Immunogen structure and cell selection. I. Cellular requirements for the in vitro response to moderately haptenated forms of the solid-phase immunogen DNP-O-Bio-Gel-P.

The cellular requirements for the in vitro response to DNP-O-Bio-Gel-P were determined. Results of these experiments indicate that: 1) splenic adherent cells are required for generation of anti-hapten PFC to the solid phase immunogen; and 2) cultured nude-mouse spleen cells have far less capacity to respond to the immunogen than do cells from wild type mice. These experiments suggest that both T and B cells are required for responsiveness to the moderately haptenated form of DNP-O-Bio-Gel-P.

Isolation of Wilms' tumor antigens by chelation.

A procedure for ethylenediaminetetraacetate extraction of minced Wilm's tumor was assessed as a method for isolating Wilm's tumor antigens. An antigen was detected by immunodiffusion using an adsorbed antiserum to this extract. This antigen was also found in ethylenediaminetetraacetate extracts of in vitro cultures of nephroblastoma cells.

In vitro response of mouse spleen cells to the solid phase immunogen DNP-O-Bio-Gel.

The in virto immunogenicity of the solid-phase hapten, dinitrophenyl-ornithine-Bio-Gel (DNP-O-Bio-Gel), was investigated in cultures of mouse spleen cells. Appropriate combinations of cells and immobilized hapten were determined. Large numbers of direct anti-hapten plaque-forming cells (PFC) were generated when 1 times 10-7 C57BL/6 or C57BL/10 spleen cells were cultured with 4 times 10-3 DNP-O-Bio-Gel beads. Specificity studies of the responses of cultured spleen cells to DNP-O-Bio-Gel yielded the following results: soluble DNP-ornithine or DNP-bovine gamma-globulin inhibited the induction of anti-hapten PFC by DNP-O-Bio-Gel; neither dinitrophenyl-Bio-gel (DNP-Bio-gel) nor ornithine-Bio-Gel (O-Bio-Gel) induced anti-hapten responsiveness; furthermore, neither DNP-Bio-Gel nor O-Bio-Gel inhibited the induction of PFC by DNP-O-Bio-Gel. It was concluded, from the results of these specificity experiments, that a spacer, ornithine, is required for immunogenicity of immobilized DNP; and that the Bio-Gel bead, itself, acts solely as a physical carrier for the hapten.

Electrophoretically homogeneous anti-DNP antibodies with restricted isoelectric points elicited in mice by immunization with the antigen papain-S-DNPL.

The primary immunization of outbred mice with the antigen Pap-S-DNPL results in the generation of low titers of anti-DNP antibody which in about one-third of the responding animals is as homogeneous as a myeloma protein by the combined criteria of (a) isoelectric focusing in gels and (b) gel electrophoresis of the antibody light chains. The electrophoretically homogeneous antibodies show a marked restriction of isoelectric points near pH 5.0. Such marked selectivity of the antihapten antibodies appears to result from the chemical and structural homogeneity of the Pap-S-DNPL antigen.