Isolation of Antigen-Binding Cells From Unprimed Mice

Abstract

The time-dependent increase in the average affinity of serum antibody after immunization (1), is said to be a result of antigen-driven selection of those antibody-forming cell precursors having the highest affinity antigen receptors (2). This interpretation infers that the specificity and affinity of antigen-binding receptors on antibody-forming cell precursors are directly correlated to the serum antibody produced by progeny antibodysecreting cells.It has been found that changes in the affinity of serum antibody with time after immunization reflect changes in the population of antibody-secreting cells. By determining the concentration of antigen required to inhibit individual plaque-forming cells (PFC) assayed at various times after immunization, a time-dependent avidity increase at the antibody-secreting cell level is seen and is comparable to the increase in affinity found in serum antibody (3). Thus, a good correlation between the (intrinsic) affinity of serum antibody and the avidity of the antibody-secreting cell products has been established.In contradistinction, evidence correlating the avidity of antigen receptors on antibodysecreting cell precursors, with the avidity of antibody secreted by their progeny cells, is meager. Indirect evidence has been reported demonstrating that memory cells with high avidity antigen-binding receptors are required to give rise to high avidity antibody-secreting cells (4). However, the putatively high avidity precursors were not isolated and tested directly in these experiments.To directly d e t e r m i n e the relationship between the avidity of secreted a n t i b o d y a n d the avidity of antigen receptors on precursors of a n t i b o d y -s e c r e t i n g cells requires the isolation of precursor cells with different antigen avidities a n d m e a s u r e m e n t of the avidities of a n t i b o d y -s e c r e t i n g cells derived from these * This work supported by NIH grants GM 17367, CA 04681, and AI 08917. ~Abbreviations used in this paper: DNP-MGG, 2,4-dinitrophenyl mouse gamma globulin; ~DNP-MGG, fluorescein-conjugated DNP-MGG; FACS, fluorescence-activated cell sorter; FCS, fetal calf serum; F/P, fluorescein per protein; KLH, keyhole limpet hemocyanin; ~KLH, rhodamineconjugated KLH; PBS, phosphate-buffered saline; PFC, plaque-forming cell(s); TNP, 2, 4, 6-trinitrophenyl.

Genetic and Small Molecule Disruption of the AID/RAD51 Axis Similarly Protects Nonobese Diabetic Mice from Type 1 Diabetes through Expansion of Regulatory B Lymphocytes

“…For Aicda -/- experiments, single cell suspensions of splenocytes were first lysed with Gey's Buffer to remove red blood cells as previously described (29). For all experiments, either propidium iodide or DAPI were used for live/dead discrimination.…”

Antigen‐specific adherence of antibody‐producing hybridoma cells: Application to neural and nonneural antigens

“…Various techniques have been developed for isolation and enrichment of antigen-specific ce!ls: purification of rosette-forming cells by velocity sedimentation [3], isolation by fluorescence activated cell sorter [4,5], adsorption of specific cells to various derivatized matrices [6], hapten-gelatin gels used as adsorbents , and hapten-modified surfaces [ 15,161. Although all of these methods are based on the same antigen-antibody reaction, they either lack simplicity, demand highly sophisticated instrumentation, or provide little information on the dynamic aspects of antibody production or on the effects of various agents on cellular processes of antibody production.…”

The role of H-2-linked genes in helper T-cell function. VI. Expression of Ir genes by helper T cells.

“…The antiserum selected for this treatment had a high cytotoxic titer on H-2 b lymphoeytes and no discernable cytotoxicity on H-2 t lymphocytes. Erythrocytes were removed from splenic T-cell preparations before antiserum and complement treatment using modified Gey's solution (14). B cells and Mq~ were obtained from the spleens of TNP-LPS-primed mice by treatment with anti-T-cell serum and complement (10).…”