining the affinities of antibody subpopulations in a complex mixture is described. The principle of this method is that antigen conc can be represented as the amount of antigen immobilized on the polystyrene surface of a microwell containing a fixed vol of diluted antibody. Thus, by measuring the proportion of antibody bound to different wells coated with varying amounts of antigen, it is a straightforward matter to calculate an affinity distribution. We have verified that: (1) the amount of antigen bound to a polystyrene plate is proportional to the concn of antigen used for sensitization and follows a typical saturation curve; (2) the antibodies bound to plates sensitized with low concns of antigen are of higher affinity than those bound to plates sensitized with high concns of antigen; (3) an apparent affinity constant (aK) is defined as the reciprocal concn of free hapten required for 50% inhibition of antibody binding to immobilized antigen; (4) the aK determined by this method is in close agreement with the intrinsic affinity constant (K) measured by fluorescence quenching or the Farr assay; and (5) that during the course of immunization in vivo there is a clear shift to higher-affinity antibody subpopulations.}, added-at = {2006-07-07T01:10:50.000+0200}, author = {Nieto, A. and Gaya, A. and Jansa, M. and Moreno, C. and Vives, J.}, biburl = {https://www.bibsonomy.org/bibtex/26b7c7a4dd65f8fe412b1b41a422bb487/biblio24}, citeulike-article-id = {570869}, doi = {10.1016/0161-5890(84)90070-1}, interhash = {203a5b420b3156eb968c02fb5d941e90}, intrahash = {6b7c7a4dd65f8fe412b1b41a422bb487}, issn = {0161-5890}, journal = {Molecular Immunology}, keywords = {methodology antibody kinetic}, month = {June}, number = 6, pages = {537--543}, priority = {2}, timestamp = {2006-07-07T01:10:50.000+0200}, title = {Direct measurement of antibody affinity distribution by hapten-inhibition enzyme immunoassay.}, url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve\&db=pubmed\&dopt=Abstract\&list_uids=6379416}, volume = 21, year = 1984 }