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The method of afkity labeling provides a general method for attaching a covalently bound label to the active sites of antibody and enzyme molecules. Previous studies with antibodies directed to the benzenearsonate hapten were entirely in accord with the predictions of the method. In the present studies, antibodies to the 2,4dinitrophenyl (DNP) hapten were investigated. The labeling reagent p-nitrophenyldiawnium fluoborate was found to react irreversibly with an excess of the unproteded antibody: (a) at a much more rapid rate than with the antibody whose active sites were protected with an excess of N-DNP-e-aminocaproic acid; (b) to give a product with stentially a pure azotyrosine spectrum; (c) at a rate which was first-order in the concentration of the reversible complex formed initially between the reagent and the antibody s i t s , in accord with the mechanism proposed for affinity labeling; and (d) to produce a loss of antibody binding sites corresponding closely to the number of azotyrosine groups formed. Related but less extensive studies were carried out with DNP-diazonium and p(carboxy)-benzenediazonium fluoborates and with other reagents. The results closely follow the predictions of the method, and provide strong evidence that a tyrosine residue is present in anti-DNP antibody sites. The results also indicate that there is a broad distribution of rates a t which the antibody sites are labeled, and therefore that the sites are heterogeneous. These studies provide the basis for an attempt to isolate and analyze labeled peptide

Abstract. Cell filtration is performed on affinity columns prepared by the chemical modification of large polyacrylamide beads. Affinity columns selectively bind cells that produce antihapten antibodies of corresponding specificity, while cells without the specific antihapten sites pass through freely and are recovered quantitatively in the eluate. The binding of the antihapten specific cells is inhibited by hapten. Such cell filtration affinity columns may be useful in specifically purifying cell populations with characteristic membrane-receptor proteins.The study of differentiation in the mammalian immune response, and probably in other complex developmental systems, would be facilitated greatly if it were feasible to separate cells according to the specificity of receptor proteins situated on cell membranes. Could this be done efficiently with cells of the immune system, several possibilities would arise: (1) one could examine lymphocyte populations depleted of, or selected for, a single-antibody specificity and isolated at different stages of (or before) response to a particular antigen; (2) such specific cell populations could be isolated from different lymphoid tissues and examined separately or in recombined pools, in vivo or in vitro, to clarify their role in induction of the immune response; (3) specifically purified populations of antigensensitive cells might be prepared as the first step in attempts ultimately to isolate and characterize membrane-bound receptor antibody.The use of immunoadsorbents should be uniquely suitable for such cell separations, and a few approaches toward the specific fractionation of cells on antigencoated columns have been reported.1 2 However, severe problems have been encountered, since large numbers of cells adhere nonspecifically to glass beads and other chromatographic matrices utilized up to now, and the specific lymphocytes one would like to bind selectively occur at low frequencies in any cell population.We report here the development of methods and materials for preparing affinity columns that bind cells with given antihapten antibody specificities, while permitting the free passage and virtually complete recovery of other cells. The columns are constituted from large polyacrylamide beads to which haptens have been covalently attached by several procedures, starting with chemical modifications of polyacrylamide recently worked out by Inman and Dintzis.3 In this paper. we describe the selective removal on affinity columns of antihapten 685

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