Electrophoretically Homogeneous Antibody Synthesized by Spleen Foci of Irradiated Repopulated Mice

Abstract

106 splenocytes from primed donors were injected, together with sheep erythrocytes (SRBC), into X-irradiated syngeneic mice. 8 days later the spleens were excised and cut into small fragments, keeping track of their location in the organ. Each fragment was cultured individually for 24 hr in the presence of 14C amino acids and the culture fluids were assayed for antibody activity. The antibody-producing fragments were found to be clustered in few restricted areas (foci) surrounded by negative tissue. The anti-SRBC antibody from single foci was purified by absorption on stroma followed by acid elution. Thereafter, it was subjected to electrophoresis and immunoelectrophoresis. The radioautography of the runs showed a considerable degree of homogeneity. Distinct and sharp spikes were localized in the beta and gamma region. The pattern of each focus is unique from the point of view of the number of spikes and their mobility. Eluates obtained from many pooled fragments gave a broad radioactive smear in beta-gamma region. Many foci synthesized antibody migrating as a single band. This homogeneous protein is probably the product of a clone of cells homogeneously differentiated. However, some foci producing two and probably more antibody bands were also encountered. Two interpretations of the finding can be given. Either more than one precursor may participate in the formation of a focus or a differentiation switch may occur during the clonal expansion.

The B Cell Specificity Repertoire: Its Relationship to Definable Subpopulations

“…Some confusion exists in the literature with regard to the homogeneity of antibody products obtained from splenic focn derived under different experimental conditions. Early studies on macroscx>pic spleen nodules and hemolytic foci measured the products of multiple stimulated B cells located in a single region, or measured the responses of B cells when T cells were limiting (Mekori & Feldman 1965, Trentin et al 1967, Kennedy et al 1965, Playfair et al 1965, Vann & Campbell 1970, Luzzati et al 1970, Cunningham 1969, Sterzl & Nordin 1971, Mitchell & Miller 1968. When conditions are employed such that the limiting cell for analysis is the B cell, the moncK:lonality of the splenic focus response, and the homogeneity of the antibody product, have been confirmed (Campbell 1972, Luzzati et al 1973.…”

Alacrity of Cells Engaged in the Immune Response

“…Simultaneously, attempts to demonstrate clonal responses have been made by visualizing clones in so‐called splenic foci (in cultures of spleen fragments) , by measuring antibody production in organisms with limiting number of lymphocytes in various systems related to repopulation of lethally irradiated mice .…”

Immunoglobulin Synthesis In Vitro by Cerebrospinal Fluid Cells in Patients with Meningoencephalitis of Presumed Viral Origin.

“…Stimulated lymphocytes have been shown by others (2, 5, 6, 10, II) to synthesize increased amounts of protein and immunoglobulins. Furthermore, lymph node and spleen fragments (3,4,9) from immunized rabbits synthesized immunoglobulins of restricted heterogeneity in vitro, even when the serum immunoglobulin and the immunoglobulin secreted by the entire lymph node showed a diffuse electrophoretic pattern over the entire IgG region (3).…”